Impact of inflammation on melanoma development and aggressiveness

Abstract

Skin cutaneous melanoma (SKCM) originates from melanocytes, neural-crest derived pigment-producing cells located in the epidermis, where their major function is to protect keratinocytes from UV-induced DNA damage. The malignant transformation of melanocytes generates this fatal form of skin cancer with a complex multigenic etiology that becomes extremely difficult to treat once it has metastasized. For that reason, SKCM is the most lethal form of skin cancer and while incidence rates are declining for most cancers, they have been steadily rising for SKCM. The relationship between inflammation process and cancer is yet considered ambiguous. Indeed, inflammation, especially chronic inflammation, may exert protumorigenic effects but, on the other hand, tumor cells spreading may be counteracted by inflammatory cells. Besides, immunosuppression is known to increase the cancer risk. The zebrafish, Danio rerio, has emerged as a new experimental organism to model cancer, thanks to the conservation of several molecular and cellular pathways involved in tumorigenesis. Also, it offers other important advantages as a tumor model organism. Zebrafish embryos are characterized by small size, external development and transparency that allow melanocyte tracking during all their developmental stages. The early developmental processes of melanocyte transformation and the methods for their early detection are important for disease eradication. In this thesis, the relevance of SPINT1 and TNFA/TNFR2 genetic alterations in the cross talk between inflammation and SKCM and in the SKCM prognosis was addressed. Furthermore, the relevance of the inflammation driven by Spint1a and Tnfr2 in early oncogenic transformation and the role played by Spint1a driven inflammation in SKCM progression and aggressiveness was also studied. To address those objectives, an in silico analysis of the occurrence and relevance of TNFA, TNFR, and SPINT1 genetic alterations of the SKCM TGCA cohort was analyzed. It was evidenced that genetic alterations in TNFA and TNFR2 genes occurred in 5 and 6 % of melanoma patients, respectively, and are associated with bad prognosis. In the SPINT1 case, we found a high prevalence of genetic alterations in SKCM patients (10%) and their association with altered tumor immune microenvironment with a concomitant poor patient survival. Functional experiments in zebrafish using morpholino gene-mediated inactivation of TNFR1 and TNFR2, and TNFR2 overexpression in transformed melanocytes using the Gal4-UAS system showed that, inflammation through the TNFA/TNFR2 signaling axis accelerates the onset of melanoma, enhancing oncogenic transformation and melanoma progression, pointing TNFR2 signaling in stromal cells as responsible of transformed cells proliferation increase. In addition, the impact of inflammation in promoting melanoma cells proliferation in vivo was evaluated by using a spontaneous model of melanoma in zebrafish expressing the oncogenic human HRASG12V driven by the melanocyte cell-specific promoter kita. Furthermore, the relevance of chronic skin inflammation in early transformation, progression and metastatic invasion of SKCM was studied using the Spint1a-deficient zebrafish model. SPINT1 is a type II transmembrane serine protease inhibitor that has been shown to be involved in the development of several types of cancer, such as squamous cell carcinoma and colorectal cancer. In addition, the Spinta-deficient zebrafish line exhibited chronic skin inflammation and areas of epidermal hyperproliferation due to the overactivation of Matriptase1, a major target of Spint1.The results reveal that Spint1a deficiency facilitates oncogenic transformation, regulates the tumor immune microenvironment crosstalk, accelerates the onset of SKCM and promotes metastatic invasion. Notably, Spint1a deficiency is required at both cell autonomous and non-autonomous levels to enhance SKCM invasiveness. These results reveal that inflammation may promote tumor cell aggresiveness, identifying a key role of TNFA/TNFR2 and SPINT1 signaling in the cross-talk between oncogenically transformed cells and the tumor microenvironment, and point to TNFA/TNFR2 and SPINT1 as novel therapeutic targets for SKCM.