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Identification of Extracellular and Intracellular Signaling Components of the Mammary Adipose Tissue and Its Interstitial Fluid in High Risk Breast Cancer Patients

Toward Dissecting The Molecular Circuitry of Epithelial-Adipocyte Stromal Cell Interactions*
  • Julio E. Celis
    Correspondence
    To whom correspondence should be addressed. Tel.: 45-35-25-73-63; Fax: 45-35-25-77-55
    Affiliations
    Department of Proteomics in Cancer, Institute of Cancer Biology, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • José M.A. Moreira
    Affiliations
    Department of Proteomics in Cancer, Institute of Cancer Biology, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • Teresa Cabezón
    Affiliations
    Department of Proteomics in Cancer, Institute of Cancer Biology, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • Pavel Gromov
    Affiliations
    Department of Proteomics in Cancer, Institute of Cancer Biology, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • Esbern Friis
    Affiliations
    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Department of Breast and Endocrine Surgery, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • Fritz Rank
    Affiliations
    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Department of Pathology, The Centre of Diagnostic Investigations, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • Irina Gromova
    Affiliations
    Department of Proteomics in Cancer, Institute of Cancer Biology, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark

    Danish Centre for Translational Breast Cancer Research, Danish Cancer Society, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
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  • Author Footnotes
    * This work was supported by the Danish Cancer Society through the budget of the Institute of Cancer Biology and by grants from the Danish Medical Research Council, the Natural and Medical Sciences Committee of the Danish Cancer Society, The Novo Research Foundation, and the John and Birthe Meyer Foundation. Support was also received from the Marketing Department at the Danish Cancer Society.
      It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease.
      During the last years there have been numerous reports indicating that growth and progression of breast as well as other tumor cells depend not only on their malignant potential but also on stromal factors present in the tumor microenvironment, the insoluble extracellular matrix as well as cell-cell interactions (Refs.
      • Wiseman B.S.
      • Werb Z.
      Stromal effects on mammary gland development and breast cancer.
      ,
      • Silberstein G.B.
      Tumour-stromal interactions. Role of the stroma in mammary development.
      ,
      • Coussens L.M.
      • Werb Z.
      Matrix metalloproteinases and the development of cancer.
      ,
      • Coussens L.M.
      • Werb Z.
      Inflammatory cells and cancer: think different!.
      ,
      • Fidler I.J.
      Regulation of neoplastic angiogenesis.
      ,
      • Fidler I.J.
      Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment.
      and references therein). Of all the cell types present in the microenvironment, which include endothelial cells and their supporting pericytes, inflammatory cells (neutrophils, macrophages, eosinophils, and mast cells), immune cells (lymphocytes and dendritic cells), smooth muscle cells, myofibroblasts, preadipocytes, and adipocytes, the last are perhaps the least well studied despite the fact that they correspond to one of the most prominent cell types surrounding the breast tumor cells (
      • Iyengar P.
      • Combs T.P.
      • Shah S.J.
      • Gouon-Evans V.
      • Pollard J.W.
      • Albanese C.
      • Flanagan L.
      • Tenniswood M.P.
      • Guha C.
      • Lisanti M.P.
      • Pestell R.G.
      • Scherer P.E.
      Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
      ).
      Until recently, adipocytes were mainly considered as an energy storage depot, but we now have clear evidence pointing to the fat tissue as an endocrine organ that produces hormones, growth factors, adipokines, and other molecules that may affect normal duct development as well as tumor growth and metastasis (Refs.
      • Howlett A.R.
      • Bissell M.J.
      The influence of tissue microenvironment (stroma and extracellular matrix) on the development and function of mammary epithelium.
      ,
      • Zangani D.
      • Darcy K.M.
      • Shoemaker S.
      • Ip M.M.
      Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells.
      ,
      • Huss F.R.
      • Kratz G.
      Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue.
      ,
      • Scherer P.E.
      • Williams S.
      • Fogliano M.
      • Baldini G.
      • Lodish H.F.
      A novel serum protein similar to C1q, produced exclusively in adipocytes.
      ,
      • Scherer P.E.
      • Bickel P.E.
      • Kotler M.
      • Lodish H.F.
      Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.
      ,
      • Bickel P.E.
      • Lodish H.F.
      • Scherer P.E.
      Use and applications of subtractive antibody screening.
      ,
      • Engelman J.A.
      • Berg A.H.
      • Lewis R.Y.
      • Lisanti M.P.
      • Scherer P.E.
      Tumor necrosis factor α-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3-L1 adipocytes.
      ,
      • Berg A.H.
      • Combs T.P.
      • Du X.
      • Brownlee M.
      • Scherer P.E.
      The adipocyte-secreted protein Acrp30 enhances hepatic insulin action.
      ,
      • Cancello R.
      • Tounian A.
      • Poitou Ch.
      • Clement K.
      Adiposity signals, genetic and body weight regulation in humans.
      ,
      • Mora S.
      • Pessin J.E.
      An adipocentric view of signaling and intracellular trafficking.
      ,
      • Trayhurn P.
      • Wood I.S.
      Adipokines: inflammation and the pleiotropic role of white adipose tissue.
      and references therein). It has been shown that normal mouse mammary ducts do not form correctly if there is no proper interaction with the fat tissue, and a number of signaling pathways that may be involved in this process have been identified (
      • Howlett A.R.
      • Bissell M.J.
      The influence of tissue microenvironment (stroma and extracellular matrix) on the development and function of mammary epithelium.
      ,
      • Zangani D.
      • Darcy K.M.
      • Shoemaker S.
      • Ip M.M.
      Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells.
      ,
      • Huss F.R.
      • Kratz G.
      Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue.
      ). Elliot and colleagues (
      • Elliott B.E.
      • Tam S.P.
      • Dexter D.
      • Chen Z.Q.
      Capacity of adipose tissue to promote growth and metastasis of a murine mammary carcinoma: effect of estrogen and progesterone.
      ), on the other hand, showed that fat tissue is able to augment the growth and ability to metastasize of the murine mammary carcinoma cell line SP1 when injected subcutaneously or peritoneally far away from fat pads, and Iyengar and colleagues (
      • Iyengar P.
      • Combs T.P.
      • Shah S.J.
      • Gouon-Evans V.
      • Pollard J.W.
      • Albanese C.
      • Flanagan L.
      • Tenniswood M.P.
      • Guha C.
      • Lisanti M.P.
      • Pestell R.G.
      • Scherer P.E.
      Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
      ) reported that factors secreted by adipocytes promote mammary tumorigenesis through induction of antiapoptotic transcriptional programs and proto-oncogene stabilization. In addition, several reports have demonstrated an association between breast cancer growth and the presence of adipose tissue (
      • Chamras H.
      • Bagga D.
      • Elstner E.
      • Setoodeh K.
      • Koeffler H.P.
      • Heber D.
      Preadipocytes stimulate breast cancer cell growth.
      ,
      • Manabe Y.
      • Toda S.
      • Miyazaki K.
      • Sugihara H.
      Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions.
      ,
      • Johnston P.G.
      • Rondinone C.M.
      • Voeller D.
      • Allegra C.J.
      Identification of a protein factor secreted by 3T3-L1 preadipocytes inhibitory for the human MCF-7 breast cancer cell line.
      ), and a connection between obesity and increased incidence of cancer has been established for breast, colorectal, endometrial, renal (renal cell), and esophageal (adenocarcinoma) malignancies (Ref.
      • Calle E.E.
      • Thun M.J.
      Obesity and cancer.
      and references therein). Presently, however, there is only limited information as to the factors produced by adipocytes that may affect normal breast duct development and tumor progression (
      • Wiseman B.S.
      • Werb Z.
      Stromal effects on mammary gland development and breast cancer.
      ,
      • Iyengar P.
      • Combs T.P.
      • Shah S.J.
      • Gouon-Evans V.
      • Pollard J.W.
      • Albanese C.
      • Flanagan L.
      • Tenniswood M.P.
      • Guha C.
      • Lisanti M.P.
      • Pestell R.G.
      • Scherer P.E.
      Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
      ,
      • Kratchmarova I.
      • Kalume D.E.
      • Blagoev B.
      • Scherer P.E.
      • Podtelejnikov A.V.
      • Molina H.
      • Bickel P.E.
      • Andersen J.S.
      • Fernandez M.M.
      • Bunkenborg J.
      • Roepstorff P.
      • Kristiansen K.
      • Lodish H.F.
      • Mann M.
      • Pandey A.
      A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.
      ).
      Most studies of adipogenesis have made use of rodent cells or primary cultures of human mesenchymal stem cells that have been induced to differentiate into adipocytes using a variety of effectors. By using cDNA microarrays (
      • Hung S.C.
      • Chang C.F.
      • Ma H.L.
      • Chen T.H.
      • Low-Tone Ho L.
      Gene expression profiles of early adipogenesis in human mesenchymal stem cells.
      ,
      • Guo X.
      • Liao K.
      Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation.
      ,
      • Burton G.R
      • Guan Y.
      • Nagarajan R.
      • McGehee Jr, R.E.
      Microarray analysis of gene expression during early adipocyte differentiation.
      ,
      • Urs S.
      • Smith C.
      • Campbell B.
      • Saxton A.M.
      • Taylor J.
      • Zhang B.
      • Snoddy J.
      • Jones Voy B.
      • Moustaid-Moussa N.
      Gene expression profiling in human preadipocytes and adipocytes by microarray analysis.
      ,
      • Boeuf S.
      • Klingenspor M.
      • Van Hal N.L.
      • Schneider T.
      • Keijer J.
      • Klaus S.
      Differential gene expression in white and brown preadipocytes.
      ,
      • Sottile V.
      • Seuwen K.
      A high-capacity screen for adipogenic differentiation.
      ,
      • Albrektsen T.
      • Richter H.E.
      • Clausen J.T.
      • Fleckner J.
      Identification of a novel integral plasma membrane protein induced during adipocyte differentiation.
      ,
      • Soukas A.
      • Socci N.D.
      • Saatkamp B.D.
      • Novelli S.
      • Friedman J.M.
      Distinct transcriptional profiles of adipogenesis in vivo and in vitro.
      ) and proteomic technologies (Refs.
      • Kratchmarova I.
      • Kalume D.E.
      • Blagoev B.
      • Scherer P.E.
      • Podtelejnikov A.V.
      • Molina H.
      • Bickel P.E.
      • Andersen J.S.
      • Fernandez M.M.
      • Bunkenborg J.
      • Roepstorff P.
      • Kristiansen K.
      • Lodish H.F.
      • Mann M.
      • Pandey A.
      A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.
      and
      • Brasaemle D.L.
      • Dolios G.
      • Shapiro L.
      • Wang R.
      Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.
      and references therein), it has been possible to identify several genes and proteins that are differentially regulated as a result of adipogenesis. These studies have been inspired by the facts that increased adiposity and a failure in adipocyte differentiation are associated with morbidity, mortality, and many disorders, including obesity, which has a strong association with type 2 diabetes (
      • Danforth Jr, E.
      Failure of adipocyte differentiation causes type II diabetes mellitus?.
      ,
      • Cederberg A.
      • Enerback S.
      Insulin resistance and type 2 diabetes—an adipocentric view.
      ), hypertension, and coronary heart disease (
      • Kopelman P.G.
      Obesity as a medical problem.
      ). The question remains, however, as to whether these experimental model systems are able to completely replicate the in vivo situation (Ref.
      • Klaus S.
      Adipose tissue as a regulator of energy balance.
      and references therein) as it has been shown that gene expression changes associated with adipogenesis in vivo and in vitro, while sharing many features in common, are in some respects rather different (
      • Soukas A.
      • Socci N.D.
      • Saatkamp B.D.
      • Novelli S.
      • Friedman J.M.
      Distinct transcriptional profiles of adipogenesis in vivo and in vitro.
      ).
      In vivo transcript profiling studies of human and murine fat tissue (
      • Urs S.
      • Smith C.
      • Campbell B.
      • Saxton A.M.
      • Taylor J.
      • Zhang B.
      • Snoddy J.
      • Jones Voy B.
      • Moustaid-Moussa N.
      Gene expression profiling in human preadipocytes and adipocytes by microarray analysis.
      ,
      • Soukas A.
      • Socci N.D.
      • Saatkamp B.D.
      • Novelli S.
      • Friedman J.M.
      Distinct transcriptional profiles of adipogenesis in vivo and in vitro.
      ,
      • Gabrielsson B.L.
      • Carlsson B.
      • Carlsson L.M.
      Partial genome scale analysis of gene expression in human adipose tissue using DNA array.
      ) have shown the complexity of the adipocyte transcriptome and have implicitly established that the biology of these cells has a degree of intricacy that was not expected. To date, however, only two studies have investigated the proteome of human and mouse adipose tissue; one resolved about 100 human proteins using wide IPG strips and identified 16 polypeptides by means of mass spectrometry (
      • Corton M.
      • Villuendas G.
      • Botella J.I.
      • San Millan J.L.
      • Escobar-Morreale H.F.
      • Peral B.
      Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide.
      ), while the other resolved a considerable number of murine white adipose tissue proteins and identified 80 unique polypeptides (
      • Lanne B.
      • Potthast F.
      • Hoglund A.
      • Brockenhuus von Lowenhielm H.
      • Nystrom A.C.
      • Nilsson F.
      • Dahllof B.
      Thiourea enhances mapping of the proteome from murine white adipose tissue.
      ).
      In our translational breast cancer program, which involves high risk patients that have undergone mastectomy (
      • Celis J.E.
      • Gromov P.
      • Gromova I.
      • Moreira J.M.
      • Cabezon T.
      • Ambartsumian N.
      • Grigorian M.
      • Lukanidin E.
      • Thor Straten P.
      • Guldberg P.
      • Bartkova J.
      • Bartek J.
      • Lukas J.
      • Lukas C.
      • Lykkesfeldt A.
      • Jaattela M.
      • Roepstorff P.
      • Bolund L.
      • Orntoft T.
      • Brunner N.
      • Overgaard J.
      • Sandelin K.
      • Blichert-Toft M.
      • Mouridsen H.
      • Rank F.E.
      Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research.
      ,
      • Celis J.E.
      • Gromov P.
      • Cabezon T.
      • Moreira J.M.
      • Ambartsumian N.
      • Sandelin K.
      • Rank F.
      • Gromova I.
      Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery.
      ,
      • Celis J.E.
      • Moreira J.M.
      • Gromova I.
      • Cabezon T.
      • Ralfkiaer U.
      • Guldberg P.
      • Straten P.T.
      • Mouridsen H.
      • Friis E.
      • Holm D.
      • Rank F.
      • Gromov P.
      Towards discovery-driven translational research in breast cancer.
      ), we have frequently detected tumor cells interdigitating with and spreading through the peripheral fat tissue suggesting a close association between these two cell types (Fig. 1) (
      • Iyengar P.
      • Combs T.P.
      • Shah S.J.
      • Gouon-Evans V.
      • Pollard J.W.
      • Albanese C.
      • Flanagan L.
      • Tenniswood M.P.
      • Guha C.
      • Lisanti M.P.
      • Pestell R.G.
      • Scherer P.E.
      Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
      ). This observation together with published data demonstrating a role for the fat tissue in mammary gland development (Refs.
      • Wiseman B.S.
      • Werb Z.
      Stromal effects on mammary gland development and breast cancer.
      and
      • Schmeichel K.L.
      • Weaver V.M.
      • Bissell M.J.
      Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype.
      and references therein) and in modulating tumor behavior (
      • Coussens L.M.
      • Werb Z.
      Matrix metalloproteinases and the development of cancer.
      ,
      • Hansen R.K.
      • Bissell M.J.
      Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones.
      ,
      • Park C.C.
      • Bissell M.J.
      • Barcellos-Hoff M.H.
      The influence of the microenvironment on the malignant phenotype.
      ,
      • Matrisian L.M.
      • Cunha G.R.
      • Mohla S.
      Epithelial-stromal interactions and tumor progression: meeting summary and future directions.
      ) prompted us to carry out a detailed proteomic analysis of fresh fat tissue and its interstitial fluid in an attempt to identify protein components and excreted factors that may shed some light on the close association between mammary epithelia and fat tissue. In the first instance and to simplify the study, we chose to analyze fat tissue located topologically distant from the tumor in high risk breast cancer patients registered at the Department of Breast and Endocrine Surgery, Copenhagen University Hospital.
      Figure thumbnail gr1
      Fig. 1Indirect immunofluorescence analysis of fat tissue peripheral to a breast tumor stained with A-FABP- (Alexa Fluor 488; green) and keratin 19 (Alexa Fluor 594; red )-specific antibodies.

      EXPERIMENTAL PROCEDURES

       Sample Collection and Handling

      Fat tissue biopsies from sites topologically distant from the tumor (more than 4–5 cm away; Fig. 2A) of high risk patients
      The criteria for high risk cancer applied by Danish Cooperative Breast Cancer Group are age below 35 years old, and/or tumor diameter of more than 20 mm, and/or histological malignancy grade 2 or 3, and/or negative estrogen and progesterone receptor status, and/or positive axillary status. Patients received no treatment prior to surgery.
      that underwent mastectomy were collected from the Pathology Department at the Copenhagen University Hospital 30–45 min after surgery. Samples for gel analysis were placed in liquid nitrogen and were rapidly transported to the Institute of Cancer Biology where they were stored at −80 °C. Samples for fluid recovery were placed in PBS, transported on ice, and processed immediately upon arrival at the Institute. On average, a total of 45 min to 1 h elapsed between surgical sample acquisition and sample preparation. The project was approved by the Scientific and Ethical Committee of the Copenhagen and Frederiksberg Municipalities (KF 01-069/03).
      Figure thumbnail gr2
      Fig. 2A, whole breast from a mastectomized high risk patient showing fat tissue topologically distant from the tumor. B, fat tissue harvested topologically distant from the tumor stained with A-FABP- (Alexa Fluor 488; green) and macrophage-specific CD68 antigen antibodies (Alexa Fluor 594; red).

       Fat Interstitial Fluid

      About 0.50 g of clean fresh fat tissue was cut to small pieces (3 mm3), dipped into 5 ml of PBS, and placed in a 10-ml conical plastic tube containing 1.0 ml of PBS. Samples were incubated for 1 h at 37 °C in a humidified CO2 incubator. Thereafter the samples were centrifuged at 1000 rpm for 2 min, and the supernatant was aspirated with the aid of an elongated Pasteur pipette. Samples were further centrifuged at 5000 rpm for 20 min in a refrigerated centrifuge (4 °C). A fraction of the fat interstitial fluid (FIF)
      The abbreviations used are: FIF, fat interstitial fluid; 2D, two-dimensional; A-FABP, adipocyte fatty acid-binding protein; TIF, tumor interstitial fluid; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; IGF, insulin-like growth factor; CSF, colony-stimulating factor; TIMP, tissue inhibitor of metalloproteinases; MAPK, mitogen-activated protein kinase; MDM2, mouse double minute 2; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; CBP, cAMP-response element-binding protein (CREB)-binding protein; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.
      (0.8 ml) was kept at −20 °C for antibody array-based analysis, while the rest was freeze-dried and resuspended in 0.5 ml of O'Farrell lysis solution and kept at −20 °C until use (
      • O’Farrell P.H.
      High resolution two-dimensional electrophoresis of proteins.
      ).

