SUMMARY The histone H3K27 methyltransferase EZH2 plays an important role in oncogenesis, by mechanisms that are incompletely understood. Here we show that the JmjC domain histone H3 demethylase NDY1 synergizes with EZH2 to silence the EZH2 inhibitor miR-101. NDY1 and EZH2 repress miR-101 by binding its promoter in concert, via a process triggered by upregulation of NDY1. Whereas EZH2 binding depends on NDY1, the latter binds independently of EZH2. However, both are required to repress transcription. NDY1 and EZH2 acting in concert, upregulate EZH2 and stabilize the repression of miR-101 and its outcome. NDY1 is induced by FGF-2 via CREB phosphorylation and activation, downstream of DYRK1A, and mediates the FGF-2 and EZH2 effects on cell proliferation, migration and angiogenesis. The FGF-2-NDY1/EZH2-miR-101-EZH2 axis described here, was found to be active in bladder cancer. These data delineate a novel oncogenic pathway that functionally links FGF-2 with EZH2 via NDY1 and miR-101.

The JmjC domain histone H3K36me2/me1 demethylase NDY1/KDM2B is overexpressed in various types of cancer. Here we show that knocking down NDY1 in a set of ten cell lines derived from a broad range of human tumors inhibited their anchorage-dependent and anchorage-independent growth by inducing senescence and/or apoptosis in some and by inhibiting G1 progression in all. We further show that the knockdown of NDY1 in mammary adenocarcinoma cell lines decreased the number, size and replating efficiency of mammospheres and downregulated the stem cell markers ALDH and CD44, while upregulating CD24. These findings combined, suggest that NDY1 is required for the self-renewal of cancer stem cells and are in agreement with additional findings showing that, tumor cells in which NDY1 was knocked down undergo differentiation and a higher number of them is required to induce mammary adenocarcinomas, upon orthotopic injection in animals. Mechanistically, NDY1 functions as a master regulator of a set of microRNAs that target several members of the polycomb complexes PRC1 and PRC2 and its knockdown results in the de-repression of these microRNAs and the downregulation of their polycomb targets. Consistent with these observations, NDY1/KDM2B is expressed at higher levels in basal-like triple negative breast cancers and its overexpression is associated with higher rates of relapse after treatment. In addition, NDY1-regulated microRNAs are downregulated in both normal and cancer mammary stem cells. Finally, in primary human breast cancer, NDY1/KDM2B expression correlates negatively with the expression of the NDY1-regulated microRNAs, and positively with the expression of their PRC targets.

Overexpression of cyclin D1 is believed to endow mammary epithelial cells (MECs) with a proliferative advantage by virtue of its contribution to pRB inactivation. Accordingly, abrogation of the kinase-dependent function of cyclin D1 is sufficient to render mice resistant to breast cancer initiated by ErbB2. Here, we report that mouse cyclin D1KE/KE MECs (deficient in cyclin D1 activity) upregulate an autophagy-like process but fail to implement ErbB2-induced senescence in vivo. In addition, immortalized cyclin D1KE/KE MECs retain high rates of autophagy and reduced ErbB2-mediated transformation in vitro. However, highlighting its dual role during tumorigenesis, downregulation of autophagy led to an increase in senescence in cyclin D1KE/KE MECs. Autophagy upregulation was also confirmed in human mammary epithelial cells (HMECs) subjected to genetic and pharmacological inhibition of cyclin D1 activity and, similar to our murine system, simultaneous inhibition of Cdk4/6 and autophagy in HMECs enhanced the senescence response. Collectively, our findings suggest a previously unrecognized function of cyclin D1 in suppressing autophagy in the mammary epithelium.