Activin-A, a transforming growth factor êµ family member, is a pleiotropic cytokine with diverse functions in development, fertility, adult tissue homeostasis, and aging. Accordingly, deregulation of Activin-A signaling has been associated with many pathological conditions, including cancer and cancer-associated muscle wasting (cachexia). Activin-A was shown to signal in endocrine, but also local autocrine and paracrine manner. Its secretion in many cancer types is associated with a poor prognosis. Previous studies suggest that Activin-A expression in melanoma promotes tumor growth and metastasis indirectly by inhibiting adaptive anti-tumor immunity, leading to a decrease in the intratumoral frequency of cytotoxic CD8+ T cells (CTLs). However, the underlying mechanisms and the relevant direct target cells of Activin-A and their significance for cancer therapies remained unknown. Similar to other TGFêµ-related ligands, proActivin-A is cleaved by proprotein convertases (PCs) to release mature ligands. However, unlike other family members whose signaling is tightly regulated, Activin-A apparently does not have latency.In this thesis, I characterize proActivin-A cleavage and show that one prodomain can be cleaved independently of known PCs. The resulting hemi-cleaved Activin-A can bind activin type II receptors and induce SMAD3 signaling in reporter cells. A protein interactome screen and analysis of cleavage intermediates in plasma revealed that the hemi-cleaved Activin-A likely interacts with extracellular matrix proteins via the remaining prodomain where it promotes local signaling and regulates trafficking. Luciferase reporter assay showed that hemi-cleaved Activin-A can avoid, at least partially, inhibition by endogenous inhibitors FSTL3 and FST, and enable parallel activation of BMP and Activin signaling pathways. Finally, analysis of B16-F1 melanoma models revealed that only fully cleaved Activin-A in source cells increased tumor growth by acting locally in the tumor microenvironment suggesting that the context of Activin-A precursor processing regulates its tumor growth-promoting effect.The second part of this thesis addresses the mechanisms of Activin-induced immunosuppression in melanoma. We found that Activin-A impairs CTL function indirectly and independently of CD4+ T cell, CSF1R+ macrophages, and IL4 signaling. At least in part, anti-tumor CTL responses are impaired by the inhibitory effects of Activin-A on the secretion of critical CTL-recruiting chemokines CXCL9 and CXCL10 by macrophages and dendritic cells (DCs). Furthermore, Activin-A conferred resistance to immune checkpoint blockade therapy. Characterization of Activin-induced changes in the gene expression of tumor-infiltrating cells by Single cell RNA sequencing revealed that INHBA expression induced the most changes in DCs and least in T cells. Interestingly, the most prominently induced hallmark in Activin-secreting tumors across cell types was the IFN signature. In vitro analyses showed that Activin-A increases IFNγ-induced activation of STAT1 and confers resistance to cytostatic IFN signaling in cancer cells.Altogether, the findings of this thesis expand our knowledge on the regulation of Activin-A signaling by precursor cleavage and offer insights into mechanisms of Activin-induced immunosuppression in melanoma. Inhibition of Activin-A maturation thus emerges as a potential new strategy to improve responses to immunotherapies in melanoma and likely other cancers
EPFL2022
