Person:

Bouillez, Audrey

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38− population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.

BMI1 is a component of the PRC1 complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16INK4a expression. In support of these results, analysis of three gene expresssion datasets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing.

The polycomb repressive complex 1 (PRC1) includes the BMI1, RING1 and RING2 proteins. BMI1 is required for survival of multiple myeloma (MM) cells. The MUC1-C oncoprotein is aberrantly expressed by MM cells, activates MYC and is also necessary for MM cell survival. The present studies show that targeting MUC1-C with (i) stable and inducible silencing and CRISPR/Cas9 editing and (ii) the pharmacologic inhibitor GO-203, which blocks MUC1-C function, downregulates BMI1, RING1 and RING2 expression. The results demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism. MUC1-C thus promotes MYC occupancy on the BMI1 promoter and thereby activates BMI1 expression. We also show that the MUC1-C→MYC pathway induces RING2 expression. Moreover, in contrast to BMI1 and RING2, we found that MUC1-C drives RING1 by an NF-κB p65-dependent mechanism. Targeting MUC1-C and thereby the suppression of these key PRC1 proteins was associated with downregulation of the PRC1 E3 ligase activity as evidenced by decreases in ubiquitylation of histone H2A. Targeting MUC1-C also resulted in activation of the PRC1-repressed tumor suppressor genes, PTEN, CDNK2A and BIM. These findings identify a heretofore unrecognized role for MUC1-C in the epigenetic regulation of MM cells.

The EZH2 histone methyltransferase is a member of the polycomb repressive complex 2 (PRC2) that is highly expressed in diverse human cancers and is associated with a poor prognosis. MUC1-C is an oncoprotein that is similarly overexpressed in carcinomas and has been linked to epigenetic regulation. A role for MUC1-C in regulating EZH2 and histone methylation is not known. Here, we demonstrate that targeting MUC1-C in diverse human carcinoma cells downregulates EZH2 and other PRC2 components. MUC1-C activates (i) the EZH2 promoter through induction of the pRB→E2F pathway, and (ii) an NF-κB p65 driven enhancer in exon 1. We also show that MUC1-C binds directly to the EZH2 CXC region adjacent to the catalytic SET domain and associates with EZH2 on the CDH1 and BRCA1 promoters. In concert with these results, targeting MUC1-C downregulates EZH2 function as evidenced by (i) global and promoter-specific decreases in H3K27 trimethylation (H3K27me3), and (ii) activation of tumor suppressor genes, including BRCA1. These findings highlight a previously unreported role for MUC1-C in activating EZH2 expression and function in cancer cells.

Immunotherapeutic approaches, particularly PD-1/PD-L1 blockade, have improved the treatment of non-small cell lung cancer (NSCLC), supporting the premise that evasion of immune destruction is of importance for NSCLC progression. However, the signals responsible for upregulation of PD-L1 in NSCLC cells and whether they are integrated with the regulation of other immune-related genes are not known. Mucin 1 (MUC1) is aberrantly overexpressed in NSCLC, activates the NF-κB p65→ZEB1 pathway and confers a poor prognosis. The present studies demonstrate that MUC1-C activates PD-L1 expression in NSCLC cells. We show that MUC1-C increases NF-κB p65 occupancy on the CD274/PD-L1 promoter and thereby drives CD274 transcription. Moreover, we demonstrate that MUC1-C-induced activation of NF-κB→ZEB1 signaling represses the TLR9, IFNG, MCP-1 and GM-CSF genes, and that this signature is associated with decreases in overall survival. In concert with these results, targeting MUC1-C in NSCLC tumors suppresses PD-L1 and induces these effectors of innate and adaptive immunity. These findings support a previously unrecognized central role for MUC1-C in integrating PD-L1 activation with suppression of immune effectors and poor clinical outcome.