Upregulated MELK Leads to Doxorubicin Chemoresistance and M2 Macrophage Polarization via the miR-34a/JAK2/STAT3 Pathway in Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (ULMS) is the most lethal gynecologic malignancy with few therapeutic options. Chemoresistance prevails as a major hurdle in treating this malignancy, yet the mechanism of chemoresistance remains largely unclear. In this study, we certified MELK as a poor prognostic marker through bioinformatic analysis of the GEO database. Cellular experiments in vitro revealed that MELK played an essential role in ULMS cells' chemoresistance and that a high expression of MELK could lead to doxorubicin resistance. mRNA profiling uncovered the pathways that MELK was involved in which led to doxorubicin resistance. MELK was found to affect ULMS cells' chemoresistance through an anti-apoptotic mechanism via the JAK2/STAT3 pathway. miRNA profiling also revealed that upregulated MELK could induce the decrease of miRNA-34a (regulated by JAK2/STAT3 pathway). We detected that MELK overexpression could induce M2 macrophage polarization via the miR-34a/JAK2/STAT3 pathway, contributing to doxorubicin chemoresistance in the tumor microenvironment. OTSSP167, a MELK inhibitor, may increase ULMS sensitivity to doxorubicin. Our investigation could propose novel targets for early diagnosis and precision therapy in ULMS patients.

MiR-509-3 augments the synthetic lethality of PARPi by regulating HR repair in PDX model of HGSOC.

BACKGROUND: PARP inhibitors have been the most promising target drugs with widely proven benefits among ovarian cancer patients. Although platinum-response, HR-related genes, or HRD genomic scar detection are acceptably used in assessment of Olaparib response, there are still evident limitations in the present approaches. Therefore, we aim to investigate more accurate approaches to predict Olaparib sensitivity and effective synergistic treatment strategies. METHODS: We probed two databases (TCGA and Qilu Hospital) in order to quest novel miRNAs associated with platinum-sensitivity or HR-related genes. Cellular experiments in vitro or in vivo and PDX models were utilized to validate their role in tumor suppression and Olaparib sensitizing. Furthermore, HR gene mutation was analyzed through WES to explore the relation between HR gene mutation and Olaparib response. RESULTS: High miR-509-3 expression indicated better response to platinum and longer progression-free and overall survival in two independent ovarian cancer patient cohorts (high vs. low miR-509-3 expression; PFS: TCGA P < 0.05, Qilu P < 0.05; OS: TCGA P < 0.05, Qilu P < 0.01). MiR-509-3 could impair the proliferation, migration, and invasion ability but enhance the sensitivity to Olaparib of ovarian cancer cell in vitro and in vivo by directly targeting HMGA2 and RAD51. In two PDX cases (PDX1 and PDX9), miR-509-3 could significantly increase the sensitivity to Olaparib along with the decrease of RAD51 positive rate (mean tumor weight NC + Olaparib vs. miR-509 + Olaparib; PDX1 P < 0.05, PDX9 P < 0.05). Additionally, in PDX8, miR-509-3 treatment dramatically reversed the Olaparib insensitivity (P < 0.05) by downregulating RAD51 expression. RAD51 functional detection revealed that all Olaparib sensitive cases exhibited low RAD51 positive rate (lesser than 50%) in treated groups. Furthermore, among the four HR gene mutation patients, three harbored HR core gene mutation and were sensitive to Olaparib while the remaining one with non-HR core gene mutation did not respond well to Olaparib. CONCLUSIONS: MiR-509-3 can sensitize ovarian cancer cells to Olaparib by impeding HR, which makes it a potential target in PARPi synergistic treatment. HR core gene analysis and RAD51 functional detection are prospectively feasible in prediction of PARPi response.

Squamous cell carcinoma transformation in mature cystic teratoma of the ovary: a systematic review.

