Promoter methylation status of key genes and its implications in the pathogenesis of endometriosis, endometrioid carcinoma of ovary and endometrioid endometrial cancer

Epigenetics refers to the stable inheritance of the phenotype of organisms or cells without changing the underlying DNA sequences which includes nuclear processes such as DNA methylation, histone modifications, and transcriptional networks.[] Epigenetic processes are involved in the regulation of various physiological processes as well as the pathogenesis of various diseases.[] DNA methylation is an important epigenetic phenomenon in which a methyl group is added at the cytosine 5' of guanine or CpG sites which are generally present in the promoter region of a gene. In general, promoter hypomethylation is associated with over-expression and promoter hypermethylation is associated with the silencing of that particular gene.[] Endometriosis is a commonly occurring, estrogen-dependent gynecological disease in which the ectopic endometrium develops outside the uterine cavity and displays symptoms such as dysmenorrhea, dyspareunia, pelvic pain, and infertility.[] Endometriosis shares many similarities with malignancies, such as metastasizing tendency, invasive, and estrogen-dependent growth with chances of reappearance. It may lead to specific histological subtypes of epithelial ovarian cancer such as endometrioid and clear cell carcinoma, also known as endometriosis-associated ovarian carcinoma (EAOC), which differs from other histological subtypes of ovarian cancer.[] Few studies also identified the possible risk of developing endometrioid endometrial cancer in patients having endometriosis.[] Therefore, it can be speculated that endometriosis increases the risk of developing endometrioid subtypes of both ovarian and endometrial cancers as all three diseases share a common molecular mechanism of etiopathogenesis such as estrogen dependence and overproduction of COX2, prostaglandins, cytokines, and chemokines.[] According to various previous reports, it has been found that DNA methylation plays an important role in the pathogenesis of endometriosis,[] endometrioid carcinoma of ovary,[] and endometrioid endometrial cancer.[] But, there is a dearth of sufficient research on promoter methylation of various genes involved in the common molecular pathogenesis of these three diseases. Several key processes such as inflammation, angiogenesis, hypoxia, reduced apoptosis, and cell proliferation are common in the pathogenesis of these three diseases. The key genes regulating these processes include COX2, VEGF, TNF, HIF1A, MYC, and TP53 which along with several other factors regulate the molecular mechanisms involved in the etiopathogenesis of these three associated diseases.[] Based on these considerations, we designed the present study to check epigenetic regulation, especially the methylation status of genes regulating common key processes in the pathogenesis of endometriosis, endometrioid carcinoma of the ovary, and endometrioid endometrial cancer, which could help to delineate the common underlying pathway in these different related disorders and could provide better interventions and therapeutic modalities.

Sample collection and ethical clearance After obtaining ethical clearance from the “Institute Ethics Committee” from the study institutions, 40 subjects were enrolled in the study. Group I comprised subjects with endometriosis (n = 10), group II comprised subjects with endometrioid carcinoma of ovary (n = 10), group III comprised subjects with endometrioid endometrial cancer (n = 10), and the control group comprised normal endometrium from patients who underwent hysterectomy for benign pathology only after obtaining histopathological confirmation for respective diseases/controls. After taking written informed consent from all the participants, tissue samples were collected in two separate vials containing PBS (Phosphate saline Buffer, pH-7.4) and RNA later (Cat # 76104, Qiagen India Private Limited, New Delhi) at the time of laparoscopy/laparotomy/hysterectomy and stored at -80°C till further experiments. Genomic DNA extraction Genomic DNA was extracted from the tissue samples of all study subjects using a spin column method with a DNeasy Blood and Tissue kit (Cat # 69504, Qiagen India Private Limited, New Delhi) according to the manufacturer's instructions. The quality and quantity of the extracted DNA were determined using agarose gel electrophoresis and a Nanodrop Spectrophotometer. The extracted DNA was stored at -20°C until further use. Methylation-Specific Polymerase Chain reaction (MS-PCR) Extracted genomic DNA was subjected to bisulfite conversion using a commercially available EZ DNA Methylation-Gold™ Kit (Cat # D5006, Zymo Research, USA) according to the manufacturer's instructions. In short, bisulfite treatment converts all the unmethylated cytosines to uracil but the methylated cytosines remain unchanged. Primer sequences for the studied genes, that is, COX2, VEGF, HIF1A, TNF, MYC, and TP53, specific for the promoter region containing CpG islands were designed using the Meth Primer software[] [Table 1]. Next, methylation-specific PCR was performed in a total volume of 20 μl in a PCR tube. Each polymerase reaction tube comprises 200 ng of bisulfite converted DNA, 100 pmol of each PCR primer (Eurofins, Pvt. Ltd. India), 10X PCR buffer, 50 mM MgCl2, 100 mM of each dNTP, 2 units of Taq DNA polymerase, and nuclease-free water. Thermal cycling conditions for every gene included: 95°C/5 min initial denaturation, followed by 35 cycles at 95°C/30 s, 45 s of annealing at their respective annealing temperatures [Table 1], 72°C/45 s, and a final extension at 72°C for 5 min. Universal unmethylated and methylated DNA (Qiagen) served