       Two-dimensional Gel Electrophoresis and Western Immunoblotting

      Twenty to thirty 6-μm cryostat sections of frozen fat tissue were resuspended in 0.1 ml of CBL1 lysis solution (Zeptosens AG, Witterswil, Switzerland) and were kept at −20 °C until use. In a few cases the sections were resuspended in 0.1 ml of O'Farrell lysis solution (
      • O’Farrell P.H.
      High resolution two-dimensional electrophoresis of proteins.
      ) with similar results. The advantages of the CBL1 lysis solution are that it yields better focused spots, and it does not dry so easily after prolonged storage. A detailed protocol of its use in the study of tumor tissue biopsies and cell lines will be the subject of a further publication.
      P. Gromov, I. Gromova, and J. E. Celis, unpublished data.
      Fat lysates and freeze-dried fluids resuspended in lysis solution were subjected to both IEF and NEPHGE two-dimensional (2D) PAGE as described previously (
      • Celis J.E.
      • Trentem⊘lle S.
      • Gromov P.
      ). Between 30 and 40 μl of sample were applied to the first dimension. Proteins were visualized using a silver staining procedure compatible with mass spectrometry analysis (
      • Gromova I.
      • Celis J.E.
      ). Gels were dried between two pieces of cellophane. Western immunoblotting was performed as described previously (
      • Celis J.E.
      • Gromov P.
      High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.
      ).

       Protein Identification by Mass Spectrometry

       In-gel Digestion Protocol—

      Protein bands were excised from the dry gels followed by rehydration in water for 30 min at room temperature. The gel pieces were detached from the cellophane film, rinsed twice with water, and cut into about 1-mm2 pieces with subsequent additional washes. Proteins were “in-gel” digested with bovine trypsin (unmodified, sequencing grade; Roche Diagnostics) for 8 h as described by Shevchenko and colleagues (
      • Shevchenko A.
      • Wilm M.
      • Vorm O.
      • Mann M.
      Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
      ). The reaction was stopped by adding TFA (up to 0.4%) followed by shaking for 20 min at room temperature to increase peptide recovery. In most cases, peptides were analyzed using the supernatant. In the few cases where the amount of peptides was too low or when no conclusive identification was achieved by peptide fingerprinting using the supernatant, the remaining amount of supernatant (approximately 10 μl) as well as the peptides additionally extracted from the gel pieces with 1% TFA and 50% ACN were concentrated on micro-ZipTip μ-C18 columns in accordance with the manufacturer's protocol (Millipore). Peptides were eluted from the column with 50% ACN, 0.2% TFA directly on the target and co-crystallized with α-cyano matrix (2 mg/ml cyano-4-hydroxycinnamic acid in 0.5% TFA/ACN, 1:2, v/v). The extraction procedure strongly increased the amount of peptides, thus allowing direct sequence analysis of low intensity peptides.

       Probe Preparation and Acquisition of the MALDI-TOF Spectra—

      Samples were prepared for analysis by applying 0.8 μl of digested supernatant or microcolumn-eluted material on the surface of a 400/384 AnchorChip target (Bruker Daltonik, GmbH) followed by co-crystallization with 0.3 μl of α-cyano matrix. After drying, the droplets were washed twice with 0.5% TFA to remove contamination from the samples.
      Mass spectrometry was performed using a Reflex IV MALDI-TOF mass spectrometer equipped with a Scout 384 ion source. All spectra were obtained in positive reflector mode with delayed extraction using an accelerating voltage of 28 kV. Each spectrum represented an average of 100–200 laser shots, depending on the signal-to-noise ratio. The resulting mass spectra were internally calibrated by using the autodigested tryptic mass values (805.417/906.505/1153.574/1433.721/2163.057/2273.160) visible in all spectra. Calibrated spectra were processed by the Xmass 5.1.1 and BioTools 2.1 software packages (Bruker Daltonik, GmbH). All spectra were analyzed manually.
      Spectra originating from parallel protein digestions were compared pairwise to discard common peaks derived either from trypsin autodigestion or from contamination with keratins. Only unique peptides present in the spectra were used in the first search. Data base searching was performed against a comprehensive non-redundant data base using MASCOT 1.8 software (
      • Perkins D.N.
      • Pappin D.J.
      • Creasy D.M.
      • Cottrell J.S.
      Probability-based protein identification by searching sequence databases using mass spectrometry data.
      ) without restriction on the protein molecular mass and taxonomy. Since proteins were recovered from gels, a number of fixed modifications (acrylamide-modified cysteine, i.e. propionamide/carbamidomethylation) as well as variable ones (methionine oxidation and protein NH2 terminus acetylation) were included in the search parameters. The peptide tolerance did not exceed 50 ppm, and as a maximum only one trypsin missed cleavage was allowed. The protein identifications were considered to be confident when the protein score of the hit exceeded the threshold significance score of 70 (p < 0.05) and not less than six peptides were recognized. The data base was checked for redundancy, and whenever it was possible the Swiss-Prot accession numbers were assigned. Additionally peptide mass fingerprinting analysis was performed using the MS-Fit program (Protein Prospector, University of California San Francisco Mass Spectrometry Facility, London, UK). We also used the Find-Mod software (Protein Prospector, University of California San Francisco Mass Spectrometry Facility, London, UK) to check any unmatched peptides for potential protein post-translational modifications. The second search was performed for all identifications as follows. 1) The predicted peptide digest was compared with the experimental one to reveal additional peptides present within the spectra; 2) the unmatched molecular weight values from the initial search were applied for extra search with the same reproducibility requirements for identification of the second and the third components in the spot. Whenever the protein score hit was close to the threshold significance score of 70, the PSD was performed as an additional means to confirm the identity of the proteins identified by post-translational modifications. The following PSD search parameters were used: peptide tolerance, 50 ppm; MS/MS tolerance, 1 Da without any restriction on the protein molecular mass and taxonomy. Since the amount of peptides extracted from the silver-stained gels did not yield overall peak intensities high enough to allow multiple peptide sequencing (prerequirement for conclusive PSD analysis), the identification of proteins was never made solely based on PSD analysis. The molecular weight and pI of the identified proteins were evaluated by analysis of mobility of the corresponding protein band in the 2D gel images. Positive protein identification was achieved in 80% of the cases with average sequence coverage of ∼33%.

       Preparation of Triton X-100 Extracts of Fat Tissue

      Fresh fat tissue cut in small pieces (about 0.5 g) was homogenized with 3 ml of 0.1% Triton X-100 in PBS for 3 min at room temperature (
      • Small J.V.
      • Celis J.E.
      Direct visualization of the 10-nm (100-Å)-filament network in whole and enucleated cultured cells.
      ). Thereafter the samples were centrifuged at 1000 rpm for 2 min, and the supernatant was aspirated with the aid of an elongated Pasteur pipette. Samples were further centrifuged at 5000 rpm for 20 min in a refrigerated centrifuge (4 °C). A fraction of the supernatant was kept at −20 °C for antibody array-based analysis, whereas the rest was freeze-dried and resuspended in lysis solution for 2D PAGE analysis (
      • O’Farrell P.H.
      High resolution two-dimensional electrophoresis of proteins.
      ,
      • Celis J.E.
      • Trentem⊘lle S.
      • Gromov P.
      ).

       Antibody Arrays for the Detection of Multiple Cytokines

      Cytokines present in FIFs were detected using the RayBio® Human Cytokine Array C Series (RayBiotech, Inc.). Each array was incubated with 0.25 ml of FIF at 4 °C overnight, and bound antigens were detected according to the manufacturer's instructions. The sensitivity of the antibodies present in the arrays ranges from 1–2000 pg/ml (for further details see www.raybiotech.com/human_array_sensitivity.pdf).

       Antibody Array-based Identification of Key Cellular Effectors and Signaling Molecules

      The relative level of cellular effectors and signaling molecules present in Triton X-100 fat tissue extracts were determined using the Panorama™ Ab Microarray-Cell Signaling array (Sigma). This array contains 224 different antibodies each spotted in two equal concentrations on nitrocellulose-coated glass slides. These antibodies represent several biological pathways including apoptosis, cell cycle, and signal transduction. Binding to a cognate antibody was detected by directly labeling the proteins in the cell extracts with a fluorescent dye according to the manufacturer's instructions.

       Antibodies

      Anti-peptide antibodies against the adipocyte fatty acid-binding protein (A-FABP) were prepared by Eurogentec. Specific antibodies recognizing Nck adaptor protein, Crk proto-oncogene, and the dual specificity MEK-2 were purchased from BD Transduction Laboratories. The monoclonal antibody (mAB 22-II-D8B), which recognizes protein 14-3-3 β and ζ, has been described previously (
      • Celis A.
      • Madsen P.
      • Nielsen H.V.
      • Rasmussen H.H.
      • Thiessen H.
      • Lauridsen J.B.
      • van Deurs B.
      • Celis J.E.
      Human proteins IEF 58 and 57a are associated with the Golgi apparatus.
      ). Specific antibodies recognizing macrophages (CD68) and vimentin were purchased from Dako Corp.

       Indirect Immunofluorescence

      Fresh tumors containing marginal fat tissue were placed in formalin fixative and paraffin-embedded for archival use. Five-micrometer sections were cut from paraffin blocks of breast tumor and fat tissue, mounted on Super Frost Plus slides (Menzel-Gläser, Braunschweig, Germany), baked at 60 °C for 60 min, deparaffinized, and rehydrated through graded alcohol rinses. Heat-induced antigen retrieval was performed by immersing slides in 10 mm citrate buffer (pH 6.0) and microwaving in a 750-watt microwave oven for 10 min. The slides were then cooled at room temperature for 20 min and rinsed abundantly in tap water. Nonspecific staining of slides was blocked by incubation with 10% normal goat serum in PBS buffer for 30 min. Antigen was detected by overnight incubation at 4 °C with a primary antibody at the appropriate dilution followed by a secondary antibody conjugated to Alexa Fluor® 488 or Alexa Fluor 594 (Molecular Probes, Eugene, OR). Sections were imaged using confocal laser scanning microscopy (Zeiss 510LSM).

