Altered Cellular Protein Levels of Tumor Suppressor Genes and Heat Shock Elements (TRAP1) Indicate Sensitivity to the Proteasome Inhibitor Bortezomib (Velcade®) in Mantle Cell Lymphoma.

Abstract

Mantle cell lymphoma (MCL) is a distinct lymphatic neoplasia characterized by a poor clinical outcome with only transient response to conventional chemotherapy. In various phase II studies the proteasome inhibitor Bortezomib (Velcade®) has demonstrated a high clinical efficacy with up to 60% remission rates in relapsed MCL, however, the target pathways of proteasome inhibition remain largely unknown. Additionally, as the mode of action involves proteasome mediated protein degradation, only analysis of the protein level may reliably identify the cellular signal pathways involved. Four MCL cell lines (HBL-2, Granta 519, Jeko-1, NCEB-1) were exposed to Bortezomib at the minimal cytotoxic concentration (25nmol) and harvested for analysis at 0, 1, 4 and 8 hours of exposure. To assess the early alterations of cellular protein levels induced by Bortezomib-effected proteasome inhibition, the whole cellular proteome was screened applying two-dimensional gel electrophoresis. A high percentage of proteins were initially affected by proteasome inhibition, increasing their cellular content. Analyzing triplicate experiments, approximately 20 % of all cellular proteins detected on the 2D-gels (201/1013) exhibited an alteration of cellular protein content by a factor of >5, while 14 % (148) of the proteins were altered by >10 x. As expected about 79 % (158/201) of these proteins showed an increase of cellular protein, and only 21 % (44/201) of the proteins were downregulated. Approximately 65 % (133/201) of these proteins were already dysregulated after 1 hour whereas the 68 remaining proteins reached the threshold level after 4 hours. Cross cell line comparison yielded 41 reproducibly altered proteins in 3 responder cell lines. Among these proteins are (putative) tumor suppressors (ENO-1, fumarate hydratase), heat shock response elements (TRAP1, HSPA8, HSP72) and structural proteins (centrosomal protein CEP290). In the non-responder cell line (NCEB-1), expression of only 22 of these 41 proteins was altered after exposure to Bortezomib. These unspecific proteins involved some heat shock response elements (HSPA8, HSP72), the respiratory chain (NADH-ubiquinon oxidoreductase 24kDa mitochondrial precursor) and signal transduction (LSP1). In contrast, the cellular protein levels of the putative tumor suppressor genes, as well as the actin cross-linking protein alpha-fodrin were altered only in the responder cell lines. RNA-expression analysis detected highly diverging expression patterns after Bortezomib treatment in all four cell lines. No down-regulation of proliferation associated genes was detected, and the expression of heat shock response related genes were increased. As predicted, due to its mode of action, there was no correlation between the detected alterations of cellular protein levels and RNA-expression after Bortezomib treatment. In summary, Bortezomib treatment specifically affected the cellular protein levels of tumor suppressor genes and distinct heat shock response elements (TRAP1) in sensitive cell lines. Currently primary patient samples are being analyzed to confirm the predictive value of these molecular markers.