as control. About 2% (w/v) agarose gel which is stained with ethidium bromide was used to analyze the results of polymerase chain reaction products and then visualized by ultraviolet light in a gel documentation system. Also, we amplified 20% of the samples repeatedly to check the validity of the results. RNA isolation and cDNA synthesis RNA was isolated from the tissue samples by using commercially available RNeasy® Mini Kit (Cat # 74104, Qiagen India Pvt. Ltd., New Delhi, India) according to the manufacturer's instructions. The quality and quantity of extracted RNA were analyzed by Nanodrop spectrophotometer and denaturing agarose gel electrophoresis. Total 1 μg/μl of RNA was reverse transcribed by Revert Aid First-strand cDNA synthesis kit (Cat # K1622, Thermo Scientific) for cDNA synthesis as per manufacturer's instructions. Quantitative RT-PCR to check mRNA levels of studied genes The mRNA levels of MYC and TP53 genes were analyzed by quantitative reverse transcriptase real-time PCR (Light Cycler LC96 Real-Time PCR system, Roche applied science, USA) by using SYBR Green I chemistry and analyzed by Livak's analysis. GAPDH and β-actin housekeeping genes were used as invariant controls. PCR was performed in a 10-μl PCR reaction mixture tube which included the following: 1 μl of cDNA, 5 μl SYBR Green Master Mix (2X), 1 μl of each forward and reverse primer, and 2 μl of nuclease-free water. The thermal cycling conditions for qRT-PCR included 5 min of initial denaturation at 95°C followed by 40 cycles of 94°C for 15 s, 57°C to 60°C for 30 s, and 70°C for 30 s. CT values were obtained followed by melting peak analysis. Primers used for MYC, TP53, β-actin, and GAPDH genes and their annealing temperatures are represented in Table 2. Statistical analysis All the statistical analysis was performed by statistical software IBM SPSS Statistical Package 22.0 (IBM, Armonk, New York, US). Data were represented as mean ± standard deviation. The normality of the data was determined by the Kolmogorov–Smirnov test. Differences in the promoter methylation frequencies of all studied groups were analyzed by the Chi-square (χ2) test. The mRNA levels of the studied genes between the control group and three disease groups as well as between methylated and unmethylated groups were compared using the Kruskal-Wallis's test and within groups by Mann–Whitney test as appropriate. A difference of P < 0.05 was considered as statistically significant.

Promoter methylation status of COX2, VEGF, HIF1A, TNF, MYC, and TP53 genes in all study groups Promoter methylation frequency of COX2, VEGF, TNF, and HIF1A genes were significantly decreased in endometriosis, endometrioid carcinoma of the ovary, and endometrial cancer study groups as compared to the control study group (P < 0.05) as per the results of methylation specific-PCR. Whereas, no statistically significant difference was observed in promoter methylation frequencies of MYC and TP53 genes in all the diseased study groups as compared to the control study group [Table 3]. Transcriptional expression of study genes in all the study subjects In the present study, mRNA levels of the MYC gene were found to be significantly increased in all diseased study subjects compared to control study subjects (P < 0.001) [Figure 1]. However, mRNA levels of TP53 genes were significantly decreased in endometriosis and endometrioid carcinoma of ovary study subjects as compared to control study subjects (P < 0.001) and significantly increased in endometrial cancer study subjects as compared to control subjects (P < 0.001) [Figure 1]. Also, mRNA levels of COX2, VEGF, TNF, and HIF1A genes were found significantly increased in endometriosis, endometrioid carcinoma of the ovary, and endometrial cancer as compared to control study subjects (P < 0.001) [http://links.lww.com/JCRT/A48] in our previous report.[] The effect of promoter methylation on the transcriptional expression of studied genes In the present study, transcriptional expressions of MYC and TP53 genes were also compared between methylated and unmethylated tissue samples of all the study groups. For the MYC gene, no significant change was observed in the promoter methylation frequency of the control group and diseased groups, however, the mRNA levels of the MYC gene were significantly increased in unmethylated tissue samples of all the diseased study groups compared to the control group (P < 0.05). Whereas, no significant difference was observed in mRNA levels of the TP53 gene between methylated and unmethylated samples of each study group. In our previous report, we observed mRNA levels of COX 2, VEGF, HIF1A, and TNF genes in all the study subjects.[] Here, in the present study, we compared the mRNA levels of the above-mentioned genes between methylated and unmethylated tissue samples of all the study groups. The results revealed that COX2 mRNA levels were significantly increased in unmethylated tissue samples of endometrioid carcinoma of ovary study subjects as compared to methylated tissue samples of the same disease tissue samples (P < 0.05). Similarly, the VEGF mRNA levels were significantly increased in unmethylated tissue samples of endometriosis and endometrioid carcinoma of ovary study subjects as compared to methylated tissue samples of the respective diseased samples (P < 0.05). Further, the transcriptional expressions of the HIF1A gene were found to be significantly increased in unmethylated tissue samples of endometrioid carcinoma of the ovary and endometrial cancer study subjects as compared to methylated tissue samples of the respective diseased study subjects (P < 0.05). However, no significant difference was observed between the unmethylated and methylated tissue samples of other study groups [Figure 2].