      RESULTS

       Protein Profiling of Whole Fat Tissue Lysates—

      Fresh fat tissue samples devoid of tumor cells were dissected from sites topologically distant from the tumor (Fig. 2A) from 21 high risk breast cancer patients and analyzed by 2D PAGE as described under “Experimental Procedures.” Figs. 3 and 4 show representative silver-stained IEF (Fig. 3) and NEPHGE (Fig. 4) gels of whole fat tissue cryostat sections (15–20 5-μm sections) dissolved in CBL1 lysis solution (Zeptosens AG). A total of 1413 well resolved and in most cases well focused proteins were detected in these gels (962 IEF and 451 NEPHGE), and of these, about 100 polypeptides migrated both in IEF and NEPHGE gels as determined by visual matching of the gels. Three of these proteins, triose-phosphate isomerase (Figs. 3 and 4; proteins IEF 147a and NEPHGE 36, respectively), α-enolase (Figs. 3 and 4; proteins IEF 26a and NEPHGE 4, respectively), and transketolase (Figs. 3 and 4; proteins IEF 144 and NEPHGE 35, respectively), indicated with blue arrows served as landmarks to align the gels (
      • Celis J.E.
      • Rasmussen H.H.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Gesser B.
      • Olsen E.
      • Gromov P.
      • Hoffmann H.J.
      • Nielsen M.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.
      • Nielsen H.
      • Andersen A.H.
      • Walbaum E.
      • Kjargaard I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      ,
      • Celis J.E.
      • Rasmussen H.H.
      • Gromov P.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Vorum H.
      • Kristensen D.B.
      • Østergaard M.
      • Hauns⊘ A.
      • Nielsen M.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.
      • Nielsen H.
      • Andersen A.H.
      • Walbaum E.
      • Kjargaard I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
      ).
      Figure thumbnail gr3
      Fig. 3IEF 2D gel of whole fat tissue proteins stained with silver nitrate. Proteins indicated with a blue arrow migrate both in IEF and NEPHGE gels and serve as landmarks to align the gels. Selected proteins indicated with red arrows are expressed at low levels or are absent in the FIF (see also ). A few proteins indicated with green arrows are enriched in the FIF (see also ). The identity of the proteins indicated with numbers is given in .
      Figure thumbnail gr4
      Fig. 4NEPHGE 2D gel of whole fat tissue proteins stained with silver nitrate. Proteins indicated with a red arrow migrate both in NEPHGE and IEF gels and served as landmarks to align the gels. Selected proteins indicated with red arrows are expressed at low levels or are absent in the FIF (see also ). A few proteins indicated with green arrows are enriched in the FIF (see also ). The identity of the proteins indicated with numbers is given in .
      Proteins were identified using a combination of procedures that included mass spectrometry of proteins recovered from 2D gels (Fig. 5 and Table I), 2D PAGE Western immunoblotting using specific antibodies (Fig. 6 and Table I), and in a few cases by comparison with the master image of the keratinocyte 2D PAGE protein data base (proteomics.cancer.dk; Refs.
      • Celis J.E.
      • Rasmussen H.H.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Gesser B.
      • Olsen E.
      • Gromov P.
      • Hoffmann H.J.
      • Nielsen M.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.
      • Nielsen H.
      • Andersen A.H.
      • Walbaum E.
      • Kjargaard I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      and
      • Celis J.E.
      • Rasmussen H.H.
      • Gromov P.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Vorum H.
      • Kristensen D.B.
      • Østergaard M.
      • Hauns⊘ A.
      • Nielsen M.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.
      • Nielsen H.
      • Andersen A.H.
      • Walbaum E.
      • Kjargaard I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
      ). A complete list of all proteins identified in whole fat tissue extracts from high risk patients is given in Table I. Only in a few instances was it possible to confirm the presence of a given protein in the fat tissue using immunohistochemistry or immunofluorescence due to the high lipid content of the tissue. As an example, Fig. 2B shows a double immunofluorescence picture of a representative paraffin section from a fat tissue biopsy from a site distant to the tumor incubated with antibodies against A-FABP (fat cells, green) and the CD68 antigen (macrophages, red). No tumor cells were detected in this preparation as ascertained by staining with keratin 19-specific antibodies (not shown).
      Figure thumbnail gr5a
      Fig. 5Protein identification.A, protein identification based solely on peptide fingerprinting (NEPHGE 11) analysis. The upper panel shows the MS spectrum obtained from spot NEPHGE 11. The result of the Mascot search is presented in the lower panel. The resulting score of 236 significantly exceeds the threshold significance score of 70 (p < 0.05), thus making further MS analysis unnecessary. B, protein identification based on peptide fingerprinting as well as PSD analysis (IEF 116). The left part of the panel shows the first step of protein identification performed by peptide fingerprinting. The score of 90 does not significantly exceed the threshold significance score of 70 (p < 0.05). PSD analysis was performed on the 1462.74 peptide to confirm the results obtained by peptide fingerprinting. The right part of the panel presents the PSD spectrum as well as the result of the Mascot search. The sequences of the two isoforms of peroxiredoxin 3 are identical except for an 18-amino acid gap within the NH2-terminal end of peroxiredoxin 3b. As a result, the accession number from Swiss-Prot was assigned because it is common for both isoforms. NCBInr, National Center for Biotechnology Information nonredundant.
      Figure thumbnail gr5b
      Fig. 5Protein identification.A, protein identification based solely on peptide fingerprinting (NEPHGE 11) analysis. The upper panel shows the MS spectrum obtained from spot NEPHGE 11. The result of the Mascot search is presented in the lower panel. The resulting score of 236 significantly exceeds the threshold significance score of 70 (p < 0.05), thus making further MS analysis unnecessary. B, protein identification based on peptide fingerprinting as well as PSD analysis (IEF 116). The left part of the panel shows the first step of protein identification performed by peptide fingerprinting. The score of 90 does not significantly exceed the threshold significance score of 70 (p < 0.05). PSD analysis was performed on the 1462.74 peptide to confirm the results obtained by peptide fingerprinting. The right part of the panel presents the PSD spectrum as well as the result of the Mascot search. The sequences of the two isoforms of peroxiredoxin 3 are identical except for an 18-amino acid gap within the NH2-terminal end of peroxiredoxin 3b. As a result, the accession number from Swiss-Prot was assigned because it is common for both isoforms. NCBInr, National Center for Biotechnology Information nonredundant.
      Table IProteins identified in whole mammary fat tissue and its interstitial fluid
      Protein nameAccession number
      Whenever possible, the Swiss-Prot accession number was used.
      Method of identification
      MS, identification of proteins separated by 2D PAGE using MALDI-TOF-MS; IB, proteins identified by Western 2D gel immunoblotting; CKD, protein identification by matching the protein patterns to the master 2D gel keratinocyte data base (proteomics.cancer.dk); AA, protein identification using the Panorama Ab Microarray Cell Signaling array; AA#, proteins identified using the RayBio Human Cytokine Array C Series. Two proteins were identified using the Bio-Rad Bioplex system.
      Spot number in 2D gelsMr/pI
      Proteins identified from 2D gels are indicated with either an IEF (Figs. 3 and 8) or an NEPHGE number (Figs. 4 and 9). Proteins migrating in both directions are indicated with both an IEF and NEPHGE number. Some proteins exhibited multiple isoforms, and these are indicated with a, b, c, etc.
      Presence in FIFGene map locusScorePeptides and PSDCoverageMolecular function
      The Mr and pI were calculated directly from the sequences. In some cases these values do not fit with the apparent molecular weights observed in the 2D gels shown in Figs. 3, 4, 8, and 9.
      Biological process
      Molecular function as well as biological process was assigned in accordance with the Human Protein Reference Database (www.hprd.org).
      %
      14-3-3 εP62258MSIEF 129/4.6Yes17p13.398838Receptor signaling complex scaffold activitySignal transduction; cell communication
      14-3-3 protein βP31946MS, IBIEF 228/4.7Yes20q13.11201145Receptor signaling complex scaffold activitySignal transduction; cell communication
      14-3-3 protein ηQ04917MS, CDKIEF 328/4.7Yes22q12.31301042Receptor signaling complex scaffold activitySignal transduction; cell communication
      14-3-3 ζ/δP29312MS, CDKIEF 427.9/4.7Yes22q121201548Receptor signaling complex scaffold activitySignal transduction; cell communication
      26 S protease regulatory subunit 8 (thyroid hormone receptor-interacting protein 1)P62195MSNEPHGE 145/7.117q24-q25107920The 26 S protease is involved in the ATP-dependent degradation of ubiquitinated proteinsProtein metabolism
      37/67 laminin receptor, 40 S ribosomal proteinP08865MSIEF 532.7/4.8Yes3p21.3100920UnknownSignal transduction; cell communication
      6-Phosphogluconolactonase (minor component in the mixture)O95336MSIEF 627/5.7Yes19p13.21151241Enzyme: hydrataseEnergy pathways
      78-kDa glucose-regulated proteinP11021MS, CDKIEF 7a, 7b72.4/5.0Yes9q33-q34.11401372ChaperoneProtein metabolism
      AAA ATPase p97 (major component)P55072MS, CKDIEF 8a, 8b89.9/5.1Yes19q133812124Transport/cargo proteinTransport
      Acetyl- and phosphohistone H3P68431AANDDNA-binding protein
      Aconitate hydrataseQ99798MSIEF 9a, 9b, 9c; NEPHGE 285.4/7.4Yes22q13.2-q13.313902739Enzyme: hydrataseEnergy pathways
      Actin, cytoplasmic 1 or 2 (γ or β actin)P02571MS, AAIEF 10a, 10b42/5.3Yes17q251261030Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      ActopaxinQ9NVD7AAND11p15.3Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      Acylaminoacyl peptidaseP13798MSIEF 1181/5.3Yes3p211851824Enzyme: hydrolaseEnergy pathways
      Acyl-CoA dehydrogenaseP45954MSIEF 1247/6.5Yes10q25-q262191638Enzyme: dehydrogenaseEnergy pathways
      Adenine phosphoribosyltransferaseP07741MSIEF 1319.5/5.8Yes16q24.396733Enzyme: ribosyltransferaseEnergy pathways
      Adenylate DE cyclase-inhibiting Gα proteinP04899MSIEF 1440/5.3Yes3p212101548GTPase activatorSignal transduction; cell communication
      Adenylyl cyclase-associated protein 1Q01518MSNEPHGE 351/8.11p34.22231837UnknownCell growth and/or maintenance
      Adipocyte complement-related protein of 30 kDaQ15848AA#Yes3q27Cytokine activityEnergy pathways
      A-FABPP15090MS, IBIEF 15a, 15b, 15c14.6/6.8Yes8q211808 (PSD: 935.48)45ChaperoneSignal transduction; cell communication
      Agrin-related proteinO00468AA#Yes1pter-p32Cytokine activityCell growth and/or maintenance
      Albumin serumP02768MS, IBIEF 16a, 16b69/5.9Yes4q11-q131281017Transport/cargo proteinTransport
      Alcohol dehydrogenase (NADP+)P14550MSIEF 1736/6.3Yes1p33-p321301027Enzyme: oxidoreductaseEnergy pathways
      Alcohol dehydrogenase β chainP00325MSNEPHGE 3739.7/8.64q21-q232301943Enzyme: dehydrogenaseEnergy pathways
      %
      Alcohol dehydrogenase class III χ chainP11766MSIEF 1839.6/7.6Yes4q21-q251331130Enzyme: oxidoreductaseEnergy pathways
      Aldehyde dehydrogenaseP49189MSIEF 19a, 19b,53/6.0Yes1q22-q232261630Enzyme: oxidoreductaseEnergy pathways
      α1-AntichymotrypsinP01011MSIEF 2047/5.3Yes14q32.11001022Protease inhibitorProtein metabolism
      α1 protease inhibitorP01009MSIEF 21a, 21b47/5.4Yes14q32.12551748Protease inhibitorProtein metabolism
      α soluble NSF attachment protein α (SNAP-α)P54920MSIEF 2233/5.219q13.321701450Protein transportTransport
      α-1B glycoproteinP04217MSIEF 23a, 23b, 23c54/5.6Yes19q13.485614UnknownUnknown
      α2-MacroglobulinP01023MSIEF 24a, 24b, 24c163/6.0Yes12p12.3-p13.3921221Protease inhibitorProtein metabolism
      α-Actinin 4 (minor component)O43707MSIEF 25105/5.3Yes9p13-p123814454Cytoskeleton-associated protein, actin binding, calcium bindingCell growth and/or maintenance
      α-Enolase, enolase 1P06733MS, CDKIEF 26a, 26b; NEPHGE 4a, 4b47.4/7.0Yes1p36.3-p36.21021125Enzyme: hydrataseEnergy pathways
      α-Tubulin 1 chainP68366MS, CKD, AAIEF 2749/4.9Yes12q13.1294818Cytoskeleton-associated proteinCell growth and/or maintenance
      Amyloid precursor proteinP05067AAND21q21.3Cell surface receptorSignal transduction; cell communication
      AngiogeninP03950AA#Yes14q11.1-q11.2Ribonuclease activityRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Angiopoietin-2O15123AA#Yes8p23.1Cytokine activitySignal transduction; cell communication
      Annexin IP04083IB, CKDIEF 2838/6.6Yes9q12-q21.2Receptor binding, calcium bindingSignal transduction; cell communication
      Annexin IIP07355MS, IBIEF 29; NEPHGE 538.7/7.5Yes15q21-q221951040Receptor binding, calcium bindingSignal transduction; cell communication
      Annexin IIIP12429MSIEF 3036/5.6Yes4q13-q221661236Enzyme: hydrolaseEnergy pathways
      Annexin IVP09525MSIEF 3135/5.8Yes2p133652455Calcium ion bindingSignal transduction; cell communication
      Annexin VP08758CKDIEF 3235.8/4.5Yes4q28-q32Calcium ion bindingSignal transduction; cell communication
      Antithrombin-IIIP01008MSIEF 3352/6.3Yes1q23-q252211836Protease inhibitorProtein metabolism
      Apolipoprotein A-IP02647MSIEF 34a, 34b30.7/5.5Yes11q23-q241501538Transport/cargo proteinTransport
      Apolipoprotein A-IVP06727MSIEF 3545/5.3Yes11q232351739Transport/cargo proteinTransport
      Apoptosis-inducing factorO95831AANDXq25-q26Enzyme: oxidoreductaseSignal transduction; cell communication
      ATP synthase D chain, mitochondrialO75947MSIEF 3618.4/5.2Yes17q25133953UnknownEnergy pathways
      Basic fibroblast growth factorO00527AA#Yes4q26-q27Cytokine activityCell growth and/or maintenance
      Bcl-10O95999AAND1p22Receptor signaling complex scaffold activityApoptosis
      Bcl-XQ07817AAND20q11.21Receptor signaling complex scaffold activityApoptosis
      β-Tubulin 1P07437CKD, AAIEF 3749/4.8Yes6p21.33Cytoskeleton-associated proteinCell growth and/or maintenance
      β-1B-glycoprotein (minor component)P02790MSIEF 3851/6.5Yes11p15.5-p15.41801020Transport/cargo proteinTransport
      %
      BetacellulinP35070AA#Yes4q13-q21Cytokine activitySignal transduction; cell communication
      β-Nerve growth factorP01138AA#Yes1p13.1Cytokine activitySignal transduction; cell communication
      Bone morphogenetic protein 4P12644AA#Yes14q22-q23Cytokine activitySignal transduction; cell communication
      Bone morphogenetic protein 6P22004AA#Yes6p24-p23Cytokine activitySignal transduction; Cell communication
      Brain-derived neurotrophic factorP23560AA#Yes11p13Cytokine activitySignal transduction; cell communication
      C1-tetrahydrofolate synthaseP11586MSIEF 39, NEPHGE 6101/6.9Yes14q243082632Enzyme: dehydrogenaseEnergy pathways
      Calgizzarin, S100C proteinP31949MSIEF 4011.7/6.5Yes1q21133956Calcium ion bindingSignal transduction; cell communication
      CalreticulinP27797CKD, AAIEF 4148/4.319p13.11Chaperone, calcium bindingProtein metabolism
      CAM kinase IV (Ay-18)Q16566AAND5q21.3Kinase: protein serine/threonine kinaseSignal transduction; cell communication
      cAMP-dependent protein kinase type II-α regulatory subunitP13861MSIEF 4245/4.93p21.3-p21.23411844Kinase: protein serine/threonine kinaseSignal transduction; cell communication
      Carbonate dehydratase IP00915MSIEF 4328/6.6Yes8q222261669Enzyme: dehydrataseEnergy pathways
      Carbonate dehydratase IIIP07451MSIEF 4429.7/6.9Yes8q222231454Enzyme: carbonic anhydraseEnergy pathways
      Carboxypeptidase BP15086MSIEF 4547/6.13q241981539Enzyme: carboxypeptidaseProtein metabolism
      Caspase 10Q92851AAND2q33-q34Cysteine proteaseApoptosis
      Caspase 11P49662AANDCysteine proteaseApoptosis
      Caspase 3P42574AAND4q34Cysteine proteaseApoptosis
      Caspase 3, activeP42574AAND4q34Cysteine proteaseApoptosis
      Caspase 6P55212AAND4q25Cysteine proteaseApoptosis
      Caspase 7P55210AAND10q25Cysteine proteaseApoptosis
      Caspase 8Q14790AAND2q33-q34Cysteine proteaseApoptosis
      CatalaseP04040MSIEF 46a, 46b; NEPHGE 7a, 7b59.6/6.9Yes11p132651835Enzyme: oxidoreductaseEnergy pathways
      Catenin αP35221AAND5q31Motor proteinCell growth and/or maintenance
      Catenin βP35222AAND3p21Adhesion moleculeSignal transduction; cell communication
      Catenin d1 (p120 catenin)O60716AAND11q11Motor proteinSignal transduction; cell communication
      Caveolin-1 (anti-VIP21)Q03135AAND7q31.1Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      CCG1-interacting factor BQ96IU4MSIEF 4722.5/5.93p21.2151844Kinase: protein serine/threonine kinaseCell growth and/or maintenance
      Cdc25P30307AAND15q24Protein tyrosine/serine/threonine phosphatase activitySignal transduction; cell communication
      CDC6Q99741AAND17q21.3Cell cycle control proteinSignal transduction; cell communication
      Cdk4Q96BE9AAND12q14Cell cycle control proteinSignal transduction; cell communication
      Cell surface glycoprotein MUC18P43121MSIEF 4871/5.6Yes11q23.32812432Adhesion moleculeSignal transduction; cell communication
      %
      Cellular tumor antigen p53P67939AAND17p13.1Cell cycle control proteinSignal transduction; cell communication
      Ceruloplasmin; ferroxidase; one out of two splice variantsP00450MSIEF 49122/5.43q23-q2414017 (PSD: 1519.76)15Enzyme: oxidoreductaseEnergy pathways
      Chaperonin containing TCP 1, subunit εP48643CKDIEF 5059/5.4Yes5p15.2Chaperone (folding of actin and tubulin)Protein metabolism
      Chaperonin containing TCP 1, subunit β (major component)P78371MS, CKDIEF 5157.7/6.0Yes12q151811437Chaperone (folding of actin and tubulin; interaction with Cyclin E)Protein metabolism
      Chaperonin containing TCP 1, subunit θP50990CKDIEF 5259/5.421q22.11Chaperone (folding of actin and tubulin)Protein metabolism
      Chloride intracellular channel protein 1O00299MSIEF 5326.7/5.0Yes6p22.1-p21.2110739Transport/cargo proteinTransport
      Chondroitin sulfateP13611AAND5q14.3Extracellular matrix proteinCell growth and/or maintenance
      Ciliary neurotrophic factorP26441AA#Yes11q12.2Cytokine activitySignal transduction; cell communication
      Citrate synthase, mitochondrialO75390MSIEF 5452/8.1Yes12q13.2-q13.323719 (PSD: 1127.67, 1167.68)30Enzyme: acyltransferaseEnergy pathways
      Clathrin light chainP09496AAND9p13Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      Coatomer b subunitP53618AAND11p15.2Transport/cargo proteinTransport
      Cofilin A, non-muscle isoformP23528MS, CKD, AANEPHGE 818/8.3Yes11q1395841Cytoskeleton-associated proteinCell growth and/or maintenance
      Collagen α 1 (VI) chainP12109MSIEF 55a, 55b108/5.3Yes but not 55b21q22.31171520Extracellular matrix proteinCell growth and/or maintenance
      Collagen α 2 (VI)P12110MSIEF 56108/5.7Yes21q22.31101010Extracellular matrix proteinCell growth and/or maintenance
      Complement component 1Q07021MSIEF 5731.8/4.7417p13.383831Complement receptorImmune response
      Complement factor HP08603MSIEF 58144/6.2Yes1q323163427Complement receptorImmune response
      Connexin 32P08034AANDXq13.1Transport/cargo proteinTransport
      Connexin 43P17302AAND6q21-q23.2Transport/cargo proteinTransport
      Creatine kinase, B chainP12277MSIEF 5943/5.3Yes14q321311130Enzyme: phosphotransferaseEnergy pathways
      CRK-LP46109AA, IBYes22q11.21Receptor signaling complex scaffold activity, adapter moleculeSignal transduction; cell communication
      Cutaneous T-cell-attracting chemokineP46092AA#Yes17q21.1-q21.3Cytokine activity (G protein-coupled receptor)Signal transduction; cell communication
      Cyclic AMP-dependent transcription factor ATF2P15336AAND2q32Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Cyclin B1P14635AAND5q12Cell cycle control proteinSignal transduction; cell communication
      Cyclin D2P30279AAND12p13Cell cycle control proteinSignal transduction; cell communication
      Cyclin-dependent kinase 4 inhibitor D p19INK4dP55273AAND19p13Cell cycle control proteinSignal transduction; cell communication
      %
      Cyclophilin AP62937MSIEF 60a, 60b, 60c; NEPHGE 9a, 9b18/7.8Yes7p131901365Transcription factorProtein metabolism
      Cyclophilin BP23284MSNEPHGE 1022/9.315q21-q221261243Transcription factorProtein metabolism
      Cytochrome b5P00167MSIEF 6115.2/4.818q23905 (PSD: 1511.72)40Enzyme: oxidoreductaseEnergy pathways
      Cytochrome c oxidase II subunitP00403MSIEF 6225/4.690733Regulatory/other subunitEnergy pathways
      Cytoplasmic antiproteinaseP35237MSIEF 6342/5.2Yes6p252051650Protease inhibitorProtein metabolism
      Cytoplasmic antiproteinase 3, Serpin B9P50453MSIEF 6442/5.66p252481842Protease inhibitorProtein metabolism
      Cytoplasmic protein NCK1/ NCK2P16333/O43639IB42.8/63q21/2q12Adapter moleculeSignal transduction, cell communication
      Death domain-associated protein 6 (DAXX)Q9UER7AAND6p21.3Receptor signaling complex scaffold activitySignal transduction, cell communication
      Δ1-Pyrroline-5-carboxylate dehydrogenaseP30038MSNEPHGE 1162/8.21p362361635Enzyme: dehydrogenaseEnergy pathways
      Δ3,5-Δ2,4-Dienoyl-CoA isomeraseQ13011MSIEF 6536/6.6Yes19q13.11101023Enzyme: hydrataseEnergy pathways
      Dihydrolipoyllysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrialP36957MSIEF 6648/9.0Yes14q24.380711Enzyme: acyltransferaseEnergy pathways
      DJ-1 proteingi‖31543380MSIEF 6720/6.3Yes1p36.33-p36.1280838RNA bindingRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Elastase inhibitorP30740MS, CDKNEPHGE 1242/5.96p25Protease inhibitorProtein metabolism
      Elongation factor 1-βP24534MSIEF 6824/4.5Yes2q33-q3488732Transcription regulationProtein metabolism
      Elongation factor 1-α 1P68104MSNEPHGE 1350/9.1Yes6q14122922Translation regulationProtein metabolism
      Elongation factor 2P13639MS, CKDIEF 69a, 69b95/6.4Yes19pter-q121281020Translation regulationProtein metabolism
      Elongation factor TuP49411MS, CKDIEF 7049/7.316p11.22221540Translation regulationProtein metabolism
      EndoplasminP14625MS, CDKIEF 7192/4.712q24.2-q24.319427 (PSD: 1176)30Heat shock proteinProtein metabolism
      Enoyl-CoA hydrataseP30084MSIEF 7231.4/8.3Yes10q26.2-q26.31351236Enzyme: hydrataseEnergy pathways
      Eotaxin precursorP51671AA#Yes17q21.1-q21.2Cytokine activity (promotes the accumulation of eosinophils, inflammatory response)Signal transduction, cell communication
      Eotaxin-2O00175AA#Yes7q11.23Cytokine activityImmune response
      Epidermal growth factorP01133AA#Yes4q25Cytokine activity (growth factor activity, calcium binding)Signal transduction, cell communication (cell proliferation, DNA replication, EGF receptor signaling pathway, activation of MAPK)
      Epidermal growth factor receptorP00533AA#Yes7p12Cytokine activity (receptor tyrosine kinase)Signal transduction, cell communication
      Epithelial neutrophil-activating protein 78P42830AA#Yes4q12-q13Cytokine activitySignal transduction, cell communication (cell proliferation, immune response)
      Esterase DP10768MSIEF 7331/6.5Yes13q14.1-q14.2100723Enzyme: esteraseEnergy pathways
      %
      ERP03372AAND6q25.1Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Eukaryotic translation initiation factor 5AP63241MSIEF 7416.7/5.0Yes17p13-p121451156Initiation factorProtein metabolism
      F-actin capping protein α-1 subunit, CapZ α-1P52907MSIEF 7532.9/5.4Yes1p13.274625Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      F-actin capping protein β subunit, Cap Z βP47756MSIEF 7631/5.3Yes1p36.180721Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      Focal adhesion kinaseQ05397AAND8q24-qterKinase: protein-tyrosine kinaseSignal transduction, cell communication
      Fas antigen APO-1/CD95Q8IUB7AA#Yes10q24.1Cytokine activitySignal transduction, cell communication
      Fatty acid-binding protein, epidermalQ01469MSIEF 7715/6.6Yes8q21.131501457Transport/cargo proteinTransport (epidermal differentiation)
      Ferritin light chainP02792MSIEF 7920/5.5Yes19q13.3-q13.41008 (PSD: 1491.7)30Storage protein (metal binding)Transport (iron homeostasis)
      Ferritin repressor proteinP21399MSIEF 8098/6.2Yes9p22-p134434048Storage protein (metal binding)Transport (iron homeostasis)
      Fibrinogen β chainP02675MSIEF 8156/8.6Yes4q2819913 (PSD: 1032.33)28Coagulation factorProtein metabolism
      Fibrinogen γ chainP02679MSIEF 8251/5.4Yes4q282961746Coagulation factorProtein metabolism
      Fibroblast growth factor-4P08620AA#Yes11q13.3Cytokine activity (growth factor activity)Signal transduction; cell communication
      Fibroblast growth factor-6P10767AA#Yes12p13Cytokine activity (growth factor activity)Signal transduction; cell communication
      Fibroblast growth factor-9P31371AA#Yes13q11-q12Cytokine activity (growth factor activity)Signal transduction; cell communication
      FK506-binding protein 8Q14318CKDNEPHGE 1438/7.6Yes19p12Adapter moleculeApoptosis
      Flavin reductaseP30043MSIEF 83a, 83b22/7.3Yes19q13.13-q13.2117947Enzyme: oxidoreductaseEnergy pathways
      Flt-3 ligand, Fms-related tyrosine kinase 3 ligandP49771AA#Yes19q13.3Receptor bindingSignal transduction; cell communication
      Fructose-bisphosphate aldolase AP04075MSNEPHGE 1539/8.4Yes16q22-q241001245Enzyme: aldolaseEnergy pathways
      Fructose-bisphosphate aldolase CP09972MSIEF 8439/6.5Yes17cen-q121361026Enzyme: aldolaseEnergy pathways
      Fumarate hydratase, mitochondrialP07954MSIEF 8554.8/8.81q42.117117 (PSD: 1763.92)29Enzyme: hydrataseEnergy pathways
      Fumarylacetoacetate hydrolaseP16930MSIEF 8646/6.5Yes15q23-q251761436Enzyme: hydrolaseEnergy pathways
      γ interferon-induced monokineQ07325AA#Yes4q21Cytokine activitySignal transduction; cell communication
