Our previous report identified two hypermethylation markers, SESN3
, but their methylation status did not predict overall or disease-free survival within endometrioid endometrial cancers [20
]. In this study, we found three additional cancer-specific methylation markers, CIDEA, HAAO
, through an evaluation of promoter microarrays containing ~27,800 CpG islands. Both COBRA and MassARRAY assays confirmed that hypermethylation of all three loci was frequent (≥63%) in endometrial carcinomas but was infrequent in normal tissues.
CIDEA is a member of the cell death-inducing DFF45-like effector (CIDE) family. DFF45 is a subunit of the DNA fragmentation factor which is cleaved by active caspase-3 during apoptosis. CIDEA was found to induce apoptosis in mammalian cells, which was inhibited by DFF45 [32
]. CIDEA also plays important roles in energy homeostasis. In an animal model, the absence of CIDEA expression may result in lean phenotypes, insulin resistance, and resistance to diet-induced obesity in mice [33
]. HAAO, known as 3-Hydroxyanthranilate-3,4-dioxygenase, is an enzyme to catalyze the biosynthetic pathway from tryptophan to quinolonate. While its function in cancer development is still unclear, we demonstrate that HAAO
is hypermethylated in ovarian cancers with high sensitivity and specificity as a biomarker [29
]. RXFP3, formerly called GPCR135 or SALPR, belongs to the relaxin family peptide receptors and can be activated by relaxin-3, a member of the insulin superfamily [34
]. Upon ligand stimulation, RXFP3 activates extracellular signal-regulated kinase signaling via various pathways including protein kinase C [34
]. The function of RXFP3 related to cancer is also unknown. Our findings have shown by RT-qPCR that RXFP3
is expressed in normal endometrium and a small subset of endometrial cancers (see ). The loss of RXFP3
expression in tumors is inversely associated with its promoter hypermethylation.
In this study we found that the hypermethylation of CIDEA
is associated with MSI+ phenotype. This observation is consistent with previous report that endometrial carcinomas with MSI+ had significantly more epigenetic alterations than MSI- tumors [35
]. Promoter hypermethylation of MLH1
contributes frequently to MSI in sporadic endometrial carcinomas [12
]. Our previous report showed that MSI+ tumors without MLH1
methylation were associated with younger age while the combined MSI/MLH1
methylation status did not predict OS or DFS [15
]. In the current study, the methylation status of HAAO
may predict DFS in patients with endometrial cancer. To definitively prove its clinical significance in predicting patient survivals, methylation analysis of a large cohort, such as the GOG-210 trial, is needed to validate this finding. Methylation markers identified in current study and others [5
] could be specific and informative for endometrial cancer only. Future development of endometrial cancer methylator phenotypes could hold great promise to improve risk assessment, diagnosis, and prognosis.
In conclusion, our findings provide three novel and cancer-specific hypermethylation markers. The methylation levels of all three loci are correlated with MSI status. Although MSI could be either a cause or a consequence of DNA methylation, high-throughput studies can be developed to establish the relationship between MSI status and DNA hypermethylation. This type of omics study may find that loss of DNA mismatch repair together with epigenetically mediated silencing of these genes can be common alterations that contribute to tumorigenesis. As such, the combined epigenetic and genetic alternations can be feasible options for predicting survival rates in cancer patients.