Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer

Abstract

Tamoxifen, an estrogen receptor-␣ (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. Molecular & Cellular

Understanding cell signaling in cancer stem cells for targeted therapy – can phosphoproteomics help to reveal the secrets?

“…In ongoing investigations by using real-time PCR, Western blot analyses, and immunofluorescence staining the overexpression of FAK2 in tamoxifen resistant cells was confirmed. Finally, siRNA knockdown of FAK2 significantly reduced the proliferation of the MCF7-tamoxifen resistant cells and thus confirmed the pivotal role of FAK2 for tamoxifen resistance in these cells [114]. …”

multiplierzv2.0: A Python-based ecosystem for shared access and analysis of native mass spectrometry data

“…Inspired by efforts in label‐free quantification , we have also implemented a sophisticated algorithm to detect and catalog MS and MS/MS spectral features; this algorithm provides a foundation for relative peptide quantification by SILAC ( multiplierz currently supports duplex and triplex labeling schemes) or label‐free analyses. We provide documentation for a full multiplierz peptide identification and SILAC quantitation workflow performed on a third‐party data set ( multiplierz Github repository); our analysis agreed well with the original quantification performed via Proteome Discoverer . For intact protein mass spectrometry applications, we implement spectral deconvolution as originally described by Fenn and Marshall (Fig.…”

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture

“…Autophagy is a self-protective mechanism to guarantee basic energy supply under nutrition-deficient conditions, such as starvation (33). The cytoprotective mechanism of autophagy against chemotherapy has also been recognized in lung cancer cells (33) and other types of cancers (9,15,34,35). Thus, autophagy has been highlighted as one of the mechanisms underlying the chemoresistance of NSCLC.…”

“…The incorporation of stable isotopes facilitates the quantitative recognition of the differences in expression profiles by tandem mass spectrometry between the 12 C-and 13 C-labeled A549 cells. SILAC has also been useful in the identification of cancer biomarkers, and chemoresistance-associated biomarkers in hepatocellular carcinoma (14), breast cancer (15) and lung cancer (16,17).…”