engleski

Hedgehog-GLI and MAPK signalling pathway activity in GANT61-resistant melanoma cells

INTRODUCTION Hedgehog-GLI (HH-GLI) signaling can be activated in a noncanonical manner due the interaction with other signaling pathways, like MAPK signaling. BRAF and NRAS are known for activating GLI proteins directly regardless of the upstream membrane events, but the exact signaling order in which this occurs, and final outcomes are still not fully understood. This interaction may be crucial in establishment and maintenance of drug resistance, which is a known issue for metastatic melanoma. MATERIAL AND METHODS We established three melanoma cell lines with different BRAF/NRAS mutation status resistant to GANT61, a GLI specific inhibitor, to explore in more depth the mechanism that underlays HH-GLI and MAPK signaling interaction. Cells were treated with increasing GANT61 concentrations in duration 8-12 months and validated with MTT viability test. HH-GLI and MAPK protein expression was examined by Western blot. Potential GLI transcription targets connected to MAPK signalling were identified with ChIP-seq for endogenous GLI1, GLI2 and GLI3 proteins in these cell lines, and validated by qPCR. Guided by the results, we focused on primary cilia visualization using immunofluorescence in parental and resistant cell lines. RESULTS AND DISCUSSION GANT61 resistant cell lines were successfully established and validated. By gaining GANT61 resistance, cell lines also changed response to other HH-GLI inhibitors beside GANT61. Resistant cell line showed upregulated MAPK signaling, downregulated HH-GLI signaling and a newly identified GLI transcription target RAB34. RAB34 is essential for primary cilia formation and plays a central role in signal transduction of several signaling pathways, including HH-GLI. We successfully induced primary cilia with serum deprivation in parental cell line, but only few primary cilia were detected in the resistant cell line, suggesting primary cilia loss in the resistant cell line. CONCLUSIONS GANT61 resistant cell lines present valuable in vitro models which can bring new insights into HH- GLI-MAPK interplay. Based on our current results, we believe that primary cilia present a potential link which can explain switching from HH-GLI on MAPK signaling. Our further experiments will be based on proteins important for regulation of ciliogenesis.

HH-GLI, MAPK, melanoma, resistance, ChIP-seq

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