      Gelsolin (major component)P06396MSIEF 87a, 87b, 87c86/6.0Yes9q332251721Cytoskeleton-associated protein (actin binding, calcium binding)Cell growth and/or maintenance
      Glial cell-derived neurotrophic factorP39905AA#Yes5p13.1-p12Cytokine activity (growth factor activity; DNA binding)Signal transduction; cell communication (TGFβ receptor signaling pathway; antiapoptosis)
      Glucocorticoid-induced TNF-related ligandQ9UNG2AA#Yes1q23Cytokine activitySignal transduction, cell communication
      Glucocorticoid-induced TNFR-related proteinQ9Y5U5AA#Yes1p36.3Cytokine activitySignal transduction, cell communication
      %
      Glucose-6-phosphate 1-dehydrogenaseP11413MSIEF 8859.6/6.4YesXq281161135Enzyme: dehydrogenaseEnergy pathways
      Glutamate dehydrogenase 1P00367MSNEPHGE 16a, 16b61.7/7.6Yes10q23.32862038Enzyme: dehydrogenaseEnergy pathways
      Glutamic acid decarboxylase (GAD65/67)Q05329AAND10p11.23/2q31UnknownUnknown
      Glutamine synthetaseP15104MS, AAIEF 8942/6.4Yes1q252031431Enzyme: transaminaseEnergy pathways
      Glutathione S-transferase PP09211MS, CDKIEF 9023.4/5.4Yes11q131971366Enzyme: glutathione transferaseEnergy pathways
      Glyceraldehyde-3-phosphate dehydrogenase, liverP04406MSNEPHGE 17a, 17b36/8.6Yes12p131251435Enzyme: oxidoreductaseEnergy pathways
      Glycerol-3-phosphate dehydrogenase (NAD+), cytoplasmicP21695MSIEF 91a, 91b37.5/5.8Yes12q12-q131951740Enzyme: dehydrogenaseEnergy pathways
      Glyoxalase 1Q04760MSIEF 9220/5.0Yes6p21.3-p21.194826Enzyme: glutathione transferaseEnergy pathways
      Granulocyte CSF receptorQ99062AA#Yes1p35-p34.3Cytokine activitySignal transduction, cell communication; cell adhesion
      Granulocyte CSFP09919AA (Bioplex)Yes17q11.2-q12Cytokine activityImmune response
      Granulocyte-macrophage CSFP04141AA (Bioplex)Yes5q31.1Cytokine activityImmune response
      Growth arrest and DNA damage-inducible protein GADD153 (CHOP-10)P14607AAND12q13.1-q13.2Transcription regulation (inhibitor of the DNA binding activity)Cell growth and/or maintenance
      Growth factor receptor-bound protein 2P62993AAND17q24-q25Receptor bindingSignal transduction, cell communication; cell adhesion
      Growth-regulated oncogeneP09341AA#Yes4q21Cytokine activity (growth factor activity)Immune response
      Growth-regulated protein αP09341AA#Yes4q21Cytokine activityImmune response
      Guanine nucleotide-binding protein G(I)/G(S)/G(T) β subunit 1P62873MSIEF 9337/5.6Yes1p36.331991438G proteinSignal transduction; cell communication
      Guanine nucleotide-binding protein G(I)/G(S)/G(T) β subunit 2P62879MSIEF 9437/5.6Yes7q211571026G proteinSignal transduction; cell communication
      Haptoglobin 1P00739MSIEF 95a, 95b, 95c, 95d39/6.4Yes16q22.11702040Secreted polypeptideImmune response
      Haptoglobin α chainP00738MSIEF 9645/6.0Yes16q22.11077 (PSD: 1708.85)15Secreted polypeptideProtein metabolism
      Histone acetyltransferase (HAT1)O14929AAND2q31.2-q33.1Acyltransferase activityEnergy pathways
      Histone deacetylase 1Q13547AAND1p34Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Histone deacetylase 2Q92769AAND6q21Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Histone deacetylase 4P56524AAND2q37.2Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      %
      Heat shock protein 27P04792MSIEF 9722.8/6.0Yes7q11.23109731Chaperone (estrogen-regulated, actin organization)Protein metabolism
      Heat shock protein 90P07900MS, CDKIEF 9885/5.0Yes14q32.331992130Chaperone (MAPK signaling pathway (Src, Raf, and Mek); approximately 100 proteins are known to be regulated by hsp90)Protein metabolism
      Heat shock protein β-5P02511MSIEF9920.2/6.7Yes11q22.3-q23.12151144Heat shock proteinProtein metabolism
      Heat shock protein β-6O14558MSIEF 10017.2/5.9Yes19q13.12140849Heat shock proteinProtein metabolism
      Heme-binding protein 1gi‖20336761MSIEF 10121/5.7Yes12p13.1117641Transport/cargo protein (hemoglobin binding)Transport
      Hemofiltrate CC chemokine-4O15467AA#Yes17q11.2Cytokine activitySignal transduction, cell communication
      Hemoglobin β chainP02023MSIEF 102a, 102b, 102c; NEPHGE 18a, 18b, 18c16/7.0Yes11p15.5981167Transport/cargo protein (oxygen binding)Transport
      Hepatocyte growth factorP14210AA#Yes7q21.1Cytokine activity (growth factor activity)Signal transduction, cell communication
      Heterogeneous nuclear ribonucleoproteins A2/B1P22626MSNEPHGE 1937/8.97p15100825RibonucleoproteinRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      High mobility group protein 1P09429MSIEF 10324/5.613q121731238DNA-binding proteinRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Histone acetyltransferase p300/CBP-associated factorQ92831AAND3p24Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Ig γ-1 chain C regionP01857MSNEPHGE 3937/8.414q32.3383723Antigen bindingImmune response
      Immunoglobulin G light chain, κIBIEF 104a, 104b, 104c; NEPHGE 20a, 20b, 20cYesImmune response
      Immunoglobulin G heavy chainIBIEF 105; NEPHGE 21YesImmune response
      Importin β-1 subunit, Importin 90Q14974MSIEF 10697/4.7Yes17q21.321011215Transport/cargo protein (nuclear import of ribosomal proteins RPL23A, RPS7, and RPL5; H1, H2A, H2B, H3, and H4 histones; GTP-dependent)Transport
      Induced myeloid leukemia cell differentiation protein (Mcl-1)Q07820AAND1q21Cell cycle control proteinSignal transduction; cell communication
      Insulin-like growth factor-binding protein 1P08833AA#Yes7p13-p12Cytokine activity (adhesion molecule; growth factor binding)Signal transduction; cell communication
      Insulin-like growth factor-binding protein 2P18065AA#Yes2q33-q34Cytokine activity (adhesion molecule; growth factor binding)Signal transduction; cell communication
      %
      Insulin-like growth factor-binding protein 3P17936AA#Yes7p13-p12Cytokine activity (adhesion molecule; growth factor binding)Signal transduction; cell communication
      Insulin-like growth factor-binding protein 6 precursorP24592AA#Yes12q13Cytokine activity (adhesion molecule; growth factor binding)Signal transduction; cell communication
      Insulin-like growth factor IAP01343AA#Yes12q22-q24.1Growth factorSignal transduction; cell communication
      Insulin-stimulated MAP2 kinase (Erk1 + Erk2)P27361AAND16p12-p11.2Kinase: protein threonine/tyrosine kinaseSignal transduction; cell communication
      Intercellular adhesion molecule-1 precursorP05362AA#Yes19p13.3-p13.2Cytokine activity (adhesion molecule; growth factor binding)Signal transduction; cell communication
      Interferon γP01579AA#Yes12q14Cytokine activityImmune response
      Interferon-inducible T-cell α chemoattractantO14625AA#Yes4q21.2Cytokine activitySignal transduction; cell communication
      IL-10P22301AA#Yes1q31-q32Cytokine activityImmune response
      Interleukin 12 p40P29460AA#Yes5q31.1-q33.1Cytokine activityImmune response
      Interleukin 12 p70P29459AA#Yes3p12-q13.2Cytokine activityImmune response
      IL-15P40933AA#Yes4q31Cytokine activityImmune response
      IL-16Q14005AA#Yes15q26.3Cytokine activityImmune response
      IL-1 αP01583AA#Yes2q14Cytokine activityImmune response
      IL-3P08700AA#Yes5q31.1Cytokine activityImmune response
      IL-4P05112AA#Yes5q31.1Cytokine activityImmune response
      IL-6P08887AA#Yes1q21Cytokine activityImmune response
      IL-8P10145AA#Yes4q13-q21Cytokine activityImmune response
      Interleukin-1 receptor antagonist proteinP18510AA#Yes2q14.2Cytokine activitySignal transduction; cell communication
      Interleukin-2 receptor α chain (CD25)P01589AA#Yes10p15-p14Cytokine activityImmune response
      Interleukin-6 receptor β chain (gp130)P40189AA#Yes1q21Cytokine activityImmune response
      Isocitrate dehydrogenaseO75874MSIEF 107; NEPHGE 2246/6.5Yes2q33.31711530Enzyme: dehydrogenaseEnergy pathways (response to oxidative stress)
      KIAA1881 protein (COOH-terminal)gi‖15620821MSNEPHGE 23134/8.9Yes13q121731820UnknownUnknown
      LeptinP41159AA#Yes7q31.3Cytokine activity (hormone activity)Signal transduction; Cell communication
      LIGHT, tumor necrosis factor ligand superfamily member 14O43557AA#Yes19p13.3Cytokine activitySignal transduction; Cell communication
      l-Lactate dehydrogenase A chainP00338MS, CDKIEF 108; NEPHGE 2436/8.4Yes11p15.41401525Enzyme: dehydrogenaseEnergy pathways
      l-Lactate dehydrogenase B chainP07195MSIEF 10936/5.7Yes12p12.2-p12.12041638Enzyme: dehydrogenaseEnergy pathways
      LymphotactinP47992AA#Yes1q23Cytokine activityImmune response
      Macrophage CSFP09603AA#Yes1p21-p13Cytokine activityImmune response
      Macrophage inflammatory protein 1 βP13236AA#Yes17q12Cytokine activityImmune response
      Macrophage inflammatory protein 1dQ16663AA#Yes17q11.2Cytokine activitySignal transduction; cell communication
      Macrophage inflammatory protein 3-αP78556AA#Yes2q33-q37Cytokine activityImmune response
      %
      Macrophage inflammatory protein 3bQ99731AA#Yes9p13Cytokine activitySignal transduction; cell communication
      Macrophage-stimulating proteinP26927AA#Yes3p21Growth factorSignal transduction; cell communication
      Macrophage-derived chemokineO00626AA#Yes16q13Cytokine activitySignal transduction; cell communication
      Malate dehydrogenase, cytoplasmicP40925MSIEF 11036/6.8Yes2p13.31327 (PSD: 1393.71)30Enzyme: dehydrogenaseEnergy pathways
      MAPK-activated protein kinase-2P49137AAND1q32Kinase serine/threonineSignal transduction; cell communication
      MAPK phosphatase-1P28562AAND5q34Protein tyrosine/serine/threonine phosphatase activitySignal transduction; cell communication
      Mesoderm-inducing factorP14174AA#Yes22q11.23Cytokine activitySignal transduction; cell communication
      Metastasis inhibition factor nm23, nucleoside diphosphate kinase AP15531MSIEF 11117/5.8Yes17q21.3927 (PSD: 1344.77)40Enzyme: phosphotransferaseEnergy pathways
      Microtubule-associated proteins 2A/2BP11137AAND2q34-q35Cytoskeleton-associated proteinCell growth and/or maintenance
      Myosin alkali light chain 3P60660MSIEF 11216/4.5Yes12q13.280735Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      Mitogen-activated protein kinase kinase kinase 2, MEK kinase 2Q9Y2U5IB69/8.52q21.1Kinase: serine/threonine kinaseSignal transduction, cell communication
      Monocyte chemoattractant protein 1P13500AA#Yes17q11.2-q21.1Cytokine activityImmune response
      Monocyte chemoattractant protein 2P80075AA#Yes17q11.2Cytokine activityImmune response
      Monocyte chemoattractant protein 3P80098AA#Yes17q11.2-q12Cytokine activityImmune response
      Monoglyceride lipase (HU-K5)Q99685MSIEF 11333.5/6.5Yes3q21.316111 (PSD: 1537.74, 1950.96)31Enzyme: hydrolaseEnergy pathways
      Myc proto-oncogene protein (c-Myc)P01106AAND8q24.12-q24.13Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Myosin Va (LE-16)Q9Y4I1AAND15q21Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      Myotrophin, V-1 proteinP58546MSIEF 11412.7/5.3Yes7q3377635Cell cycle control proteinProtein metabolism
      Neurofilament, heavy polypeptide 200 kDaP12036AAND22q12.2Cytoskeleton-associated protein: structural proteinCell growth and/or maintenance
      Neurotrophin 3P20783AA#Yes12p13Receptor bindingSignal transduction; cell communication
      Neurotrophin 4P34130AA#Yes19q13.3Growth factorSignal transduction; cell communication
      Neutrophil-activating protein 2P02775AA#Yes4q12-q13Cytokine activitySignal transduction; cell communication
      NicastrinQ92542AAND1q22-q23Integral membrane proteinSignal transduction; cell communication
      Nitric-oxide synthase, inducibleP35228AAND17q11.2-q12Enzyme: synthetaseEnergy pathways
      Nitric-oxide synthase, brainP29475AAND12q24.2-q24.31Enzyme: synthetaseEnergy pathways
      %
      Nitric-oxide synthase, endothelialP29474AAND7q36Enzyme: synthetaseEnergy pathways
      Nucleoside diphosphate kinase BP22392MSNEPHGE 2517/8.5Yes17q21.3991061Transcription factorSignal transduction; cell communication
      Oncostatin MP13725AA#Yes22q12.2Cytokine activitySignal transduction; cell communication
      OsteoprotegerinO00300AA#Yes8q24Cytokine activitySignal transduction; cell communication
      Outer mitochondrial membrane protein porin 2 (VDAC-2)P45880MSNEPHGE 2638/6.3Yes10q22116927Voltage-gated channelTransport
      p21Waf1P38936AAND6p21.2Cell cycle control proteinSignal transduction; cell communication
      p34cdc2P06493AAND10q21.1Kinase serine/threonineSignal transduction; cell communication
      p35P51948AAND14q23Cell cycle control proteinSignal transduction; cell communication
      p38MAPKQ16539AAND6p21.3-p21.2Kinase serine/threonine
      Peroxiredoxin 2; thioredoxin peroxidase 1P32119MSIEF 11522/5.6Yes19p13.22409 (PSD: 1211.68)45Enzyme: peroxidaseEnergy pathways
      Peroxiredoxin 3, antioxidant protein 1P30048MSIEF 11627/7.710q25-q26906 (PSD: 1462.80)19Enzyme: peroxidaseEnergy pathways
      Peroxiredoxin 6P30041MSIEF 117a, 117b25/6.0Yes1q24.214012 (PSD: 1409.66)43Enzyme: peroxidaseEnergy pathways
      Phosphatidylserine receptorQ8IUM5AAND17q25Receptor activitySignal transduction; cell communication
      Phosphoglycerate kinaseP00558MS, CDKNEPHGE 2744/8.3YesXq13811227Enzyme: kinaseEnergy pathways
      Phosphoglycerate mutase isomerase BP18669MSIEF 118a, 118b28/6.710q25.31501245Enzyme: hydrolaseEnergy pathways
      PIGFQ07326AA#Yes2p21-p16Integral membrane proteinEnergy pathways
      PKB/AKTP31749AAND14q32.32Kinase serine/threonineSignal transduction; cell communication
      PKDQ15139AAND14q11Kinase serine/threonineSignal transduction; cell communication
      Platelet-derived endothelial cell growth factorP19971MSIEF 11950/5.4Yes22q13.331601221Growth factorSignal transduction; cell communication
      Platelet-derived growth factor BBP01127AA#Yes22q13.1Growth factorSignal transduction; Cell communication
      Profilin IP07737MS, CDKNEPHGE 2814.9/8.4Yes17p13.3142973Cytoskeleton-associated protein (actin binding; inhibits the polymerization of actin)Cell growth and/or maintenance
      Progesterone membrane-binding proteinO15173MSIEF 12024/4.7Yes4q2685622Integral membrane proteinSignal transduction; cell communication
      Prostate apoptosis response 4, WT1-interacting proteinQ86TG5AAND12q21Transcriptional repressorSignal transduction; cell communication
      Proteasome activator 28-αQ06323MSIEF 12128/5.8Yes14q11.210710 (PSD: 971.47)40Ubiquitin proteasome system protein (γ-interferon-inducible activator of multicatalytic protease; ATP-dependent protease)Protein metabolism
      Proteasome activator 28-βQ9UL46MSIEF 12227/5.4Yes14q11.2118930Ubiquitin proteasome system protein (γ-interferon-inducible activator of multicatalytic protease; ATP-dependent protease)Protein metabolism
      %
      Proteasome subunit α type 2 (C3)P25787MSNEPHGE 2925/7.1Yes7p14.194823Ubiquitin proteasome system protein (γ-interferon-inducible activator of multicatalytic protease; ATP-dependent protease)Protein metabolism
      Proteasome θ chainP49720MSIEF 12323/6.12q351581046Ubiquitin proteasome system protein (γ-interferon-inducible activator of multicatalytic protease; ATP-dependent protease)Protein metabolism
      Protein-disulfide isomeraseP07237MS, CDKIEF 12457/4.7Yes17q252351831Enzyme: isomeraseProtein metabolism
      Protein-disulfide isomerase A3P30101MSIEF 125a, 125b57/6.0Yes15q153412447Enzyme: isomeraseProtein metabolism
      Protein-Tyr kinase Pyk2Q14289AAND8p22-p11.2Kinase: protein-tyrosine kinaseSignal transduction; cell communication
      PTENP60484AAND10q23Enzyme: hydrolaseSignal transduction; cell communication
      Pulmonary and activation-regulated chemokineP55774AA#Yes17q11.2Cytokine activityImmune response
      Pyridoxine kinaseO00764MSIEF 12635/5.7Yes21q22.32151442Enzyme: phosphotransferaseEnergy pathways
      Pyruvate kinase, isozymes M1/M2P14618MSNEPHGE 30a, 30b
      Molecular function as well as biological process was assigned in accordance with the Human Protein Reference Database (www.hprd.org).
      58/7.9Yes15q223142348Enzyme: phosphotransferaseEnergy pathways
      Rab GDI βP50395MSIEF 12750/6.1Yes10p151311433GTPase activatorTransport
      Raf kinase-binding proteinP30086MSIEF 128; NEPHGE 3121/7.4Yes12q24.23100745Protease inhibitor (binds ATP; binds to RAF1, MEK, and ERK; overexpression interferes with the activation of MEK and ERK and induction of AP1)Signal transduction; cell communication
      RAF1P04049AAND3p25Kinase serine/threonineSignal transduction; cell communication
      RANTESP13501AA#Yes17q11.2-q12Cytokine activitySignal transduction; cell communication
      Ras-related protein Rab-2AP61019MSIEF 12923/6.18q12.11881152Protein transportTransport
      Receptor of activated protein kinase C 1P63244MSNEPHGE 3235/7.6Yes5q35.31321032Adapter moleculeSignal transduction; cell communication
      Retinoblastoma-binding protein 4Q09028MSIEF 130a, 130b47/4.7Yes1p35.11361536Transcription regulation (Ras signal transduction pathway; mediates chromatin assembly in different process)Regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Retinoblastoma-binding protein P46Q16576MSIEF 13147.8/4.9Xp22.23411834Transcription regulationRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Rho GDP dissociation inhibitor 1P52565MSIEF 13223/5.0Yes17q25.3103729GTPase activatorCell growth and/or maintenance
      Rho GDP dissociation inhibitor 2 βP52566MSIEF 13323/5.0Yes12p12.3103933GTPase activatorSignal transduction; cell communication
      %
      Ribonuclease/angiogenin inhibitorP13489MSIEF 13449/4.7Yes11p15.51481231Translation regulationRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Selenium-binding protein 1 (minor component)Q13228MSIEF 135a, 135b52/6.1Yes1q21-q22133920UnknownProtein metabolism
      Serotransferrin, transferrinP02787IBIEF 78a, 78b, 78c, 78d77/6.8Yes3q22.1Transport/cargo proteinTransport
      Serum amyloid P-componentP02743MSIEF 13625.5/6.1Yes1q21-q23135931Secreted polypeptideProtein metabolism
      Serum- and glucocorticoid-inducible kinaseO00141AAND6q23Kinase serine/threonineSignal transduction; cell communication
      Skeletal muscle LIM-protein 1 (SLIM 1) isoform 1Q13642MSNEPHGE 3836/9.2Xq262681758UnclassifiedCell growth and/or maintenance
      SMAC/DIABLOQ9NR28AAND12q24.31Cell cycle control proteinSignal transduction; cell communication
      SMAD4Q13485AAND18q21.1Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Small inducible cytokine A28Q9NRJ3AA#Yes5p12Cytokine activitySignal transduction; cell communication
      Small inducible cytokine A3 (MIP-1α)P10147AA#Yes17q11-q21Cytokine activityImmune response
      Smooth muscle protein 22-α, SM22-αQ01995MSIEF 137; NEPHGE 3322/8.811q23.21701041Cytoskeleton-associated protein (actin binding; muscle development)Cell growth and/or maintenance
      Spermidine synthaseP19623MSIEF 13834.5/5.3Yes1p36-p2285818Enzyme: synthetaseEnergy pathways
      Stem cell factorP21583AA#Yes12q22Growth factorSignal transduction; cell communication
      Stromal cell-derived factor 1P48061AA#Yes10q11.1Cytokine activitySignal transduction; cell communication
      Succinyl-CoA synthetase, βA chainQ9P2R7MSIEF 13950/7.013q12.22211835Enzyme: synthetaseEnergy pathways
      Succinyl-CoA:3-ketoacid-coenzyme A transferase 1P55809MSIEF 14057/7.15p13.11101233Enzyme: CoA transferaseEnergy pathways
      Superoxide dismutase (Cu-Zn)P00441MS, IBIEF 14115/5.7Yes21q22.112081256Enzyme: superoxide dismutase (antioxidant; metal binding)Energy pathways
      Synaptosomal-associated protein 25P60880AAND20p11.2NeurotransmitterTransport
      SynaptotagminP21579AAND12cen-q21Calcium ion bindingSignal transduction; cell communication
      Synthase ATP, β chainP06576CKDIEF 14256/5.212q13.13Transport/cargo proteinEnergy pathways
      Syntrophin a1Q13424AAND20q11.2Adapter moleculeSignal transduction; cell communication
      Synuclein aP37840AAND4q21ChaperoneProtein metabolism
      Synuclein bQ16143AAND5q35UnknownSignal transduction; cell communication
      Thioredoxin peroxidase 1P10599MSIEF 14311/4.8Yes9q3188858Enzyme: reductaseEnergy pathways
      ThrombopoetinP40225AA#Yes3q27Growth factorSignal transduction; cell communication
      %
      Thymus and activation-regulated chemokineQ92583AA#Yes16q13Cytokine activitySignal transduction; cell communication (inflammatory response)
      Thymus-expressed chemokineO15444AA#Yes19p13.2Cytokine activityImmune response
      TIMP-1P01033AA#YesXp11.3-p11.23Extracellular matrix proteinCell growth and/or maintenance (metalloprotease inhibitor)
      TIMP-2P16035AA#Yes17q25Extracellular matrix proteinCell growth and/or maintenance (metalloprotease inhibitor)
      TRAIL receptor-3O14798AA#Yes8p22-p21Cell surface receptorUnknown
      TRAIL receptor-4Q9UBN6AA#Yes8p21Cell surface receptorSignal transduction; cell communication
      Transcription factor activator protein 1 (proto-oncogene c-Jun)P05412AAND1p32-p31Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      Transcription factor E2F1Q01094AAND20q11.2Transcription factorRegulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism
      TGF-β1P01137AA#Yes19q13.1Receptor binding (cytokine activity)Signal transduction; cell communication
      TGF-β3P10600AA#Yes14q24Growth factor (cytokine activity)Signal transduction; cell communication
      TransketolaseP29401MSIEF 144; NEPHGE 3467.8/7.6Yes3p14.31001021Enzyme: transketolaseEnergy pathways
      Translationally controlled tumor proteinP13693MS, CDKIEF 14519.5/4.8Yes13q12-q14855 (PSD: 1241.66)21UnknownUnknown
      TransthyretinP02766MSIEF 14616/5.5Yes18q11.2-q12.1907 (PSD: 1394.58)35Transport/cargo proteinTransport
      Triose-phosphate isomeraseP60174MS, CKDIEF 147a, 147b; NEPHGE 3526/6.5Yes12p131421245Enzyme: isomeraseEnergy pathways
      Tropomyosin 4 α chainP07226MSIEF 14828/4.6Yes19p13.12402046Cytoskeleton-associated protein, actin bindingCell growth and/or maintenance
      Tropomyosin 1 α chain (NH2-terminal part)P09493MSIEF 14933/4.7Yes15q22.180930Cytoskeleton-associated protein, actin bindingCell growth and/or maintenance
      Tropomyosin α 3 chainP06753MS, CDKIEF 15033/4.7Yes1q21.22401850Cytoskeleton-associated proteinCell growth and/or maintenance
      Tubulin γP23258AAND17q21Cytoskeleton-associated protein (GTP binding)Cell growth and/or maintenance
      Tumor necrosis factor receptor 1P19438AA#Yes12p13.2Cell surface receptor (cytokine activity)Signal transduction; cell communication
      Tumor necrosis factor receptor 2P20333AA#Yes1p36.3-p36.2Cell surface receptor (cytokine activity)Signal transduction; cell communication
      TNF-αP01375AA#Yes6p21.3Membrane-bound ligand (cytokine activity)Signal transduction; cell communication
      TYRO3 protein-tyrosine kinase 3Q86VR3AA#Yes15q15.1-q21.1Cytokine activity (receptor tyrosine kinase)Signal transduction; cell communication
      Tyrosine-protein kinase receptor UFOP30530AA#Yes19q13.1-q13.2Cytokine activity (receptor tyrosine kinase)Signal transduction; cell communication
      %
      Ubiquinol-cytochrome-c reductase complex core protein IP31930MSIEF 15153/5.9Yes3p21.33552661Enzyme: reductaseEnergy pathways
      UbiquitinP02248MS, CDKIEF 1528.5/6.5Yes12q24.31771085Ubiquitin-specific protease activityProtein metabolism
      Ubiquitin thiolesterase protein OTUB1Q96FW1MSIEF 15331/4.811q13.11841135Ubiquitin-specific protease activityProtein metabolism
      Ubiquitin-like protein NEDD8 (Nedd8)Q15843AAND14q11.2Ubiquitin-specific protease activityProtein metabolism
      Ubiquitin-protein ligase E3 Mdm2 (MDM2)Q00987AAND12q14.3-q15Ubiquitin-specific protease activityProtein metabolism
      UDP-glucose pyrophosphorylase 2Q16851MSNEPHGE 36a, 36b55.8/7.7Yes2p14-p132201433Enzyme: nucleotidyltransferaseEnergy pathways
      Urokinase plasminogen activator surface receptor precursorQ03405AA#Yes19q13Cytokine activity (receptor activity, GPI anchor)Signal transduction; cell communication
      Vascular endothelial growth factorP15692AA#Yes6p12Growth factorSignal transduction; cell communication
      Vascular endothelial growth factor DO43915AA#YesXp22.31Growth factorSignal transduction; cell communication
      VimentinP08670MS, IBIEF 154a, 154b, 154c53.5/5.0Yes10p133493347Cytoskeleton-associated proteinCell growth and/or maintenance
      VinculinP18206MSIEF 155123/5.5Yes10q22.1-q233413128Cytoskeleton-associated proteinCell growth and/or maintenance
      Vitamin D-binding proteinP02774MSIEF 15653/5.4Yes4q11-q133962950Transport/cargo proteinTransport
      f This protein migrates in a much higher molecular weight area of the 2D gel than expected. Since this observation was validated by the analysis of several samples and it may represent an interesting feature of this protein, it has been included in the table.
      a Whenever possible, the Swiss-Prot accession number was used.
      b MS, identification of proteins separated by 2D PAGE using MALDI-TOF-MS; IB, proteins identified by Western 2D gel immunoblotting; CKD, protein identification by matching the protein patterns to the master 2D gel keratinocyte data base (proteomics.cancer.dk); AA, protein identification using the Panorama Ab Microarray Cell Signaling array; AA#, proteins identified using the RayBio Human Cytokine Array C Series. Two proteins were identified using the Bio-Rad Bioplex system.
      c Proteins identified from 2D gels are indicated with either an IEF (Figs. 3 and 8) or an NEPHGE number (Figs. 4 and 9). Proteins migrating in both directions are indicated with both an IEF and NEPHGE number. Some proteins exhibited multiple isoforms, and these are indicated with a, b, c, etc.
      d The Mr and pI were calculated directly from the sequences. In some cases these values do not fit with the apparent molecular weights observed in the 2D gels shown in Figs. 3, 4, 8, and 9.
      e Molecular function as well as biological process was assigned in accordance with the Human Protein Reference Database (www.hprd.org).
      Figure thumbnail gr6
      Fig. 6Protein identification by 2D PAGE Western immunoblotting. 2D immunoblot of whole fat tissue proteins incubated with antibodies against vimentin, 14-3-3 β, and Crk (A); MEK-2 (B); A-FABP (C); and Nck (D).

