Ruifen Dong

A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA

OBJECTIVE Epithelial ovarian cancer (EOC) remains the most lethal disease among gynecological malignancies. Prompt diagnosis is challenging because of the non-specific symptoms exhibited during the early stage of the disease. As a result, there is an urgent need for improved detection methods. In this study, we established a multiplex methylation-specific PCR (MSP) assay to improve the early detection of ovarian cancer, via identification of the methylation status of cell-free serum DNA. METHODS After screening, we chose seven candidate genes (APC, RASSF1A, CDH1, RUNX3, TFPI2, SFRP5 and OPCML) with a high frequency of methylation to construct the multiplex-MSP assay. When methylation of at least one of the seven genes was observed, the multiplex-MSP assay was considered positive. We performed retrospective and screening studies to verify the specificity and sensitivity of the assay in the detection of EOC. RESULTS The methylation status of cell-free serum DNA was examined in the preoperative serum of 202 patients, including 87 EOC patients (stage I, n=41; stage II-IV, n=46), 53 patients with benign ovarian tumors and 62 healthy controls. As expected, the multiplex MSP assay achieved a sensitivity of 85.3% and a specificity of 90.5% in stageI EOC, strikingly higher rates compared with a single CA125, which produced a sensitivity of 56.1% at 64.15% specificity [P=0.0036]. CONCLUSION A multiplex MSP assay that analyzes the methylation status of cell-free serum DNA is a suitable and reliable approach to improve the early detection of ovarian cancer, potentially benefiting a broad range of applications in clinical oncology.

MiR-106a represses the RB tumor suppressor p130 to regulate cellular proliferation and differentiation in High-grade serous ovarian carcinoma

The degree of differentiation in human cancers generally reflects the degree of malignancy, with the most undifferentiated cancer being also the highest grade and the most aggressive. High-grade serous ovarian carcinomas (HGSOC) are poorly differentiated and fast-growing malignancies. The molecular mechanisms underlying the poor differentiation of HGSOC has not been completely characterized. Evidence suggests that miRNA, miR are dysregulated in HGSOC. Therefore, we focused on those miRNAs that are relevant to tumor differentiation. Expression profiling of miRNAs in HGSOC, indicated miR-106a and its family members were significantly upregulated. Upregulation of miR-106a was further validated by real-time reverse transcriptase PCR (qRT-PCR) and miRNA in situ hybridization in a large cohort of HGSOC specimens. Overexpression of miR-106a in benign and malignant ovarian cells significantly increased the cellular proliferation rate and expanded the side-population fraction. In particular, SKOV3 cells with miR-106a overexpression had significantly higher tumor initial/stem cell population (CD24- and CD133-positive cells) than control SKOV3 cells. Among many miR-106a predicated target genes, p130 (RBL2), an retinoblastoma (Rb) tumor suppressor family member, was not only confirmed as a specific target of miR-106a but also related to tumor growth and differentiation. The importance of mir-106a and RBL2 was further demonstrated in vivo, in which, SKOV3 cells overexpressing miR-106a formed poorly differentiated carcinomas and had reduced RBL2 levels. To our knowledge, this is the first study of miR-106a mediating proliferation and tumor differentiation in HGSOC. Implications: The current study suggests that the RB tumor suppressor pathway is a critical regulator of growth and differentiation in HGSOC. Mol Cancer Res; 11(11); 1314–25. ©2013 AACR.

Histologic and molecular analysis of patient derived xenografts of high-grade serous ovarian carcinoma.

BackgroundPatient derived xenografts (PDX) are generated by transplanting the original patient’s tumor tissue into immune-deficient mice. Unlike xenograft models derived from cell lines, PDX models can better preserve the histopathology from the original patient and molecular pathways. High-grade serous carcinoma (HGSC) is a deadly form of ovarian/fallopian tube cancer whose response to current chemotherapies varies widely due to patient variability. Therefore, a PDX model can provide a valuable tool to study and test treatment options for each individual patient.MethodsIn this study, over 200 PDX tumors from nine HGSC were analyzed to investigate the nature and behavior of PDX tumors originating from HGSC. PDX tumors were serially passaged (from P0 to P4) and tumors were grafted orthotopically under the ovarian bursa or subcutaneously.ResultsComparative analysis of the histology and molecular markers of tumors from over 200 PDX tumor-bearing mice, revealed that the tumors maintained similar histologies, stem cell populations, and expression for the majority of the tested oncogenic markers, compared to the primary tumors. However, a significant loss of steroid hormone receptors and altered expression of immunoresponsive genes in PDX tumors were also noted. ConclusionOur findings provide substantial new information about PDX tumor characteristics from HGSC which will be valuable towards the development of personalized therapy and new drug development for HGSC.

miR-130a upregulates mTOR pathway by targeting TSC1 and is transactivated by NF-κB in high-grade serous ovarian carcinoma

