Abstract 5425: MITF/TFE regulatory programs in neural crest migration are co-opted in melanoma metastasis

Abstract Introduction: Microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte differentiation and melanoma oncogenesis. Melanoma expression profiling has revealed a strong trend between low MITF expression levels and invasive behavior. However, it is unclear how low levels of MITF are connected to invasive behavior. Preliminary data from our lab has identified transcription factor E3 (TFE3), a MITF paralog, as a transcriptional regulator in MITF-low melanoma states. We hypothesize that in melanoma cells with low MITF levels, TFE3 promotes invasion. To assess the possibility that the antagonistic regulatory interaction between MITF and a paralog was coopted from the embryo, we examine migratory behavior of neural crest cells in zebrafish embryos that are wild-type, mitfa loss-of-function (LOF) mutants, tfec (TF3 ortholog) LOF mutants, or mitfa/tfec double LOF embryos. Methods: We evaluated TFE3 levels by Western blot in a panel of melanoma cell lines known to express a range of MITF expression levels. We mutated TFE3 exon 4, which encodes the DNA binding domain, using a single guide RNA, and assessed cellular invasive potential using an in vitro transwell invasion assay. CUT&RUN sequencing was used to profile TFE3 genomic binding. Zebrafish embryos were generated in which the mitfa promoter drove expression of RNA encoding green fluorescent protein (GFP), and of the genotypes mentioned above. From transgenic wild-type and mitfa LOF mutant embryos we isolated GFP-expressing cells and conducted single cell-RNA sequencing. We assessed migration of GFP-labeled cells by fluorescent microscopy in 20-48 hour post fertilization zebrafish embryos of all genotypes mentioned above. Results: We found that melanoma cell lines with high levels of MITF (SKMEL3 and SKMEL28) had low levels of TFE3 protein, and those with low levels of MITF (A375 and RPMI 7952) had high levels of TFE3 protein. The TFE3-high cell lines were more invasive in vitro than the TFE3-low cell lines. TFE3-high cell lines depleted of TFE3 were less in invasive in vitro than parental cells. CUT&RUN profiling of TFE3 demonstrates that TFE3 binds mesenchymal-like enhancers in MITF-low cell lines. Single cell RNA sequencing revealed that in a melanocyte precursor cluster present in both wild types and in mitfa mutants, expression of tfec was higher in the latter. Finally, migration of GFP-expressing cells was higher in mitfa LOF mutant embryos than in mitfa/tfec double LOF mutant embryos. Conclusion: These data indicate that reduction of MITF expression up-regulates TFE3 expression in melanoma and tfec in zebrafish melanocytes, although the mechanism of this regulation is unclear. They also show that TFE3 and Tfec promote migration of metastatic melanoma and melanocyte precursors, respectively. Because regulatory mechanisms in cancer are often coopted from embryonic cells, studying the embryo may illuminate cancer pathogenesis. Citation Format: Jeremy Chang, Katelyn Campbell, Rachel Moore, Robert Cornell, Colin Kenny. MITF/TFE regulatory programs in neural crest migration are co-opted in melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5425.