Supplementary Figure S1. Test of the methylation status of candidate genes used pooled DNA from endometrial tissues and cervical scrapings. Supplementary Figure S2. Verification of 14 candidate genes in the pilot set of cervical scrapings. Supplementary Table S1: Demographics relating to the two data sets of genome-wide DNA methylation. Supplementary Table S2. Demographics related to the clinical samples. Supplementary Table S3: Summary the area under the receiver-operating characteristic curve for distinction between endometrial cancer and noncarcinoma samples in pilot testing. Supplementary Table S4: Summary of cophenetic correlation results for analysis of consensus clustering of DNA methylation patterns. Supplementary Table S5: List of 180 hypermethylated genes in four clusters. Supplementary Table S6. Summary of the methylation levels of four candidate genes in cervical scrapings from endometrial cancer and noncarcinoma samples.

PDF file - 1049K, Supplementary Figure S1. Summarized sequencing reads and coverage of MBDCap-seq Supplementary Figure S2. Reproducibility test of MBDCap-seq Supplementary Figure S3. Validation of MBDCap-seq by MassARRAY analysis Supplementary Figure S4. Distribution of differentially methylated regions (DMRs) in endometrial tumors relative to normal control Supplementary Figure S5. Network analysis of differential methylation loci by using Steiner-tree analysis Supplementary Figure S6. DNA methylation distribution in early and late stage of the TCGA endometrial cancer cohort Supplementary Figure S7. Relative expression of BMP genes with EGF treatment in the presence or absence of PI3K/AKT and Raf (MAPK) inhibitors in endometrial cancer cells Supplementary Figure S8. Induction of invasion by EGF in AN3CA and HEC1A cell lines Supplementary Figure S9. Knockdown expression of BMP4 and BMP7 in RL95-2 cells Supplementary Figure S10. Relative expression of BMPs and BMPRs in normal endometrial cell and endometrial cancer cell lines Supplementary Figure S11. Microfluidics-based PCR analysis of EMT gene panel in RL95-2 cells with or without EGF treatment Supplementary Figure S12. Knockdown expression of EpCAM by different shRNA sequences in RL95-2 cells Supplementary Figure S13. Proposed model for EpICD mediated regulation of BMP genes by EGF Supplementary Table 1. Characteristics of endometrial cancer patients Supplemental Table 2. Summary of sequencing reads in endometrial tumors an normal controls Supplementary Table 3. Differentially methylated promoter CpG islands in recurrent relative to non-recurrent tumors Supplementary Table 4. List of primer sequences Supplementary Table 5. List of siRNA and shRNA sequences

Adenomyosis is linked to dysmenorrhea and infertility. The pathogenesis of adenomyosis remains unclear, and little is known of the genetic and epigenetic changes in the eutopic endometrium in adenomyosis, which may predispose patients to the invasion and migration of endometrial tissues into the myometrium. Transcriptome studies have identified genes related to various cell behaviors but no targets for therapeutic intervention. The epigenetics of the eutopic endometrium in adenomyosis have rarely been investigated. Endometrial tissue was obtained from premenopausal women with (n = 32) or without adenomyosis (n = 17) who underwent hysterectomy aged 34–57 years at a tertiary hospital. The methylome and transcriptome were assessed by using a Methylation 450 K BeadChip array and Affymetrix expression microarray. Protein expression was examined by immunohistochemistry. Differential methylation analysis revealed 53 lowly methylated genes and 176 highly methylated genes with consistent gene expression in adenomyosis, including three genes encoding potassium ion channels. High expression of KCNK9 in the eutopic and ectopic endometria in patients with adenomyosis but not in normal controls was observed. Hormone-free, antibody-based KCNK9 targeting is a potential therapeutic strategy for adenomyosis-related dysmenorrhea, menorrhagia, and infertility.

