Targeting MUC1-C suppresses polycomb repressive complex 1 in multiple myeloma

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.


INTRODUCTION
Mucin 1 (MUC1) is a cell membrane heterodimeric complex that is aberrantly expressed in primary multiple myeloma (MM) cells and MM cell lines [1][2][3][4][5]. The transmembrane MUC1 C-terminal (MUC1-C) subunit of the heterodimer functions as an oncoprotein that is necessary for the proliferation and survival of MM cells [5][6][7][8]. In this respect, the MUC1-C cytoplasmic domain is an intrinsically disordered 72-amino acid structure that has the plasticity to act as a node and intersect with multiple signaling pathways linked to self-renewal, inflammation and transformation [9,10]. MUC1-C activates the proinflammatory IKK → NF-κB pathway, binds directly to NF-κB p65 and promotes the induction of NF-κB target genes, including MUC1 itself in an autoinductive loop [11][12][13]. The MUC1-C cytoplasmic domain also binds directly to β-catenin, inhibits β-catenin degradation and activates the WNT/β-catenin/TCF4 pathway [14,15]. Studies in MM cells have demonstrated that MUC1-C increases occupancy of β-catenin on the MYC promoter and drives MYC transcription [8]. Moreover, analysis of microarray datasets from primary MM cells showed that MUC1 expression positively correlates with that of MYC [8]. By extension, silencing MUC1-C in MM cells results in downregulation of MYC and thereby MYC target genes [8]. MM cells are addicted to MYC [16][17][18]. Thus, targeting MUC1-C with the downregulation of MYC explains, at least in part, why MM cells are dependent on MUC1-C for their proliferation and survival
The polycomb repressive complex 1 (PRC1) includes the ring domain-containing BMI1, RING1 and RING2 proteins [19,20]. BMI1 and RING1 bind to the catalytic RING2 subunit, and both contribute to the RING2 ubiquitin E3 ligase function [21]. BMI1 is also necessary for maintaining integrity of the complex [22]. In the prevailing hierarchical model, PRC1 is recruited to sites of H3K27 trimethylation (H3K27me3) generated by the polycomb repressive complex 2 (PRC2) [23], which includes enhancer of zeste homolog 2 (EZH2) and suppressor of zeste 12 homolog (SUZ12) components [24]. In turn, PRC1 catalyzes the mono-ubiquitination of histone H2A on K119 and promotes repression of homeobox (HOX) genes, among others [19][20][21][22]25]. Of the PRC1 subunits, BMI1 has been linked to the self-renewal of normal stem cells and the tumorigenic potential of cancer stem-like cells (CSCs) [26][27][28][29][30][31]. BMI1 contributes to selfrenewal and stemness by repressing the CDNK2A locus, which encodes the p16 INK4a and p14 ARF tumor suppressors [26,28]. In carcinoma cells, BMI1 has also been linked to downregulation of the PTEN tumor suppressor [28,32,33]. Additionally, in MM cells, BMI1 suppresses the expression of multiple proapoptotic proteins, including BIM, and in this way is essential for MM self-renewal [34]. BMI1 also activates the WNT pathway by repressing the Dickkopf (DKK) family of WNT inhibitors [35]. Repression of DKK proteins contributes to activation of the MYC gene and thereby a positive-feedback loop linking the WNT pathway to induction of BMI1 expression [35]. BMI1 is thus a potentially important target for the treatment of MM; however, to date, there are no clinically available BMI1 inhibitors [29]. Therefore, targeting upstream effectors that drive expression of BMI1 and other PRC1 components represents an attractive approach for reprogramming PRC1-mediated gene repression in MM.
The present studies demonstrate that MUC1-C drives expression of BMI1, RING1 and RING2 in MM cells. We show that MUC1-C activates the BMI1 promoter by a MYC-mediated mechanism. In addition, we report that MUC1-C induces (i) RING2 by MYC-dependent signaling, and (ii) RING1 by activation of the NF-κB p65 pathway. In concert with these findings, targeting MUC1-C results in suppression of H2A ubiquitylation and induction of the PTEN, p14 ARF and BIM tumor suppressors in MM cells.

