Abstract #5489: Sorafenib inhibits IL-6 induced STAT3 signaling in cholangiocarcinoma cells and demonstrates in vivo efficacy

Introduction and aims: Cholangiocarcinoma (CCA) is a devastating neoplasm with no proven medical therapy. IL-6 signaling pathways play a pivotal role in the biology of this cancer. Sorafenib is a multikinase inhibitor approved by the FDA for the treatment of hepatocellular carcinoma. However, its effect on the IL-6 signaling cascade and its in vivo efficacy in CCA is unknown. Our aims were, therefore, to evaluate sorafenib as a therapeutic agent for CCA. Methods: Studies were performed using the human CCA cell lines HuCCT-1 and KMCH-1. Expression and activation of the IL-6 signaling axis were evaluated by immunoblot analysis and STAT3 nuclear translocation by immunofluorescence studies. Apoptosis was morphologically quantified by DAPI-staining and fluorescence microscopy; apoptosis was confirmed by caspase-3/7 analysis. In vivo efficacy analysis was performed in a recently described novel orthotopic, syngeneic CCA rat model (Sirica et al.: Hepatology 2008; 47: 1178-1190). Results: Sorafenib (10 \#956;M) does not interfere with IL-6 secretion by CCA cells as quantified by an ELISA assay. Likewise, it does not alter expression of gp130, gp80, JAK 1 and 2, key proteins of the IL-6 signaling complex. In contrast, sorafenib completely abrogates STAT3 phosphorylation, a key mediator of IL-6 signaling, without altering total cellular STAT3 levels. Sorafenib treatment also reduced nuclear localization of this transcription factor four-fold. The Sorafenib-mediated effect on the phosphorylated form of STAT3 was inhibited in the presence of the phosphatase inhibitor sodium pervanadate. Further analysis revealed that the effect of sorafenib on STAT3 dephosphorylation was mediated by Raf-kinase inhibition. Sorafenib treatment also reduced cellular levels of the anti-apoptotic protein Mcl-1, a transcriptional target of STAT3. As Mcl-1 downregulation is known to sensitize cells to TRAIL-mediated cytotoxicity, we examined TRAIL-mediated apoptosis. Treatment with sorafenib markedly sensitized HuCCT-1 and KMCH-1 CCA cells to TRAIL killing. Finally, our in vivo data demonstrate that sorafenib elicits a significant tumor suppressive effect in an orthotopic, syngeneic rat CCA model. Treatment with 10 mg/kg Sorafenib achieved significant tumor suppression. 21 days after tumor implantation, 60% of Sorafenib treated animals were alive versus 0% in the treatment group. Conclusions: In cholangiocarcinoma cells, Sorafenib-mediated Raf-kinase inhibition interferes with IL-6 activation of STAT3, reducing cellular expression of Mcl-1, and, thereby, sensitizing CCA cells to TRAIL-cytotoxicity. Significant therapeutic effects were observed with Sorafenib treatment in an orthotopic, syngeneic CCA rat model. Sorafenib merits evaluation for the treatment of human CCA. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5489.