RASSF2 hypermethylation is present and related to shorter survival in squamous cervical cancer

Abstract

Ras association (RalGDS/AF-6) domain family member 2 (RASSF2) is a gene involved in the progression of several human cancers, including breast, colorectal and lung cancer. The aims of this study were to determine the hypermethylation of the gene in squamous cervical cancer and precursor lesions, along with that of RASSF1 and the recently described EPB41L3, and to analyze the potential prognostic role of these genes. Methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status of RASSF2 and EPB41L3 gene in 60 squamous cervical cancer, 76 cervical intraepithelial neoplasias grade III, 16 grade II, 14 grade I and 13 cases of normal tissue adjacent to cervical intraepithelial neoplasia. RASSF2 expression was evaluated by immunohistochemistry and the re-expression of RASSF2 and EPB41L3 was analyzed by quantitative reverse-transcription PCR in HeLa, SiHa, C33A and A431 cell lines treated with 5-aza-2'-deoxycytidine and/or trichostatin. RASSF1 hypermethylation and human papillomavirus type were also analyzed in all the cases by methylation-specific PCR and reverse line blot, respectively. RASSF2 hypermethylation was predominant in squamous cervical cancer (60.9%) compared with cervical intraepithelial neoplasias (4.2%) and was associated with a lower level of RASSF2 expression and vascular invasion in squamous cervical cancer. EPB41L3 and RASSF1 hypermethylations were also more frequent in cancer than in precursor lesions. Patients with RASSF2 hypermethylation had shorter survival time, independent of tumor stage (hazard ratio: 6.0; 95% confidence interval: 1.5-24.5). Finally, the expressions of RASSF2 and EPB41L3 were restored in several cell lines treated with 5-aza-2'-deoxycytidine. Taken together, our results suggest that RASSF2 potentially functions as a new tumor-suppressor gene that is inactivated through hypermethylation in cervical cancer and is related to the bad prognosis of these patients.

Differential gene hypermethylation in genital lichen sclerosus and cancer: a comparative study

“…RASSF1A (RAS‐association domain family 1) is a potential effector of the Ras oncoprotein, which is involved in the cell cytoarchitecture, apoptosis and cell cycle pathways . RASSF2A is one of the three isoforms encoded by the RASSF2 gene, which participates in Ras signalling and is inactivated by hypermethylation in breast, lung cancer, and, as recently described by our group, squamous cervical carcinoma . The thrombospondin‐1 gene ( TSP1 ) encodes an angiogenesis inhibitor, and its silencing is associated with neovascularization in cancer .…”

RASSF tumor suppressor gene family: Biological functions and regulation

“…RASSF2 methylation was also detected in colorectal cancer (CRC), gastric cancer and oral squamous cell carcinoma (please see Table 2 for the complete list and references). In squamous cervical cancer RASSF2 hypermethylation was associated with shorter survival time [146]. Interestingly, in CRC patients the histone modification epigenetic mechanism was probably also involved in silencing of RASSF2, because treating cells with a histone deacetylase inhibitor augmented RASSF2 expression [58].…”

Epigenomic Characterization of Locally Advanced Anal Cancer

“…100 In cervical cancer, methylation of RASSF2 was associated with increased tumor vascular invasion and shorter survival time, independent of tumor stage. 100 Such differences in methylation provide biological insight into the mechanisms of carcinogenesis.…”

“…For example, differential methylation patterns have been reported in the progression of several malignancies, including prostate 93,94 bladder, 95,96 renal cell, 47 esophageal, 97 HPV-positive and HPV-negative head and neck, 98,99 and cervical cancers. 100,101 In general, these papers reported that increased DNA methylation within CpG islands was associated with increasing tumor aggressiveness 94,101 and some epigenetic events appear to be early markers of progression. 100 In cervical cancer, methylation of RASSF2 was associated with increased tumor vascular invasion and shorter survival time, independent of tumor stage.…”

“…100,101 In general, these papers reported that increased DNA methylation within CpG islands was associated with increasing tumor aggressiveness 94,101 and some epigenetic events appear to be early markers of progression. 100 In cervical cancer, methylation of RASSF2 was associated with increased tumor vascular invasion and shorter survival time, independent of tumor stage. 100 Such differences in methylation provide biological insight into the mechanisms of carcinogenesis.…”