Article Figures & Data
Figures
- Fig. 1.
Tamoxifen-resistant MCF7 cell line exhibits resistance to tamoxifen and increase in global phosphotyrosine levels. A, Inhibitory effect of different concentrations of tamoxifen on the proliferation of MCF7 tamoxifen-resistant (MCF7-TamR) cell line and MCF7 sensitive control (MCF7-CTRL) cell line. Cell proliferation was assessed using MTT assay after 7 days of treatment. Quantification was done relative to vehicle-treated MCF7-CTRL. Student's t test was performed for statistical analysis. B, Tumor size of xenograft on mice grown with either MCF7-TamR or MCF7-CTRL cell lines with estrogen (E2) supplementation and treated with or without tamoxifen (TAM). Two-way Anova test was performed for statistical analysis. C, Immunoblot of tyrosine phosphorylated proteins using the anti-phosphotyrosine antibody (pTyr) on the cell lysates or after immunoprecipitation (IP) with pTyr antibody. β-actin, heavy (H) chain and light (L) chain serve as loading control.
- Fig. 2.
Workflow of phosphoproteomic profiling of tamoxifen-resistant and sensitive cell line. MCF7 tamoxifen-resistant (MCF7-TamR) cell line was labeled with light amino acids and MCF7 tamoxifen-sensitive control (MCF7-CTRL) cell line was labeled with heavy amino acids, mixed in equal amounts, digested with trypsin and subjected to either anti-phosphotyrosine antibody enrichment or strong cation exchange (SCX) fractionation prior to TiO2-based phosphopeptide enrichment. The enriched phosphopeptides were then subjected to LC-MS/MS analysis.
- Fig. 3.
Phosphoproteomic profiling data analysis reveals global elevation of protein phosphorylation. A, Distribution of identified phosphorylated serine, threonine, and tyrosine residues in the combined data sets of anti-phosphotyrosine antibody- and TiO2- based phosphopeptide enrichments from all biological replicate samples. B, Density scatter plot of log2-transformed phosphopeptide ratios (MCF7-TamR versus MCF7-CTRL) from two biological replicates. Pearson coefficient correlation (R) is indicated. C, Distribution of phosphopeptides comparing the fold changes of phosphorylation levels of MCF7-TamR over MCF7-CTRL cells. Red dots: hyperphosphorylated peptides (>twofold), green dots: hypophosphorylated peptides (<0.5-fold). D, Top signaling pathways identified from pathway enrichment analysis of the hyperphosphorylated proteins (>twofold) in the MCF7-TamR cells. E, Cell morphology of MCF7-CTRL and MCF7-TamR viewed under phase contrast microscopy. F, Matrigel invasion assay of MCF7-CTRL and MCF7-TamR. Student's t test was performed for statistical analysis.
- Fig. 4.
Activation of focal adhesion pathways in tamoxifen resistance. A, Hyperphosphorylated and hypophosphorylated (MCF7-TamR versus MCF7-CTRL) proteins identified in the MCF7-TamR cells that are involved in the focal adhesion pathways. Protein phosphorylation sites and phospho-regulation patterns were specified with color-coded circles. Diagram adapted from Pathvisio, a pathway analysis tool. B-E, Representative MS spectra of hyperphosphorylated peptides of FAK2, FAK1, PXN and BCAR1.
- Fig. 5.
FAK2 is overexpressed and hyperphosphorylated in tamoxifen resistant cells. A, Western blot validation of phosphorylation sites of proteins involved in the focal adhesion pathway identified in phosphoproteomic profiling. β-actin serves as loading control. B, Increase in expression of FAK2 in MCF7 cells treated with tamoxifen short term or long term. C, Real-time RT-PCR analysis of FAK2 mRNA expression in MCF7-TamR and MCF7-CTRL cells. Student's t test was used for statistical analysis. D, Immunofluorescence staining of pY402 FAK2 in MCF7-CTRL and MCF7-TamR cells. Green: FAK2 pY402; Blue: DAPI staining of nuclei. E, Focal adhesions of each cell were counted and Student's t test was used for statistical analysis.
- Fig. 6.