Background: We describe a DNA methylation assay, named MPap test, using cervical scraping as an alternative technique for endometrial cancer detection. Methods: A multicenter hospital-based, two-stage validation study was conducted to validate the cancer detection performance of the MPap test. The MPap value was determined from the DNA methylation status of two genes (BHLHE22, CDO1) and combined with two other clinical variables (age, BMI). The cutoff threshold of the MPap value was established in stage 1 and validated in stage 2. A total of 592 women with abnormal uterine bleeding were enrolled from five medical centers throughout Taiwan. Results: In stage 1, the sensitivity, specificity, and positive and negative predictive values of the MPap test for detecting endometrial cancer were 92.9%, 71.5%, 39.8%, and 98.0%, respectively. These values were validated in stage 2, being 92.5%, 73.8%, 40.2%, and 98.1%. Moreover, MPap outperformed transvaginal ultrasound in sensitivity and negative predictive values for detecting endometrial cancer. When we applied the algorithm for triage of endometrial cancer detection by MPap in the Taiwan National Health Insurance dataset, we found that it may reduce invasive procedures by 69~73%. Conclusions: MPap may provide a feasible alternative for endometrial cancer detection and can be considered as a triage test to reduce unnecessary invasive procedures.

Endometrial cancer (EC) rates are rising annually. Additional prediction markers need to be evaluated because only 10–20% of EC cases show an objective response to immune-checkpoint inhibitors (ICIs). Our previous methylomic study found that BHLHE22 is hypermethylated in EC tissues and can be detected using a Pap-smear sample. BHLHE22, a basic helix loop helix transcription factor family member, is known as a transcriptional repressor and is involved in cell differentiation. However, the role of BHLHE22 in EC remains poorly understood. Herein, we analyzed BHLHE22 expression in 54 paired cancer and normal endometrial tissue samples, and confirmed with databases (TCGA, GTEx, and human protein atlas). We found that BHLHE22 protein expression was significantly downregulated in EC compared with normal endometrium. High BHLHE22 expression was associated with microsatellite-instable subtype, endometrioid type, grade, and age. It showed a significant favorable survival. BHLHE22 overexpression inhibited the proliferation and migration of EC cells. Functional enrichment analysis showed that BHLHE22 was significantly associated with immune-related pathways. Furthermore, BHLHE22 was positively correlated with proinflammatory leukocyte infiltration and expression of chemokine genes in EC. In conclusion, BHLHE22 regulates immune-related pathways and modulates the immune microenvironment of EC.

Gynecological cancer is a grave threat to women’s health. The human papillomavirus (HPV) infection is the main risk of cervical cancer. The incidence and mortality of cervical cancer have gradually decreased since the clinical use of pap smears. However, the sensitivity of pap smears still varies with different screening equipment, which seriously affects the effectiveness of screening for cervical cancer. On the other hand, the etiology of endometrial cancer and ovarian cancer is not clear. Although the treatment response is good in the early stage, the late diagnosis results in poor treatment outcomes and high mortality. Therefore, developing biomarkers for early detection and effective screening methods of these three diseases is necessary and urgent. The DNA methylomic studies of these diseases can have the opportunity to discover genes related to diseases and serve as indicators of disease diagnosis and prognosis biomarkers.

Intraperitoneal metastasis is a challenging clinical scenario in epithelial ovarian cancer (EOC). As they are distinct from hematogenous metastasizing tumors, epithelial ovarian cancer cells primarily disseminate within the peritoneal cavity to form superficially invasive carcinomas. Unfavorable pharmacokinetics for peritoneal tumors and gut toxicity collectively lead to a narrow therapeutic window and therefore limit the opportunities for a favorable clinical outcome. New insights into tumor metastasis in the peritoneal microenvironment are keenly awaited to develop new therapeutic strategies. Epithelial ovarian cancer stem cell (OCSC) seeding is considered to be a critical component of the peritoneal spread. Using a unique and stepwise process of the OCSC differentiation model may provide insight into the intraperitoneal metastasis. The transcriptome and epigenome of OCSC differentiation were characterized by expression array and MethylCap-Seq. The TCGA, AOCS, and KM-Plotter databases were used to evaluate the association between survival outcomes and the methylation/expression levels of candidate genes in the EOC datasets. The STRING database was used to investigate the protein–protein interaction (PPI) for candidates and their associated genes. The infiltration level of immune cells in EOC patients and the association between clinical outcome and OCSCs differentiation genes were estimated using the TIDE and TIME2.0 algorithms. We established an EOC differentiation model using OCSCs. After an integrated transcriptomics and methylomics analysis of OCSCs differentiation, we revealed that the genes associated with earlier OCSC differentiation were better able to reflect the patient’s outcome. The OCSC differentiation genes were involved in regulating metabolism shift and the suppressive immune microenvironment. High GPD1 expression with high pro-tumorigenic immune cells (M2 macrophage, and cancer associated fibroblast) had worst survival. Moreover, we developed a methylation signature, constituted by GNPDA1, GPD1, GRASP, HOXC11, and MSLN, that may be useful for prognostic prediction in EOC. Our results revealed a novel role of epigenetic plasticity OCSC differentiation and suggested metabolic and immune intervention as a new therapeutic strategy.