MUC1-C induces BMI1 expression in MM cells
MUC1-C was stably silenced in MM cells to investigate whether this oncoprotein is involved in the regulation of BMI1 expression. In studies of RPMI8226 cells, MUC1-C silencing was associated with substantial downregulation of BMI1 mRNA and protein ( Figure  1A, left and right). Similarly, silencing MUC1-C in OPM-2, KMS-12-E and U266 cells resulted in marked decreases in BMI1 expression ( Figure 1B, left and right; Supplementary Figures 1A and 1B). In extending these observations to primary MM cells from 2 patients with refractory disease, we also found that targeting MUC1-C significantly reduces BMI1 levels ( Figure 1C, left and right; Supplementary Figure 1C). To confirm these results, we stably transduced RPMI8226 cells to express a control Tet-CshRNA or a Tet-MUC1shRNA. DOX treatment of RPMI8226/Tet-MUC1shRNA, but not RPMI8226/ Tet-CshRNA, cells was associated with significant downregulation of BMI1 mRNA and protein ( Figure  1D, left and right). In concert with these results, the enforced overexpression of MUC1-C in OPM-2 cells was associated with an increase in BMI1 expression ( Figure  1E, left and right), clearly indicating that MUC1-C and not the shed MUC1-N subunit is responsible for driving BMI1 expression.
The MUC1-C subunit consists of a 72-amino acid (aa) cytoplasmic domain with a CQC motif that is necessary and sufficient for the formation of MUC1-C homodimers and for MUC1-C-mediated transformation ( Figure 2A). Accordingly, the cell-penetrating peptide inhibitor, GO-203, was developed to block the MUC1-C CQC motif in MM and other cancer cells ( Figure 2A). Notably, treatment of RPMI8226 and OPM-2 cells with GO-203, but not the control peptide CP-2, induced a marked decrease in BMI1 mRNA and protein ( Figure  2B and 2C, left and right). Downregulation of BMI1 expression was also observed in studies of GO-203treated KMS-12-PE and NCIH929 cells (Supplementary Figure 2A and 2B). Furthermore, targeting MUC1-C with CRISPR/Cas9 gene editing was associated with a marked reduction in the BMI1 expression ( Figure 2D, left and right; Supplementary Figure 2C). These findings collectively indicated that MUC1-C activates BMI1 expression in MM cells.

MUC1-C drives BMI1 transcription by a MYCdependent mechanism
MUC1-C induces MYC expression in MM cells by activating the WNT/β-catenin/TCF-4 pathway [8]. The BMI1 promoter contains a consensus E-Box (CACGTG) Oncotarget 69239 www.impactjournals.com/oncotarget at position -177 to -182 upstream to the transcription start site ( Figure 3A), invoking the possibility that MUC1-C induces BMI1 expression through the MYC pathway in MM cells. To address this potential mechanism, we assessed the effects of targeting MYC on BMI1 expression. RPMI8226 cells were thus transduced to stably express a Tet-inducible MYC shRNA (Tet-MYCshRNA) or a Tetinducible control shRNA (Tet-CshRNA). Treatment of RPMI8226/Tet-MYCshRNA cells with DOX resulted in the marked suppression of MYC and BMI1 levels as compared to that in DOX-treated RPMI8226/Tet-CshRNA cells ( Figure 3B, left, middle and right). These results were confirmed in DOX-treated OPM-2/Tet-CshRNA and OPM-2/Tet-MYCshRNA cells ( Figure 3C, left, middle and right). In extending these observations, we found that treatment of RPMI8226 cells with the BET bromodomain inhibitor, JQ1, was associated with the marked suppression of MYC, MUC1-C and BMI1 expression ( Figure 3D, left, middle and right). Similar results were obtained in studies of JQ1-treated OPM-2 cells ( Figure 3E, left, middle and right), supporting the notion that the MUC1-C → MYC pathway induces BMI1 expression in MM cells.

