Abnormally High Levels of E- and N-Cadherin Expression Add to Altered Regulation of b-Catenin In Multiple Myeloma

Abstract 2982

Gene expression profiling (GEP) of normal and malignant plasma cells and B-cells, revealed that MM is uniquely characterized by elevated expression of E- or N-cadherin. Classical cadherins are integral plasma membrane proteins, that together with a- and b-catenin form calcium-dependent adherent junctions. Homotypic interaction of N-cadherin+ hematopoietic stem cells and N-cadherin+ bone lining cells in the endosteal niche regulates HSC function. Adherent junctions contribute to the regulation of Wnt/b-catenin signaling by modulating the balance between membrane-bound and free cytosolic b-catenin, the latter of which is required for TCF transcriptional activity. Overexpression of DKK1 and suppression of Wnt/b-catenin osteoblasts causes a loss in self-renewal of HSC and only stromal cells with active nuclear b-catenin can support hematopoiesis in-vitro. On the other hand, disruption of adherent junctions and release of b-catenin contributes to epithelial-to-mesenchymal transition and solid tumor metastases. We, and others, have demonstrated that Wnt/β-catenin signaling is active in MM. However, emerging evidence suggests that loss of Wnt/b-catenin activity, rather than its activation, is central to MM pathogenesis. Nearly 90% of primary MM cells express and secrete DKK1 and/or SFRP3 or SFRP2, potent inhibitors of Wnt/b-catenin signaling. Moreover, loss-of-function mutations of APC or axin genes or gain-of-function mutations in the β-catenin gene common in colon cancer have not been found in MM. We therefore hypothesized that elevated expression of N/E-cadherin in MM cells contributes to the abnormally increase of b-catenin in MM. We first assessed the steady-state levels of β-catenin protein in MMCL with immunoblotting analysis. β-Catenin protein was expressed in all tested MMCL, with variable levels in individual lines. Interestingly, relative levels of β-catenin protein were comparable to N-cadherin expression in all eight tested myeloma cell lines. CD138-enriched plasma cells from the BM of 72 patients newly diagnosed MM revealed β-catenin protein levels are highly variable. After normalization of β-catenin with β-tubulin levels we segregated cases into three groups: 39% had low, 23% moderate, and 38% high levels of β-catenin. Analysis of correlation of b-catenin protein levels with U133Plus microarray data revealed there are striking positive correlations between N- or E-cadherin mRNA levels with levels of b-catenin protein. Importantly, b-catenin levels were not correlated with known Wnt/b-catenin target genes. To evaluate the role of N-cadherin in regulating β-catenin signaling in MM, we used a lentiviral expression system to express wild-type N-cadherin (NCadW/MMS1) or empty vector (EV/MMS1) in MMS1 cells. Significant increases in total and free b-catenin correlated with N-cadherin protein expression. These results indicate that N-cadherin protein modulates b-catenin levels MM cells. Results of experiments to determine whether N-cadherin-mediated regulation of b-catenin translates into altered TCF/b-catenin transcriptional activity in MM cells will be reported.