Dissecting the hindrance of Hedgehog pathway in Intrahepatic Cholangiocarcinoma: a step towards targeted therapy

Cholangiocarcinoma (CCA) comprises a heterogeneous group of cancers affecting the biliary tree and is considered one of the deadliest cancers. CCA incidence and mortality are increasing dramatically worldwide in the last decades. CCA prognosis is dismal (5-year survival rate <20%) due to various aspects including late diagnosis, poor response to chemotherapy and lack of well-established targeted therapeutic options. One of the foremost patho-biological features of CCA is the high inter- and intra-tumoral heterogeneity, which has limited the discovery of biomarkers and novel therapeutic alternatives, hampering the development of tools for early diagnosis and effective treatment, with unfavorable implications on patient outcome. From an anatomical outlook, CCA is classified into intrahepatic (iCCA), perihilar (pCCA) and distal (dCCA). Furthermore, iCCA shows distinct histological subtypes which led the World Health Organization (ICD-O-3.2) to categorize it into small and large bile duct-type. Noteworthy, thus far no data exist concerning the correlation of molecular traits and specific iCCA subtypes. Indeed, this marked heterogeneity strikingly restricts the possibility of translational research and represents an utmost aspect to be scrutinized. The Hedgehog (Hh) pathway is one of the molecular networks involved in the pathogenesis of iCCA. As a matter of fact, it is fundamental in various hallmarks of iCCA, including tumor growth, survival, migration, cancer stem cells activation and epithelial-mesenchymal transition. In the light of these observations, targeting this cascade for therapeutic purposes in iCCA is possible owing to evidence of Hh's pathogenetic role in the disease. This study aims to evaluate, in vitro, in primary and established cell lines, a novel natural chemical compound called Glabrescione B (GlaB) that specifically inhibits Gli1, a transcriptional factor downstream of Hh pathway. Using Trypan Blue Exclusion test, the dose- and time-dependent response of free GlaB and hyaluronic acid (HA)-encapsulated GlaB (HA-GlaB) has been evaluated. MTS assay has been performed to determine the IC50. Western blot has been used to analyze the target protein expression levels. Wound healing assay has been achieved to assess the migratory activity of cells. Colony formation assay has been performed to rate the colony-forming ability of cancer cells. Flow cytometry analysis has been accomplished to investigate cell death (apoptosis and necrosis). The results were confirmed in at least three independent experiments and the quantitative data were reported as the mean ± SEM. Student’s t-tests for paired samples were used to assess differences among two groups. A p-value of <0.05 was considered statistically significant (p>0.05;*p<0.05;**p<0.01;***p<0.001). Our findings demonstrate that both free GlaB and HA-GlaB treatments significantly reduce iCCA cell rate viability, migration, colony-forming ability, and Gli1 levels in a dose- and time-dependent manner (0.05<0.001), leading to a decisive decrease in cancer cells invasiveness. Ultimately, compared to controls, early evidence from flow cytometry reveals an induction of necrosis and apoptosis as a result of drug administration. Hedgehog pathway dysregulation is well known to be correlated with the development and progression of various cancers, including iCCA. A novel natural compound, named Glabrescione B, is able to inhibit cancer cells growth, survival, invasiveness and to induce cancer cell death. Taken together, these data provide insight into a new and potentially beneficial natural compound for the management of iCCA in vivo.