Role of DNA methylation in the regulation of SLC22A17 in cutaneous melanoma. Identification of SLC22A17 methylation hotspot as potential epigenetic biomarker

Authors: Alessandro Lavoro; Luca Falzone; Giuseppe Gattuso; Giuseppe Nicolò Conti; Rosario Caltabiano; Gabriele Madonna; Mariaelena Capone; James Andrew McCubrey; Paolo Antonio Ascierto; Massimo Libra; Saverio CandidoCorresponding author: Dr Massimo Libra

Abstract 

Cancer onset and progression are driven by genetic and epigenetic alterations leading to oncogene activation and silencing of tumor suppressor genes. Among epigenetic mechanisms, DNA methylation (methDNA) has attracted growing interest in cancer research, playing a critical role in the regulation of gene expression. As widely reported, promoter hypomethylation is strictly associated with tumor-related gene activation. Moreover, the intragenic methDNA is involved in transcriptional elongation, alternative spicing, and activation of cryptic start sites. However, the exact role of intragenic methDNA has not been completely elucidated yet. In this context, several tumor microenvironment genes are modulated by methDNA, including the Solute Carrier Family 22 Member 17 (SLC22A17), which is involved along with the Gelatinase-Associated Lipocalin (NGAL) ligand in iron trafficking and extracellular matrix remodeling.

On these bases, this study aimed to investigate the role of methDNA in the regulation of SLC22A17 in cutaneous melanoma (CM). To this purpose, correlation and differential analyses between SLC22A17 expression and methDNA levels in CM patients and nevi samples were performed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Functional studies on melanoma cell lines treated with 5-Azacytidine (5-Aza) were conducted to assess the correlation between methDNA and SLC22A17 expression. Validation study was finally performed analyzing the in silico identified SLC22A17 methDNA hotspot in Formalin-Fixed Paraffin-Embedded (FFPE) samples from a consecutive cohort of CM patients and healthy controls to test its potential application as a diagnostic and prognostic biomarker. The FFPE tissues were collected at the Melanoma Cancer Immunotherapy and Innovative Therapy Unit of the National Cancer Institute ‘Fondazione ‘G. Pascale’ (Naples, Italy), retrospectively retrieving the FFPE samples from the archives of the Pathology Unit (years 2008 – 2020) . The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the National Cancer Institute “Fondazione G. Pascale” (protocol n. 33/17 and 37/22 oss). 

The computational analyses revealed that SLC22A17 was significantly downregulated in CM and its expression levels were strictly related to promoter hypomethylation and body hypermethylation. Moreover, SLC22A17 overexpression, as well as hypermethylation of 2 intragenic methDNA hotspots, were associated with a better clinical outcome in CM patients. The correlation between SLC22A17 methDNA and expression was confirmed in 5-Aza treated cell lines. In agreement with in silico analyses, the selected SLC22A17 hotspot showed higher methDNA levels in CM samples compared to nevi. In addition, the methDNA levels of this hotspot were positively correlated with advanced CM.

Overall, the SLC22A17 methDNA hotspot could represent a promising biomarker for CM, highlighting the regulatory role of methDNA on SLC22A17 expression. In conclusion, these results pave the way to the identification of novel epigenetic biomarkers and therapeutic targets for the management of CM patients.