Influence of Fibronectin,(FN), Adhesion on Gene Expression Profiles, (GEP), of Isolated Patient Myeloma Cells.

Myeloma cells proliferate preferentially in the bone marrow and the latter has been shown to confer a survival advantage and drug resistance, (DR), through a process known as Cell Adhesion Mediated Drug Resistance or CAM-DR, a form of de-novo drug DR. Therefore, in studying the mechanisms of drug resistance in myeloma, the contribution of the microenvironment needs to be considered. Recently, we compared the genotypic and phenotypic profiles of CAM-DR melphalan resistance to acquired melphalan resistance. (Hazlehurst et al Cancer Res. 63: 7900–7906, 2003,)Briefly, we reported that de novo melphalan resistance associated with CAM-DR was significantly less complex, both phenotypically and genotypically, compared to acquired melphalan resistance in 8226 cells. GEP of adherent 8226 myeloma cells demonstrated fewer changes in gene expression compared to acquired melphalan resistance with no changes related to genes involved with DNA damage or cell cycle; however, changes were noted for genes involved in mitochondrial perturbation and caspase activation. Studies examining GEP profiles in myeloma patients, have been performed using isolated myeloma cells without considering the contribution of the tumor microenvironment and its influence on the phenotype or genotype of these cells. We hypothesize that the interactions of the myeloma cells with the microenvironment affects the myeloma GEP and needs to be considered when developing gene signatures that would ultimately predict level of response to treatment. In this study, we examined GEP of CD138+ myeloma cells isolated from 18 patients with relapsed, (n=8) or newly diagnosed (n=10), myeloma. The CAM-DR phenotype in CD-138+ cells was analyzed by exposing patient bone marrow specimens to Dexamethasone when cells were in suspension versus adhered to FN. We showed myeloma cells to be significantly more sensitive to Dexamethasone when kept in suspension vs adhered to FN in 5/6 specimens studied. For GEP studies, CD138+ were maintained in suspension or adhered to FN for 24 hours, then harvested for RNA extraction and hybridized to GeneChip arrays (Affymetrix HG-133A chip). Differential exression of genes was performed using a chi square test to compare observed vs expected. Only genes that were consistently differentially expressed in >50 % of patients with p value <.001 were considered for the final analysis. Among all the patients, FN adhesion of myeloma cells resulted in the overexpression of 24 genes and suppression of 45 genes.. In relapsed patients, 16 genes were found to be overexpressed and 56 suppressed when cells were adhered to fibronectin. In newly diagnosed patients, 123 genes were found to be overexpressed and 83 supressed on FN. Genes found to be overexpressed on FN vs suspension include adhesion related genes,(cathepsisn, integrin alpha 4 and 8), growth and proliferation related genes,(syndecan, TNF-induced protein), cytokines and chemokines (IL-16, CXC R4 and 2), and antiapoptotic proteins(MADS family).

In summary, we have shown that fibronectin adhesion of patient myeloma cells alters gene expression. As we expand the number of patients studied, we should be able to develop a gene signature associated with adhesion and determine whether these genotypes are better predictors of tumor behavior and response to therapy. Identification of genes uniquely associated with cell adhesion may lead to the development on novel therapies for myeloma.