Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma.
OBJECTIVE
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.
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Nataliya
N
Razumilava
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Sergio A
SA
Gradilone
Department of General Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Rory L
RL
Smoot
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Division of Gastroenterology and Hepatology, University Hospital Zurich, Switzerland.
Joachim C
JC
Mertens
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Steven F
SF
Bronk
Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA.
Alphonse E
AE
Sirica
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Electronic address: gores.gregory@mayo.edu.
Gregory J
GJ
Gores
D001650:Bile Duct Neoplasms / Q000473:pathology*; D001653:Bile Ducts, Intrahepatic; D045744:Cell Line, Tumor; D002633:Chemotaxis; D018281:Cholangiocarcinoma / Q000473:pathology*; D002923:Cilia / Q000502:physiology; D053823:Hedgehog Proteins / Q000502:physiology*; D006801:Humans; D009362:Neoplasm Metastasis; D015398:Signal Transduction / Q000502:physiology*
D053823:Hedgehog Proteins
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