The role of Hedgehog signaling in Cholangiocarcinogenesis: An in vitro and in vivo (mouse model) study

Abstract

Cholangiocarcinoma (CC) is the second most frequently occurring primary liver tumor worldwide and is receiving medical importance because of its rising number of incidences, poor diagnosis and substandard response to the therapy. It is associated not only with genetic alterations, but other essential modifications of tumour microenvironment, which lead to the activation of various signaling pathways involved in tumour induction and progression. Thus, further exploration of the molecular mechanisms involved in the regulation of CC phenotype is needed for a better understanding of tumor progression and designing of more effective therapies. Hedgehog pathway is one of them, and its augmentation has been observed in various human malignant neoplasms, but its involvement in cholangiocarcinogenesis is still unclear. In this pursuit, this study was conducted in Alb- Cre/LSL-KRASG12D/p53L/L mice and human CC cell lines (TFK-1 and HUCCT-1) to gain a better understanding about the underlying mechanisms by which Hedgehog pathway activation triggers and maintains the process of cholangiocarcinogenesis. The study revealed CC development by 19-20 weeks of age in our mouse model, which was followed by lung metastasis via upregulation of mesenchymal markers. The expression of Hedgehog components like SHH and its downstream elements, SMO and GLI1, was found to be significantly elevated in liver tissue during CC development. Consequently, the expression of CSC transcription factors SOX-2, OCT-4 and NANOG, and CD133 were remarkably enhanced, resulting in stem- like properties and tumor progression in the liver tissue. Blockade of Hedgehog pathway using Cylopamine inhibited the growth of endogenous Hedgehog pathway- dependent tumors by suppressing SMO protein expression and GLI1 nuclear translocation. Moreover, systemic Cyclopamine treatment also inhibited metastasis by inhibiting the expression of invasive markers, thereby prolonging the survival of these mice. Additionally, Hedgehog pathway inhibition by Cyclopamine abrogated NANOG, Oct4, SOX2 and CD133 augmentation. Further, to validate our findings from mouse model studies, we investigated the expression of Hh pathway elements in human TMAs. Our data showed that in the study cohort of 49 patients, more than 80 % of CC samples were SHH and GLI1 positive, thereby implicating the role of Hedgehog pathway in cholangiocarcinogenesis. Based on previously obtained results, we analysed the fraction of CD133+ cells in HUCCT-1 cell line and assessed their tumor forming capacity by implanting 100 or 1000 CD133+ cells in NOD/SCID mice. We observed that CD133+ cells had a remarkably high tumorigenic potential, since as few as 100 cells could result in 100% tumor incidence as compared to unsorted cells. To further validate our hypothesis, we evaluated the expression of Hedgehog pathway elements, SHH and GLI1, in xenografted tumors derived from implantation of CSC marker- based sorted cells. We found that the expression of SHH and GLI1 was markedly upregulated in CD133+ derived tumors as compared with the tumors developed from unsorted cells. Intra-tumoral hypoxia is known to contribute towards therapeutic resistance through modulatory effects on various pathways. In this study, we investigated the relationship between hypoxia and SHH pathway activation and the effect of this interplay on cancer stemness and EMT during cholangiocarcinogenesis. Hypoxia promoted SHH pathway activation, evidenced by upregulated SHH and SMO levels, and enhanced GLI1 nuclear translocation; whereas silencing of HIF-1α impaired SHH upregulation. Hypoxia also enhanced the expression of CSC transcription factors (NANOG, Oct4, SOX2), CD133 and EMT markers (N-cadherin, Vimentin), thereby supporting invasion. Cyclopamine treatment suppressed hypoxia induced SHH pathway activation, consequently reducing invasiveness by downregulating the expression of CSC transcription factors, CD133 and EMT. Cyclopamine also induced apoptosis in CC cells under hypoxia, suggesting that hypoxia induced activation of SHH pathway has modulatory effects on CC progression. Taken together, the results presented here indicate that aberrant activation of Hedgehog signaling has an important early role in tumor induction, metastasis and maintenance of CC cancer stemness. Therefore, it suggests that Hedgehog signaling may hold promise for new diagnostic and therapeutic approaches for CC treatment.