Immunomodulatory actions of NIDOGEN-1 in the Tumor Microenvironment: Impact of its ADAMTS1-mediated Proteolysis

Over last decades, tumor microenvironment (TME) has emerged as a critical focus on cancer research. This dynamic ecosystem, shaped by interactions between stromal cells, cancer cells and non-cellular components, drives all hallmarks of cancer. Central to this landscape is the extracellular matrix (ECM), whose composition and extremely dynamism directly regulate tumor evolution. Of particular importance are extracellular proteases such as the ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) family, which remodel the ECM and release bioactive factors that regulate key cellular processes, including proliferation, migration, differentiation, immune activation, and angiogenesis. For years, our laboratory has focused on studying the role of ADAMTS1 in tumor progression. Data from our group, as well as other research teams, have demonstrated that ADAMTS1 displays either pro- or anti-tumorigenic roles depending on the specific tumor context. Classically, ADAMTS1 has been associated with processes such as tumor cell migration and angiogenesis, but we recently uncovered its ability to modulate the immune compartment of tumors. In two different immunocompetent mouse models, we observed that absence of Adamts1 correlated with inhibition of tumor progression, accompanied with increased deposition of one of its substrates, the basement membrane glycoprotein NIDOGEN-1 (NID1). This finding pointed NID1 as a downstream effector of ADAMTS1 activity on tumor modulation. Based on these previous findings, in this thesis we investigate the role of NID1 on tumor progression, considering its proteolytic processing by ADAMTS1, and how it can influence to tumor progression, and others specific processes modulated by the protease. Using B16F1 murine melanoma model, we have found that intact NID1 inhibited tumor progression, however, its ADAMTS1-proteolyzed fragments failed to block tumor growth. Interestingly, tumors overexpressing intact NID1 showed an enhanced infiltration of immune cells and a global inflammatory landscape, emphasizing the presence of an anti-tumorigenic M1-like population of macrophages. Mechanistically, we showed NID1 drove M1-like macrophage polarization via αvβ3 integrin. Importantly, the NID1-overexpressing tumor model disclosed an overlapping phenotype with the Adamts1-deficient model, in terms of increased immune infiltration and inhibition of tumor growth. These results pointed NID1 as a tumor suppressor molecule whose function depends on its proteolytic state, underscoring the ADAMTS1- NID1 axis as a key regulatory circuit in tumor progression. Recognizing the translational potential of these findings, we extended our investigation to human melanoma. By integrating transcriptomic data from NID1-overexpressing murine tumors with datasets from melanoma patients, we identified a conserved “M1- associated gene signature” induced by NID1 overexpression. This signature was strongly correlated with the abundance of M1-like macrophages, as predicted by CIBERSORT analysis, and was able to predict overall survival in melanoma patients. The robustness of this signature was corroborated in two independent melanoma cohorts including 1100 patients. These results not only validate our preclinical findings but also underscore the relevance of ECM-immune interactions in human cancer. Collectively, this thesis uncovers a direct association between ADAMTS1 and its substrate NID1, demonstrating that the proteolytic regulation of NID1 is a key determinant of tumor progression through its effects on the immune microenvironment. We reveal novel tumor-suppressive function of intact NID1, mediated by the induction of M1-like macrophage polarization. Furthermore, the discovering of the “M1-associated gene signature” with prognostic value highlights the clinical significance of these findings. Overall, this work contributes to advance the understanding of how ECM remodeling, and in particular, extracellular proteases and their substrates, shape the TME and immune landscape, opening new avenues for the development of targeted therapies and biomarkers in cancer.