N-Cadherin Stabilizes β-Catenin and Promotes β-Catenin/TCF Transcriptional Activation and Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma

Introduction:

Inappropriate activation of Wnt/β-catenin signaling plays a role in some cancers. β-catenin (β-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive β-cat activation seen in other cancers, we and others have demonstrated that Wnt/β-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving β-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/β-catenin signaling in MM.

Materials and Methods:

We detected different forms of β-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate β-cat transcriptional activity as previously described.

Results:

We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active β-cat with an antibody that specifically recognizes the unphosphorylated active form of β-cat. Higher levels of cytosolic and/or nuclear β-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of β-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high β-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/β-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of β-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with β-cat protein but not β-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and β-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of β-cat both total and unphosphorylated β-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and β-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that β-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and β-cat protein levels in MS versus non-MS HMCLs. TCF activity and active β-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated β-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/β-cat complexes can contribute to adhesion-mediated drug resistance in MM.

Conclusion: Taken together, these findings establish that β-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of β-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance.