Targeting PKC: A Novel Role for Beta-catenin in ER Stress and Apoptotic Signaling

Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising pre-clinical activity in a wide range of tumor cells. In this study, we further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of b-catenin in regulating growth and survival of tumor cells. Inhibition of PKC leads to rapid accumulation of b-catenin by preventing the phosphorylation required for its proteasomal degradation. Specifically, b-catenin was dephosphorylated at Ser^33,37,41 and accumulated in a dose- and time-dependent manner in all cell lines tested (including primary MM cells and 10 MM cell lines, 3 colon cancer, HeLa, as well as HEK 293 cells). Microarray analysis and siRNA-mediated gene silencing in MM cells revealed that accumulated b-catenin activates early ER stress signaling via eIF2a, CHOP and p21, leading to immediate inhibition of proliferation. Conversely, knock-down of components of the ER stress response pathway by siRNA (i.e., CHOP) abrogated the inhibitory effect of enzastaurin on MM cell proliferation. Importantly, accumulated b-catenin also contributes to enzastaurin-induced cell death, since inhibition of b-catenin by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a b-catenin -dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; b-catenin induction by enzastaurin led to p73 (but not p53) activation, which was also abrogated by b-catenin -specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of b-catenin in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. In summary, our data reveal a novel role of b-catenin in ER stress-mediated growth inhibition and a new pro-apoptotic mechanism triggered by b-catenin upon inhibition of PKC isoforms, and further demonstrate that p73 represents a novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies including MM.