Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma

Abstract

Background & aims: The Hedgehog signaling pathway contributes to cholangiocarcinoma biology. However, canonical Hedgehog signaling requires cilia, and cholangiocarcinoma cells often do not express cilia. To resolve this paradox, we examined non-canonical (G-protein coupled, pertussis toxin sensitive) Hedgehog signaling in cholangiocarcinoma cells.

Methods: Human [non-malignant (H69), malignant (HuCC-T1 and Mz-ChA-1)] and rat [non-malignant (BDE1 and NRC), and malignant (BDEneu)] cell lines were employed for this study. A BDE(ΔLoop2) cell line with the dominant-negative receptor Patched-1 was generated with the Sleeping Beauty transposon transfection system.

Results: Cilia expression was readily identified in non-malignant, but not in malignant cholangiocarcinoma cell lines. Although the canonical Hh signaling pathway was markedly attenuated in cholangiocarcinoma cells, they were chemotactic to purmorphamine, a small-molecule direct Smoothened agonist. Purmorphamine also induced remodeling of the actin cytoskeleton with formation of filopodia and lamellipodia-like protrusions. All these biological features of cell migration were pertussis toxin sensitive, a feature of G-protein coupled (Gis) receptors. To further test the role of Hedgehog signaling in vivo, we employed a syngeneic orthotopic rat model of cholangiocarcinoma. In vivo, genetic inhibition of the Hedgehog signaling pathway employing BDE(ΔLoop2) cells or pharmacological inhibition with a small-molecule antagonist of Smoothened, vismodegib, was tumor and metastasis suppressive.

Conclusions: Cholangiocarcinoma cells exhibit non-canonical Hedgehog signaling with chemotaxis despite impaired cilia expression. This non-canonical Hedgehog signaling pathway appears to contribute to cholangiocarcinoma progression, thereby, supporting a role for Hedgehog pathway inhibition in human cholangiocarcinoma.

Keywords: 4′-6-diamidino-2-phenylindole; Biliary tract cancer; CAFs; CCA; DAPI; Dominant-negative Ptch1; FBS; G-protein coupled receptor; Gli; Hedgehog; Hh; PBS; PTX; Patched-1; Ptch1; Shh; Smo; Smoothened; Sonic Hh; cancer-associated fibroblasts; cholangiocarcinoma; fetal bovine serum; glioma-associated transcriptional factor; pertussis toxin; phosphate-buffered saline; qRT-PCR; quantitative reverse transcription polymerase chain reaction.