MUC1-C activates the BMI1 promoter by enhancing MYC occupancy
To determine whether MUC1-C drives BMI1 transcription by a MYC-mediated mechanism, we transfected MM cells to express a pBMI1-Luc reporter that includes the MYC binding site ( Figure 4A). Using this approach, we found that activation of pBMI1-Luc is decreased in RPMI8226/MUC1shRNA, as compared to RPMI8226/CshRNA, cells ( Figure 4B). Silencing MUC1-C in OPM-2 cells was also associated with suppression of pBMI1-Luc activity ( Figure 4C). Mutation of the MYC binding site (CACGTG→CGCGTG) in Mut-pBMI1-Luc ( Figure 4A) resulted in downregulation of

MUC1-C differentially activates RING1 and RING2
Notably, little is known about the regulation of RING1 and RING2. However, based on the identification of a MUC1-C → MYC → BIM1 pathway, we asked if MUC1-C induces the expression of RING1 and RING2 by a similar mechanism. In this context, inhibiting MYC with JQ1 was associated with marked downregulation of RING2 and a modest decrease in RING1 expression in RPMI8226 ( Figure 6A, left and right) and OPM-  Figure 5B) cells also decreased RING2, and to a lesser extent RING1, mRNA and protein levels. Notably, the RING2, but not the RING1, promoter contains putative E-boxes for potential MYC binding ( Figure 6C). Moreover and in support of these findings, targeting MUC1-C decreased MYC occupancy on the RING2 promoter ( Figure 6C, left and  right). MUC1-C also activates NF-κB p65 in association with epigenetic regulation [36]. In this regard, silencing NF-κB p65 in RPMI8226 ( Figure 6D, left and right) and OPM-2 (Supplementary Figure 5C) cells was associated with significant decreases in RING1, but not RING2, mRNA and protein. Moreover, BAY-11-7085 treatment of RPMI8226 ( Figure 6E, left and right) and OPM-2 (Supplementary Figure 5D) cells suppressed RING1 and not RING2 expression. Furthermore, analysis of RING1 promoter revealed consensus sites for potential NF-κB p65 binding ( Figure 6F). Targeting MUC1-C also decreased NF-κB p65 occupancy on the RING1 promoter ( Figure  6F, left and right). These results collectively supported a model in which MUC1-C drives (i) RING2 by a MYCmediated mechanism and (ii) RING1 predominantly by the NF-κB p65 pathway.   Table 2) for the MYC binding site in the BMI1 promoter. The results (mean±SE of 3 determinations) are expressed as the relative fold enrichment compared with that obtained for the IgG control (assigned a value of 1).

Oncotarget 69243 www.impactjournals.com/oncotarget
Targeting MUC1-C derepresses PTEN, p14 ARF and BIM expression PRC1 possesses H2A-K119 ubiquitin E3 ligase activity and catalyzes the ubiquitylation of H2A [32]. As expected from the suppression of BMI1, RING1 and RING2, we also found that targeting MUC1-C downregulates H2A ubiquitylation in RPMI8226 and OPM-2 cells ( Figure 7A, left and right). PRC1 has been linked to the repression of certain tumor suppressor genes, such as PTEN and INK4a/ARF (CDKN2A), in carcinoma cells [26,28,32]. By extension, we found that silencing MUC1-C in RPMI8226 ( Figure 7B, left and right) and OPM-2 (Supplementary Figure S6A) cells is associated with the derepression of PTEN expression. Targeting MUC1-C with CRISPR/Cas9 was also associated with significant upregulation of PTEN levels (Supplementary Figure 6B). In addition, targeting MUC1-C resulted in the induction of p14 ARF ( Figure 7C, left and right; Supplementary Figure S6C). Other work has shown that BMI1 suppresses the proapoptotic BIM protein in MM cells [34]. Along these lines, silencing MUC1-C and thereby downregulation of BMI1 was associated with the upregulation of BIM ( Figure 7D, left and right; Supplementary Figure S6D). These findings thus support a model in which (i) MUC1-C drives expression of BMI1, RING1 and RING2, and (ii) targeting MUC1-C suppresses PRC1 function in MM cells.