DNA methylation is a promising and continuously evolving area of research in epigenetics. Since it is restricted to a limited region of DNA (CpG islands), it is more stable, sensitive, and specific than protein or DNA mutations and it holds a biomarker potential. The profiling of promoter methylation of a panel of genes can provide new insight into their use as early diagnosis, risk evaluation, prognosis, and personalized treatment. The science of DNA methylation has picked up steam in the past decade as its implications directly address the diseases of the surgical oncology realm. It is a known fact that cancers are characterized by the inappropriate activation of oncogenes caused due to global hypomethylation, while localized hypermethylation of CpGs at the promoter region of tumor suppressor genes prevents their activation. Currently, cancer-related studies are creating a buzz in the scientific field but methylation-based biomarkers can also be utilized in other fields such as neurodegenerative and psychiatric disorders including Alzheimer's, Schizophrenia, and so on. In our study, it was seen that the hypomethylation of promoter regions of pro-inflammatory and pro-angiogenic genes leading to their enhanced gene expression is shared by all three diseases. Information about how cancer originates from a non-malignant form through molecular events could enable a clinician to predict its medical outcome more accurately. The present study observed the promoter methylation frequencies of the key genes such as COX2, VEGF, HIF1A, TNF, MYC, and TP53 involved in the common molecular pathophysiology of endometriosis, endometrioid carcinoma of the ovary, and endometrioid endometrial cancer were analyzed by methylation specific-PCR. Also, the effect of promoter methylation on transcriptional expression of the above-mentioned genes was compared in all the study groups. In the present study, the COX2 gene promoter region was found to be hypomethylated in all three diseased study subjects, that is, endometriosis (20%), endometrioid carcinoma of the ovary (30%), and endometrioid endometrial cancer (30%) compared to the control study (80%) subjects (P < 0.05). Previous studies have identified the association between increased levels of COX2 with DNA hypomethylation of NFIL-6 site (Nuclear factor for interleukin-6) within the promoter of this gene in endometriosis,[] which were further corroborated by the observations of the present study. Thus, overexpression of the COX2 gene associated with endometriosis could be directly associated with the work of Zhang et al.,[] which is in accordance with our present study results. In ovarian cancer, as per literature, no study has been found on the promoter methylation status of COX2 gene. Ours is the first study to report decreased promoter methylation frequencies in endometrioid carcinoma of ovary study subjects (P < 0.05) compared to control subjects. Also, the mRNA levels of the COX2 gene were significantly increased in unmethylated samples (P < 0.05) compared to methylated samples of endometrioid carcinoma of the ovary which indicates that COX2 promoter hypomethylation could be responsible for the over-expression of COX2 gene in these study subjects as observed in case of endometriosis. Thus, the present study results provide an important lead to the fact that promoter hypomethylation of the COX2 gene is one of the major reasons for its elevated transcriptional levels in the pathogenesis of endometriosis and associated ovarian and endometrial cancers. The potential role of VEGF promoter methylation status has been linked with increased VEGF expression levels in many diseases such as retinal disorders[] as well as reproductive system disorders, such as preeclampsia.[] As per our present results, a significant difference was observed in the mRNA levels of the VEGF gene in unmethylated samples of endometriosis and endometrioid carcinoma of the ovary as compared to methylated samples of the same diseases (P < 0.05). Since, the promoter region of the VEGF gene displayed hypomethylation as per our observations in endometriosis (30%), endometrioid carcinoma of the ovary (20%), and endometrioid endometrial cancer (20%), it can be speculated that promoter hypomethylation could be one of the governing factors for over-expression of VEGF gene in all these three diseases, and molecular mechanism for the same can be explored to establish it as a potential therapeutic target. Further, present study observed that tumor necrosis factor-alpha (TNF) gene promoter methylation frequencies were significantly decreased from 80% in control subjects to 20% in subjects with endometriosis (P < 0.05), 10% in endometrioid carcinoma of ovary study subjects (P < 0.05), and 30% in endometrioid endometrial cancer study subjects (P < 0.05). Promoter hypomethylation has a significant effect on the transcriptional expression of a gene and TNF promoter hypomethylation was found to be an important player in the over-expression of TNF in many diseases such as Alzheimer's disease, chronic periodontitis, and rheumatoid arthritis.