Inhibition of Sonic Hedgehog signaling reduces tumor growth in pancreatic cancer in vitro and in vivo

Abstract

Introduction: Activation of the hedgehog signal transduction pathway, triggered by hedgehog binding to the transmembrane receptor PTCH or by mutations in the PTCH gene, plays an important role in the development of various tumors including cutaneous basal cell carcinomas and cerebellar medulloblastomas. Recent findings indicate that digestive tract tumors including pancreatic adenocarcinomas also display increased hedgehog signaling activity due to an abnormal expression of the secreted hedgehog ligand sonic hedgehog (Shh). Material: To investigate if alterations of PTCH may also be involved in the pathogenesis of pancreatic adenocarcinoma we screened human pancreatic carcinomas and 3 cell lines for mutations using single-strand conformation polymorphism analysis (SSCP) of all coding exons and direct sequencing. Furthermore, the expression levels of known hedgehog target genes were determined using the competitive RT-PCR method. For Inhibition of the pathway in vitro we used the small — molecule inhibitor Hh-Antag. In vivo experiments were performed using a standardized heterotopic tumor model (nu/nu nude mouse) for treatment with the steroidal alkaloid cyclopamine and cyclopamine in combination with gemcitabine. Results: In the majority of pancreatic cancers we found significant overexpression of the target genes GLI-1 and PTCH compared to the corresponding normal pancreatic tissue. Additionally the effects of the small molecule inhibitor of the Hedgehog-Patched pathway Hh-Antag were investigated. Hh-Antag inhibits the function of Smoothened in pancreatic cancer cells; this results in inhibition of cell proliferation. Combination of cyclopamine+gemcitabine showed a significant reduction of tumor growth in vivo. Expression of target gens after treatment with cyclopamine showed a reduced expression of PTCH indicating an inactivation of the pathway in vivo as well. Conclusion: Overexpression of the taget genes GLI-1 and PTCH indicate an activation of hedgehog signaling in human pancreatic carcinoma. Inhibition of the pathway reduces tumor growth in vitro and in vivo. Therefore, inhibition of hedgehog signaling is a promising approach for treatment of pancreatic carcinoma.