The Knock-out of HDAC7 in MCF10A cells negatively impacts on proliferation, stem cells maintenance and neoplastic transformation.

The mammary gland is a complex tissue which undergoes different morphological changes during women’s life. In this thesis, we wanted to clarify the involvement of HDAC7 in the regulation of cell proliferation, stemness and neoplastic transformation that occurs in mammary epithelial cells, using the MCF10A cell line as a model. Histone deacetylases are important modifiers of the epigenetic status in response to developmental and environmental cues. The subfamily class IIa HDACs comprises four deacetylases (HDAC4, HDAC5, HDAC7 and HDAC9) which are characterized by: poor activity against acetyl-lysine, nuclear-cytoplasmic shuttling and the ability to bind MEF2 transcription factors. In particular, it was demonstrated that HDAC7 is the most expressed member in MCF10A cells, pointing to an important role in the homeostasis of these cells. With the aim to investigate the involvement of HDAC7 in mammary epithelial homeostasis, morphogenesis and proliferation we knocked out HDAC7 in MCF10A cells with CRISPR/Cas9 system. MCF10A HDAC7-/- cells show an inhibition of cell cycle progression, through p21 up regulation. This data confirms the role of HDAC7 as a repressor of the p21 expression in MCF10A. Through microarray analysis we describe the gene expression profile of MCF10A lacking HDAC7 to describe the genes directly regulated by the HDAC7 expression. Moreover, we investigated the HDAC7 involvement in stem cells growth and maintenance by applying the mammosphere assay. As a matter of fact, we propose a double role of HDAC7 in the regulation of stemness. First, an intrinsic role of HDAC7 was demonstrated in stem cells growth. As a matter of fact, we verified the reduced ability of MCF10A HDAC7-/- cells to form spheres in ultra-low adhesion conditions. This could be explained by a reduced presence of progenitors in a population where HDAC7 is absent. The second, an extrinsic role, was proved by the ability of HDAC7 to release factors important for the stem cells renewal. In fact, conditionated medium by the MCF10A WT cells increase the number of mammospheres, confirming that HDAC7 could play a fundamental role in the regulation of factors released by the mammary stem cell niches. Finally, we also investigated the contribution of HDAC7 in neoplastic transformation. We obtained MCF10A HDAC7-/- transformed with the RAS oncogene. We discovered that the knock-out of HDAC7 negatively impacts on the RAS transformed phenotype. This observation opens new hypothesis on the cooperation between HDAC7 and oncogenes in promoting proliferation, invasiveness and altered morphogenic processes. Moreover, we described that HDAC7 loss can impact on stemness also in transformed cells. All these findings consolidate a pro-oncogenic role of HDAC7 in mammary epithelial cells, promoting the identification of new therapeutic strategies to target HDAC7 in breast cancer.