Function and targeting of the Hedgehog signaling in human cholangiocarcinoma and melanoma

ABSTRACT Hedgehog (HH) signaling is a conserved pathway that plays a pivotal role during embryonic development, tissue homeostasis and regeneration. Aberrant activation of the HH pathway has been reported to drive tumor progression in numerous cancers, including those of the skin, brain, lung and breast. The key signaling components of the HH signaling are the ligands, the cell membrane receptor Smoothened and the three transcription factors GLI1, GLI2 and GLI3, which upon activation of the HH signaling translocate into the nucleus where they activate the transcription of specific target genes. In the last few years, the Smoothened receptor has become an interesting target for the development of anticancer agents. In the first part of this work, we tested the requirement of Hedgehog signaling in intrahepatic cholangiocarcinoma (iCCA), a group of aggressive hepatobiliary malignancies with still poorly understood molecular background. Due to the lack of effective therapeutic strategies, CCA is mostly considered an intractable disease. We show that the Hedgehog signaling pathway is upregulated in intrahepatic cholangiocarcinoma (iCCA) cell lines compared to normal cholangiocytes, and it drives and regulates iCCA cell proliferation and survival. Furthermore, we present evidence that pharmacological inhibition of the HH signal transducer Smoothened with novel acylguanidine derivative compounds negatively affects iCCA in vitro growth and viability, leading to cellular apoptosis as a result of the induction of double strand break (DSB) DNA damage. Altogether, these data report new findings in cholangiocarcinoma biology and point to a possible therapeutic potential of targeting the Hedgehog signaling in iCCA. In the second part of this thesis, we identified a novel axis downstream of Hedgehog signaling, that drives in vitro and in vivo melanoma cell invasiveness and metastatic behavior through the induction of a group of enzymes belonging to the O-glycan biosynthesis pathway. In particular, we find that GLI1 regulates transcription of the sialyltransferase ST3GAL1, through direct binding to regulatory regions in the enhancer II of ST3GAL1. Functional assays show that ST3GAL1 is a critical mediator of GLI1-induced melanoma cell invasiveness. In addition, our biochemical data suggest that the receptor tyrosine kinase AXL is a substrate of ST3GAL1, which is required to induce dimerization and activation of AXL in melanoma cells. Taken together, our results describe a novel HH-GLI1-ST3GAL1-AXL axis as crucial driver of melanoma invasiveness, proposing new potential therapeutic targets for management of metastatic melanoma.