TMEFF2 is a transmembrane protein in the tomoregulin family that has been demonstrated to be hypermethylated and underexpressed in various tumor types. It has been reported that in gastric cancer, TMEFF2 deregulation may play an important role in the progression of gastric carcinogenesis[21]. However, it was also proposed that in prostate cancer, the extracellular domain of TMEFF2 would shed from the cell membrane and promote cell proliferation by combining the ErbB1 receptor[22]. In the current study, we assessed the expression and promoter methylation of TMEFF2 and explored its clinical implication in adult diffuse glioma.
TMEFF2 has been reported to be highly expressed in human brain tissues[17]. In our current study, we confirmed that in comparison to healthy tissue, TMEFF2 expression is significantly decreased in glioma tumor tissues and GBM cells. In addition, from the data of TCGA GBM and LGG cohorts, we ascertained that TMEFF2 expression is progressively downregulated during the progression from Grade II to Grade IV glioma. Our data are also consistent with previous reports that TMEFF2 expression is downregulated and negatively correlated with tumor histologic grade in gastric cancer and coral cancer[18, 28].
High TMEFF2 expression levels have been reported to be associated with both growth-promoting and growth-suppressing functions in various studies in multiple cancers. In most studies, TMEFF2 has been found to suppress growth[19–21], while Nazim Ali and Vera Knauper proposed that shedding of the soluble TMEFF2 ectodomain would induce proliferation by inducing ERK1/2 phosphorylation in an ErbB1-dependent manner in prostate cancer cells[22]. In our study, cell biological function assays showed increased proliferation in TMEFF2 knockdown GBM cells, indicating that TMEFF2 acted as an inhibitor of proliferation of GBM cells. These data suggest that TMEFF2 may play important roles in suppressing the growth of adult diffuse glioma. Downregulation of TMEFF2 may be related to glioma tumor progression.
We performed bisulfite amplicon sequencing (BSAS) in GBM cells compared with SVG p12 cells and provided the first identification of hypermethylated CG sites in the TMEFF2 promoter of GBM cells. Our MSP work also illustrated TMEFF2 hypermethylation in GBM cells. Moreover, we confirmed the negative correlation between TMEFF2 methylation and mRNA expression. Taken together, low TMEFF2 expression in gliomas may be due to the methylation regulation of its promoter. It was demonstrated that histone deacetylases as well as c-Myc, STAT1 and STAT3 may contribute independently to the transcriptional suppression of TMEFF2 in colon cancer, prostate cancer and gastric cancer[29–32]. However, more detailed studies need to be undertaken to better understand the regulatory mechanism of promoter methylation and transcription of TMEFF2 in gliomas.
However, we failed to clearly clarify the expression of TMEFF2 in glioma tumor tissues or cells by Western blot (WB) using commercial antibodies (data not shown). We noticed that O-methylguanine-DNA methyltransferase (MGMT) is an important biomarker for the chemosensitivity of gliomas, and methylation detection (not IHC) is more precise for the clinical testing of MGMT in glioma patients[1, 33, 34]. Andreas Herbst et al. and Su Man Lee et al. detected methylated free-circulating DNA (ctDNA) for TMEFF2 in the blood of metastatic colorectal cancers and non-small cell lung cancer, respectively[35, 36]. It has also been reported that sequencing ctDNA in cerebrospinal fluid (CSF) can provide a landscape of the tumor genome for glioma patients and is associated with disease outcome[37]. In this study, we successfully and stably detected TMEFF2 DNA methylation levels in GBM cells and in glioma patients’ tumor tissues. Thus, methylated TMEFF2 DNA could also be a used as a detectable indicator in glioma patients’ tumor specimens or cerebrospinal fluid in the future.
Mounting evidence has confirmed that mutation of IDH1 is a hallmark of favorable patient outcomes in adult diffuse gliomas. In our previous research, we demonstrated that mutation of IDH1 occurs frequently and predicts favorable prognosis in Chinese glioma patients[38]. Mutant IDH1 in gliomas causes broad epigenetic alterations, including DNA hypermethylation, and results in a subtype of glioma with a CpG island methylator phenotype (G-CIMP)[39, 40]. In our study, we observed a small subset (27/427) of IDH1 mutant gliomas with a high degree of TMEFF2 methylation that showed a poor prognosis compared to the large subset (400/427), which had less TMEFF2 methylation. Due to tumor heterogeneity between glioma patients, TMEFF2 methylation may be a biomarker of poor prognosis in IDH1 mutant glioma patients. Genome-wide methylation of G-CIMP gliomas shifts substantially in tumor recurrence[41]. Our data have not shown whether TMEFF2 methylation is associated with malignant tumor transformation during tumor recurrence. More studies could be performed to clarify the role of TMEFF2 methylation in tumor recurrence.
Gliomas with mutant IDH and 1p/19q noncodeletion mostly harbor loss-of-function mutations in ATRX and gain of new function mutations in TP53[2]. In low-grade gliomas, this subtype is mainly composed of astrocytoma and anaplastic astrocytoma and displays a favorable prognosis. In glioblastomas, mutated IDH is present in only < 10% of all cases, but in those cases there is a better prognosis[10, 42]. The molecular profiles of glioblastoma with mutated IDH are similar to those of astrocytoma with mutated IDH, including frequent ATRX and TP53 mutations and MGMT hypermethylation. In our study, through TCGA database analysis and clinical specimen verification, we found that TMEFF2 promoter methylation is negatively correlated with mutations in IDH1, ATRX and TP53. The IDH1, ATRX and TP53 combined mutant (IDH1+/ATRX+/TP53+) samples in our glioma cohort presented lower levels of TMEFF2 promoter methylation and higher levels of TMEFF2 expression than other samples. Thus, low TMEFF2 methylation may be a new detectable molecular marker used to identify IDH1+/ATRX+/TP53 + gliomas.