BACKGROUND: 0.17-2% of mature cystic teratoma of the ovary (MCTO) undergo malignant transformation, of which 80% are squamous cell carcinoma (SCC) transformation in MCTO. We aim to investigate the clinical characteristics and treatment of SCC transformation in MCTO METHODS: We systematically searched PubMed database and individual patient data about SCC transformation in MCTO were extracted. The published cases were combined with 6 cases of SCC transformation in MCTO from Qilu Hospital, Shandong University. RESULTS: The incidence of SCC transformation in MCTO was 0.3%. A total of 435 cases of SCC transformation in MCTO were enrolled in the analysis. The mean age of diagnosis was 53.5 (range 19-87) years old. The most common clinical manifestations were abdominal pain (47.3%) and abdominal mass (26.0%). StageI,II, III and IV accounted for 50.0, 18.8, 26.8 and 4.4% of all cases, respectively. Patients with stage I had significantly better prognosis than stage II, III and IV patients (P < 0.01). Hysterectomy can improve overall survival (P < 0.01). For patients younger than 45 years old with stageIA orIC, there was no difference in mortality between fertility-sparing and radical surgery (P = 1.00). Adjuvant chemotherapy can improve survival in patients with advanced stage (P = 0.02), and chemotherapy with platinum was related to better prognosis (P = 0.02). CONCLUSION: SCC transformation in MCTO is a rare malignancy mainly occurs in older age. FIGO stage is an independent prognostic factor. Hysterectomy and platinum-based chemotherapy are associated with better survival. Fertility-sparing surgery is feasible for young patients with early stage.

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.

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.

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

BACKGROUND: Patient 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. METHODS: In 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. RESULTS: Comparative 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. CONCLUSION: Our 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.

Overexpression and oncogenic function of HMGA2 in endometrial serous carcinogenesis.

The high-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor highly expressed in fetal tissue and malignant tumors but rarely detected within normal adult tissues. The clinical implications and biological functions of HMGA2 in endometrial carcinoma are largely unknown. Here we report that HMGA2 expression was barely detected in benign endometrium samples (2 of 28 samples). However, HMGA2 expression increased significantly from precancerous lesion endometrial glandular dysplasia (7 of 17, 41.2%), to serous endometrial intraepithelial carcinoma (5 of 8, 62.5%) and to full blown endometrial serous carcinoma (39 of 59, 66.1%). Functional characterization of HMGA2 revealed that the gene has both tumor growth promotion and metastasis. In addition, HMGA2 induced epithelial-mesenchymal transition (EMT) through modulation vimentin and ß-catenin. Furthermore, HMGA2 overexpression started from endometrial serous precancers, non-invasive cancers, as well as in full blown carcinomas in a p53 knockout mouse model we recently established in our laboratory. Our findings suggest that HMGA2 may serve as a useful diagnostic marker in the assessment of endometrial serous cancer and its precursor lesions.

Role of miR-182 in response to oxidative stress in the cell fate of human fallopian tube epithelial cells.

High grade serous ovarian carcinoma (HGSC) is a DNA instable tumor and its precursor is commonly found originating from the fimbriated end of the fallopian tube secretory epithelial (FTSE) cells. The local stresses via ovulation and related inflammation are risks for HGSC. In this study, we examined the cellular and molecular responses of FTSE cells to stress. We found that excess intracellular reactive oxygen species (ROS) in normal FTSE cells upregulated a subset of microRNA expression (defined as ROSmiRs). Most ROSmiRs' expression and function were influenced and regulated by p53, and together they drove the cells into stress-induced premature senescence (SIPS). However, ROS-induced miR-182 is regulated by ß-catenin, not by p53. In normal FTSE cells, miR-182 overexpression triggers cellular senescence by p53-mediated upregulation of p21. Conversely, in cells with p53 mutations, miR-182 overexpression no longer enhances p21 but functions as an "Onco-miR". p53 dysfunction is a prerequisite for miR-182-mediated tumorigenesis. In addition, we found that human follicular fluid could significantly induce intracellular ROS in normal FTSE cells. These findings suggest that ROS and p53 mutations may trigger a series of events, beginning with overexpressing miR-182 by ROS and ß-catenin, impairing the DNA damage response, promoting DNA instability, bypassing senescence and eventually leading to DNA instable tumors in FTSE cells.

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.

miR-145 inhibits tumor growth and metastasis by targeting metadherin in high-grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSOC), the most common and aggressive subtype of epithelial ovarian cancer, is characterized by TP53 mutations and genetic instability. Using miRNA profiling analysis, we found that miR-145, a p53 regulated miRNA, was frequently down-regulated in HGSOC. miR-145 down-regulation was further validated in a large cohort of HGSOCs by qPCR. Overexpression of miR-145 in ovarian cancer cells significantly suppressed proliferation, migration and invasion in vitro and inhibited tumor growth and metastasis in vivo. Metadherin (MTDH) was subsequently identified as a direct target of miR-145, and was found to be significantly up-regulated in HGSOC. Furthermore, overexpression of MTDH rescued the inhibitory effects of miR-145 in ovarian cancer cells. Finally, we found that high level of MTDH expression correlated with poor prognosis of HGSOC. Therefore, lack of suppression of MTDH by miR-145 when p53 is dysfunctional leads to increased tumor growth and metastasis of HGSOC. Our study established a new link between p53, miR-145 and MTDH in the regulation of tumor growth and metastasis in HGSOC.

