Sorafenib Inhibits Signal Transducer and Activator of Transcription-3 Signaling in Cholangiocarcinoma Cells by Activating the Phosphatase Shatterproof 2

Cholangiocarcinoma (CCA) is the most common biliary malignancy. Unfortunately, its incidence is increasing in several Western countries.1 Therapeutic options for CCA are limited, and the overall prognosis for patients with CCA is dismal, with median survival being less than 24 months. New insights into the biology of this cancer may provide a framework for targeted therapeutic approaches. CCA, similar to hepatocellular carcinoma and colorectal cancer, often arises in an inflammatory environment.2,3 Inflammation-associated carcinogenesis is, in part, mediated by dysregulated cytokine signaling pathways,4 which potentially can be therapeutically targeted for treatment of this cancer. Members of the signal transducers and activators of transcription (STAT) family are key signal transducers in cytokine and growth factor signaling. Especially STAT3 has been shown to be an essential signaling molecule in carcinogenesis through its transcriptional activity on genes regulating apoptosis, proliferation, differentiation, and angiogenesis.5,6 One of the main activators of STAT3 in CCA cells is interleukin-6 (IL-6) via Janus kinases (JAK).7 IL-6 is a cytokine secreted by inflammatory cells (i.e., macrophages) but also by CCA cells where it activates STAT3 by autocrine and paracrine mechanisms.2,3,8,9 Under physiologic conditions, the JAK/STAT3 signaling pathway is tightly regulated through negative feedback mechanisms including protein phosphatases.7 In CCA, IL-6–mediated JAK/STAT3 signaling was found to be dysregulated due to epigenetic silencing of suppressor of cytokine signaling-3.10 Interestingly, interruption of JAK/STAT3 signaling either through JAK inhibitors or STAT3 small interfering RNA (siRNA) resulted in sensitization to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through down-regulation of the STAT3 transcriptional target Mcl-1, an antiapoptotic Bcl-2 family protein.8 Sorafenib was developed as a c-Raf kinase inhibitor, although it also targets several other Raf-kinases (wild-type b-Raf, b-Raf V600) as well as other receptor tyrosine kinases.11 Sorafenib prolongs survival of patients with hepatocellular carcinoma.12 Recently, inhibition of STAT3 phosphorylation by sorafenib was observed in medulloblastoma and esophageal carcinoma.13,14 These studies were predominantly observational, and the mechanisms by which sorafenib inhibits STAT3 phosphorylation were not elucidated. Given this information, the effect of sorafenib on STAT3 regulation in CCA warrants exploration as a potential therapeutic agent. The objective of this study was to examine the effect of sorafenib on the JAK/STAT3 signaling cascade in CCA cells. The results of this study suggest that sorafenib inhibits the JAK/STAT3 signaling axis at the level of STAT3 phosphorylation, resulting in down-regulation of Mcl-1, thereby sensitizing human CCA cells to TRAIL-mediated apoptosis. The inactivation of phospho-STAT3 occurs by a phosphatase shatterproof 2 (SHP2)-dependent mechanism which appears to be stimulated by Raf-kinase inhibition. Furthermore, in an orthotopic, syngeneic rodent CCA model, sorafenib achieves significant tumor suppression.