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.

Calcium-Dependent Enhancement by Extracellular Acidity of the Cytotoxicity of Mitochondrial Inhibitors against Melanoma.

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Because acidity potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic mechanisms in an acidic microenvironment should be effective. We report here that drugs which abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E) mutations, and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH, and in vivo systemic alkalinization with NaHCO3 enhanced subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. Intracellular calcium (Ca2+) was significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca2+ chelator, BAPTA/AM, inhibited cytoplasmic Ca2+ as well as melanoma cell death. Surprisingly, ROS scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors, suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven mitochondrial addiction, from therapy-induced senescence, and from slow, JARID1B-high-associated cell cycling, all of which have been shown to promote vulnerability to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the cytotoxicity of mitochondrial inhibitors against cancer cells. Mol Cancer Ther; 16(5); 936-47. ©2017 AACR.

Cancer susceptibility and embryonic lethality in Mob1a/1b double-mutant mice.

Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1a(Δ/Δ)1b(tr/+) or Mob1a(Δ/+)1b(tr/tr) mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated double-mutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway.

Hic-5 affects proliferation, migration and invasion of B16 murine melanoma cells.

Hic-5 is a shuttling protein between the cell membrane and the nucleus which functions as a focal adhesion adaptor protein and a nuclear receptor coactivator. Although several studies have shown its involvement in other types of cancer, the role of Hic-5 in melanoma is unknown. Herein, we show for the first time that Hic-5 is expressed in B16-F1 murine melanoma cells. To determine its function in melanoma cells, we used shRNA-mediated RNA interference and established stable clones with down-regulated Hic-5 expression. These clones had impaired growth and metastatic potential compared with controls in vivo, which correlated with decreased proliferation, migration and invasion in vitro. Moreover, silencing of Hic-5 expression in B16-F1 activated RhoA with an amoeboid phenotypic change, indicating that Hic-5 is a key regulator of B16-F1 metastasis in the context of Rho-dependent motility. These results provide new evidence that Hic-5 is a possible molecular target for treatment of melanoma.