The role of POLE4 in melanoma progression

• Grant number: 24/03987-5
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: September 01, 2024
• End date: August 31, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Silvya Stuchi Maria-Engler
• Grantee: Adriana Hiromi Uehara
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Melanoma is the most lethal type of skin cancer, characterized by its high metastatic potential. The MAPK pathway, frequently activated by mutations in BRAF(V600E), plays a central role in its pathogenesis. Despite significant progress with BRAF inhibitors and immunotherapies, treatment resistance amplified by intra-tumoral heterogeneity underscores the urgent need for new biomarkers and therapeutic targets. POLE4 is part of the DNA polymerase µ complex and emerges as a promising candidate due to its function, along with POLE3, in promoting DNA supercoiling during replication. POLE4 high expression has been reported to have a correlation with adverse prognosis in metastatic melanomas. To investigate the function of POLE4 in melanoma, we will follow a multifaceted approach, combining in-vitro methodologies with bioinformatic analyses. A melanoma cell line panel will be analysed for POLE4 expression using Real Time quantitative PCR (RT-qPCR) and Western Blotting to evaluate its expression in critical cellular phenotypes such as proliferative, invasive, and treatment resistant phenotypes. Functional assays, including cell proliferation assays (MTT assay, Trypan Blue exclusion growth curve), migration and invasion assays (wound healing and transwell assays), and drug resistance evaluation (Trypan Blue exclusion survival curve, clonogenic assay), will be conducted to explore the effects of POLE4 gene silencing using techniques such as siRNA or CRISPR-Cas9. POLE4 expression will also be evaluated in different melanoma subtypes, e.g. acral melanoma, through immunohistochemistry (IHC) and RT-qPCR in patient tissue samples, aiming to identify variations. Computational analyses will be conducted for assessing POLE4 and POLE3 gene expression in different datasets containing information about various stages of melanoma progression available in public databases. Along with POLE4, co-expressed genes will be evaluated to further understand underlying biological processes linked to gene cluster alterations and potential interaction networks. Furthermore, responsiveness to specific therapies in patients with different levels of POLE4 expression will be evaluated, focusing on immunotherapy and BRAF inhibitors. This project aims not only to establish POLE4 as a prognostic biomarker but also to uncover new therapeutic opportunities that may overcome current treatment limitations, benefiting a wide range of patients from those with cutaneous melanoma to those with less common subtypes, such as acral melanoma.