Proteasome Inhibitors Induce Death but Activate Nf-kb on Endometrial Carcinoma Cell Lines and Primary Culture Explants

ENDOMETRIAL CARCINOMA CELL LINES AND PRIMARY CULTURE EXPLANTS. Xavier Dolcet, David Llobet, Mario Encinas***, Judit Pallares, Albert Cabero*, Joan Antoni Schoenenberger**, Joan X Comella***, Xavier Matias-Guiu Departments of Pathology and Molecular Genetics, Gynecology*, and Pharmacy**, Hospital Universitari Arnau de Vilanova, Cell signalling and Apoptosis Group***. Laboratori de Recerca de l’Hospital Universitari Arnau de Vilanova, Departament de Ciencies Mèdiques Bàsiques i Cirurgia, Universitat de Lleida, IRBLLEIDA Running title: Proteasome inhibitors and NF-kB in endometrial cancer. Address for correspondence: Xavier Dolcet, Department of Pathology and Molecular Genetics Hospital Universitari Arnau de Vilanova. Av Alcalde Rovira Roure 80. 25198 Lleida, Spain. Email:[email protected] SUMMARY Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-kB, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-kB, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKKα/β, phosphorylation and degradation of IkBα and phosphorylation of p65 NF-kB subunit on serine 536. Proteasome inhibitorinduced NF-kB activity can be blocked by a non-degradable form of IkBα or dominant negative forms of either IKKα or IKKβ. Lentiviral delivery of shRNAs to either IKKα or IKKβ cause blockade of NF-kB transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IkBα degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-kB activation. Accordingly, siRNA knock-down of p65 inhibits proteasome inhibitor-induced NF-kB transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-kB instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NFkB activation should be carefully examined in particular cell types. INTRODUCTION The proteasome represents a novel putative target for cancer therapy. PS-341 (Velcade/bortezomib) is a dipeptidyl boronic acid inhibitor with high specificity for the proteasome (1,2). It is currently used in the treatment of patients with multiple myeloma (3,4,5,6,7). Preclinical studies have suggested that proteasome inhibitors show antitumour activity against solid tumours, including carcinomas of the breast (8), lung (9), colon (10), bladder (11), ovary, prostate (12), pancreas (13) and glioblastoma multiforme. Furthermore, evidence has shown that transformed cells appear to be more susceptible to proteasome inhibitorinduced apoptosis than nontransformed cells. Finally, these inhibitors can sensitize cancer cells to death induced by members of the TNF family such as TRAIL . The antitumoural effects of bortezomib have been extensively studied in multiple myeloma, being inactivation of NF-kB one of the proposed mechanisms of action. NF-kB is a pleiotropic transcription factor, which is activated by a broad variety of stimuli such as growth factors, cytokines, ionizing radiation, ultraviolet light or chemotherapeutic drugs (14, 15) NF-kB regulates the expression of a large number of genes, which carry important functions in inflammation, apoptosis, proliferation, and angiogenesis. NF-kB shows constitutive or increased activity in a wide variety of tumours (16, 17), including endometrial carcinoma (18) and plays a crucial role in neoplastic transformation (16, 19). In resting cells, NF-kB is held inactive in the cytoplasm, bound to the