Identificazione e Validazione di Hotspot di Metilazione del DNA come Biomarcatori per il Melanoma Cutaneo

Cutaneous melanoma represents the most aggressive skin cancer due to its high invasive and metastatic behavior. Despite the adoption of novel screening programs and the development of new pharmacological treatments, the incidence and mortality rates of cutaneous melanoma are constantly increasing highlighting the need for novel diagnostic and prognostic biomarkers as well as new therapeutic targets for the management of this pathology. Recently, several studies have demonstrated that the development of cutaneous melanoma is not only prompted by gene mutations affecting key oncogenes but is also associated with several epigenetic modifications responsible for the alterations of different cellular and molecular processes underlying the neoplastic transformation of melanocytes. Among the epigenetic alterations, DNA methylation represents the non-structural modification of DNA mainly involved in the alteration of the expression levels of genes potentially responsible for the loss of apoptotic processes and uncontrolled cell proliferation responsible for the onset and progression of tumors. It was also hypothesized that DNA methylation could represent an early event of neoplastic transformation and that the identification of DNA methylation hotspots could be used as a promising strategy for the early diagnosis of cutaneous melanoma. On these bases, the aim of the present study was to evaluate the overall DNA methylation status in cutaneous melanoma in order to identify DNA methylation hotspots involved in the development and progression of this tumor. Particular attention was paid to the study of DNA methylation phenomena affecting genes involved in melanocyte differentiation and epithelial to mesenchymal transition. For these purposes, a bioinformatics analysis was first performed using the EpiMethEx R-package to evaluate the correlation between DNA methylation and gene expression data contained in The Cancer Genome Atlas (TCGA) and GTEx databases thus identifying genes whose methylation status correlated positively or negatively with gene expression. Subsequently, Gene Ontology analyses were performed to establish the functional role of these genes and to identify a subgroup of transcription factors involved in embryonic development and neural crest formation as well as in the melanocytic differentiation potentially involved in the development of melanoma. Through these computational analyses, it was possible to identify a set of DNA methylation hotspots affecting dysregulated genes involved in the development and progression of cutaneous melanoma. Among these genes, DNA methylation hotspots affecting RARB and ISL1 were selected for the validation analyses performed on melanoma cell lines and melanoma FFPE samples. In particular, the expression and methylation levels of these two transcription factors were evaluated in five melanoma cell lines, A375, A2058, M14, MeWo and SK-23-MEL. The expression levels of both RARB and ISL1 were evaluated by using droplet digital PCR (ddPCR) while the methylation levels of the DNA methylation hotspots computationally identified were assessed using a custom protocol developed by the Laboratory of Experimental Oncology of the University of Catania defined Methylation Sensitive Restriction Enzyme droplet digital PCR (MSRE-ddPCR). The in vitro findings were further validated in a pilot cohort of melanoma patients through the analysis of the methylation levels of the DNA methylation hotspots of RARB and ISL1 detected in FFPE melanoma tissues and normal controls. The in vitro and clinical evaluations of RARB and ISL1 methylation levels confirmed the bioinformatic results obtained through EpiMethEx demonstrating a negative correlation between RARB promoter methylation and gene expression and a positive correlation between ISL1 intragenic methylation and gene expression. Therefore, the bioinformatics and experimental data obtained in this study demonstrate the high predictive value of the analyzes performed with the EpiMethEx tool as well as the reproducibility of the results obtained by the highly sensitive MSRE-ddPCR methylation analysis protocol here developed. Overall, the findings of this study pave the way for the development of novel strategies for the identification of diagnostic and prognostic melanoma biomarkers. The results obtained so far need to be clinically validated in a series of patients with cutaneous melanoma, healthy individuals and individuals at risk for this pathology.

Grantor dc:publisher

Università degli studi di Catania

Year dc:date

2021

Author dc:creator

• FALZONE, LUCA

Contributors dc:contributor

• LIBRA, Massimo

• Melanoma, DNA methylation, Methylation hotspots, ddPCR, Epigenetics
• Melanoma, Metilazione del DNA, Hotspot di Metilazione, ddPCR, Epigenetica

Statement dc:rights

• info:eu-repo/semantics/openAccess
• license:PUBBLICO - Pubblico con Copyright
• license uri:iris.PUB02

Language dc:language

ita

Handle dc:identifier

https://hdl.handle.net/20.500.11769/581902

OAI identifier oai:identifier

oai:www.iris.unict.it:20.500.11769/581902