GABA regulates electrical activity and tumor initiation in melanoma

Oncogenes can initiate tumors only in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors in keratinocytes. Electron microscopy demonstrates specialized cell–cell junctions between keratinocytes and melanoma cells, and multielectrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte cocultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that GABAergic signaling across the skin microenvironment regulates the ability of oncogenes to initiate melanoma. Significance: This study shows evidence of GABA-mediated regulation of electrical activity between melanoma cells and keratinocytes, providing a new mechanism by which the microenvironment promotes tumor initiation. This provides insights into the role of the skin microenvironment in early melanomas while identifying GABA as a potential therapeutic target in melanoma

Supplementary Table 2 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

RNA-seq and GSEA results in switched vs non-switched keratinocytes</p

Supplementary Video 1 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

3D rendering of melanoma/keratinocyte contacts in zebrafish embryos</p

Supplementary Video 3 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

3D rendering of gephyrin positive clusters in melanoma/keratinocyte co-cultures</p

Supplementary Video 2 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

3D rendering of melanoma/switched keratinocyte contacts and stained vesicles in human melanoma/keratinocyte co-cultures</p

Supplementary Table 1 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

LOPAC Screen results in melanoma keratinocyte co-cultures</p

Supplementary Figures S1-S17 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

Combined supplementary figures and figure legends. Supplementary Fig. 1: Switching requires direct contact between nascent melanoma cells and keratinocytes in vivo. Supplementary Fig. 2: Switching requires direct contact between melanoma cells and keratinocytes in vitro. Supplementary Fig. 3: Switching does not involve cell fusion between keratinocytes and melanoma cells. Supplementary Fig. 4: Exosome-like vesicles are transferred from melanoma cells to keratinocytes. Supplementary Fig. 5: GABAergic signaling is active in skin and melanoma cells. Supplementary Fig. 6: Melanoma cells express GAD1 and produce GABA. Supplementary Fig. 7: Disruption of GABA signaling blocks melanoma/keratinocyte communication. Supplementary Fig. 8: Specialized cell-cell junction signatures are enriched in melanoma cells and keratinocytes. Supplementary Fig. 9: Melanoma vesicles are present near specialized GABAergic cell-cell junctions in co-cultures. Supplementary Fig. 10: gad activation correlates with higher tumor burden in melanoma. Supplementary Fig. 11: GABA producing enzymes are expressed in zebrafish melanoma cells. Supplementary Fig. 12: Disruption of GABA synthesis blocks tumor initiation in melanoma. Supplementary Fig. 13: GAD expression/GABA treatment is pro-tumorigenic in a non cellautonomous manner. Supplementary Fig. 14: Switched keratinocytes are pro-tumorigenic in melanoma. Supplementary Fig. 15: LIF expression in keratinocytes drives melanoma cell proliferation. Supplementary Fig. 16: Zebrafish melanoma scRNAseq identifies GABA and LIF associated components in the TME. Supplementary Fig. 17: MYCN drives LIF expression in keratinocytes.</p

Supplementary Video 4 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

Representative trace videos of spike activity in melanoma/keratinocyte co-cultures +/- GABA signaling</p