[] However, in endometriosis and associated endometrioid subtypes of ovarian and endometrial cancer, the role of TNF promoter methylation status has not been explored much. Since, endometriosis is considered an inflammatory disease and elevated levels of cytokines were associated with its pathogenesis. Therefore, epigenetic regulation of one of the main inflammatory cytokine genes points toward the imperial role of promoter hypomethylation in elevated expression levels of the TNF gene in these three associated diseases. More directed research toward this field may help in finding better therapeutic targets focusing on epigenetic regulation of inflammatory cytokine genes in the treatment of these three debilitating diseases. As per our present study observations, promoter methylation frequencies of the HIF1A gene were found to be significantly downregulated from 90% in control subjects to 20% in subjects with endometriosis (P < 0.05), 30% in subjects with endometrioid carcinoma of the ovary (P < 0.05) and 20% in endometrioid endometrial cancer study subject. HIF1A mRNA levels were usually increased in many types of cancers and control the expression of several genes that have been associated with the growth and development of tumors.[] According to the studies of Kim et al.,[] tumor-associated CpG demethylation aids positive autoregulation of the HIF1A gene. The promoter region of the HIF1A gene has a hypoxia response element which is normally repressed by methylation of a CpG dinucleotide located in the core element. They also found that aberrant demethylation of the core element results in the binding of HIF1A to its promoter and results in auto-activation of HIF1A gene expression. In line with previous studies, our results also confirm hypomethylation in the promoter region of HIF1A in endometriosis as well as endometrioid carcinoma of the ovary and endometrioid endometrial cancer study subjects. Hence, the present observations corroborate the fact that promoter hypomethylation of the HIF1A gene results in positive regulation of transcriptional over-expression of the HIF1A gene. Also, in the present study, no significant difference was observed in promoter methylation frequencies of MYC and TP53 gene in all three diseased study subjects in comparison to control subjects in this present study. Whereas, the mRNA levels of MYC gene were observed significantly increased in all three diseased study subjects compared to control subjects (P < 0.001), and mRNA levels of TP53 gene were significantly decreased in endometriosis and endometrioid carcinoma of ovary study subjects (P < 0.001) and significantly increased in endometrioid endometrial cancer study subjects (P < 0.001) compared to control subjects as per our present study results. This might be because MYC is a well-known oncogene overexpressed in many types of cancers as well as endometriosis. Numbers of mechanisms are involved in MYC gene expression involving many transcriptional regulatory motifs in the promoter region.[] Similarly, TP53 mutations are involved in 40% to 80% of tumors[] and our present study also observed significantly altered expression levels of the TP53 gene in all three diseased study subjects compared to control subjects. Thus, we can say that DNA methylation at the promoter has less effect on MYC and TP53 gene regulation in all of these three associated diseases. Moreover, DNA methylation of TP53 does not seem convincing because of its involvement in cell cycle control and DNA repair in addition to its role as a tumor suppressor gene.

In conclusion, we can assert that epigenetic changes such as aberrant DNA methylation levels at the promoter region play a crucial role in the regulation of genes such as COX2, VEGF, TNF, and HIF1A regulating key processes in the pathogenesis of endometriosis, endometrioid carcinoma of the ovary, and endometrioid endometrial cancer. The continuum of genomic alterations connecting the endometriosis to endometrioid carcinoma of ovary/endometrium needs to be explored at full length, likewise the recommendations of the Alliance for the Human Epigenome and Disease (AHEAD) project and the International Human Epigenome Consortium (IHEC). Concludingly, evaluation of genome-wide methylation in patients may help create massive sets of data which may boost the early disease prognosis and eventually aid in deciphering better treatment modalities. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship DST-SERB (ECR/2016/000359), UGC JRF (22/06/2014(i) EU-V) New Delhi, INDIA and UGC CAS-II (F.4-28/2015(CAS-II) (SAP-II)) New Delhi, INDIA. Conflicts of interest There are no conflicts of interest. Acknowledgments Mr. Puneet Bhardwaj, Ph.D. Scholar, Department of Zoology Panjab University for support in sample collection.

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