UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma.

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2's K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.

ZBTB7A governs estrogen receptor alpha expression in breast cancer.

ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer.

An EZH2-mediated epigenetic mechanism behind p53-dependent tissue sensitivity to DNA damage.

Renewable tissues exhibit heightened sensitivity to DNA damage, which is thought to result from a high level of p53. However, cell proliferation in renewable tissues requires p53 down-regulation, creating an apparent discrepancy between the p53 level and elevated sensitivity to DNA damage. Using a combination of genetic mouse models and pharmacologic inhibitors, we demonstrate that it is p53-regulated MDM2 that functions together with MDMX to regulate DNA damage sensitivity by targeting EZH2 (enhancer of zeste homolog 2) for ubiquitination/degradation. As a methyltransferase, EZH2 promotes H3K27me3, and therefore chromatin compaction, to determine sensitivity to DNA damage. We demonstrate that genetic and pharmacologic interference of the association between MDM2 and MDMX stabilizes EZH2, resulting in protection of renewable tissues from radio-/chemotherapy-induced acute injury. In cells with p53 mutation, there are diminished MDM2 levels, and thus accumulation of EZH2, underpinning the resistant phenotype. Our work uncovers an epigenetic mechanism behind tissue sensitivity to DNA damage, carrying important translation implications.

YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer.

The transcriptional co-activator Yes-associated protein 1 (YAP1), a key nuclear effector of the Hippo pathway, is a potent oncogene, and yet, the interaction between YAP1 and androgen receptor (AR) remains unexplored. Here we identify YAP1 as a physiological binding partner and positive regulator of AR in prostate cancer. YAP1 and AR co-localize and interact with each other predominantly within cell nuclei by an androgen-dependent mechanism in a hormone naive and an androgen-independent mechanism in castration-resistant prostate cancer cells. The growth suppressor MST1 kinase modulates androgen-dependent and -independent nuclear YAP1-AR interactions through directly regulating YAP1 nuclear accumulation. Disruption of YAP1 signalling by genetic (RNAi) and pharmacological (Verteporfin) approaches suppresses AR-dependent gene expression and prostate cancer cell growth. These findings indicate that the YAP1-AR axis may have a critical role in prostate cancer progression and serves as a viable drug target.

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression.

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells' resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.

SIK2 is involved in the negative modulation of insulin-dependent muller cell survival and implicated in hyperglycemia-induced cell death.

PURPOSE: To investigate the role of the serine/threonine kinase SIK2, a member of the salt-inducible kinase (SIK) family, in insulin-dependent cell survival and hyperglycemia-induced cell death in Müller glia. METHODS: Expression studies were performed by RT-PCR, immunostaining, Northern blotting, and immunoblotting. Insulin-dependent changes in SIK2 activity were investigated by in vitro kinase assays in MIO-M1 Müller cell line. Akt activation was studied by immunoblotting and cell death by TUNEL assay. The potential role of SIK2 in insulin signaling was explored by overexpression and sh-RNA knock-down approaches. Effects of hyperglycemia were studied in vitro and in vivo in streptozotocin-injected rats. RESULTS: SIK2 expression was detected throughout adult retina, except for the outer nuclear layer. Insulin stimulation of MIO-M1 cells resulted in a rapid 2-fold increase of SIK2 activity, increased insulin receptor substrate 1 (IRS1)-SIK2 interaction, and reduced cell death. pAkt levels following insulin treatment were modulated by SIK2 activity. Under hyperglycemia, increased SIK2 activity/expression was concomitant to decreased Akt activation and enhanced apoptosis; whereas knockdown of SIK2 under normo- and hyperglycemic conditions resulted in a rapid increase in pAkt levels and blunted cell death. SIK2 overexpression under normoglycemia had an opposite effect. SIK2 activity increased significantly within 2 weeks of induction of hyperglycemia in the rat retina. CONCLUSIONS: Results indicate that SIK2 functions as a negative modulator of the insulin-dependent survival pathway and contributes to hyperglycemia-induced cell death of Müller glia in vitro. Although still hypothetical at this point, our study suggests that SIK2 could serve a similar role during the development of diabetic retinopathy in vivo and that it represents a potential target to control disease progression.