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

by Selleckchem | Sep 08 2023

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.

Comments:

The passage you provided highlights a research study that investigated cellular heterogeneity in melanoma and its relationship with melanin pigment content. The researchers compared high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs) using RNA sequencing (RNAseq) data and identified EZH2 as a master regulator of these distinct cellular states.

They observed that EZH2 protein was upregulated in LPCs and showed an inverse correlation with melanin deposition in melanoma patients. The researchers then tested conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, but found that these inhibitors had no effect on LPC survival, clonogenicity, and pigmentation, despite fully inhibiting the methyltransferase activity of EZH2.

However, when the researchers silenced EZH2 using siRNA or targeted its degradation using DZNep or MS1943, they observed inhibition of LPC growth and induction of HPCs. This indicated that EZH2 itself was necessary for LPC survival and that inhibiting EZH2 through other means, such as degradation or silencing, had an impact on cellular states.

Further investigation revealed that the proteasomal inhibitor MG132 induced EZH2 protein levels in HPCs. The researchers then examined the involvement of the ubiquitin pathway proteins in regulating EZH2 levels between HPCs and LPCs. They found that in LPCs, the E2-conjugating enzyme UBE2L6, in cooperation with UBR4, an E3 ligase, depleted EZH2 protein through ubiquitination at EZH2's K381 residue. Additionally, UBE2L6 was downregulated in LPCs due to UHRF1-mediated CpG methylation.

The findings suggest that targeting the UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 could be a potential strategy for modulating the activity of this oncoprotein in situations where conventional EZH2 methyltransferase inhibitors are ineffective.

Overall, this research sheds light on the mechanisms underlying cellular heterogeneity in melanoma and identifies EZH2 and its regulation as a potential therapeutic target for modulating melanoma cell states.