Sonic hedgehog pathway as a new target in cholangiocarcinoma therapy

Introduction:

The therapeutic resistance of cancer stem cells (CSCs), a small subset in the tumor micro environment known for its self renewal and differentiating capacity, has remained an enigma until now. Various factors, including hypoxia, have been known to support tumor growth by facilitating the proliferation of CSCs as well as activating and deregulating various signaling pathways. One of such pathways, the Sonic hedgehog pathway (Shh), has been shown to be aberrantly activated in a variety of human cancers, however, its involvement in cholangiocarcinogenesis has not been explored till now.

Objective:

We investigated the interplay between hypoxia and Shh pathway activation and its effect on CSCs during cholangiocarcinoma (CCA) progression.

Methodology:

HUCCT-1 (Intrahepatic) and TFK-1 (Extrahepatic) cell lines were cultured under hypoxic (1% oxygen) and normoxic (21% oxygen) conditions for up to 48hr in the presence or absence of Cyclopamine (an inhibitor of Smoothened, a G- protein coupled receptor). The protein and mRNA expression of HIF-1 α, Shh, Gli-1, stem cell transcription factors (OCT-4, NANOG, SOX-2) and CD133 was determined by immunoblot and RT- qPCR respectively, nuclear translocation of Gli-1, OCT-4 and NANOG was investigated by immunofluorescence microscopy and induction of apoptosis was assessed by Annexin V- PI assay for up to 72 hrs.

Results:

Hypoxia led to the transcriptional activation of Shh gene and induced Gli-1 nuclear translocation in a time dependent manner. Expression of NANOG, OCT-4 and SOX-2 transcription factors, which regulate the self renewal capacity of CSCs, also increased significantly, which was associated with a concomitant increase in the CD133 marker level. Inhibition of Shh pathway by Cyclopamine led to the suppression of Shh and Gli-1 protein expression and impaired GLi-1 nuclear translocation, which resulted in enhanced induction of apoptosis. Furthermore, it decreased the protein levels of NANOG, OCT-4 and SOX-2 significantly, leading to a substantially abrogated CD133 expression.

Conclusion:

We propose Shh pathway as a novel target for the treatment of CCA, which is refractory to standard chemotherapy.