IDENTIFICATION OF NOVEL EPIGENETIC TARGETS THAT SUSTAIN BREAST CANCER GROWTH

Breast cancer is the second leading cause of tumor-related death in women, mainly due to resistance to first line therapy, high risk of relapse and metastatic dissemination. Breast cancer is a highly heterogeneous disease, which displays diverse biological characteristics, clinical behaviour and prognosis. For these reasons, it has become challenging the identification and characterization of novel genes responsible for breast cancer initiation and progression. To identify new targets that sustain breast cancer growth, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model. We screened two libraries targeting several chromatin remodeling enzymes (around 200 in total), which are essential genes in breast cancer maintenance and represent optimal druggable candidates. We identified approximately 70 genes that were depleted in our screens, and among them, we selected five hits to validate the screens. Remarkably, the silencing of each target gene significantly reduced tumor growth in vivo and decreased proliferation, colony formation and migration in vitro, thus validating our screens. We deeply investigated the Chromodomain Helicase DNA binding Domain 4 (CHD4) gene, whose silencing in breast cancer cells greatly reduces tumor growth, but does not affect normal mammary epithelial proliferation and migration. We examined the role of CHD4 in primary cells derived from spontaneous mammary tumors of MMTV/NeuT transgenic mice. Upon CHD4 depletion, we confirmed a significant decrease of tumor growth in vivo and cell proliferation and migration in vitro. Intriguingly, we demonstrated that CHD4 silencing reduced tumor growth in vivo in a patient-derived xenopatient (PDX) model of Luminal B drug-resistant breast carcinoma. Moreover, we investigated the mechanism through which CHD4 promotes breast cancer cell proliferation and we showed that CHD4 regulates cell cycle progression of breast cancer cells. CHD4 depletion provokes a consistent accumulation of cells in the G0/G1 phase and a strong reduction of the S phase of the cell cycle, and an upregulation of p21. In summary, RNAi screens allowed us to identify CHD4 as a critical target that sustains human breast cancer. Importantly, we showed that CHD4 modulation does not modify normal mammary cell proliferation and migration, suggesting that its targeting in tumor cells might not impact on the surrounding normal tissues. Moreover, CHD4 is crucial in almost any subtype of breast cancer, as shown by its effect on MMTV/NeuT and PDX tumorigenesis. Finally, we demonstrated that CHD4 is a key regulator of breast cancer cell cycle.