Post-translational modifications of proteins in human breast cancer : proteomics studies of phosphorylation and nitration and implication of these PTMs in tumorigenesis

Breast cancer is the most common cancer in women. Even though improvements in diagnosis and treatment of breast cancer have been made, it is still the most common cause of cancer death in women. There is a great need to find biomarkers for early detection of the disease and novel drug targets to fight the cancer.

Cell immortalization is the prerequisite step in tumorigenesis, and identification of the biomarkers of immortalized cells may be helpful for early detection of cancer. In this thesis, we described identification of 71 immortalization-related proteins. We used proteomics and conditionally immortalized human breast epithelial cells. Identified proteins showed involvement in immortalization of such functional domains as cell proliferation and growth, death, cell assembly and organization, cellular movement, cell-to-cell signaling, and cell morphology. Kinase MAP2K3 was identified as down-regulated in immortalized cells. Overexpression of MAP2K3 in immortal human breast epithelial cells was sufficient to induce senescence. p38, p53 and pRB were modulated by MAP2K3. We also identified KSR2 as up-regulated in immortalized human breast epithelial cells and in human breast tumors.

Twenty-four proteins affected by hyperthermia of human primary breast epithelial cells were also identified. Among the proteins, TGF-β2 was found up-regulated. It induced HSP27 expression, and protected cells from cell death.

Aberrant protein tyrosine nitration has been associated with different diseases, including cancer. We explored changes in protein tyrosine nitration during the cell cycle, and observed that tyrosine nitration affected a number of cell cycle regulators.

Cross talk of different regulatory pathways may contribute to the resistance to the anti-cancer treatment. Targeting of multiple pathways has been regarded as a novel anti-cancer strategy. We showed that combined action of TGF-β1 and EGF involves changes in phosphorylation of 47 proteins. We observed that the convergence components of TGF-β1 and EGF, e.g., MEK1, CK1, can influence cell proliferation in the context of TGF-β1 and EGF signaling. Interestingly, we observed that the strongest inhibitory effect of Gefitinib (Iressa), EGFR kinase inhibitor, would be only when both EGF and TGF-β are highly active, and MEK1 and CK1 are inhibited. ZAK kinase was identified as a convergent target of TGF-β and EGF signaling, and was found contributing to the positive feedback regulation of cell migration upon combined TGF-β and EGF action.

We also studied effects of a long term exposure to EGF and estrogen on tumorigenesis of breast epithelial cells. We observed that the long-term exposure to EGF and 17β-estradiol may affect proliferation rate, colony formation, vessel formation, and stem cell features of human breast epithelial cells. Thus, our findings provided insights into different mechanisms of tumorigenesis, and impact of cross-talk of signaling pathways on tumor development.

List of scientific papers

I. Min Jia, Nazariy Souchelnytskyi, Ulf Hellman, Michael O‟Hare, Parmjit S. Jat, Serhiy Souchelnytskyi. Proteome profiling of immortalization-to-senescence transition of human breast epithelial cells identified MAP2K3 as a senescence promoting protein which is down regulated in human breast cancer. Proteomics Clin Appl. 2010 Nov; 4(10-11):816-28.
https://doi.org/10.1002/prca.201000006

II. Min Jia, Serhiy Souchelnytskyi. Proteome profiling of heat-shock of human primary breast epithelial cells, a dataset report. Cell Stress Chaperones. 2011 Jul; 16(4):459-67.
https://doi.org/10.1007/s12192-010-0253-3

III. Min Jia, Serhiy Souchelnytskyi. Kinase suppressor of ras 2 is involved in regulation of cell proliferation and is up-regulated in human invasive ductal carcinomas of breast. Exp Oncol. 2010 Sep; 32(3):209-12.
https://doi.org/10.1016/j.bbrc.2011.08.084

IV. Min Jia, Claudia Mateoiu, Serhiy Souchelnytskyi. Protein tyrosine nitration in the cell cycle. Biochem Biophys Res Commun. 2011 Sep 23; 413(2): 270-6.
https://doi.org/10.1016/j.bbrc.2011.08.084

V. Min Jia, Andrey Alexeyenko, Claudia Mateoiu, Serhiy Souchelnytskyi. Network signaling by TGFβ1 and EGF in regulation of the cell proliferation as predictor of application of Iressa. [Manuscript]

VI. Min Jia, Serhiy Souchelnytskyi. Role of sterile alpha motif and leucine zipper containing kinase AZK (ZAK) in combined signaling by TGFβ1 and EGF. [Manuscript]

VII. Min Jia, Sara Cunha, Serhiy Souchelnytskyi. Transformation and stemness of human breast epithelial cells exposed to EGF and 17β-Estradiol. [Manuscript]