PROTEOMICS OF INVASIVENESS OF HUMAN BREAST EPITHELIAL CELLS

Mechanisms of malignant transformation and cancer invasion and factors controlling them in response to various stimuli remain elusive. We used proteomics and systems biology to explore these mechanisms in human breast epithelial cells. The objective has been to identify a set of biomarkers for diagnostics and prognostics of breast cancer. Acquiring of high proliferation by cells is a major hallmark of malignant transformation. Using global expression proteome profiling approach, we identified a set of proteins associated with high proliferation rate of human breast epithelial cells upon carcinogenic transformation (paper I). In this study, we described a proteome signature of cells with enhanced proliferation rate, and observed that deregulation of CDK4 and cyclin D3 may be among the early malignant transformation events. Distal metastasis is the leading cause of death among breast cancer patients, and invasion of cancer cells is the first step in metastatic process. We established a highly invasive clone of MCF7 cells from non-invasive MCF7 cells (paper II). Using proteome profiling, we identified key regulators of invasiveness. Systemic analysis suggested that the invasive-specific network has features of a scale-free network, with TGF, EGFRB, TAF1, HNF4, MYC and RB1 as key nodes. Analysis of TGF and EGF-centered network showed more than 30 key nodes which may define how TGF and EGF cooperate. Among these nodes were identified insulin, VEGF, HNF4 and NFB. This result indicates that the insulin signaling disturbance may interfere with the invasiveness, thus explain the clinical observation of the increased risk of breast cancer metastasis in diabetes patients. The correlation between protein translation and breast cancer is crucial in understanding of breast carcinogenesis. In paper III, we identified eukaryortic elongation factor 1 A1 (eEF1A1) as a direct substrate of type I transforming growth factor--receptor (TRI). We showed that the phosphorylation of eEF1A1 at Ser300 by TR-I mediates a direct inhibitory effect of TGF on protein synthesis, and contributes to effects on cell proliferation, anchorage-dependent and anchorage-independent cell growth. Furthermore, we showed that the phosphorylation of Ser300 is decreased in human breast tumors. In paper IV, we showed that eEF1A1 itself contributed to the increased proliferation of human breast epithelial cells by promoting transition of cells through the Sand G2/Mphases of the cell cycle. Therefore, our identification of eEF1A1 as a substrate of TR-I unveiled novel translation-related regulatory pathway downstream of TR-I, which is involved in breast tumorigenesis. Breast cancer metastatic suppressor I (BRMS1) was identified by us as an invasiveness-related protein. In paper V, we showed that expression of BRMS1 resulted in a shift to epithelial morphology of otherwise mesenchymal morphology MDA-MB-231 cells. Our study concluded that TGF and EGF may modulate BRMS1-dependent breast cancer invasion by regulating focal adhesion and cytoskeletal rearrangement, and that Smad2 and Erk1/2 phosphorylation are involved in molecular mechanisms engaged by BRMS1. Thus, presented here studies delivered a proteome signature of invasiveness and enhanced proliferation, and explored roles of eEF1A1 and BRMS1 in breast tumorigenesis. We described proteome signatures and proteins which may be considered as markers for diagnostics and prognostics of human breast cancer. LIST OF PUBLICATIONS I. Nimesh Bhaskaran*, Kah Wai Lin*, Aude Gautier*, Hanna Woksepp, Ulf Hellman, Serhiy Souchelnytskyi. Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of cdk4 and cyclin D3 in cell proliferation. Proteomics Clinical Applications. 2009; 3(1): 68-77. II. Kah Wai Lin, Serhiy Souchelnytskyi. Proteome signature of invasiveness of human breast epithelial cells. (Manuscript) III. Kah Wai Lin*, Ihor Yakymovych*, Min Jia, Mariya Yakymovych, Serhiy Souchelnytskyi. Phosphorylation of eukaryotic elongation factor eEF1A at Ser300 by type I transforming growth factor--receptor results in inhibition of mRNA translation. Current Biology. 2010; 20(18): 1615-1625. IV. Kah Wai Lin, Serhiy Souchelnytskyi. Eukaryotic elongation factor eEF1A1 promotes and Ser300 mutants of eEF1A1 inhibit transition through the S and G2/M phases of the cell cycle. Journal of Cell and Molecular Biology. 2010; 8(2): 125-130. V. Kah Wai Lin, Serhiy Souchelnytskyi. TGF and EGF coordinately modulate BRMS1-mediated breast cancer invasion by activation of focal adhesion and cytoskeleton rearrangement. (Manuscript) ADDITIONAL PUBLICATIONS I. Kah Wai Lin, Serhiy Souchelnytskyi. Translational connection of TGF signaling: Phosphorylation of eEF1A1 by TR-I inhibits protein synthesis. Small GTPase. 2011; 2(2): 104-108. (Invited Extra View) II. Kah Wai Lin, Min Jia, Serhiy Souchelnytskyi. Application of bioinformatics tools in gel-based proteomics. In Proteomics / Book 2 (Eastwood Leung, ed, ISBN 979-953-307-693-4, InTech, 2011. (In Press) (Invited Book Chapter) III. Min Jia, Kah Wai Lin, Serhiy Souchelnytskyi. Phosphoproteomics: detection, identification and importance of protein phosphorylation. In Proteomics / Book 2 (Eastwood Leung, ed), ISBN 979-953-307-693-4, InTech, 2011. (In Press) (Invited Book Chapter) * contributed equally # corresponding author