Activation of mammalian target of rapamycin (mTOR) signaling pathway is associated with poor prognosis of epithelial ovarian cancer. The TSC1-TSC2 complex is a critical negative regulator of mTOR signaling. Here, we demonstrated that TSC1 was frequently downregulated in high-grade serous ovarian carcinoma (HGSOC) and low TSC1 expression level is associated with advanced tumor stage. We next identified miR-130a to be a negative regulator of TSC1 by targeting its 3’UTR. miR-130a was overexpressed in HGSOC and could drive proliferation and invasion/metastasis of ovarian cancer cells. miR-130a could also attenuate rapamycin/starvation-induced autophagy. Ectopic TSC1 expression could block the effects of miR-130a on cell proliferation, migration and autophagy. Finally, we found that miR-130a expression could be upregulated by inflammatory factors and was transactivated by NF-κB. Therefore, our findings establish a crosstalk between inflammation and mTOR signaling that is mediated by miR-130a, which might have a pivotal role in the initiation and progression of HGSOC.

MDC1 promotes ovarian cancer metastasis by inducing epithelial-mesenchymal transition.

Ovarian cancer is a highly invasive cancer with poor prognosis. Previous studies have revealed lots of connections between the invasiveness and epithelial-mesenchymal transition (EMT), which is common during the progression of ovarian cancer. MDC1, a mediator of DNA damage checkpoint, has recently been implicated as a potential oncogene. Here, in this article, we studied the role of MDC1 in ovarian cancer metastasis. First, in tissue samples, we found that high expression level of MDC1 was correlated with poor prognosis. Furthermore, MDC1 overexpression in ovarian cancer cells significantly increased migration and invasion. In contrast, silencing MDC1 reversed these processes. Consistently, nude mice xenograft confirmed that silencing MDC1 suppressed tumor metastasis in vivo. We further demonstrated that MDC1 induced EMT through modulation EMT markers such as E-cadherin, N-cadherin, and vimentin. Taken together, our findings suggest that MDC1 promotes ovarian cancer metastasis through the induction of EMT.

Anti-angiogenesis effect of Neferine via regulating autophagy and polarization of tumor-associated macrophages in high-grade serous ovarian carcinoma

High-grade serous ovarian carcinoma (HGSOC) is one of the most lethal gynecologic malignancies. Currently, anti-angiogenesis therapy is the most promising strategy for the successful treatment of HGSOC. In this study, we found Neferine could inhibit the angiogenesis of ovarian cancer cells both in vitro and in vivo. Further analysis revealed that its suppressive effect on human umbilical vein endothelial cell (HUVEC) proliferation correlated with promoting cell cycle arrest and autophagy. The cell cycle genes were dose-dependently reduced and the level of LC3II/LC3I (microtubule associated protein 1 light chain 3) was increased. Using a specific marker for macrophages (CD206 and Mrc1), we indicated that Neferine could inhibit M2-macrophage in vivo. Finally, CD206 was stained in 150 HGSOC samples and its high expression predicted inferior overall survival. Our current study is the first to demonstrate the anti-angiogenesis mechanism of Neferine by inducing autophagy via mTOR/p70S6K pathway inhibition and suppressing M2-macrophage polarization. Our findings suggest that Neferine is an attractive reagent with great potential in HGSOC therapy, especially in standard-therapy resistant cases.

Comparison among fertility-sparing therapies for well differentiated early-stage endometrial carcinoma and complex atypical hyperplasia

Objective To compare fertility-sparing therapies including oral progestogens, hysteroscopic resection (HR), and the levonorgestrel- releasing intrauterine system (LNG-IUS) in achieving disease regression, recurrence and live birth rate in well differentiate early-stage endometrial carcinoma (eEC) and complex atypical hyperplasia(CAH). Study Design This was a meta-analysis of previous studies focus on the fertility-sparing therapy for well differentiate early-stage endometrial carcinoma (eEC) and complex atypical hyperplasia (CAH). Date Sources Medline, the Cochrane Library and Embase was searched with the terms and Synonyms: words similar to eEC and CAH with therapies associated with fertility-sparing. Main Outcome Measures The number of all patients accepted fertility sparing therapies, patients got regressed, relapsed and delivered were extracted from each study, and the regression, recurrence, and live birth rate of each study were calculated. The regression, recurrence and live birth rates between each two interventions were compared with the aid of meta-regression in packages of “meta” and ”meta for” written in R. Results Fifty-four studies reported fertility sparing therapies in young women with eEC and CAH were included. Meta-analysis showed that HR followed by progestogens achieved a higher pooled regression (98.06% vs 77.20% P < 0.0001) and live birth rate (52.57% vs 33.38%, P = 0.0944) and a lower recurrence rate compared with oral progestogens alone (4.79% vs 32.17% P = 0.0004). At the same time, the pooled live birth rate (52.57% vs 18.09% P =0.0399) of HR followed by progestogens are significantly higher than the LNG-IUS alone. Which no statistical difference in regression (98.06% vs 94.24%; P = 0.4098) and recurrence rates (4.79% vs 3.90% P = 0.8561) was seen. Conclusions Of the available fertility-sparing therapeutic options, HR followed by progestogens may be a more effective one.