Mucin-1 (MUC1) Oncoprotein in Multiple Myeloma Cells Inhibits the Th1 Responses By Down Regulating the Expression of Mir-200c and up-Regulating the PDL1 Expression

Introduction: The PDL1/PD-1 pathway is a critical mediator of immune escape in patients with multiple myeloma (MM). Regulation of this pathway has not been well characterized. MicroRNAs (miRNAs) are a conserved class of small (~22 nucleotides) RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) and, in some settings, coding regions of target mRNAs. MiRNAs suppress gene expression by promoting mRNA degradation or inhibiting translation. Of note, the 3’UTR of the PDL1 gene contains putative binding sites for miR-200 family of micro-RNA’s, suggesting a possible role of miR-200’s in regulation of PDL1 expression. We have previously demonstrated that miR-200c is suppressed by the MUC1 oncoprotein, and hypothesized that MUC1 expression on myeloma cells upregulates the expression of PDL1, via suppressing miR-200c. In the present study, we investigated the relationship between MUC1, miR-200c and PDL1 in multiple myeloma.

Methods and Results: Lentivirus vectors expressing miR-200c or a control vector with green fluorescence protein (GFP) were transduced in two different MM cell lines (MM-RPMI, MM-U266). Cells were harvested sorted by Fluorescence-Activated Cell Sorting (FACS) after 72 hours of transduction, using a dual fluorescence for GFP and anti-PDL1 antibody to analyze the changes in PDL1 expression. MiR-200c transduction of U266 cells resulted in a decrease in mean expression of PDL1 from 69.55% to 1.4% (n=2). Similarly, RPMI cells demonstrated a reduction in mean expression of PDL1 from 62.5% to 1.9% (n=2) following miR-200c transduction. The abrogation of PDL1 expression in MM cells by ectopic expression of miR-200c was confirmed using western immunoblot analysis. Having previously demonstrated that miR-200c is suppressed by MUC1 in a solid tumor model, we evaluated the effect of silencing MUC1 in U266 and RPMI cell lines on miR-200c and PDL1 expression. MUC1 silenced stable cell lines of RPMI and U266 cells were generated using lentivirus shRNA vectors against MUC1 or a scrambled vector control. MUC1 silenced cells demonstrated an increase in miR-200c expression (> 2 fold, p value <0.05). Notably, PDL1 expression decreased from 52% to 3.7% and from 62.5% to 6.1% following silencing of MUC1 on U266 and RPMI cells respectively.

Conclusions: Ectopic expression of micro-RNA miR-200c in RPMI-MM and U266-MM cell lines results in down regulation of PDL1 expression. Silencing MUC1 in RPMI-MM and U266-MM cell lines results in both increased expression of miR-200c and downregulation of PDL1 expression. These results support the hypothesis that MUC1 expression on myeloma cells contributes to tumor mediated immunosuppression, by suppressing miR-200c thereby enhancing PDL1 expression. Interfering with MUC1 mediated signaling represents a novel approach towards augmenting immune mediated targeting of myeloma.