       Identification of Cellular Effectors and Signaling Molecules in Triton X-100 Fat Extracts—

      Given the limitations inherent to 2D PAGE, we used multianalyte protein-based technologies to complement the gel-based proteomic analysis in an effort to detect lesser abundant cellular effectors and components of signaling pathways. To this end we treated fat tissue with Triton X-100 in PBS and incubated the extracts with the Panorama Ab Microarray-Cell Signaling array that contains 224 different antibodies against components of various biological pathways (see Fig. 7) as described under “Experimental Procedures.” Fig. 7 shows an array with the presence of 80 components detected in this particular case. A list of proteins identified by this approach is given in Table I.
      Figure thumbnail gr7
      Fig. 7Detection of signaling molecules in fat tissue Triton X-100 extracts. Shown is a representative picture of the results obtained with the Panorama signaling antibody array (Sigma). PKB, protein kinase B; ATF2, activating transcription factor 2; MAP, microtubule-associated protein; PCAF, p300/CBP-associated factor; CAM, calmodulin; EGF, epidermal growth factor; PKC, protein kinase C; PKD, protein kinase D; e-NOS, endothelial nitric-oxide synthase; i-NOS, inducible nitric-oxide synthase; b-NOS, brain-derived nitric-oxide synthase; HSP, heat shock protein; DAPK, death-associated protein kinase; CUG-BP1, CUG repeat-binding protein 1; GAP1, GTPase-activating protein 1; NMDAR, N-methyl-d-aspartate receptor; MAP Kinase, mitogen-activated protein kinase.

       Protein Profiling of the FIF—

      FIF recovered from fresh fat tissue specimens dissected from sites topologically distant from the tumor of 20 high risk breast cancer patients (Fig. 2A) was analyzed by 2D PAGE as described under “Experimental Procedures.” Figs. 8 and 9 show representative IEF (Fig. 8) and NEPHGE (Fig. 9) gels of FIF proteins from patient 48 stained with silver nitrate. A total of 1040 proteins was detected (786 IEF and 254 NEPHGE), and of these about 70 migrated both in IEF and IF 48; Fig. 10). Proteins present in whole fat tissue extracts that are either absent or present at very low levels in the FIF are indicated with red arrowheads in Figs. 3, 4, 8, and 9, whereas proteins enriched in the FIF are indicated with green arrows. Where appropriate, FIF proteins are indicated in Figs. 8 and 9 with the same numbers as in Figs. 3 and 4 and are listed in Table I.
      Figure thumbnail gr8
      Fig. 8IEF 2D PAGE of FIF proteins stained with silver nitrate. Selected proteins indicated with red arrows are expressed at low levels or are absent in the FIF (see also ). A few proteins indicated with green arrows are enriched in the FIF (see also ). The identity of the proteins indicated with numbers is given in .
      Figure thumbnail gr9
      Fig. 9NEPHGE 2D PAGE of FIF proteins stained with silver nitrate. Selected proteins indicated with red arrows are expressed at low levels or are absent in the FIF (see also ). A few proteins indicated with green arrows are enriched in the FIF (see also ). The identity of the proteins indicated with numbers is given in .
      Figure thumbnail gr10
      Fig. 10Cytokine profiling of FIF. Cytokine-specific antibody arrays (RayBio Human Cytokine Array Series 1000, RayBiotech, Inc.) were incubated with 0.5 ml of FIF 48 and TIF 48, respectively, according to the manufacturer's instructions. POS, positive; NEG, negative; PDGF, platelet-derived growth factor; RANTES, regulated on activation normal T cell expressed and secreted; MCP, monocyte chemotactic protein; CNTF, ciliary neurotrophic factor; BMP, bone morphogenic protein; FGF, fibroblast growth factor; SDF, stromal cell-derived factor; M-CSF, macrophage CSF; GM-CSF, granulocyte-macrophage CSF; BDNF, brain-derived neurotrophic factor; BLC, B lymphocyte chemoattractant; SCF, stem cell factor; MDC, macrophage-derived chemokine; MIG, γ interferon-induced monokine, MIP, macrophage inflammatory protein; IGFBP, IGF-binding protein; NT, neurotrophin; TARC, thymus and activation-regulated chemokine; PARC, pulmonary and activation-regulated chemokine; IFN, interferon; CTACK, cutaneous T-cell-attracting chemokine; ICAM, intercellular adhesion molecule; I-TAC, interferon-γ-inducible T-cell α chemoattractant; TECK, thymus-expressed chemokine; EGF-R, epidermal growth factor receptor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; VEGF, vascular endothelial growth factor; GITR, glucocorticoid-induced TNF receptor; HCC, human CC chemokine; PIGF, phosphatidylinositol-glycan biosynthesis, class F protein; bFGF, basic fibroblast growth factor; uPAR, urokinase plasminogen activator surface receptor precursor; R, receptor; sTNF, soluble TNF; HGF, hepatocyte growth factor; MSP, macrophage-stimulating protein; GRO, growth-related oncogene; ENA-78, epithelial neutrophil-activating protein 78.
      Low abundance cytokines and growth factors were detected by incubating FIF preparations with a multiple cytokine antibody array as described under “Experimental Procedures” (RayBio Human Cytokine Array C Series, RayBiotech, Inc.). As an illustrative example, Fig. 9 depicts arrays showing the presence of 98 cytokines and growth factors in FIF 48. Similar analysis of FIFs recovered from the fat tissue of several patients (results not shown) indicated that the cytokine patterns are quite similar among each other, albeit with some changes in their levels, but distinctive from the tumor interstitial fluid (compare FIF 48 with TIF 48; Fig. 9). Two proteins (granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte CSF) were identified using the Bio-Rad Bioplex cytokine system as described previously (
      • Celis J.E.
      • Moreira J.M.
      • Gromova I.
      • Cabezon T.
      • Ralfkiaer U.
      • Guldberg P.
      • Straten P.T.
      • Mouridsen H.
      • Friis E.
      • Holm D.
      • Rank F.
      • Gromov P.
      Towards discovery-driven translational research in breast cancer.
      ). A complete list of proteins detected in the FIF, including cytokines and growth factors, is given in Table I.

       Functional Classification—

      All in all, the study identified 359 primary translation products present in the fat tissue and its interstitial fluid (Table I). The molecular functions of these proteins as well as the biological process in which they participate were assigned in accordance with the Human Protein Resource Database (www.hprd.org) and are given in Table I. The list includes, but is not limited to, polypeptides involved in various biological processes such as signal transduction and cell communication (34%); energy metabolism (19%); protein metabolism (12%); cell growth and/or maintenance (10%); immune response (10%); transport (6%); regulation of nucleobase, nucleoside, and nucleic acid metabolism (5%); and apoptosis (3%). Approximately 1% of the proteins are of unknown function (Fig. 11).
      Figure thumbnail gr11
      Fig. 11Functional categories of proteins identified in the fat tissue and its interstitial fluid recovered from biopsies topologically distant from the breast tumor.

      DISCUSSION

      A growing body of research indicates that tumor growth in vivo is dependent, in part, on adipose tissue. In addition, large epidemiology studies have found that obese women are at increased risk of developing postmenopausal breast cancer (
      • Vainio H.
      • Bianchini F.
      ,
      • Harvie M.
      • Hooper L.
      • Howell A.H.
      Central obesity and breast cancer risk: a systematic review.
      ), and there is some evidence that environmental cues from adipocytes affect tumor cell survival, proliferation, differentiation, and migration. However, the exact mechanisms by which adipose tissue can promote an aggressive breast cancer phenotype are poorly understood. The results presented here provide for the first time a broad overview of the proteome of fresh mammary fat tissue and its interstitial fluid. The gel-based analysis resolved more than 1000 well focused proteins of which 183 unique polypeptides were identified using mass spectrometry (169 polypeptides), immunoblotting (7 polypeptides), and comparison with the keratinocyte protein database (7 polypeptides). Due to the inherent limitations of the gel-based approach, we extended these studies by using antibody arrays, which led to the identification of an additional 178 unique proteins, mainly signaling molecules, hormones, cytokines, and growth factors. Considering that one of the main functions of adipocytes is to act as an energy reservoir by storing lipids (
      • Klaus S.
      Adipose tissue as a regulator of energy balance.
      ,
      • Reidy S.P.
      • Weber J.
      Leptin: an essential regulator of lipid metabolism.
      ), it is not surprising that many of the proteins identified corresponded to components that play a role in energy metabolism (Fig. 11 and Table I). These include many enzymes involved in lipid metabolism as well as the hormones leptin and adiponectin, both of which are components of the FIF. These hormones have been the subject of extensive research (
      • Mora S.
      • Pessin J.E.
      An adipocentric view of signaling and intracellular trafficking.
      ,
      • Zhang Y.
      • Proenca R.
      • Maffei M.
      • Barone M.
      • Leopold L.
      • Friedman J.M.
      Positional cloning of the mouse obese gene and its human homologue.
      ,
      • Friedman J.M.
      • Halaas J.L.
      Leptin and the regulation of body weight in mammals.
      ,
      • Sierra-Honigmann M.R.
      • Nath A.K.
      • Murakami C.
      • Garcia-Cardena G.
      • Papapetropoulos A.
      • Sessa W.C.
      • Madge L.A.
      • Schechner J.S.
      • Schwabb M.B.
      • Polverini P.J.
      • Flores-Riveros J.R.
      Biological action of leptin as an angiogenic factor.
      ,
      • Bouloumie A.
      • Drexler H.C.
      • Lafontan M.
      • Busse R.
      Leptin, the product of Ob gene, promotes angiogenesis.
      ,
      • Liu Z.
      • Uesaka T.
      • Watanabe H.
      • Kato N.
      High fat diet enhances colonic cell proliferation and carcinogenesis in rats by elevating serum leptin.
      ,
      • Laud K.
      • Gourdou I.
      • Pessemesse L.
      • Peyrat J.P.
      • Djiane J.
      Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line.
      ,
      • Dieudonne M.N.
      • Machinal-Quelin F.
      • Serazin-Leroy V.
      • Leneveu M.C.
      • Pecquery R.
      • Giudicelli Y.
      Leptin mediates a proliferative response in human MCF7 breast cancer cells.
      ,
      • Somasundar P.
      • McFadden D.W.
      • Hileman S.M.
      • Vona-Davis L.
      Leptin is a growth factor in cancer.
      ,
      • Yamauchi T.
      • Kamon J.
      • Waki H.
      • Terauchi Y.
      • Kubota N.
      • Hara K.
      • Mori Y.
      • Ide T.
      • Murakami K.
      • Tsuboyama-Kasaoka N.
      • Ezaki O.
      • Akanuma Y.
      • Gavrilova O.
      • Vinson C.
      • Reitman M.L.
      • Kagechika H.
      • Shudo K.
      • Yoda M.
      • Nakano Y.
      • Tobe K.
      • Nagai R.
      • Kimura S.
      • Tomita M.
      • Froguel P.
      • Kadowaki T.
      The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.
      ,
      • Iyengar P.
      • Scherer P.E.
      Adiponectin/Acrp30, an adipocyte-specific secretory factor: physiological relevance during development.
      ,
      • Gil-Campos M.
      • Canete R.R.
      • Gil A.
      Adiponectin, the missing link in insulin resistance and obesity.
      ,
      • Guerre-Millo M.
      Adipose tissue hormones.
      ,
      • Hukshorn C.J.
      • Saris W.H.
      Leptin and energy expenditure.
      ,
      • Fasshauer M.
      • Paschke R.
      • Stumvoll M.
      Adiponectin, obesity, and cardiovascular disease.
      ), and we will therefore restrict the discussion of our results to cytokines and growth factors secreted by adipocytes as well as to signaling pathways present in these cells.