FAK2 is a novel therapeutic target in tamoxifen-resistant breast cancer. A, Western blot analysis of siRNA knockdown of FAK2 (siFAK2) or control (siCTRL). β-actin serves as a loading control. B, Proliferation assay following siRNA knockdown of FAK2 (siFAK2) or control siRNA (siCTRL) with tamoxifen or vehicle. Student's t test was performed for statistical analysis. C, Western blot analysis of MCF7-CTRL and MCF7-TamR cells treated with two different siRNA against FAK2. Protein expression and phosphorylation levels of the proteins involved in the focal adhesion pathway were examined. D, The inhibitory effect of PF562271 on cell growth of MCF7-CTRL and MCF7-TamR cell. The IC50 curve was plotted using a nonlinear regression dose-response (variable slope) curve fit. E, Tumor volumes of mice xenograft grown with either MCF7-TamR or MCF7-CTRL cells with the treatment of PF562271 or DMSO control. Two-way Anova was performed for statistical analysis. F, Kaplan-Meier curve of metastasis-free survival of ER+ patients treated with tamoxifen. The patients were stratified based on expression of FAK2.
Tables
Gene symbol Phosphopeptide sequence Protein name Site TamR/CTRL PTK2B YIEDEDYyKASVTRLPIK focal adhesion kinase 2 (FAK2) Y580 25.74 RHsMREEDFIQPSSR S778 8.91 PTK2 YMEDSTYyKASK focal adhesion kinase 1 (FAK1) Y662 2.20 GSIDREDGSLQGPIGNQHIyQPVGKPDPAAPPK Y946 2.74 SRC Y439 FYN WTAPEAALyGR v-src viral oncogene Y440 4.21 YES Y446 BRAF sPQKPIVR v-raf oncogene homolog B S151 0.45 BCAR1 RPGPGTLyDVPR breast cancer anti-estrogen resistance 1 Y433 7.41 AQQGLyQVPGPSPQFQSPPAK Y174 8.35 PXN VGEEEHVySFPNK paxillin Y124 3.42 FIHQQPQSSsPVyGSSAK Y94, S91 3.64 TLN1 STVLQQQyNR talin 1 Y436 4.61 TMQFEPSTMVyDACR Y26 5.94 FYN KLDNGGyYITTR FYN oncogene Y213 2.80 GAySLSIR Y185 4.14 CRK YRPAsASVSALIGGR v-crk avian sarcoma virus CT10 oncogene S194 5.89 JUN LAsPELER jun proto-oncogene S73 3.68 MAPK8 MAPK10 TAGTSFMMTPyVVTR mitogen-activated protein kinase 8, 10 Y185 0.37 Y226 ACTG1 EITALAPsTMK actin, gamma 1 S323 3.18 ACTB actin, beta ACTN1 HRPELIDyGK actinin, alpha 1 Y193 4.81 ARHGAP5 GGIDNPAITsDQELDDKK Rho GTPase activating protein 5 S1218 4.41 RTHsDAsDDEAFTTSK S1173, S1176 3.91 BAD RMsDEFVDSFKK BCL2-associated agonist of cell death S118 4.99 FLNA CSGPGLsPGMVR filamin A, alpha S1459 5.06 APsVANVGSHCDLSLK S2152 4.92 ILK NGtLNKHSGIDFK integrin-linked kinase T181 2.25 PAK2 FYDsNTVK p21 protein (Cdc42/Rac)-activated kinase 2 S132 2.79 DGFPsGTPALNAK S152 2.04 PARVA SPSVPKsPTPKSPPSR parvin, alpha S54 12.72 PPP1CA yGQFSGLNPGGRPITPPR protein phosphatase 1, catalytic subunit, alpha isozyme Y317 3.53 PPP1CB YQYGGLNSGRPVtPPR protein phosphatase 1, catalytic subunit, beta isozyme T316 2.49 PPP1R12A LAsTSDIEEK protein phosphatase 1, regulatory subunit 12A S507 5.55 FPTTATKIsPK S422 6.37 VASP VQIYHNPtANSFR vasodilator-stimulated phosphoprotein T43 2.13 VAV2 ASsRsPVFTPR vav 2 guanine nucleotide exchange factor S769, S771 0.50 VCL ILLRNPGNQAAyEHFETMK vinculin Y693 3.48 DPSAsPGDAGEQAIR S291 28.40 ZYX FsPGAPGGSGSQPNQK zyxin S341 4.23 sPGAPGPLTLK S404 7.30
Supplemental Data
- Supplemental Table S1 - A list of phosphoPSM identified by Sequest HT
- Supplemental Table S2 - A list of identified phosphopeptides quantified by SILAC
- Supplemental Table S3 - A list of identified phosphoproteins quantified by SILAC
- Supplemental Figure S1 - Western blot analysis of key regulators and markers of EMT in MCF7-CTRL and MCF7-TamR cells. MDA-MB-231 and U2OS cells serve as positive control and &#61538;-actin serves as loading control.
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