Objective: To study whether the methylation status of cervical secretions can reflect the ability of the endometrium to allow embryo implantation. Design: Case-control study. Setting: In vitro fertilization centers. Patient(s): Women undergoing embryo transfer cycles, in which at least 1 good-quality embryo was transferred. Intervention(s): Collection of cervical secretions during the procedure of embryo transfer. Main Outcome Measure(s): Methylation profiles of cervical secretions in relation to pregnancy outcomes. Result(s): Genome-wide methylation profiles differ between cervical secretions from pregnancy and nonpregnancy cycles. Clustering analysis on the basis of the top 2,000 differentially methylated probes of cervical secretions from 28 pregnancy and 29 nonpregnancy cycles correctly categorized 86.0% of the samples in terms of conceptional status, which was verified in selected genes by quantitative methylation-specific polymerase chain reaction and validated in another independent sample set. The combination of selected genes was estimated to predict pregnancy outcomes with a maximal area under the receiver operating characteristic curve of 0.83. Conclusion(s): The methylation profiles of cervical secretions were associated with pregnancy outcomes in embryo transfer cycles. Although not clinically useful at present, deoxyribonucleic acid methylation in cervical secretions may shed new light on the less invasive assessment of endometrial receptivity. (Fertil Steril Sci (R) 2022;3:74-83. (c) 2021 by American Society for Reproductive Medicine.)

Abstract Study question Is cervical secretion gene methylation profile different between receptive and non-receptive endometrium and associated with implantation outcome in frozen-embryo transfer (FET) cycle? Summary answer The combination of candidate genes methylation profiles obtained from cervical secretion showed significant associations with pregnancy outcomes. What is known already Implantation failure remains a black box in reproductive medicine, and the exact mechanism of how endometrial receptivity is regulated is still unknown. Epigenetic modifications play a role in the gene expression pattern and may alter the endometrial receptivity in the human endometrium. Cervical secretion containing various implantation-related cytokines, and the gene methylation change can be used as a non-invasive molecular source that reflects the endometrium condition. Study design, size, duration In this retrospective case-control study, sixty-two women who entered the FET cycle (30 pregnant and 32 non-pregnant women) were enrolled. Participants/materials, setting, methods Cervical secretion was collected before embryo transfer from women enrolled in multicenter university-affiliated reproductive units. The DNA methylation status of six candidate genes was measured using quantitative methylation-specific PCR (qMSP). The correlation between methylation change and the pregnancy outcome was analyzed. Main results and the role of chance The candidate genes were selected from that associated with implantation with literature review and the original genome-wide DNA methylation data from NCBI GEO DataSets (GSE90060) which processed using bioinformatics analysis. Six candidate genes whose CpG-level methylation analysis with β-value statistically higher in receptive endometrium than in a pre-receptive endometrium were selected. All six candidate genes showed different degrees of correlation with the pregnancy outcomes. Among them, PRKAG2 methylation changes showed the highest correlation with the pregnancy outcome. A logistic regression model was used to evaluate the performance of a single gene or a combination of genes for implantation prediction. The results showed a statistically significant association between the methylation status of a combination of genes (PRKAG2, KRS1, HAND2) and the pregnancy outcome (p = 0.008), resulting in an optimal AUC of 0.7 (95% CI: 0.57 - 0.81) for implantation prediction. Limitations, reasons for caution The results obtained from a relatively small cohort size. A larger study and further comprehensive methylome investigations are warranted. Wider implications of the findings: This study is the first proof-of-concept study that cervical secretion methylation profile is associated with implantation outcome in a FET cycle, and showed potential as a non-invasive method for implantation prediction. Trial registration number non applicable

