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

Abstract

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.

Keywords: BMI1; MUC1-C; RING1; RING2; multiple myeloma.

Figure 1. Targeting MUC1-C downregulates BMI1 expression

A.-C. RPMI8226 (A), OPM-2 (B), and primary MM (C) cells from Patient #1 stably expressing a control shRNA (CshRNA) or a MUC1 shRNA were analyzed for BMI1 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). D. RPMI8226 cells were stably transduced to express a Tetracycline-inducible control shRNA (Tet-CshRNA) or a MUC1shRNA (Tet-MUC1shRNA). Cells treated with 200 ng/ml DOX for 5 d were analyzed for BMI1 mRNA levels by qRT-PCR (left). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared with that obtained for control DOX-treated Tet-CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). E. OPM-2 cells transiently transduced to express an empty vector or one expressing MUC1-C were analyzed for BMI1 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 vector cells (assigned a value of 1). Lysates from were immunoblotted with the indicated antibodies (right).

Figure 2. Targeting the MUC1-C cytoplasmic domain downregulates BMI1 expression

A. Schema of the MUC1-C subunit with the 58-aa extracellular domain (ED), the 28-aa transmembrane domain (TM) and the 72-aa cytoplasmic domain (CD). Highlighted is the CQC motif, which is necessary and sufficient for MUC1-C homodimerization, and is targeted by GO-203 and not the control peptide CP-2. Also highlighted are interactions of the MUC1-C cytoplasmic domain with effectors of the NF-κB and MYC pathways. B.-C. RPMI8226 (B) and OPM-2 (C) cells were treated with 5 μM CP-2 or GO-203 for 48 h. The cells were analyzed for BMI1 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 CP-2-treated cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). D. RPMI8226 cells were silenced for MUC1 using CRISPR/Cas9 gene editing. The parental WT and CRISPR cells were analyzed for BMI1 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 WT cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right).

Figure 3. MUC1-C regulates BMI1 expression by a MYC-dependent mechanism

A. Schema of the BMI1 promoter with highlighting of a MYC binding site (CACGTG) at positions -177 to -182. B.-C. RPMI8226/Tet-CshRNA and RPMI8226/Tet-MYCshRNA (B) and OPM-2/Tet-CshRNA and OPM-2/Tet-MYCshRNA (C) cells treated with DOX for 5 d were analyzed for MYC and BMI1 mRNA levels by qRT-PCR (left and middle). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared with that obtained for the Tet-CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). D.-E. RPMI8226 (D) and OPM-2 (E) cells treated with JQ1 or vehicle control for 48 h were analyzed for MYC and BMI1 mRNA levels by qRT-PCR (left and middle). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared with that obtained for the control cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right).

Figure 4. MUC1-C activates BMI1 gene transcription by a MYC-dependent mechanism

A. Schema of the pBMI1-Luc reporter with highlighting of the wild-type (WT) and mutated MYC binding site. B.-C. The designated RPMI8226 (B) and OPM-2 (C) cells expressing a CshRNA or MUC1shRNA were transfected with pGL3-Luc or pBMI1-Luc and SV40-Renilla-Luc as an internal control. Luciferase activity was measured at 24 h after transfection. The results (mean±SE of 3 determinations) are expressed as the relative luciferase activity compared with that obtained with cells expressing pGL3-Luc. D. RPMI8226 cells were transfected with (i) pGL3-Luc, (ii) pBMI1-Luc or Mut-pBMI1-Luc and (iii) SV40-Renilla-Luc. Luciferase activity was measured at 24 h after transfection. The results (mean±SE of 3 determinations) are expressed as the relative luciferase activity compared with that obtained with cells expressing pGL3-Luc. E. RPMI8226 cells were treated with JQ1 or vehicle control for 48 h and then transfected with the pGL3-Luc or pBMI1-Luc and SV-40-Renilla-Luc. Luciferase activity was measured at 24 h after transfection. The results (mean±SE of 3 determinations) are expressed as the relative luciferase activity compared with that obtained with cells expressing pGL3-Luc. F. Soluble chromatin from the RPMI8226/CshRNA and RPMI8226/MUC1shRNA cells was precipitated with anti-MYC or a control IgG antibody. The final DNA samples were amplified by qPCR with pairs of primers (Supplementary 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).

Figure 5. Targeting MUC1-C downregulates RING1 and RING2 expression

A.-B. The designated RPMI8226 (A) and OPM-2 (B) cells expressing a CshRNA or MUC1shRNA were analyzed for RING1 and RING2 mRNA levels by qRT-PCR (left and middle). 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). C.-E. Lysates from DOX-treated RPMI8226/Tet-CshRNA and RPMI8226/Tet-MUC1shRNA (C), RPMI8226/WT and RPMI8226/CRISPR (D), and CP-2- and GO-203-treated RPMI8226 cells (E) were immunoblotted with the indicated antibodies.

Figure 6. MUC1-C regulates RING2 and RING1 by MYC- and NF-κB p65-dependent mechanisms, respectively

A. RPMI8226 cells treated with JQ1 or vehicle control for 48 h were analyzed for RING2 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 vehicle treated cells (assigned a value of 1, left). Lysates were immunoblotted with the indicated antibodies (right). B. RPMI8226/Tet-CshRNA and RPMI8226/Tet-MYCshRNA cells treated with DOX for 5 d were analyzed for RING2 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 Tet-CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). C. Schema of the RING2 promoter with highlighting of putative MYC 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-MYC or a control IgG antibody. The final DNA samples were amplified by qPCR with pairs of primers (Supplementary 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).

Figure 7. Targeting the MUC1-C → PRC1 pathway derepresses PTEN, p14ARF and BIM

A. Lysates from the designated RPMI8226 (left) and OPM-2 (right) cells expressing a CshRNA or MUC1shRNA were immunoblotted with the indicated antibodies. B.-D. RPMI8226/CshRNA and RPMI8226/MUC1shRNA cells were analyzed for PTEN (B), p14^ARF (C) and BIM (D) 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. 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.