       Cytokines and Growth Factors Secreted by Adipocytes

      There is compelling evidence indicating that adipocytes play a role in normal mammary epithelia development (
      • Wiseman B.S.
      • Werb Z.
      Stromal effects on mammary gland development and breast cancer.
      ,
      • Schmeichel K.L.
      • Weaver V.M.
      • Bissell M.J.
      Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype.
      ) as well as in tumorigenesis (
      • Wiseman B.S.
      • Werb Z.
      Stromal effects on mammary gland development and breast cancer.
      ,
      • Iyengar P.
      • Combs T.P.
      • Shah S.J.
      • Gouon-Evans V.
      • Pollard J.W.
      • Albanese C.
      • Flanagan L.
      • Tenniswood M.P.
      • Guha C.
      • Lisanti M.P.
      • Pestell R.G.
      • Scherer P.E.
      Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
      ,
      • Elliott B.E.
      • Tam S.P.
      • Dexter D.
      • Chen Z.Q.
      Capacity of adipose tissue to promote growth and metastasis of a murine mammary carcinoma: effect of estrogen and progesterone.
      ,
      • Johnston P.G.
      • Rondinone C.M.
      • Voeller D.
      • Allegra C.J.
      Identification of a protein factor secreted by 3T3-L1 preadipocytes inhibitory for the human MCF-7 breast cancer cell line.
      ), and we have frequently detected tumor cells interdigitating with, and spreading through, the peripheral fat tissue in high risk breast cancer patients suggesting a close association between these cell types (Fig. 1). These observations prompted us to search for growth factors and cytokines that may be produced by adipocytes in vivo and that may lead to a better understanding of the mechanisms underlying this close association. To this end, we took advantage of a simple protocol that we devised for recovering the interstitial fluid that bathes the breast tumor microenvironment (TIF; Refs.
      • Celis J.E.
      • Gromov P.
      • Cabezon T.
      • Moreira J.M.
      • Ambartsumian N.
      • Sandelin K.
      • Rank F.
      • Gromova I.
      Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery.
      and
      • Celis J.E.
      • Moreira J.M.
      • Gromova I.
      • Cabezon T.
      • Ralfkiaer U.
      • Guldberg P.
      • Straten P.T.
      • Mouridsen H.
      • Friis E.
      • Holm D.
      • Rank F.
      • Gromov P.
      Towards discovery-driven translational research in breast cancer.
      ). The TIF is composed of hundreds of proteins that are either secreted, shed by membrane vesicle-like exosomes (
      • Nickel W.
      The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.
      ,
      • Stoorvogel W.
      • Kleijmeer M.J.
      • Geuze H.J.
      • Raposo G.
      The biogenesis and functions of exosomes.
      ,
      • Peng X.
      • Wu Y.
      • Chen J.
      • Wang S.
      Proteomic approach to identify acute phase response-related proteins with low molecular weight in loach skin following injury.
      ,
      • Pisitkun T.
      • Shen R.F.
      • Knepper M.A.
      Identification and proteomic profiling of exosomes in human urine.
      ), and/or externalized due to cell death, and preliminary results indicated that the procedure could also be applied to fat tissue (
      • Celis J.E.
      • Gromov P.
      • Cabezon T.
      • Moreira J.M.
      • Ambartsumian N.
      • Sandelin K.
      • Rank F.
      • Gromova I.
      Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery.
      ,
      • Celis J.E.
      • Moreira J.M.
      • Gromova I.
      • Cabezon T.
      • Ralfkiaer U.
      • Guldberg P.
      • Straten P.T.
      • Mouridsen H.
      • Friis E.
      • Holm D.
      • Rank F.
      • Gromov P.
      Towards discovery-driven translational research in breast cancer.
      ).
      Apart from providing a first glance at the in vivo mammary adipocyte secretome, our studies revealed proinflammatory cytokines (interleukin (IL)-6, IL-8, IL-10, transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, and nerve growth factor) (
      • Trayhurn P.
      • Wood I.S.
      Adipokines: inflammation and the pleiotropic role of white adipose tissue.
      ,
      • Hotamisligil G.S.
      • Shargill N.S.
      • Spiegelman B.M.
      Adipose expression of tumor necrosis factor-α: direct role in obesity-linked insulin resistance.
      ), growth factors (insulin-like growth factor (IGF)-I, IGF-binding proteins, TNF-α, angiotensin II, and macrophage colony-stimulating factor) that are known to stimulate cell proliferation (Refs.
      • Trayhurn P.
      • Wood I.S.
      Adipokines: inflammation and the pleiotropic role of white adipose tissue.
      and
      • Hausman D.B.
      • DiGirolamo M.
      • Bartness T.J.
      • Hausman G.J.
      • Martin R.J.
      The biology of white adipocyte proliferation.
      and references therein), angiogenic factors (vascular endothelial growth factor, angiogenin, angiopoietin-2, granulocyte CSF, epidermal growth factor, fibroblast growth factors, hepatocyte growth factor, TGF-α and -β, and leptin) needed for fat expansion (Refs.
      • Bouloumie A.
      • Drexler H.C.
      • Lafontan M.
      • Busse R.
      Leptin, the product of Ob gene, promotes angiogenesis.
      ,
      • Beecken W.D.
      • Kramer W.
      • Jonas D.
      New molecular mediators in tumor angiogenesis.
      , and
      • Rupnick M.A.
      • Panigrahy D.
      • Zhang C.Y.
      • Dallabrida S.M.
      • Lowell B.B.
      • Langer R.
      • Folkman M.J.
      Adipose tissue mass can be regulated through the vasculature.
      and references therein), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) that hamper matrix metalloproteinase activity and invasion of tumor cells (Refs.
      • Lee Y.K.
      • So I.S.
      • Lee S.C.
      • Lee J.H.
      • Lee C.W.
      • Kim W.M.
      • Park M.K.
      • Lee S.T.
      • Park D.Y.
      • Shin D.Y.
      • Park C.U.
      • Kim Y.S.
      Suppression of distant pulmonary metastasis of MDA-MB 435 human breast carcinoma established in mammary fat pads of nude mice by retroviral-mediated TIMP-2 gene transfer.
      and
      • Brew K.
      • Dinakarpandian D.
      • Nagase H.
      Tissue inhibitors of metalloproteinases: evolution, structure and function.
      and references therein). In addition, we identified several cytokines and growth factors that have not been previously associated with the fat tissue (Fig. 7 and Table I).
      From our immunofluorescence analysis it seems likely that breast tumor cells and adipocytes provide mutual growth support to each other via the secretion of substances that may offer common benefits. For example, we often observed isolated preadipocytes in the stroma surrounding the breast tumor cells (Fig. 12, inset), suggesting that the latter secrete factors that directly, or indirectly through other cell types present in the microenvironment, commit pluripotent mesenchymal stem cells to adipocyte differentiation (
      • Gregoire F.
      • De Broux N.
      • Hauser N.
      • Heremans H.
      • Van Damme J.
      • Remacle C.
      Interferon-γ and interleukin-1β inhibit adipoconversion in cultured rodent preadipocytes.
      ,
      • Dani C.
      Embryonic stem cell-derived adipogenesis.
      ,
      • MacDougald O.A.
      • Mandrup S.
      Adipogenesis: forces that tip the scales.
      ). Preadipocytes recruited in this way differentiate into mature adipocytes under suitable conditions and may provide the tumor cells with a friendly environment in which to spread (Fig. 12). Expanding adipose tissue requires active angiogenesis, and sites of neovascular angiogenesis are critical for tumor progression (
      • Giatromanolaki A.
      • Sivridis E.
      • Koukourakis M.I.
      Tumour angiogenesis: vascular growth and survival.
      ,
      • Dass C.R.
      Tumour angiogenesis, vascular biology and enhanced drug delivery.
      ). The nature of the interplay between adipocytes and tumor cells is at present unknown, although the availability of the FIF may allow studying the effect of the adipokine mixture on breast epithelial cells using three-dimensional cultures of both normal and malignant breast tissue (
      • Huss F.R.
      • Kratz G.
      Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue.
      ,
      • Celis J.E.
      • Gromov P.
      • Gromova I.
      • Moreira J.M.
      • Cabezon T.
      • Ambartsumian N.
      • Grigorian M.
      • Lukanidin E.
      • Thor Straten P.
      • Guldberg P.
      • Bartkova J.
      • Bartek J.
      • Lukas J.
      • Lukas C.
      • Lykkesfeldt A.
      • Jaattela M.
      • Roepstorff P.
      • Bolund L.
      • Orntoft T.
      • Brunner N.
      • Overgaard J.
      • Sandelin K.
      • Blichert-Toft M.
      • Mouridsen H.
      • Rank F.E.
      Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research.
      ,
      • Schmeichel K.L.
      • Weaver V.M.
      • Bissell M.J.
      Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype.
      ). Alternatively there are various model systems that have been described to study adipocyte differentiation (
      • Kratchmarova I.
      • Kalume D.E.
      • Blagoev B.
      • Scherer P.E.
      • Podtelejnikov A.V.
      • Molina H.
      • Bickel P.E.
      • Andersen J.S.
      • Fernandez M.M.
      • Bunkenborg J.
      • Roepstorff P.
      • Kristiansen K.
      • Lodish H.F.
      • Mann M.
      • Pandey A.
      A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.
      ,
      • Wabitsch M.
      • Brenner R.E.
      • Melzner I.
      • Braun M.
      • Moller P.
      • Heinze E.
      • Debatin K.M.
      • Hauner H.
      Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation.
      ), although these may not completely duplicate the in vivo situation (Refs.
      • Soukas A.
      • Socci N.D.
      • Saatkamp B.D.
      • Novelli S.
      • Friedman J.M.
      Distinct transcriptional profiles of adipogenesis in vivo and in vitro.
      and
      • Klaus S.
      Adipose tissue as a regulator of energy balance.
      and references therein).
      Figure thumbnail gr12
      Fig. 12Presence of preadipocytes in the stroma surrounding tumor cells as determined by indirect immunocytochemistry. Preadipocytes and adipocytes are labeled green (A-FABP staining), whereas tumor cells are red (cytokeratin 19 staining). The inset shows a higher magnification of an area of the section distal to the fat-tumor interface depicting a single preadipocyte.
      It should be stressed that, in addition to the cytokines and growth factors mentioned above, the FIF also contains proteins that may have important functions in the tumor microenvironment. For example, we detected the presence of type VI collagen that has been described previously by Scherer and colleagues (
      • Scherer P.E.
      • Bickel P.E.
      • Kotler M.
      • Lodish H.F.
      Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.
      ) in adipocyte tissue as an adipocyte-enriched secretory protein that promotes pro-oncogenic pathways in breast cancer cells and that may play a key role in the regulation of normal and transformed mesenchymal cell proliferation in vitro (
      • Atkinson J.C.
      • Ruhl M.
      • Becker J.
      • Ackermann R.
      • Schuppan D.
      Collagen VI regulates normal and transformed mesenchymal cell proliferation in vitro.
      ) as well as in preventing apoptosis (
      • Ruhl M.
      • Sahin E.
      • Johannsen M.
      • Somasundaram R.
      • Manski D.
      • Riecken E.O.
      • Schuppan D.
      Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax.
      ). It has also been shown that type VI collagen promotes the phosphorylation of glycogen synthase kinase-3β in malignant ductal epithelial cells, leading to increased β-catenin stabilization and transcriptional activity (
      • Iyengar P.
      • Combs T.P.
      • Shah S.J.
      • Gouon-Evans V.
      • Pollard J.W.
      • Albanese C.
      • Flanagan L.
      • Tenniswood M.P.
      • Guha C.
      • Lisanti M.P.
      • Pestell R.G.
      • Scherer P.E.
      Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
      ).

       Signaling Pathways in Mammary Fat Tissue

      By using Triton X-100 extracts of fat tissue in combination with multianalyte protein-based technologies for the detection of low abundance and phosphorylation-dependent key regulatory proteins it was possible to perform signal pathway profiling of mammary adipocytes focusing on discovery and quantitative analysis of proteins involved in a variety of biological processes that include apoptosis, cell cycle, stress response, transcription, and signal transduction. The Panorama Ab Microarray-Cell Signaling array used for this purpose contains over 224 antibodies (for a description, see www.sigmaaldrich.com/img/assets/5941/Antibody_List.pdf) with some probes being specific for the phosphorylated protein of interest (e.g. focal adhesion kinase, MAPK, Raf, p38, Pyk2, p21-activated kinase 1, and death-associated protein kinase). In this way we were able to detect the presence of many low abundance signaling proteins in all biological processes examined, providing some insight into the cellular circuitry of adipocytes. We present below an overall functional view of the obtained results.

       Cell Cycle—

      Adipose tissue is a dynamic body that changes in response to metabolic cues (
      • Camp H.S.
      • Ren D.
      • Leff T.
      Adipogenesis and fat-cell function in obesity and diabetes.
      ). Following key extracellular and intracellular signals, adipose tissue cell number increases through fat cell formation or adipogenesis, a process in which preadipocytes differentiate to become mitotically quiescent adipocytes (
      • Gregoire F.M.
      Adipocyte differentiation: from fibroblast to endocrine cell.
      ). Adipocyte withdrawal from the cell cycle is presumably regulated by cell cycle inhibitors (
      • Naaz A.
      • Holsberger D.R.
      • Iwamoto G.A.
      • Nelson A.
      • Kiyokawa H.
      • Cooke P.S.
      Loss of cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obesity.
      ). We identified several cell cycle regulators, positive as well as negative (e.g. p53, MDM2, p21Waf1, p34cdc2, p19INK4d, and Cdk4), and cyclins (cyclins B1, D2, and D3) reflecting the mixed nature of the samples, which contain both differentiated adipocytes as well as preadipocytes.

       Apoptosis—

      Regulation of adipocyte cell number by apoptosis is thought to play an important role in adipose tissue homeostasis and to be part of a normal physiological cycle in adipocyte growth and development that can be altered under a variety of physiological and pathological conditions. Adipokines such as leptin, TNF-α, and ciliary neurotrophic factor can induce adipose tissue apoptosis (
      • Gullicksen P.S.
      • Della-Fera M.A.
      • Baile C.A.
      Leptin-induced adipose apoptosis: Implications for body weight regulation.
      ,
      • Duff E.
      • Li C.L.
      • Hartzell D.L.
      • Choi Y.H.
      • Della-Fera M.A.
      • Baile C.A.
      Ciliary neurotrophic factor injected icv induces adipose tissue apoptosis in rats.
      ,
      • Warne J.P.
      Tumour necrosis factor α: a key regulator of adipose tissue mass.
      ), suggesting that adipose tissue cell number is regulated, at least in part, by an apoptotic signaling pathway involving caspase activation. Our cytokine array analysis showed that leptin, TNF-α, and ciliary neurotrophic factor were all present in the samples examined (Fig. 7 and Table I), and we identified several components of the caspase cascade including active effector caspase-3, and caspase-6, -7, -8, -10, and -11 as well as other apoptotic factors such as apoptosis-inducing factor, Smac/DIABLO, Bcl-x, and Bcl-10 (Fig. 7 and Table I). We also identified components of the death receptor signaling pathway such as Fas and DAXX.

       Transcription Factors—

      Several transcription factors were detected in the fat tissue extracts analyzed (Fig. 7 and Table I). Some of these such as c-Jun, c-Myc, and E2F1 are known to regulate the adipogenic program (
      • Ninomiya-Tsuji J.
      • Torti F.M.
      • Ringold G.M.
      Tumor necrosis factor-induced c-myc expression in the absence of mitogenesis is associated with inhibition of adipocyte differentiation.
      ,
      • Zeng G.
      • Dave J.R.
      • Chiang P.K.
      Induction of proto-oncogenes during 3-deazaadenosine-stimulated differentiation of 3T3-L1 fibroblasts to adipocytes: mimicry of insulin action.
      ,
      • Freytag S.O.
      • Geddes T.J.
      Reciprocal regulation of adipogenesis by Myc and C/EBP α.
      ,
      • Fajas L.
      • Landsberg R.L.
      • Huss-Garcia Y.
      • Sardet C.
      • Lees J.A.
      • Auwerx J.
      E2Fs regulate adipocyte differentiation.
      ,
      • Smyth M.J.
      • Sparks R.L.
      • Wharton W.
      Proadipocyte cell lines: models of cellular proliferation and differentiation.
      ), lending some support to our experimental approach. We also detected the NEDD8 ubiquitin-like protein, a polypeptide that controls the activity of stem cell factor ubiquitin ligase complexes and that can promote modification of the p53 tumor suppressor protein and Mdm2 (
      • Xirodimas D.P.
      • Saville M.K.
      • Bourdon J.C.
      • Hay R.T.
      • Lane D.P.
      Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.
      ), both of which were found in our samples (Fig. 7 and Table I).
      One factor we identified that is of great interest in the context of breast cancer is the estrogen receptor (ER) (Fig. 7 and Table I). Several findings indicate that mature human adipocytes possess ERs and thus might be an estrogen-responsive tissue (
      • Pedersen S.B.
      • Hansen P.S.
      • Lund S.
      • Andersen P.H.
      • Odgaard A.
      • Richelsen B.
      Identification of oestrogen receptors and oestrogen receptor mRNA in human adipose tissue.
      ,
      • Mizutani T.
      • Nishikawa Y.
      • Adachi H.
      • Enomoto T.
      • Ikegami H.
      • Kurachi H.
      • Nomura T.
      • Miyake A.
      Identification of estrogen receptor in human adipose tissue and adipocytes.
      ). Recent work by Manabe and colleagues (
      • Manabe Y.
      • Toda S.
      • Miyazaki K.
      • Sugihara H.
      Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions.
      ) has shown that mature adipocytes may be involved in the mechanisms regulating the growth of breast tumors through their growth-promoting effect on ER-positive tumor cells. Furthermore increased leptin levels in breast cancer patients are associated with enhanced blood plasma concentrations of progesterone and estradiol as well as enhanced tissue levels of ER and progesterone receptor suggesting that leptin stimulates the production of sex hormones (
      • Tessitore L.
      • Vizio B.
      • Pesola D.
      • Cecchini F.
      • Mussa A.
      • Argiles J.M.
      • Benedetto C.
      Adipocyte expression and circulating levels of leptin increase in both gynaecological and breast cancer patients.
      ). In view of these data and considering that breast cancer is a disease where treatment is largely based on antiestrogen therapy, the presence of large amounts of adipose tissue should be of major therapeutical concern and must be taken into consideration. Another class of transcription factors present in substantial amounts in the samples examined included histone acetylases (HAT1 and p300/CBP-associated factor) and deacetylases (histone deacetylases 1, 2, and 4), which most likely reflect a central role in the adipose cell differentiation program (
      • Wiper-Bergeron N.
      • Wu D.
      • Pope L.
      • Schild-Poulter C.
      • Hache R.J.
      Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex.
      ,
      • Lagace D.C.
      • Nachtigal M.W.
      Inhibition of histone deacetylase activity by valproic acid blocks adipogenesis.
      ).

