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Signaling Switch from Hedgehog-GLI to MAPK Potentially Drives Primary Cilia Loss in NRAS Mutated GANT61-Resistant Melanoma Cell Line

Hedgehog-GLI signaling pathway is extremely important for normal embryonal development and often deregulated in cancer. Its activity is highly dependent on the primary cilia and can be non- canonically modified through the interaction with other signaling pathways, like MAPK signaling. BRAF and NRAS can activate GLI proteins directly regardless of the upstream membrane events, but the exact order in which this occurs, mediators of these interactions and final outcomes are still not fully understood. These interactions may be crucial in establishment and maintenance of drug resistance, a known issue for metastatic melanoma treatment. To investigate in more depth the interaction between HH-GLI and MAPK in resistance, we established two melanoma cell lines MEL224 (NRAS Q61R) and CHL-1 (NRAS WT) resistant to GANT61, a specific GLI protein inhibitor. Cell lines were treated 8- 12 months with increased GANT61 concentrations and afterwards validated with the MTT test. To characterize the established cell lines, we examined if the response to other HH-GLI and MAPK inhibitors has changed after resistance development, colony formation and migration capacity. HHGLI, MAPK protein signaling components and autophagy markers were examined by western blot. In our previous study (Kurtović et al. 2022), using a combined ChIP-seq and RNA-seq approach we identified novel GLI transcription targets involved in MAPK signaling. Therefore, by using qPCR we examined if any of these potential targets were changed in the resistant cell lines. Our results suggest that a signaling switch from HH-GLI to MAPK signaling has occurred in the resistant NRAS mutated cell line MEL224. Both cell lines exhibit a higher colony formation and migration capacity, but they differ in HH-GLI and MAPK signaling activity. A newly identified GLI2 transcription target and potential MAPK substrate, RAB34 essential for ciliogenesis, was downregulated in MEL224 resistant cell lines. For that reason, we checked primary cilia formation using immunofluorescence and detected primary cilia loss in resistant cell line. Kuonen et al. have previously reported cilia loss after HH-GLI and MAPK signaling switch in basocellular carcinomas resistant to SMO inhibitors. We believe that primary cilia represent a potential link between HH-GLI and MAPK signaling in GANT61 resistant NRAS mutated melanoma. Therefore, our future studies will focus on ciliogenesis regulation via MAPK signaling and its function in drug resistance.

Melanoma ; Hedgehog-GLI ; RAS/RAF/MAPK ; therapy resistance ; primary cilia

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