Exploring the effects of Anti-Cancer Drugs and Transglutaminase Type 2 in Skin Cutaneous Melanoma and Endothelium

Tumor angiogenesis differs from physiological angiogenesis, resulting in abnormal branching and vessel leakiness that led to uneven distribution of anti-cancer drugs and an inadequate elicitation of the immune response within the tumor microenvironment (TME). At a cellular level, tumor endothelial cells become unresponsive to inflammatory stimuli, the so-called “endothelial anergy”, by the downregulation of specific adhesion molecules. This impedes the recruitment and extravasation of specific immune cell subtypes, establishing an immune-privileged TME that facilitates tumor progression. In recent years, anti-angiogenic therapy and vessel normalization, have been implemented to overcome endothelial anergy. The goal is to restore proper vascularization and establish an immune-activated TME within the tumor. However, it is noteworthy that these drugs, along with chemotherapy, have been shown to induce vascular toxicity. This underscores the importance of monitoring the impact of anti-cancer agents on the primary tumor and on the endothelium. Therefore, in this work we evaluated the effects of several anti-cancer drugs on endothelial cells, both in vitro and in vivo. We observed that specific compounds at sublethal doses exerted an impact on endothelial migration and tubule formation in vitro and they influenced the development of Intersegmental Vessels in a Zebrafish model. Taking advantage of a proteomic and transcriptomic approach, we elucidated a new NF-B/JNK signaling axis that in turn upregulated endothelial adhesion molecules expression upon the treatment. These findings could establish a connection between our anticancer drugs and the reversal of endothelial anergy. We therefore evaluated the effect of one of the compounds also in vivo, in a mouse melanoma model, showing a decrease in lung metastases formation. Furthermore, we evaluated the effects of the compounds on mitochondrial physiology, demonstrating an increase in mitochondrial membrane depolarization, and a decrease in respiration. Together, these findings provide a better comprehension of the interplay between cancer, endothelium, and anticancer drugs, and presents a chance to formulate innovative therapeutic strategies that account for their influence also on the TME. Transglutaminase type 2 (TG2) is a versatile enzyme with different cellular localizations and with implications in regulating different cellular pathways, positioning the enzyme as a significant contributor to the development of diseases. The aim of our research has been to study the role of TG2 in a tumor context and in the surrounding TME, in particular in angiogenesis. Indeed, TG2 has been recognized both as a tumor promoting and inhibiting factor. We recently demonstrated that TG2 has a positive prognostic value in only one out of 32 different cancer types, the Skin Cutaneous Melanoma (SKCM). Given this preliminary background a first aim of this work was to better elucidate its role in SKCM tumorigenesis. Adopting an omic approach along with in vitro and in vivo validations, we unveiled the existence of a TG2-MITF signaling axis, which plays a pivotal role in the regulation of melanoma intratumor heterogeneity and modulates the so-called “phenotype switching”. Also, TG2 exerts a still debated role in the TME. For example in the angiogenic process, where it has been defined both as pro- and anti-angiogenic factor. Therefore, we had a deeper insight on its role also in the endothelium, where we demonstrated that its pharmacological and genetical targeting impaired fundamental endothelial properties in vitro, and Intersegmental Vessels development in vivo. Altogether these findings set up the preliminary background on melanoma intratumor heterogeneity and in the relationship among the tumor and the TME. Our findings also pave the way for the development of new therapies that consider TG2 role both in tumor progression and in angiogenesis