       TGF-β/Smad Signaling—

      One of the most prominent features we observed in our array analysis was the relatively high level expression of Smad4 (Fig. 7), suggesting that TGF-β signaling (detected by cytokine array; Fig. 10) might play a key regulatory role in the mammary fat tissue of breast cancer patients given that repression of the activity of the key adipogenic transcription factors, CCAAT/enhancer-binding proteins, by Smad3/4 at CCAAT/enhancer-binding protein binding sites is known to block adipogenesis (
      • Choy L.
      • Derynck R.
      Transforming growth factor-β inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function.
      ).

       Nitric Oxide Signaling—

      Nitric oxide is involved in adipose tissue biology by influencing adipogenesis, insulin-stimulated glucose uptake, and lipolysis (
      • Engeli S.
      • Janke J.
      • Gorzelniak K.
      • Bohnke J.
      • Ghose N.
      • Lindschau C.
      • Luft F.C.
      • Sharma A.M.
      Regulation of the nitric oxide system in human adipose tissue.
      ). We observed the presence of several enzymes responsible for nitric oxide formation (e.g. endothelial and inducible nitric-oxide synthases) in adipose cells (Fig. 7).

       MAPK Signaling—

      Of the three MAPK pathways (p38MAPK, ERK1/2, and c-Jun NH2-terminal kinase (JNK)) we could identify components of two of them (p38MAPK and ERK1/2). However, although we could detect the presence of p38MAPK, we failed to detect the activated phosphorylated form of this protein, suggesting that p38 is present in mammary fat tissue in a latent form. This is consistent with the main known role that p38MAPK plays in adipose tissue metabolism as a central mediator of the cAMP signaling mechanism of brown fat that promotes thermogenesis by phosphorylating activating transcription factor 2 (Fig. 7, ATF2) (
      • Cao W.
      • Daniel K.W.
      • Robidoux J.
      • Puigserver P.
      • Medvedev A.V.
      • Bai X.
      • Floering L.M.
      • Spiegelman B.M.
      • Collins S.
      p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene.
      ). The presence of the phosphorylated form of the other MAPK pathway effector we observed, ERK1/2, as well as an epistatic component of the pathway (c-Raf) suggests that this pathway is active in mammary fat tissue. Signaling by the ERK pathway is reportedly involved in adipocyte cellular responses to cell size sensing (
      • Farnier C.
      • Krief S.
      • Blache M.
      • Diot-Dupuy F.
      • Mory G.
      • Ferre P.
      • Bazin R.
      Adipocyte functions are modulated by cell size change: potential involvement of an integrin/ERK signalling pathway.
      ), presence of adipokines (
      • Brown J.M.
      • Boysen M.S.
      • Chung S.
      • Fabiyi O.
      • Morrison R.F.
      • Mandrup S.
      • McIntosh M.K.
      Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines.
      ,
      • Valladares A.
      • Porras A.
      • Alvarez A.M.
      • Roncero C.
      • Benito M.
      Noradrenaline induces brown adipocytes cell growth via β-receptors by a mechanism dependent on ERKs but independent of cAMP and PKA.
      ), and differentiation (
      • Tanabe Y.
      • Koga M.
      • Saito M.
      • Matsunaga Y.
      • Nakayama K.
      Inhibition of adipocyte differentiation by mechanical stretching through ERK-mediated downregulation of PPARγ2.
      ,
      • Prusty D.
      • Park B.H.
      • Davis K.E.
      • Farmer S.R.
      Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα gene expression during the differentiation of 3T3-L1 preadipocytes.
      ). Consequently activity of this pathway in mammary fat tissue is consistent with a normal physiological cycle in adipocyte growth and development.
      These data also suggest that ERK might be the most prominent of the MAPK signaling pathways in adipose tissue homeostasis in non-pathological conditions. Recent findings have shown that chronic activation of ERK, p38, or JNK can induce insulin resistance with the contribution of ERK being the strongest (
      • Fujishiro M.
      • Gotoh Y.
      • Katagiri H.
      • Sakoda H.
      • Ogihara T.
      • Anai M.
      • Onishi Y.
      • Ono H.
      • Abe M.
      • Shojima N.
      • Fukushima Y.
      • Kikuchi M.
      • Oka Y.
      • Asano T.
      Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes.
      ), lending some support to our observation.
      Another important observation is the lack of JNK MAPK protein in the fat tissue samples. It has been shown that JNK is activated during obesity (
      • Hirosumi J.
      • Tuncman G.
      • Chang L.
      • Gorgun C.Z.
      • Uysal K.T.
      • Maeda K.
      • Karin M
      • Hotamisligil G.S.
      A central role for JNK in obesity and insulin resistance.
      ), and recent genetic and pharmacological data indicate that activated JNK could be critical in causing diabetes and insulin resistance (
      • Bennett B.L.
      • Satoh Y.
      • Lewis A.J.
      JNK: a new therapeutic target for diabetes.
      ). Thus, it would appear that the presence and subsequent activation of JNK in adipose tissue occurs only in response to adipose tissue metabolism-related stimuli, which would account for the lack of JNK expression in our samples.

       Protein Kinase C and Phospholipase Signaling—

      Several reports demonstrated a functional role for the protein kinase B signaling pathway in adiposity (
      • Peng X.D.
      • Xu P.Z.
      • Chen M.L.
      • Hahn-Windgassen A.
      • Skeen J.
      • Jacobs J.
      • Sundararajan D.
      • Chen W.S.
      • Crawford S.E.
      • Coleman K.G.
      • Hay N.
      Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2.
      ,
      • Whiteman E.L.
      • Cho H.
      • Birnbaum M.J.
      Role of Akt/protein kinase B in metabolism.
      ). Consistent with these observations we found the presence of the inactive non-phosphorylated form of protein kinase B/AKT but not the phosphorylated forms (Thr308 and Ser473) in mammary fat tissue. We also identified several other proteins involved in phospholipase signaling such as MAPK phosphatase-1, PTEN, serum- and glucocorticoid-inducible kinase, and protein kinase D but not protein kinase C (α, β, or γ).

       Cytoskeletal Signaling—

      We also found a significant number of molecules involved in cytoskeletal cell signaling, both structural, such as connexin 43, microtubule-associated proteins 2A/2B, caveolin, catenins (α and β), stathmin, cofilin, chondroitin sulfate, and associated molecules like Grb2 or focal adhesion kinase.
      To conclude, our proteomic analysis is the most extensive carried out to date, and although the DNA microarray studies have identified many more genes, proteomics provided us with a glance at the gene products that are actually present in these cells. In particular, the large number of cytokines and growth factors secreted by adipocytes add to the complex mix of factors present in the fluid that bathes the tumor microenvironment (TIF) (
      • Hansen R.K.
      • Bissell M.J.
      Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones.
      ). Understanding how all these factors converge and regulate the social behavior of tumor cells represents a daunting scenario that may not be easy to recapitulate using current in vitro systems (
      • Soukas A.
      • Socci N.D.
      • Saatkamp B.D.
      • Novelli S.
      • Friedman J.M.
      Distinct transcriptional profiles of adipogenesis in vivo and in vitro.
      ,
      • Ntambi J.M.
      • Young-Cheul K.
      Adipocyte differentiation and gene expression.
      ,
      • Phillips B.W.
      • Vernochet C.
      • Dani C.
      Differentiation of embryonic stem cells for pharmacological studies on adipose cells.
      ).
      We would like to stress the fact that the large number of proteins present in the FIF (Table I) can have major implications for programs aiming at biomarker discovery in the blood because molecules secreted by adipocytes, and in the present study cytokines and growth factors in particular, add to the complex mixture of factors present in the tumor microenvironment, which is presumably the major source of molecules of predicted value that end up in the blood stream. Finally we would like to emphasize that the results presented here open new possibilities to the study of obesity and by association to type 2 diabetes (
      • Danforth Jr, E.
      Failure of adipocyte differentiation causes type II diabetes mellitus?.
      ,
      • Cederberg A.
      • Enerback S.
      Insulin resistance and type 2 diabetes—an adipocentric view.
      ), hypertension, and coronary heart disease (
      • Kopelman P.G.
      Obesity as a medical problem.
      ).

      Acknowledgments

      We are grateful to Dorrit Lützhøft, Hanne Nors, Michael Radich Johansen, Britt Olesen, and Signe Trentemøller for expert technical assistance. We also thank H. Mouridsen and D. Holm for helpful discussion.

