Quantitative Proteomics with siRNA Screening Identifies Novel Mechanisms of Trastuzumab Resistance in HER2 Amplified Breast Cancers

Abstract

HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO 2 chromatography utilizing a dual trap configuration, ϳ1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2؉ breast cancer patients who develop trastuzumab resistance. Molecular & Cellular Proteomics

Comprehensive molecular tumor profiling in radiation oncology: How it could be used for precision medicine

“…Several studies highlight the importance of the analysis of protein phosphorylation in elucidating the radio- and chemoresistance mechanisms and identifying promising drug combinations [6,30,31,96–103]. For example, a phosphoproteome array in HNSCC cells treated with cetuximab showed activation of the c-Jun N-terminal kinase (JNK) pathway which attenuated the efficacy of cetuximab itself [30].…”

Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members

“…EFS and other proteins involved in SRC kinase signaling (CDCP1/Trask and Paxillin) were showed to have increased expression in a study of breast cancer using trastuzumab (Herceptin)-resistant versus -sensitive BT474 cell line derivatives [93]. Importantly, EFS knockdown with siRNA restored trastuzumab sensitivity [93].…”

“…Importantly, EFS knockdown with siRNA restored trastuzumab sensitivity [93]. Reflecting the importance of post-translational modification of CAS proteins, in a study of cell lines and tumor tissue in malignant melanoma, EFS phosphorylation and activity significantly decreased (p<0.05) in response to vemurafenib treatment in BRAF wild-type melanoma tumors compared to tumors with a BRAF V600E mutation with additional resistance to vemurfenib [94].…”

New crossroads for potential therapeutic intervention in cancer - intersections between CDCP1, EGFR family members and downstream signaling pathways

“…Another powerful example of the role of CDCP1 in mediating poor response to therapy involves breast cancer resistance to the HER2 targeted therapeutic antibody trastuzumab. A quantitative proteomics screen comparing the phospho-tyrosine (p-Y) content of trastuzumab-sensitive and -resistant breast cancer cell lines identified 12 fold higher levels of p-Y-CDCP1 in resistant cells [20]. Confirming the role of CDCP1 in resistance in this model, siRNA mediated silencing of CDCP1 restored cell line responsiveness in vitro to trastuzumab [20].…”

“…A quantitative proteomics screen comparing the phospho-tyrosine (p-Y) content of trastuzumab-sensitive and -resistant breast cancer cell lines identified 12 fold higher levels of p-Y-CDCP1 in resistant cells [20]. Confirming the role of CDCP1 in resistance in this model, siRNA mediated silencing of CDCP1 restored cell line responsiveness in vitro to trastuzumab [20]. Further supporting that CDCP1 is important in breast cancer resistance to trastuzumab, a more recent study demonstrated that CDCP1 and HER2 are co-overexpressed in 12% of primary and 30% of metastatic breast tumors, and that co-expression correlates with poorest disease free and overall survival [21].…”