Role of RKIP and Pirin in the malignant progression of cutaneous melanoma. New diagnosis and prognosis biomarkers

Résumé

Melanoma is an extremely lethal skin cancer that arises as a result of the malignant transformation of melanocytes. The incidence of this pathology worldwide has increased in the last 30 years in greater proportion than the rest of the cancer, between 4-6% yearly, and shows substantial disparities between populations. To ensure appropriate treatment and a successful outcome, a timely and accurate diagnosis and prognosis of malignant melanoma is essential. Because of this, molecular alterations in the pathogenesis of melanoma are the subject of more active research. In terms of histology, melanoma show a wide variety of characteristics including epithelial, hematological, mesenchymal, and neural features, which can often make the diagnosis of the disease challenging. As new biomarkers candidates, in thisthesis we examined the expression of RKIP (Raf Kinase Inhibitor Protein) and Pirin. RKIP has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancer, and Pirin originally was considered to act as a transcriptional co-factor, but it has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. However, these studies were performed with a small cohort of patients, so a larger study is required for further evaluation of this marker's diagnostic or prognostic value. In this context, this doctoral thesis¿ goals were to evaluate the potential value of RKIP and Pirin as melanoma markers and their implication on melanocytic cell biology.Regarding RKIP, immunohistochemistry analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I-II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. In relation to Pirin, the immunohistochemistry multivariate analysis revealed that early melanoma with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. On the other hand, transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis.To summarize, the results obtained in this thesis support the diagnostic utility of RKIP staining due to the significantly lower RKIP protein levels in melanoma samples, even at early stages (I¿II) of the disease, and the use of Pirin staining along with the Breslow index as a prognostic marker at early stages (I-II) of melanoma. Moreover, we propose that RKIP could play a role in the maintenance of the differentiation state by negatively regulating NANOG gene expression and, that Pirin could play an important role in modulating the proliferative state of melanoma cells by regulating JARID1B gene expression.