DISCUSSION
The findings that MUC1-C is invariably expressed in MM cells [1][2][3][4] and is necessary for their growth and survival [6][7][8] have supported MUC1-C Oncotarget 69244 www.impactjournals.com/oncotarget as a potential target for MM treatment. In addition, the recent demonstration that MUC1-C drives MYC in MM cells provided additional support for the importance of MUC1-C in the pathogenesis of MM [8]. In this respect, there is abundant evidence that MYC is essential for the progression and survival of MM cells [16][17][18]37]. What was not clear, however, is how the MUC1-C → MYC pathway functions in promoting the malignant  Table S2) encompassing the MYC binding sites in the RING2 promoter. The results (mean±SE of 3 determinations) are expressed as the relative fold enrichment compared with that obtained for the IgG control (assigned a value of 1). D. RPMI8226/CshRNA and RPMI8226/NF-κBshRNA were analyzed for RING1 mRNA levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). E. RPMI8226 cells treated with the 5 μM BAY-11-7085 or vehicle control for 24 h were assessed for RING1 levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the vehicle treated cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). F. Schema of the RING1 promoter with highlighting of putative NF-κB p65 binding sites. Soluble chromatin from the RPMI8226/CshRNA and RPMI8226/MUC1shRNA (left) and OPM-2/CshRNA and OPM-2/MUC1shRNA (right) cells was precipitated with anti-NF-κB p65 or a control IgG antibody. The final DNA samples were amplified by qPCR with pairs of primers (Supplementary  Table 2) encompassing the NF-κB p65 binding sites in the RING1 promoter. The results (mean±SE of 3 determinations) are expressed as the relative fold enrichment compared with that obtained for the IgG control (assigned a value of 1).
Oncotarget 69245 www.impactjournals.com/oncotarget MM phenotype. The present studies show that MUC1-C induces expression of the BMI1 component of the PRC1 complex and that this response is mediated by a MYC-dependent mechanism. Notably, BMI1 was identified based on its role in accelerating Myc-induced lymphomagenesis [38] and has been linked to promoting self-renewal of cancer and leukemic stem cells [27,30,31]. In addition, BMI1 is overexpressed in MM cells [39][40][41] and is essential for their growth [38]. Our results show that MUC1-C promotes MYC occupancy on the BMI1 promoter and thereby activates BMI1 transcription in MM cells. In this way, targeting MUC1-C with (i) inducible and stable silencing, or (ii) the GO-203 inhibitor suppressed activation of the BMI1 promoter and BMI1 expression. These findings do not exclude the possibility that MUC1-C regulates BMI1 by other mechanisms. For instance, studies in carcinoma cells have shown that MUC1-C induces miR-200c by an NF-κB p65-mediated signaling [42] and that miR-200c inhibits BMI1 translation [43,44]. Recent work has further shown that MUC1-C forms a complex with BMI1 in cancer cells [44], invoking the possibility that MUC1-C also affects BMI1 expression by post-translational mechanisms.
PRC1 is formed by binding of BMI1 to RING1 and the active RING2 subunit, which catalyzes the ubiquitylation of H2A and promotes gene silencing [19][20][21]. Surprisingly, we found that targeting MUC1-C in MM cells is also associated with suppression of RING1 and RING2. Based on our findings with BMI1 and a lack of available information regarding the regulation of RING1 The results (mean±SD of 3 determinations) are expressed as relative mRNA levels as compared with that obtained for the CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). E. Schema depicting the proposed involvement of MUC1-C in driving expression of the PRC1 components, BMI1, RING2 and RING1. MUC1-C activates the MYC gene in MM cells [8]. In turn, MYC occupies the BMI1 promoter by a MUC1-C-dependent