Pre-Operative Evaluation of DNA Methylation Profile in Oral Squamous Cell Carcinoma Can Predict Tumor Aggressive Potential

Abstract

Background: Prognosis of oral squamous cell carcinoma (OSCC) is difficult to exactly assess on pre-operative biopsies. Since OSCC DNA methylation profile has proved to be a useful pre-operative diagnostic tool, the aim of the present study was to evaluate the prognostic impact of DNA methylation profile to discriminate OSCC with high and low aggressive potential. Methods: 36 OSCC cases underwent neoplastic cells collection by gentle brushing of the lesion, before performing a pre-operative biopsy. The CpG islands methylation status of 13 gene (ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MiR193, LINC00599, MiR296, TERT, GP1BB) was studied by bisulfite Next Generation Sequencing (NGS). A Cox proportional hazards model via likelihood-based component-wise boosting was used to evaluate the prognostic power of the CpG sites. Results: The boosting estimation identified five CpGs with prognostic significance: EPHX3-24, EPHX3-26, ITGA4-3, ITGA4-4, and MiR193-3. The combination of significant CpGs provided promising results for adverse events prediction (Brier score = 0.080, C-index = 0.802 and AUC = 0.850). ITGA4 had a strong prognostic power in patients with early OSCC. Conclusions: These data confirm that the study of methylation profile provides new insights into the molecular mechanisms of OSCC and can allow a better OSCC prognostic stratification even before surgery.

Detection of oncometabolites 1-methylnicotinamide, nicotine imine and N-Methylnicotinium in nails of oral cancer patients and prediction of them as modulators of DNMT1

“…There are limited attempts that show the connection between metabolic and epigenetic landscape including the use of SAM to drive the need of NNMT enzyme that leads to the less availability of SAM for methylation activity of DNMT1 upon the DNA (Wu et al, 2008; Ulanovskaya et al, 2013; Kraus et al, 2014; Cui et al, 2020). Data support that inhibition of DNMT1 is linked with the global hypomethylation and associated with the pro-tumor transcriptional state in cancer cells (Feinberg and Vogelstein, 1983; Christmane et al., 2002; Lopez-Serra and Esteller, 2008; Palii et al, 2008; Gissi et al, 2020). In spite of knowledge on the existence of intracellular signaling axis that links the use of SAM, activation of NNMT and DNA hypomethylation, molecular and clinical relevance of NNMT-induced abundance of oncometabolite 1-MNA in oral cancer is completely lacking.…”

“…In these cells, intracellular metabolic adaptations including activation of NNMT, methylation of nicotine are linked with the consumption of methyl group from SAM to methylate nicotinamide and nicotine derived products. Due to these changes, cancer cells achieve altered epigenetic state as global hypomethylation to promote expression of pro-tumorigenic genes (Feinberg and Vogelstein, 1983; Christmane et al., 2002; Lopez-Serra and Esteller, 2008; Palii et al, 2008; Wu et al, 2008; Ulanovskaya et al, 2013; Kraus et al, 2014; Cui et al, 2020; Gissi et al, 2020). However, there is a significant gap on the suitability and availability of the nicotine metabolites and 1-MNA that influence the global DNA methylation and in turn pro-tumor microenvironment.…”

“…Upon performing molecular docking and visualization using Discovery Studio Viewer, it is revealed that nicotine imine interacts with the key amino acid residues namely ARG690, PRO574, VAL658, PRO692 and ALA695 of DNMT1. Existing data support that various known DNMT1 inhibitors including 5’-Aza-2’-deoxycytidine also bind to the similar regulatory domain spanning from 550 to 700 amino acid residues that actually influence the methylation activity of DNMT1 (Feinberg and Vogelstein, 1983; Christmane et al., 2002; Lopez-Serra and Esteller, 2008; Palii et al, 2008; Gissi et al, 2020). A comparison of molecular interactions and similar amino acid residues between nicotine imine and 5’-Aza-2’-deoxycytidine clearly convinces the possibility that nicotine imine is capable of inhibiting the activity of DNMT1.…”

“…Among plethora of detoxifying systems, there are reports on the overexpression of NNMT in OSCC and this cytoplasmic methyltransferase family of protein is known to consume SAM to produce 1-MNA from nicotinamide (Feinberg and Vogelstein, 1983; Christmane et al., 2002; Lopez-Serra and Esteller, 2008; Palii et al, 2008; Wu et al, 2008; Ulanovskaya et al, 2013; Kraus et al, 2014; Cui et al, 2020; Gissi et al, 2020). Interestingly, the utilization of SAM by NNMT is suggested to lead to the DNA hypomethylation as a pro-tumor epigenetic landscape.…”

“…In fact, metabolic reprogramming is different in cancer cells that lead to pro-tumor intracellular landscape. Among various metabolic adaptations in cancer cells, involvement of various classes of methyltransferases including cytoplasmic nicotine methyltransferase (NNMT) and nuclear methyltransferases that include DNA methyltransferase and histone methyltransferase is imperative (Feinberg and Vogelstein, 1983; Christmane et al., 2002; Lopez-Serra and Esteller, 2008; Palii et al, 2008; Wu et al, 2008; Ulanovskaya et al, 2013; Kraus et al, 2014; Cui et al, 2020; Gissi et al, 2020). There are limited attempts that show the connection between metabolic and epigenetic landscape including the use of SAM to drive the need of NNMT enzyme that leads to the less availability of SAM for methylation activity of DNMT1 upon the DNA (Wu et al, 2008; Ulanovskaya et al, 2013; Kraus et al, 2014; Cui et al, 2020).…”