BACKGROUND:Leiomyosarcoma (LMS), the most common soft tissue sarcoma, exhibits heterogeneous and complex genetic karyotypes with severe chromosomal instability and rearrangement and poor prognosis.METHODS:Clinical variables associated with NKX6-1 were obtained from The Cancer Genome Atlas (TCGA). NKX6-1 mRNA expression was examined in 49 human uterine tissues. The in vitro effects of NXK6-1 in LMS cells were determined by reverse transcriptase PCR, western blotting, colony formation, spheroid formation, and cell viability assays. In vivo tumor growth was evaluated in nude mice.RESULTS:Using The Cancer Genome Atlas (TCGA) and human uterine tissue datasets, we observed that NKX6-1 expression was associated with poor prognosis and malignant potential in LMS. NKX6-1 enhanced in vitro tumor cell aggressiveness via upregulation of cell proliferation and anchorage-independent growth and promoted in vivo tumor growth. Moreover, overexpression and knockdown of NKX6-1 were associated with upregulation and downregulation, respectively, of stem cell transcription factors, including KLF8, MYC, and CD49F, and affected sphere formation, chemoresistance, NOTCH signaling and Sonic hedgehog (SHH) pathways in human sarcoma cells. Importantly, treatment with an SHH inhibitor (RU-SKI 43) but not a NOTCH inhibitor (DAPT) reduced cell survival in NKX6-1-expressing cancer cells, indicating that an SHH inhibitor could be useful in treating LMS. Finally, using the TCGA dataset, we demonstrated that LMS patients with high expression of NKX6-1 and HHAT, an SHH pathway acyltransferase, had poorer survival outcomes compared to those without.CONCLUSIONS:Our findings indicate that NKX6-1 and HHAT play critical roles in the pathogenesis of LMS and could be promising diagnostic and therapeutic targets for LMS patients.

Background Patients with ovarian clear cell carcinoma (OCCC) have a poor prognosis because they show low sensitivity to platinum-based chemotherapy. New treatments for refractory OCCC are urgently needed. Case presentation We present a patient with refractory OCCC in whom conventional chemotherapy failed. Cachexia was induced by the disseminating recurrent tumors. Tumor tissue staining and genomic analysis revealed PD-L1 negativity, a low tumor burden, stable microsatellite instability, and two mutations in ARID1A . The patient was administered pembrolizumab combined with bevacizumab triweekly. Her serum CA-125 level decreased dramatically after the first cycle. A computerized tomography scan showed marked regression of the recurrent masses after 3 cycles, and the patient reached complete remission after 9 cycles. She showed good recovery from cachexia. We observed no marked side effects except for mild polyarthritis of the small joints. Conclusions The therapeutic effect of checkpoint inhibitors combined with angiogenesis inhibitors is very promising in our patient with OCCC. Further clinical trials of tumors including ARID1A mutations are warranted.

Ten‐eleven translocation methylcytosine dioxygenase‐1, TET1, takes part in active DNA demethylation. However, our understanding of DNA demethylation in cancer biology and its clinical significance remain limited. This study showed that TET1 expression correlated with poor survival in advanced‐stage epithelial ovarian carcinoma (EOC), and with cell migration, anchorage‐independent growth, cancer stemness, and tumorigenicity. In particular, TET1 was highly expressed in serous tubal intraepithelial carcinoma (STIC), a currently accepted type II EOC precursor, and inversely correlated with TP53 mutations. Moreover, TET1 could demethylate the epigenome and activate multiple oncogenic pathways, including an immunomodulation network having casein kinase II subunit alpha (CK2α) as a hub. Patients with TET1 high CK2α high EOCs had the worst outcomes, and TET1‐expressing EOCs were more sensitive to a CK2 inhibitor, both in vitro and in vivo . Our findings uncover the oncogenic and poor prognostic roles of TET1 in EOC and suggest an unexplored role of epigenetic reprogramming in early ovarian carcinogenesis. Moreover, the immunomodulator CK2α represents a promising new therapeutic target, warranting clinical trials of the tolerable CK2 inhibitor, CX4945, for precision medicine against EOC. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Ovarian cancer (OC) is the most lethal gynecological cancer, worldwide, largely due to its vague and nonspecific early stage symptoms, resulting in most tumors being found at advanced stages. Moreover, due to its relative rarity, there are currently no satisfactory methods for OC screening, which remains a controversial and cost-prohibitive issue. Here, we demonstrate that Papanicolaou test (Pap test) cervical scrapings, instead of blood, can reveal genetic/epigenetic information for OC detection, using specific and sensitive DNA methylation biomarkers. Results We analyzed the methylomes of tissues (50 OC tissues versus 6 normal ovarian epithelia) and cervical scrapings (5 OC patients versus 10 normal controls), and integrated public methylomic datasets, including 79 OC tissues and 6 normal tubal epithelia. Differentially methylated genes were further classified by unsupervised hierarchical clustering, and each candidate biomarker gene was verified in both OC tissues and cervical scrapings by either quantitative methylation-specific polymerase chain reaction (qMSP) or bisulfite pyrosequencing. A risk-score by logistic regression was generated for clinical application. One hundred fifty-one genes were classified into four clusters, and nine candidate hypermethylated genes from these four clusters were selected. Among these, four genes fulfilled our selection criteria and were validated in training and testing set, respectively. The OC detection accuracy was demonstrated by area under the receiver operating characteristic curves (AUCs) in 0.80–0.83 of AMPD3 , 0.79–0.85 of AOX1 , 0.78–0.88 of NRN1 , and 0.82–0.85 of TBX15 . From this, we found OC-risk score, equation generated by logistic regression in training set and validated an OC-associated panel comprising AMPD3 , NRN1 , and TBX15 , reaching a sensitivity of 81%, specificity of 84%, and OC detection accuracy of 0.91 (95% CI, 0.82–1) in testing set. Conclusions Ovarian cancer detection from cervical scrapings is feasible, using particularly promising epigenetic biomarkers such as AMPD3/NRN1/TBX15 . Further validation is warranted.

Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long-term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen-mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c-Kit, by epigenetic silencing the tumor suppressor miR-193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c-KIT, but downregulated miR-193a. Luciferase assays further confirmed that microRNA-193a targets both E2F6 and c-Kit. Interestingly, ChIP-PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR-193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR-193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR-193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen-mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA-193a activity, promoting ovarian cancer stemness and tumorigenesis.

Background Endometrial cancer is a common gynecologic cancer. Noninvasive molecular biomarkers for triage of high-risk patients for invasive procedures are needed. Based on the success of cytological Pap smear screening, cervical scrapings are a good source of DNA for molecular testing. In addition to genetic lesions, DNA methylation is a promising biomarker. We assessed the usefulness of combining genetic and epigenetic biomarkers from cervical scrapings to detect endometrial carcinomas. Methods We performed a retrospective case–control study of 96 consecutive cervical scrapings from patients with abnormal uterine bleeding who underwent surgery for diagnostic evaluation. Thirty and 16 cases were diagnosed with type I and type II endometrial cancers, respectively. The remaining non-cancer cases included normal endometrium ( n = 12), benign uterine lesions ( n = 20), and endometrial hyperplasia (n = 18). Quantitative methylation-specific PCR and mass spectrometry were used for DNA methylation and genetic mutation analysis. Logistic regression was used to evaluate the clinical performance of these candidate biomarkers. Results We tested the effectiveness of the methylation status of four genes ( BHLHE22 , CDO1 , TBX5 , and HAND2 ) in endometrial cancer detection. The area under the receiver operating characteristic curves ranged from 0.703 to 0.878, and panels of hypermethylated BHLHE22 / CDO1 / HAND2 (87.0% sensitivity and 86.0% specificity) and BHLHE22 / CDO1 / TBX5 (89.1% sensitivity and 80.0% specificity) showed significant differences and could distinguish benign from malignant endometrial lesions. The sensitivity and specificity in endometrial cancer detection for BHLHE22 / CDO1 were 84.8% and 88.0%, respectively. Both type I and II endometrial carcinomas could be detected using a BHLHE22 / CDO1 -based methylation profile, suggesting that they may have common epigenomes. Moreover, PTEN and TP53 mutations were found in 63.3% of type I and 93.6% of type II endometrial cancers. Unexpectedly, PTEN and TP53 mutations were commonly found in cervical scrapings of the normal endometrium (25% and 33.3%, respectively) and in cases with benign uterine lesions (10% and 50%, respectively). Finally, combinations of any one mutation of PTEN and TP53 mutations had a sensitivity of 91.3%, but a specificity of only 42.0%. Conclusions Adding PTEN / TP53 mutation testing to BHLHE22 / CDO1 -based methylation testing did not improve the detection of endometrial cancer.

Cancer cells exhibit phenotypic plasticity during epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat further reveal cell state-dependent plasticity upon GRHL2 overexpression. Overall, we demonstrate that epithelial genes are subject to epigenetic control during intermediate phases of EMT/MET involving GRHL2.