mechanism and induces BMI1 expression. Targeting MUC1-C and thereby MYC also resulted in the downregulation of RING2, supporting a similar pathway for the regulation of BMI1 and RING2. MUC1-C binds directly to NF-κB p65 and promotes activation of NF-κB p65-target genes, including MUC1 itself in an autoinductive circuit [12]. Along these lines, targeting MUC1-C and NF-κB p65 resulted in the suppression of RING1 expression, indicating that MUC1-C regulates these three PRC1 components by a least two pathways. In contrast to BMI1 and RING2, targeting MYC was also associated with partial downregulation of RING1. In concert with the involvement of MUC1-C in regulating PRC1 components, the results further support a model in which MUC1-C activates PRC1 function and thereby the upregulation of H2AUb1 levels and suppression of PTEN, p14 ARF and BIM expression in MM cells. www.impactjournals.com/oncotarget and RING2, we asked whether the MUC1-C → MYC pathway also drives expression of these additional PRC1 components. We found that, like BMI1, targeting MYC is associated with marked downregulation of RING2. In concert with a potential role for MUC1-C → NF-κB p65 signaling in regulating PRC1 function, we also found that targeting MUC1-C and NF-κB p65 suppresses RING1 expression. ChIP studies further demonstrated that MUC1-C promotes (i) MYC occupancy on the RING2 promoter, and (ii) NF-κB p65 occupancy on the RING1 promoter. Other studies will be needed to address the observation that targeting MYC is associated with partial downregulation of RING1 expression, which may be related to cross-talk between MUC1-C and MYC signaling. Our findings nonetheless underscore the notion that MUC1-C drives the expression of multiple components of the PRC1 complex, albeit by at least two distinct mechanisms, and thereby can integrate the regulation of BMI1, RING1 and RING2 in MM cells ( Figure 7E). Our findings further support the premise that MUC1-C activates PRC1 function in epigenetic regulation. Indeed, targeting MUC1-C was associated with a marked decrease in PRC1-mediated H2A ubiquitylation and the induction of the PRC1-targeted tumor suppressor genes, PTEN and INK4a/ARF ( Figure 7E). In concert with the demonstration that BIM1 suppresses the BIM gene in MM cells [34], we also found that targeting MUC1-C is associated with induction of BIM expression ( Figure 7E). Our findings have thus identified a previously unrecognized role for MUC1-C in MM cells by activating the PRC1 complex and thereby the repression of certain TSGs.
With regard to translational relevance, PTC-209 is a small molecule inhibitor of BMI1 expression that has shown preclinical activity against colorectal and lung adenocarcinomas [29,45]; however, to our knowledge, there are no clinically available BMI1 inhibitors. Our results indicate that targeting MUC1-C represents an alternative strategy for suppressing the expression of BMI1, as well as RING1 and RING2. In this regard, the MUC1-C inhibitor GO-203 has completed a Phase I trial in patients with advanced solid tumors and, based on the findings that GO-203 is highly synergistic with decitabine [46], is presently under study in a Phase II trial in combination with decitabine for patients with AML (NCT02204085). Therefore, the present results provide the basis for targeting MUC1-C and thereby reprogramming of the MM epigenome alone or in combination with other agents.

Real-time quantitative reverse-transcription PCR (qRT-PCR)
Total RNA was isolated using the RNAeasy Mini Kit (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) [8]. The cDNA samples were amplified using the SYBR Green qRT-PCR assay kit and the ABI Prism 7000 sequence detector (Applied Biosystems). Primers used for qRT-PCR analysis are listed in Supplementary Table 1. The results were analyzed using the ΔΔ cycle threshold (DDCt) method as described [53].