University thesis:
Dissertation, Universität Freiburg, 2021
Footnote:
Description:
Abstract: Breast cancer is a heterogeneous, malignant disease that will be encountered by one out of eight women during her lifetime. Proteases are linked to this malignancy due to their necessity for physiological cellular function. The complete set of proteases is frequently defined as the degradome, which is currently combining 588 human and 672 murine genes. Because of the known association of many proteases with breast cancer, I aimed in this PhD thesis to further deepen the understanding of the role of proteases in breast cancer progression. Using degradome-wide knockdown screens I investigated the functional influence of genetic protease targeting on survival and growth of breast cancer cell lines derived from the MMTV-PyMT model of metastasizing breast cancer. Furthermore, I investigated the influence of combined genetic protease targeting and pharmacological inhibition of the oncogenic Phosphatidylinositol 3 kinase (PI3K) pathway. <br>The degradome-wide knockdown screens revealed 253 degradome-encoded proteins that significantly influenced proliferation and/or survival of murine breast cancer cells. Furthermore, targeting of 221 proteins sensitized breast cancer cells to PI3K inhibition or mediated resistance against this treatment. By applying strict selection criteria and comparing different independent screens, 16 candidates were chosen for validation regarding cell growth, viability and sensitivity to PI3K-inhibitors. Besides the two murine breast cancer cell lines also one normal murine breast epithelial cell line was used. A step-wise validation based on several in vitro assays yielded in Ubiquitin-specific protease 7 (Usp7), OTU domain-containing protein 5 (Otud5), Cathepsin A (Ctsa), Methionine aminopeptidase1 (Metap1) and Methionine aminopeptidase2 (Metap2) as promising candidates for detailed investigation. <br>A general tumor-promoting function for Ctsa, Usp7, Metap1 and Metap2 was validated in murine breast cancer cells, which is in line with current literature. Although a tumor-suppressive function for Otud5 was described in other cancer entities, tumor-promoting function could be shown in breast cancer cells. The reduced growth of murine breast cancer cells upon knockdown of Usp7, Metap1 or Metap2 was linked to general impairment of cell cycle progression and was transferrable to human MCF7 and MDAMB-231 breast cancer cells. Knockdown of Usp7 induced the transcriptional upregulation of the cell cycle-inhibitor p21 in cells with wildtype p53 which was not observed in p53 mutant MDAMB-231 breast cancer cells. Moreover, increased inhibitory phosphorylation of eIF2α was shown for murine Metap1 and Metap2 knockdown breast cancer cells that might be linked to reduced cell growth. <br>Importantly, combined genetic targeting of either Metap1, Metap2 or Usp7 with PI3K inhibition by the kinase-inhibitors BKM-120 or BEZ-235 (EC10 or EC20) at low doses mediated prominent combinatory and partly synergistic effects on viability and growth of murine and human breast cancer cells. The combinatory effects of METAP1 or METAP2 knockdown and PI3K inhibition might be linked to an obstructed MAPK signaling in human breast cancer cells. <br>Due to these results, the proteases Metap1, Metap2 and Usp7 might be candidates for the development of protease/kinase combinatory therapies. Sensitizing cancer cells to PI3K pathway-inhibitors would secure therapeutic success and limit dose-dependent side effects by reducing doses