Objective: To share our experience of transition from multiport to single-site robotic surgery for benign gynecological conditions as well as to assess the selection criteria of candidates for robotic single-site supracervical hysterectomy (RSSH). Materials and methods: A retrospective review was conducted on patients undergoing robotic supracervical hysterectomy by a single surgeon in a single institute between June 2014 and December 2017. Patients who underwent additional procedures along with supracervical hysterectomy and who had unexpectant corpus malignancy proved pathologically were excluded from comparisons between patients undergoing RSSH and robotic multiport supracervical hysterectomy (RMSH). Results: Between June 2014 and December 2017, we accomplished 26 RSSH and 57 RMSH. There were no conversions, intraoperative complications, and readmissions within 30 days after surgery. In the RSSH group, the mean uterine weight was 264.6 +/- 140.9 g with mean docking time of 15.8 +/- 5.5 min, mean console time of 61.1 +/- 35.6 min and mean operative time of 140.3 +/- 34.4 min. In comparison to the RMSH group, the percentage of overweight/obese patients was lower (p = 0.018) and the uterine size was smaller (p < 0.001) with adenomyosis diagnosed more frequently (p = 0.002) in the RSSH group. While the operative time in the RSSH group was significantly shorter (p = 0.002), the RSSH group took longer time in docking (p < 0.001) and comparable time in console (p = 0.254). In view of chronological change, docking time and console time in the RMSH group remained steady, whereas steep decreases were observed in the RSSH group. The intraoperative blood loss and hemoglobin drop were comparable. The length of hospital stay was significantly shorter in the RSSH group (p = 0.005). Conclusion: Transition from multiport to single-site surgery can be smooth for a surgical team experienced in the conventional multiport robotic system. RSSH is safe and feasible in properly selected patients. (C) 2019 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V.

DNA hypermethylation is a driving force in carcinogenesis. However, the role of active DNA hypomethylation in cancer remains largely unknown. This process, facilitated by ten-eleven translocation methylcytosine dioxygenase 1 (TET1), which oxidizes 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), has never been studied in cervical cancer. Here, we found that TET1 and 5hmC correlative increases from normal cervix to Low-grade squamous intraepithelial lesion (LSIL), maximizing in High-grade squamous intraepithelial lesion (HSIL), and decreasing in invasive cancer. Full-length HPV-immortalized HSIL cells demonstrated higher TET1/5hmC levels, and stemness properties, compared to invasive cancer cells. TET1 silencing promoted the epithelial-mesenchymal transition (EMT), to transform precancerous cells in vivo. TET1 increased 5hmC in the ZEB1 and VIM promoters, surprisingly, silencing both genes. TET1 interaction with the histone modifiers, LSD1 and EZH2, on the ZEB1 promoter, resulted in gene silencing, via loss of histone H3K4 trimethylation, and gain of histone H3K27 trimethylation. Taken together, TET1 promotes stemness properties, and inhibits EMT, in HSIL cells, through 5hmC-dependent and -independent mechanisms.

DNA methylation alteration, such as global hypomethylation and localized hypermethylation, within the promoters of tumor suppressor genes, is an important risk factor in cervical cancer. The potential use of DNA methylation detection, in cervical cancer screening or triage of mildly abnormal cytology, has recently been demonstrated. In particular, PAX1 DNA methylation testing was approved as an adjunct to cytology, in Taiwan, and is now undergoing registration trials in China. However, the function of PAX1 in cancer biology remains largely unknown. Here, we show that PAX1 inhibits malignant phenotypes upon oncogenic stress. Specifically, PAX1 expression inhibited the phosphorylation of multiple kinases, after challenges with oncogenic growth factors such as EGF and IL-6. Analogously, PAX1 activated a panel of phosphatases, including DUSP1, 5, and 6, and inhibited EGF/MAPK signaling. PAX1 also interacted with SET1B, increasing histone H3K4 methylation and DNA demethylation of numerous phosphatase-encoding genes. Furthermore, hypermethylated PAX1 associated with poor prognosis in cervical cancer. Taken together, this study reveals, for the first time, the functional relevance of PAX1 in cancer biology, and further supports the prospect of targeting multifold oncogenic kinase cascades, which jointly contribute to multiresistance, via epigenetic reactivation of PAX1.