engleski

Primary cilia - a potential link between hedgehog- GLI and MAPK signaling in GANT61 -resistant melanoma cell lines

Hedgehog-GLI and MAPK signaling are known signaling partners. BRAF and NRAS can activate GLI proteins directly regardless of the upstream membrane events, but the exact signaling order in which this occurs, other components involved in this interaction and final outcomes are still not fully understood. This interaction may be crucial in establishment and maintenance of drug resistance, a known issue for metastatic melanoma. We established three melanoma cell lines with different BRAF/NRAS mutation background 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 transcription targets connected to MAPK signaling were identified with ChIP-seq for endogenous GLI1, GLI2 and GLI3 proteins in parental cell lines and validated by qPCR. By gaining GANT61 resistance, cell lines changed response to other HH-GLI inhibitors beside GANT61, migration and colony forming capacity. In comparison to the BRAF/NRAS wild type cell line, the NRAS mutated 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 and MAPK. Therefore, we focused on primary cilia visualisation using immunofluorescence. We successfully induced primary cilia with serum deprivation in the NRAS mutated parental cell line, but only few primary cilia were detected in the resistant cell line, suggesting primary cilia loss in the resistant cell line. Based on our current results, we believe that primary cilia present a potential link which can explain the switching from HH-GLI on MAPK signaling. Established GANT61- resistant cell lines present valuable in vitro models which can bring new insights into HHGLI-MAPK interplay. Our future work will be based on proteins involved in ciliogenesis to see if primary cilia restoration can resenzitize cells to GANT61.

HH-GLI ; melanoma ; GANT61, MAPK ; resistance

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