Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression

Originally considered to act as a transcriptional co‑factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow‑up. 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. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow‑cycling JARID1B gene.This project was supported by grants from the Basque Government (KK2017-041 and KK2020-00069 to M.D.B.), the UPV/EHU (GIU17/066 to M.D.B.), H2020-ESCEL JTI (15/01 to M.D.B.) and MINECO (PCIN-2015-241 to M.D.B.). CP holds a predoctoral fellowship from the Basque Government. Part of this project is under European patent No. EP3051291 (EP14796149.4): “Method for diagnosis and prognosis of cutaneous melanoma”, Univer- sity of the Basque Country (UPV/EHU). The authors acknowledge the technical support SGIker resources at the UPV/EHU for the computational calculations, which were carried out in the Arina Informatics Cluster. The authors are grateful to the Basque Biobank for providing the biopsy samples and in particular, to María Jesús Fernández and Arantza Perez Dobaran for their technical support with the immunohistochemistry

Role of Rkip and pirin in the malignant progression of cutaneous melanoma. New diagnosis and prognosis biomarkers.

192 p.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