      REFERENCES

        • Wiseman B.S.
        • Werb Z.
        Stromal effects on mammary gland development and breast cancer.
        Science. 2002; 296: 1046-1049
        • Silberstein G.B.
        Tumour-stromal interactions. Role of the stroma in mammary development.
        Breast Cancer Res. 2001; 3: 218-223
        • Coussens L.M.
        • Werb Z.
        Matrix metalloproteinases and the development of cancer.
        Chem. Biol. 1996; 3: 895-904
        • Coussens L.M.
        • Werb Z.
        Inflammatory cells and cancer: think different!.
        J. Exp. Med. 2001; 193: F23-F26
        • Fidler I.J.
        Regulation of neoplastic angiogenesis.
        J. Natl. Cancer Inst. Monogr. 2001; 28: 10-14
        • Fidler I.J.
        Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment.
        J. Natl. Cancer Inst. 2001; 93: 1040-1041
        • Iyengar P.
        • Combs T.P.
        • Shah S.J.
        • Gouon-Evans V.
        • Pollard J.W.
        • Albanese C.
        • Flanagan L.
        • Tenniswood M.P.
        • Guha C.
        • Lisanti M.P.
        • Pestell R.G.
        • Scherer P.E.
        Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization.
        Oncogene. 2003; 22: 6408-6423
        • Howlett A.R.
        • Bissell M.J.
        The influence of tissue microenvironment (stroma and extracellular matrix) on the development and function of mammary epithelium.
        Epithelial Cell Biol. 1993; 2: 79-89
        • Zangani D.
        • Darcy K.M.
        • Shoemaker S.
        • Ip M.M.
        Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells.
        Exp. Cell Res. 1999; 247: 399-409
        • Huss F.R.
        • Kratz G.
        Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue.
        Cells Tissues Organs. 2001; 169: 361-367
        • Scherer P.E.
        • Williams S.
        • Fogliano M.
        • Baldini G.
        • Lodish H.F.
        A novel serum protein similar to C1q, produced exclusively in adipocytes.
        J. Biol. Chem. 1995; 45: 26746-26749
        • Scherer P.E.
        • Bickel P.E.
        • Kotler M.
        • Lodish H.F.
        Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.
        Nat. Biotechnol. 1998; 16: 581-586
        • Bickel P.E.
        • Lodish H.F.
        • Scherer P.E.
        Use and applications of subtractive antibody screening.
        Biotechnol. Genet. Eng. Rev. 2000; 17: 417-430
        • Engelman J.A.
        • Berg A.H.
        • Lewis R.Y.
        • Lisanti M.P.
        • Scherer P.E.
        Tumor necrosis factor α-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3-L1 adipocytes.
        Mol. Endocrinol. 2000; 14: 1557-1569
        • Berg A.H.
        • Combs T.P.
        • Du X.
        • Brownlee M.
        • Scherer P.E.
        The adipocyte-secreted protein Acrp30 enhances hepatic insulin action.
        Nat. Med. 2001; 7: 947-953
        • Cancello R.
        • Tounian A.
        • Poitou Ch.
        • Clement K.
        Adiposity signals, genetic and body weight regulation in humans.
        Diabetes Metab. 2004; 30: 215-227
        • Mora S.
        • Pessin J.E.
        An adipocentric view of signaling and intracellular trafficking.
        Diabetes Metab. Res. Rev. 2002; 18: 345-356
        • Trayhurn P.
        • Wood I.S.
        Adipokines: inflammation and the pleiotropic role of white adipose tissue.
        Br. J. Nutr. 2004; 92: 347-355
        • Elliott B.E.
        • Tam S.P.
        • Dexter D.
        • Chen Z.Q.
        Capacity of adipose tissue to promote growth and metastasis of a murine mammary carcinoma: effect of estrogen and progesterone.
        Int. J. Cancer. 1992; 51: 416-424
        • Chamras H.
        • Bagga D.
        • Elstner E.
        • Setoodeh K.
        • Koeffler H.P.
        • Heber D.
        Preadipocytes stimulate breast cancer cell growth.
        Nutr. Cancer. 1998; 32: 59-63
        • Manabe Y.
        • Toda S.
        • Miyazaki K.
        • Sugihara H.
        Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions.
        J. Pathol. 2003; 201: 221-228
        • Johnston P.G.
        • Rondinone C.M.
        • Voeller D.
        • Allegra C.J.
        Identification of a protein factor secreted by 3T3-L1 preadipocytes inhibitory for the human MCF-7 breast cancer cell line.
        Cancer Res. 1992; 52: 6860-6865
        • Calle E.E.
        • Thun M.J.
        Obesity and cancer.
        Oncogene. 2004; 23: 6365-6378
        • Kratchmarova I.
        • Kalume D.E.
        • Blagoev B.
        • Scherer P.E.
        • Podtelejnikov A.V.
        • Molina H.
        • Bickel P.E.
        • Andersen J.S.
        • Fernandez M.M.
        • Bunkenborg J.
        • Roepstorff P.
        • Kristiansen K.
        • Lodish H.F.
        • Mann M.
        • Pandey A.
        A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.
        Mol. Cell. Proteomics. 2002; 1: 213-222
        • Hung S.C.
        • Chang C.F.
        • Ma H.L.
        • Chen T.H.
        • Low-Tone Ho L.
        Gene expression profiles of early adipogenesis in human mesenchymal stem cells.
        Gene (Amst.). 2004; 340: 141-150
        • Guo X.
        • Liao K.
        Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation.
        Gene (Amst.). 2000; 251: 45-53
        • Burton G.R
        • Guan Y.
        • Nagarajan R.
        • McGehee Jr, R.E.
        Microarray analysis of gene expression during early adipocyte differentiation.
        Gene (Amst.). 2002; 293: 21-31
        • Urs S.
        • Smith C.
        • Campbell B.
        • Saxton A.M.
        • Taylor J.
        • Zhang B.
        • Snoddy J.
        • Jones Voy B.
        • Moustaid-Moussa N.
        Gene expression profiling in human preadipocytes and adipocytes by microarray analysis.
        J. Nutr. 2004; 134: 762-770
        • Boeuf S.
        • Klingenspor M.
        • Van Hal N.L.
        • Schneider T.
        • Keijer J.
        • Klaus S.
        Differential gene expression in white and brown preadipocytes.
        Physiol. Genomics. 2001; 7: 15-25
        • Sottile V.
        • Seuwen K.
        A high-capacity screen for adipogenic differentiation.
        Anal. Biochem. 2001; 293: 124-128
        • Albrektsen T.
        • Richter H.E.
        • Clausen J.T.
        • Fleckner J.
        Identification of a novel integral plasma membrane protein induced during adipocyte differentiation.
        Biochem. J. 2001; 359: 393-402
        • Soukas A.
        • Socci N.D.
        • Saatkamp B.D.
        • Novelli S.
        • Friedman J.M.
        Distinct transcriptional profiles of adipogenesis in vivo and in vitro.
        J. Biol. Chem. 2001; 276: 34167-34174
        • Brasaemle D.L.
        • Dolios G.
        • Shapiro L.
        • Wang R.
        Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.
        J. Biol. Chem. 2004; 279: 46835-46842
        • Danforth Jr, E.
        Failure of adipocyte differentiation causes type II diabetes mellitus?.
        Nat. Genet. 2000; 26: 13
        • Cederberg A.
        • Enerback S.
        Insulin resistance and type 2 diabetes—an adipocentric view.
        Curr. Mol. Med. 2003; 3: 107-125
        • Kopelman P.G.
        Obesity as a medical problem.
        Nature. 2000; 404: 635-643
        • Klaus S.
        Adipose tissue as a regulator of energy balance.
        Curr. Drug. Targets. 2004; 5: 241-250
        • Gabrielsson B.L.
        • Carlsson B.
        • Carlsson L.M.
        Partial genome scale analysis of gene expression in human adipose tissue using DNA array.
        Obes. Res. 2000; 8: 374-384
        • Corton M.
        • Villuendas G.
        • Botella J.I.
        • San Millan J.L.
        • Escobar-Morreale H.F.
        • Peral B.
        Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide.
        Proteomics. 2004; 4: 438-441
        • Lanne B.
        • Potthast F.
        • Hoglund A.
        • Brockenhuus von Lowenhielm H.
        • Nystrom A.C.
        • Nilsson F.
        • Dahllof B.
        Thiourea enhances mapping of the proteome from murine white adipose tissue.
        Proteomics. 2001; 1: 819-828
        • Celis J.E.
        • Gromov P.
        • Gromova I.
        • Moreira J.M.
        • Cabezon T.
        • Ambartsumian N.
        • Grigorian M.
        • Lukanidin E.
        • Thor Straten P.
        • Guldberg P.
        • Bartkova J.
        • Bartek J.
        • Lukas J.
        • Lukas C.
        • Lykkesfeldt A.
        • Jaattela M.
        • Roepstorff P.
        • Bolund L.
        • Orntoft T.
        • Brunner N.
        • Overgaard J.
        • Sandelin K.
        • Blichert-Toft M.
        • Mouridsen H.
        • Rank F.E.
        Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research.
        Mol. Cell. Proteomics. 2003; 2: 369-377
        • Celis J.E.
        • Gromov P.
        • Cabezon T.
        • Moreira J.M.
        • Ambartsumian N.
        • Sandelin K.
        • Rank F.
        • Gromova I.
        Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery.
        Mol. Cell. Proteomics. 2004; 3: 327-344
        • Celis J.E.
        • Moreira J.M.
        • Gromova I.
        • Cabezon T.
        • Ralfkiaer U.
        • Guldberg P.
        • Straten P.T.
        • Mouridsen H.
        • Friis E.
        • Holm D.
        • Rank F.
        • Gromov P.
        Towards discovery-driven translational research in breast cancer.
        FEBS J. 2005; 272: 2-15
        • Schmeichel K.L.
        • Weaver V.M.
        • Bissell M.J.
        Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype.
        J. Mammary Gland Biol. Neoplasia. 1998; 3: 201-213
        • Hansen R.K.
        • Bissell M.J.
        Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones.
        Endocr. Relat. Cancer. 2000; 7: 95-113
        • Park C.C.
        • Bissell M.J.
        • Barcellos-Hoff M.H.
        The influence of the microenvironment on the malignant phenotype.
        Mol. Med. Today. 2000; 6: 324-329
        • Matrisian L.M.
        • Cunha G.R.
        • Mohla S.
        Epithelial-stromal interactions and tumor progression: meeting summary and future directions.
        Cancer Res. 2001; 6: 3844-3846
        • O’Farrell P.H.
        High resolution two-dimensional electrophoresis of proteins.
        J. Biol. Chem. 1975; 250: 4007-4021
        • Celis J.E.
        • Trentem⊘lle S.
        • Gromov P.
        Celis J.E. Carter N. Hunter T. Shotton D. Simons K. Small J.V. Cell Biology. A Laboratory Handbook. 4. Academic Press, San Diego2005 (in press)
        • Gromova I.
        • Celis J.E.
        Celis J.E. Carter N. Hunter T. Shotton D. Simons K. Small J.V. Cell Biology. A Laboratory Handbook. 4. Academic Press, San Diego2005 (in press)
        • Celis J.E.
        • Gromov P.
        High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.
        EXS. 2000; 88: 55-67
        • Shevchenko A.
        • Wilm M.
        • Vorm O.
        • Mann M.
        Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
        Anal. Chem. 1996; 68: 850-858
        • Perkins D.N.
        • Pappin D.J.
        • Creasy D.M.
        • Cottrell J.S.
        Probability-based protein identification by searching sequence databases using mass spectrometry data.
        Electrophoresis. 1999; 20: 3551-3567
        • Small J.V.
        • Celis J.E.
        Direct visualization of the 10-nm (100-Å)-filament network in whole and enucleated cultured cells.
        J. Cell Sci. 1978; 31: 393-409
        • Celis A.
        • Madsen P.
        • Nielsen H.V.
        • Rasmussen H.H.
        • Thiessen H.
        • Lauridsen J.B.
        • van Deurs B.
        • Celis J.E.
        Human proteins IEF 58 and 57a are associated with the Golgi apparatus.
        FEBS Lett. 1988; 227: 14-20
        • Celis J.E.
        • Rasmussen H.H.
        • Madsen P.
        • Leffers H.
        • Honore B.
        • Dejgaard K.
        • Gesser B.
        • Olsen E.
        • Gromov P.
        • Hoffmann H.J.
        • Nielsen M.
        • Celis A.
        • Basse B.
        • Lauridsen J.B.
        • Ratz G.
        • Nielsen H.
        • Andersen A.H.
        • Walbaum E.
        • Kjargaard I.
        • Puype M.
        • Van Damme J.
        • Vandekerckhove J.
        The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
        Electrophoresis. 1992; 13: 893-959
        • Celis J.E.
        • Rasmussen H.H.
        • Gromov P.
        • Olsen E.
        • Madsen P.
        • Leffers H.
        • Honore B.
        • Dejgaard K.
        • Vorum H.
        • Kristensen D.B.
        • Østergaard M.
        • Hauns⊘ A.
        • Nielsen M.
        • Celis A.
        • Basse B.
        • Lauridsen J.B.
        • Ratz G.
        • Nielsen H.
        • Andersen A.H.
        • Walbaum E.
        • Kjargaard I.
        • Puype M.
        • Van Damme J.
        • Vandekerckhove J.
        The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
        Electrophoresis. 1995; 16: 2177-2240
        • Vainio H.
        • Bianchini F.
        IARC Handbooks of Cancer Prevention. 6. IARC Press, Lyon, France2002
        • Harvie M.
        • Hooper L.
        • Howell A.H.
        Central obesity and breast cancer risk: a systematic review.
        Obes. Rev. 2003; 4: 157-173
        • Reidy S.P.
        • Weber J.
        Leptin: an essential regulator of lipid metabolism.
        Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2000; 125: 285-298
        • Zhang Y.
        • Proenca R.
        • Maffei M.
        • Barone M.
        • Leopold L.
        • Friedman J.M.
        Positional cloning of the mouse obese gene and its human homologue.
        Nature. 1994; 372: 425-432
        • Friedman J.M.
        • Halaas J.L.
        Leptin and the regulation of body weight in mammals.
        Nature. 1998; 395: 763-770
        • Sierra-Honigmann M.R.
        • Nath A.K.
        • Murakami C.
        • Garcia-Cardena G.
        • Papapetropoulos A.
        • Sessa W.C.
        • Madge L.A.
        • Schechner J.S.
        • Schwabb M.B.
        • Polverini P.J.
        • Flores-Riveros J.R.
        Biological action of leptin as an angiogenic factor.
        Science. 1998; 281: 1683-1686
        • Bouloumie A.
        • Drexler H.C.
        • Lafontan M.
        • Busse R.
        Leptin, the product of Ob gene, promotes angiogenesis.
        Circ. Res. 1998; 83: 1059-1066
        • Liu Z.
        • Uesaka T.
        • Watanabe H.
        • Kato N.
        High fat diet enhances colonic cell proliferation and carcinogenesis in rats by elevating serum leptin.
        Int. J. Oncol. 2001; 19: 1009-1014
        • Laud K.
        • Gourdou I.
        • Pessemesse L.
        • Peyrat J.P.
        • Djiane J.
        Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line.
        Mol. Cell. Endocrinol. 2002; 188: 219-226
        • Dieudonne M.N.
        • Machinal-Quelin F.
        • Serazin-Leroy V.
        • Leneveu M.C.
        • Pecquery R.
        • Giudicelli Y.
        Leptin mediates a proliferative response in human MCF7 breast cancer cells.
        Biochem. Biophys. Res. Commun. 2002; 293: 622-628
        • Somasundar P.
        • McFadden D.W.
        • Hileman S.M.
        • Vona-Davis L.
        Leptin is a growth factor in cancer.
        J. Surg. Res. 2004; 116: 337-349
        • Yamauchi T.
        • Kamon J.
        • Waki H.
        • Terauchi Y.
        • Kubota N.
        • Hara K.
        • Mori Y.
        • Ide T.
        • Murakami K.
        • Tsuboyama-Kasaoka N.
        • Ezaki O.
        • Akanuma Y.
        • Gavrilova O.
        • Vinson C.
        • Reitman M.L.
        • Kagechika H.
        • Shudo K.
        • Yoda M.
        • Nakano Y.
        • Tobe K.
        • Nagai R.
        • Kimura S.
        • Tomita M.
        • Froguel P.
        • Kadowaki T.
        The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.
        Nat. Med. 2001; 7: 941-946
        • Iyengar P.
        • Scherer P.E.
        Adiponectin/Acrp30, an adipocyte-specific secretory factor: physiological relevance during development.
        Pediatr. Diabetes. 2003; 4: 32-37
        • Gil-Campos M.
        • Canete R.R.
        • Gil A.
        Adiponectin, the missing link in insulin resistance and obesity.
        Clin. Nutr. 2001; 23: 963-974
        • Guerre-Millo M.
        Adipose tissue hormones.
        J. Endocrinol. Investig. 2002; 25: 855-861
        • Hukshorn C.J.
        • Saris W.H.
        Leptin and energy expenditure.
        Curr. Opin. Clin. Nutr. Metab. Care. 2004; 7: 629-633
        • Fasshauer M.
        • Paschke R.
        • Stumvoll M.
        Adiponectin, obesity, and cardiovascular disease.
        Biochimie (Paris). 2004; 86: 779-784
        • Nickel W.
        The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.
        Eur. J. Biochem. 2003; 270: 2109-2119
        • Stoorvogel W.
        • Kleijmeer M.J.
        • Geuze H.J.
        • Raposo G.
        The biogenesis and functions of exosomes.
        Traffic. 2002; 3: 321-330
        • Peng X.
        • Wu Y.
        • Chen J.
        • Wang S.
        Proteomic approach to identify acute phase response-related proteins with low molecular weight in loach skin following injury.
        Proteomics. 2004; 4: 3989-3997
        • Pisitkun T.
        • Shen R.F.
        • Knepper M.A.
        Identification and proteomic profiling of exosomes in human urine.
        Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13368-13373
        • Hotamisligil G.S.
        • Shargill N.S.
        • Spiegelman B.M.
        Adipose expression of tumor necrosis factor-α: direct role in obesity-linked insulin resistance.
        Science. 1993; 259: 87-91
        • Hausman D.B.
        • DiGirolamo M.
        • Bartness T.J.
        • Hausman G.J.
        • Martin R.J.
        The biology of white adipocyte proliferation.
        Obes. Rev. 2001; 2: 239-254
        • Beecken W.D.
        • Kramer W.
        • Jonas D.
        New molecular mediators in tumor angiogenesis.
        J. Cell. Mol. Med. 2000; 4: 262-269
        • Rupnick M.A.
        • Panigrahy D.
        • Zhang C.Y.
        • Dallabrida S.M.
        • Lowell B.B.
        • Langer R.
        • Folkman M.J.
        Adipose tissue mass can be regulated through the vasculature.
        Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10730-10735
        • Lee Y.K.
        • So I.S.
        • Lee S.C.
        • Lee J.H.
        • Lee C.W.
        • Kim W.M.
        • Park M.K.
        • Lee S.T.
        • Park D.Y.
        • Shin D.Y.
        • Park C.U.
        • Kim Y.S.
        Suppression of distant pulmonary metastasis of MDA-MB 435 human breast carcinoma established in mammary fat pads of nude mice by retroviral-mediated TIMP-2 gene transfer.
        J. Gene Med. 2004; 7: 145-157
        • Brew K.
        • Dinakarpandian D.
        • Nagase H.
        Tissue inhibitors of metalloproteinases: evolution, structure and function.
        Biochim. Biophys. Acta. 2000; 1477: 267-283
        • Gregoire F.
        • De Broux N.
        • Hauser N.
        • Heremans H.
        • Van Damme J.
        • Remacle C.
        Interferon-γ and interleukin-1β inhibit adipoconversion in cultured rodent preadipocytes.
        J. Cell. Physiol. 1995; 151: 300-309
        • Dani C.
        Embryonic stem cell-derived adipogenesis.
        Cells Tissues Organs. 1999; 165: 173-180
        • MacDougald O.A.
        • Mandrup S.
        Adipogenesis: forces that tip the scales.
        Trends Endocrinol. Metab. 2002; 13: 5-11
        • Giatromanolaki A.
        • Sivridis E.
        • Koukourakis M.I.
        Tumour angiogenesis: vascular growth and survival.
        APMIS. 2004; 112: 431-440
        • Dass C.R.
        Tumour angiogenesis, vascular biology and enhanced drug delivery.
        J. Drug Target. 2004; 12: 245-255
        • Wabitsch M.
        • Brenner R.E.
        • Melzner I.
        • Braun M.
        • Moller P.
        • Heinze E.
        • Debatin K.M.
        • Hauner H.
        Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation.
        Int. J. Obes. Relat. Metab. Disord. 2001; 25: 8-15
        • Atkinson J.C.
        • Ruhl M.
        • Becker J.
        • Ackermann R.
        • Schuppan D.
        Collagen VI regulates normal and transformed mesenchymal cell proliferation in vitro.
        Exp. Cell Res. 1996; 228: 283-291
        • Ruhl M.
        • Sahin E.
        • Johannsen M.
        • Somasundaram R.
        • Manski D.
        • Riecken E.O.
        • Schuppan D.
        Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax.
        J. Biol. Chem. 1999; 274: 34361-34368
        • Camp H.S.
        • Ren D.
        • Leff T.
        Adipogenesis and fat-cell function in obesity and diabetes.
        Trends Mol. Med. 2002; 8: 442-447
        • Gregoire F.M.
        Adipocyte differentiation: from fibroblast to endocrine cell.
        Exp. Biol. Med. (Maywood). 2001; 226: 997-1002
        • Naaz A.
        • Holsberger D.R.
        • Iwamoto G.A.
        • Nelson A.
        • Kiyokawa H.
        • Cooke P.S.
        Loss of cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obesity.
        FASEB J. 2004; 18: 1925-1927
        • Gullicksen P.S.
        • Della-Fera M.A.
        • Baile C.A.
        Leptin-induced adipose apoptosis: Implications for body weight regulation.
        Apoptosis. 2003; 8: 327-335
        • Duff E.
        • Li C.L.
        • Hartzell D.L.
        • Choi Y.H.
        • Della-Fera M.A.
        • Baile C.A.
        Ciliary neurotrophic factor injected icv induces adipose tissue apoptosis in rats.
        Apoptosis. 2004; 9: 629-634
        • Warne J.P.
        Tumour necrosis factor α: a key regulator of adipose tissue mass.
        J. Endocrinol. 2003; 177: 351-355
        • Ninomiya-Tsuji J.
        • Torti F.M.
        • Ringold G.M.
        Tumor necrosis factor-induced c-myc expression in the absence of mitogenesis is associated with inhibition of adipocyte differentiation.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9611-9615
        • Zeng G.
        • Dave J.R.
        • Chiang P.K.
        Induction of proto-oncogenes during 3-deazaadenosine-stimulated differentiation of 3T3-L1 fibroblasts to adipocytes: mimicry of insulin action.
        Oncol. Res. 1997; 9: 205-211
        • Freytag S.O.
        • Geddes T.J.
        Reciprocal regulation of adipogenesis by Myc and C/EBP α.
        Science. 1992; 256: 379-382
        • Fajas L.
        • Landsberg R.L.
        • Huss-Garcia Y.
        • Sardet C.
        • Lees J.A.
        • Auwerx J.
        E2Fs regulate adipocyte differentiation.
        Dev. Cell. 2002; 3: 39-49
        • Smyth M.J.
        • Sparks R.L.
        • Wharton W.
        Proadipocyte cell lines: models of cellular proliferation and differentiation.
        J. Cell Sci. 1993; 106: 1-9
        • Xirodimas D.P.
        • Saville M.K.
        • Bourdon J.C.
        • Hay R.T.
        • Lane D.P.
        Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.
        Cell. 2004; 118: 83-97
        • Pedersen S.B.
        • Hansen P.S.
        • Lund S.
        • Andersen P.H.
        • Odgaard A.
        • Richelsen B.
        Identification of oestrogen receptors and oestrogen receptor mRNA in human adipose tissue.
        Eur. J. Clin. Investig. 1996; 26: 262-269
        • Mizutani T.
        • Nishikawa Y.
        • Adachi H.
        • Enomoto T.
        • Ikegami H.
        • Kurachi H.
        • Nomura T.
        • Miyake A.
        Identification of estrogen receptor in human adipose tissue and adipocytes.
        J. Clin. Endocrinol. Metab. 1994; 78: 950-954
        • Tessitore L.
        • Vizio B.
        • Pesola D.
        • Cecchini F.
        • Mussa A.
        • Argiles J.M.
        • Benedetto C.
        Adipocyte expression and circulating levels of leptin increase in both gynaecological and breast cancer patients.
        Int. J. Oncol. 2004; 24: 1529-1535
        • Wiper-Bergeron N.
        • Wu D.
        • Pope L.
        • Schild-Poulter C.
        • Hache R.J.
        Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex.
        EMBO J. 2003; 22: 2135-2145
        • Lagace D.C.
        • Nachtigal M.W.
        Inhibition of histone deacetylase activity by valproic acid blocks adipogenesis.
        J. Biol. Chem. 2004; 279: 18851-18860
        • Choy L.
        • Derynck R.
        Transforming growth factor-β inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function.
        J. Biol. Chem. 2003; 278: 9609-9619
        • Engeli S.
        • Janke J.
        • Gorzelniak K.
        • Bohnke J.
        • Ghose N.
        • Lindschau C.
        • Luft F.C.
        • Sharma A.M.
        Regulation of the nitric oxide system in human adipose tissue.
        J. Lipid Res. 2004; 45: 1640-1648
        • Cao W.
        • Daniel K.W.
        • Robidoux J.
        • Puigserver P.
        • Medvedev A.V.
        • Bai X.
        • Floering L.M.
        • Spiegelman B.M.
        • Collins S.
        p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene.
        Mol. Cell. Biol. 2004; 24: 3057-3067
        • Farnier C.
        • Krief S.
        • Blache M.
        • Diot-Dupuy F.
        • Mory G.
        • Ferre P.
        • Bazin R.
        Adipocyte functions are modulated by cell size change: potential involvement of an integrin/ERK signalling pathway.
        Int. J. Obes. Relat. Metab. Disord. 2003; 27: 1178-1186
        • Brown J.M.
        • Boysen M.S.
        • Chung S.
        • Fabiyi O.
        • Morrison R.F.
        • Mandrup S.
        • McIntosh M.K.
        Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines.
        J. Biol. Chem. 2004; 279: 26735-26747
        • Valladares A.
        • Porras A.
        • Alvarez A.M.
        • Roncero C.
        • Benito M.
        Noradrenaline induces brown adipocytes cell growth via β-receptors by a mechanism dependent on ERKs but independent of cAMP and PKA.
        J. Cell. Physiol. 2000; 185: 324-330
        • Tanabe Y.
        • Koga M.
        • Saito M.
        • Matsunaga Y.
        • Nakayama K.
        Inhibition of adipocyte differentiation by mechanical stretching through ERK-mediated downregulation of PPARγ2.
        J. Cell Sci. 2004; 117: 3605-3614
        • Prusty D.
        • Park B.H.
        • Davis K.E.
        • Farmer S.R.
        Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα gene expression during the differentiation of 3T3-L1 preadipocytes.
        J. Biol. Chem. 2002; 277: 46226-46232
        • Fujishiro M.
        • Gotoh Y.
        • Katagiri H.
        • Sakoda H.
        • Ogihara T.
        • Anai M.
        • Onishi Y.
        • Ono H.
        • Abe M.
        • Shojima N.
        • Fukushima Y.
        • Kikuchi M.
        • Oka Y.
        • Asano T.
        Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes.
        Mol. Endocrinol. 2003; 17: 487-497
        • Hirosumi J.
        • Tuncman G.
        • Chang L.
        • Gorgun C.Z.
        • Uysal K.T.
        • Maeda K.
        • Karin M
        • Hotamisligil G.S.
        A central role for JNK in obesity and insulin resistance.
        Nature. 2002; 420: 333-336
        • Bennett B.L.
        • Satoh Y.
        • Lewis A.J.
        JNK: a new therapeutic target for diabetes.
        Curr. Opin. Pharmacol. 2003; 3: 420-425
        • Peng X.D.
        • Xu P.Z.
        • Chen M.L.
        • Hahn-Windgassen A.
        • Skeen J.
        • Jacobs J.
        • Sundararajan D.
        • Chen W.S.
        • Crawford S.E.
        • Coleman K.G.
        • Hay N.
        Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2.
        Genes Dev. 2003; 17: 1352-1365
        • Whiteman E.L.
        • Cho H.
        • Birnbaum M.J.
        Role of Akt/protein kinase B in metabolism.
        Trends Endocrinol. Metab. 2002; 13: 444-451
        • Ntambi J.M.
        • Young-Cheul K.
        Adipocyte differentiation and gene expression.
        J. Nutr. 2000; 130: 3122S-3126S
        • Phillips B.W.
        • Vernochet C.
        • Dani C.
        Differentiation of embryonic stem cells for pharmacological studies on adipose cells.
        Pharmacol. Res. 2003; 47: 263-268