Bortezomib-Induced Reversal of CAM-DR Phenotype in Myeloma Is Associated with AKT and ERK1/2 Signaling.

An increasing body of evidence suggests that interactions between myeloma cells and the microenvironment are critical for tumor cell survival, proliferation and drug resistance. These pro-survival interactions include binding of soluble factors, and direct contact with stromal cells, or extra cellular matrix (ECM) components such as fibronectin (FN). More specifically, adhesion of multiple myeloma (MM) to FN via ν1 integrins has been demonstrated to confer resistance to a host of chemotherapeutic agents (Shain et al., 2000). This anti-apoptotic phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), is an intrinsic pathway for evading the therapy-induced apoptosis (Damiano et al., 2000). Bortezomib (PS-341), a specific inhibitor of proteosome, has been shown to cause apoptosis of several tumor cell lines including multiple myeloma. In contrast to conventional cytotoxics, Bortezomib, showed more apoptosis in 24 hours for 8226 myeloma cells adhered to FN (25 % 10 nM) compared to cells in suspension (10 % 10 nM) in a dose -dependent manner. To investigate the signal transduction mechanism responsible for the reversal of CAM-DR phenotype by Bortezomib, we conducted Affymetrix GeneChip^Ò Expression of 8226 cells cultured either in suspension, or adhered to FN in the presence and absence of Bortezomib (50 nM) for 4 and 24 hours using the Affymetrix U133A chips. A total of 197 genes were upregulated on FN compared to suspension, among which only 12 genes were increased with Bortezomib and 105 genes were decreased with PS-341. A total of 870 genes were downregulated on FN adhesion, among which the majority of them were increased with Bortezomib. Similar patterns of gene expression profiles were observed after 24 hrs of treatment with Bortezomib. To examine how Bortezomib is reversing CAMDR phenotype, we chose to examine the genes that were changed with FN adhesion and reversed when treated with Bortezomib. The adhesion of 8226 cells to FN enhanced both ERK1/2 and AKT signaling pathways, and both pathways were down regulated when treated with Bortezomib. The main indicators of the AKT signaling on FN are upregulation of PYK2, AKT2, MDM2 and downregulation of p21 and BAD. In conformation with our micro-array data, PYK2 phosphorylation and ERK1/2 phosphorylation were enhanced when 8226 cells were adhered to FN. The mechanisms to understand role of PYK2 phosphorylation and downstream AKT signaling in Bortezomib-induced reversal of CAMDR are under investigation.