DNMT1–MicroRNA126 Epigenetic Circuit Contributes to Esophageal Squamous Cell Carcinoma Growth via ADAM9–EGFR–AKT Signaling

Abstract

Purpose: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)-based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)-specific miRNAs and miRNArelated epigenetic modulations.Experimental Design: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo.Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor-AKT signaling.Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel "DNMT1-miR-126 epigenetic circuit" is involved in ESCC progression. Consequently, miR-126-based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics.

Overexpression of ADAM9 in oral squamous cell carcinoma

“…Though particularly in squamous cell carcinoma, ADAM9 protein overexpression is also present in cancer of the cervix, the esophagus, the pharynx, the larynx, and the skin around the head and neck region (9,21,25,26). However, few previous studies have demonstrated no significant difference in ADAM9 mRNA expression between OSCC and normal tissues, or between oral cancer cell lines and normal oral keratinocytes (9,28), a finding that differs from our results on ADAM9 protein overexpression.…”

“…In this study, a significant increase in ADAM9 protein expression in OSCC tissues, compared with in normal oral tissues, was demonstrated by immunohistochemistry, a finding that agrees with the results of other prior studies, which demonstrated ADAM9 protein overexpression in various types of cancer (9,15,17,18,(20)(21)(22)(23)25,26). Though particularly in squamous cell carcinoma, ADAM9 protein overexpression is also present in cancer of the cervix, the esophagus, the pharynx, the larynx, and the skin around the head and neck region (9,21,25,26).…”

“…Then, the average percentage of positive cells in each section was determined. Finally, the average percentage of positive cells was multiplied by the staining intensity score, and the IHC score (0-3) was obtained for each section (25).…”

“…Therefore, ADAM9 can function in both physiological and pathological conditions. Regarding its involvement in pathologic conditions, ADAM9 overexpression has been identified in a variety of cancer types, including breast cancer (16), renal cancer (17), prostate cancer (18), skin melanoma (19), uterine cervical cancer (20,21), hepatocellular carcinoma (22), non-small cell lung cancer (23), colon cancer (24), gastric cancer (15), esophageal cancer (25) and head and neck squamous cell carcinoma (HNSCC) (26). In the oral cavity, conflicting results of chromosomal aberrations in the ADAM9-containing region have been identified by the array comparative genomic hybridization technique (9,27).…”

MicroRNA-126 is Dysregulated in Cancer- Free Females Harboring Methylated BRCA1 Promoter through Up-Regulation of DNMTs

“…However, the lack of this correlation in the breast cancer patients also suggests that during the carcinogenesis process miR-126 is post-transcriptionally regulated. Indeed, it has been previously shown that pri-miRNA and mature expression is positively correlated in normal tissue but not in breast tumors [36]. These results suggested a multistep model for miRNA dysregulation.…”