Transcriptional Response and Immune Activation after Morphogen Signaling in Melanoma

Abstract

TGFb and Wnt developmental signaling pathways are commonly dysregulated in cancer; this includes melanoma, the deadliest form of skin cancer. These pathways terminate in signaling transcription factors (TFs) binding to chromatin and modulating transcription. Here, for the first time, we visualized dynamic TGFb and Wnt responses in the melanoma tumor microenvironment using inducible and specific zebrafish reporters. These reporters were created using human melanoma enhancer regions with unique open chromatin and signaling TF binding upon stimulation. While TGFb and Wnt reporters were not expressed in normal melanocytes or early melanoma, both reporters are active in mature zebrafish melanomas and macrophages phagocytose both TGFb and Wnt responsive tumor cells. Single cell RNA-sequencing revealed that TGFb+ melanoma cells down-regulate interferon alpha and gamma, likely having an immune suppressive effect, while Wnt responding melanoma cells up-regulate interferon alpha and gamma. These conflicting phenotypes suggest that Wnt and TGFb responding melanoma cells are likely distinct subsets. We found that this heterogenous cellular response is maintained at the chromatin level. ChIP-seq for TGFb and Wnt signaling TFs revealed that they occupy distinct regulatory regions. Wnt factors are more likely to occupy distal regulatory regions than TGFb TFs SMAD2/3, which bind both promoters and exons. The overlap between TGFb and Wnt responsive regions was surprisingly minimal, only 3%. SMAD2/3 bound regions were greatly enriched for AP-1 motifs and ChIP-seq confirmed that ATF3 and JUNB are bound at SMAD2/3 responsive elements before stimulus. Loss of AP-1 motifs in a TGFb inducible enhancer revealed that AP-1 binding is required for TGFb signaling response, suggesting that AP-1 factors facilitate the binding of TGFb TFs by opening chromatin and exposing SMAD binding motifs. Together this work identifies novel divergent TGFb and Wnt transcriptional, cellular, and immune responses and highlights the need for more work examining combination immune checkpoint, TGFb, and Wnt inhibitors in future melanoma treatment.