Integrated analysis of long noncoding RNAs and mRNAs reveals their potential roles in the pathogenesis of uterine leiomyomas.

Global expression profiling studies showed that miRNAs are aberrantly expressed in uterine leiomyomas (ULMs) and are involved in ULM pathogenesis. Long noncoding RNAs (lncRNAs) are another group of regulatory RNA whose expression and roles in ULMs have not been explored. In this study, we examined the global expressions of lncRNAs and mRNAs in ULMs using microarray and interrogated their interrelationship through co-expression analysis. We found that lncRNAs and mRNAs were dysregulated in ULMs and the degree of dysregulation was positively correlated with tumor size. Further analysis showed that lncRNAs correlate to their cis mRNAs in expression levels depending on genomic locations and orientations. Moreover, we identified several dysregulated pathways that were correlated to dysregulated lncRNAs. We validated several aberrantly expressed lncRNAs in extended samples and confirmed that AK023096 was down-regulated and chromatin-associated RNA (CAR) Intergenic 10 was up-regulated in the majority of leiomyomas. Knockdown of Intergenic 10 inhibited the proliferation of leiomyoma cells in vitro, indicating its functional importance in ULM pathogenesis. The neighboring protein-coding gene ADAM12 was also downregulated in Intergenic 10 knockdown leiomyoma cells. We showed for the first time that lncRNAs were dysregulated in uterine leiomyomas. Aberrantly expressed lncRNAs may contribute to the pathogenesis of uterine leiomyomas.

miR-1236-3p represses the cell migration and invasion abilities by targeting ZEB1 in high-grade serous ovarian carcinoma.

Ovarian cancer, particularly high-grade serous ovarian carcinoma (HG-SOC), is still the main cause of death among gynecological malignancies. However, the molecular mechanisms related to its malignant biological behavior are still unclear. Recent studies indicate that microRNAs (miRNAs) play an important role in tumor metastasis. Here, we report that miR-1236-3p expression was downregulated in HG-SOC when compared to that in normal fallopian tube tissue. Manipulation of miR-1236-3p significantly influenced the morphology, migration and invasion of ovarian cancer cell lines (A2780 and SKOV3). With dual-luciferase reporter assay, we demonstrated that miR­1236-3p binds to the 3'UTR of zinc-finger E-box binding homeobox 1 (ZEB1) mRNA, and functions as a negative regulator of ZEB1. Furthermore, we revealed that manipulation of miR-1236-3p modulates ZEB1 expression and influences expression of its downstream genes E-cadherin and N-cadherin at both the mRNA and protein levels. We also found an inverse relationship between miR­1236-3p and ZEB1 expression in the HG-SOC tissue samples. Taken together, our results indicate that miR-1236-3p regulates ovarian cancer metastasis by directly targeting ZEB1, and it may play an important role in the diagnosis and treatment of ovarian cancer.

miR-106a represses the Rb tumor suppressor p130 to regulate cellular proliferation and differentiation in high-grade serous ovarian carcinoma.

UNLABELLED: 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.

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.

Intracellular metabolism, subcellular localization and phototoxicity of HMME/HB in ovarian cancer cells.

BACKGROUND: Photodynamic therapy (PDT) is considered a promising new strategy for ovarian cancer treatment. As the key component in PDT, photosensitizer metabolism and localization in cancer cells is particularly important. MATERIALS AND METHODS: The localization of the photosensitizers hematoporphyrin monomethyl ether (HMME) and hypocrellin B (HB) were determined in the ovarian cancer cell lines SKOV3 and NuTu-19 by fluorescence microscopy and laser scanning confocal microscopy(LSCM). A JD801 image analysis system was used to analyze the fluorescence intensity of the photosensitizers in the cells. The phototoxicity of both drugs to the cancer cells was determined by MTT assay. RESULTS: Both photosensitizers were mainly distributed in the cytoplasm. Drug uptake reached a peak after 4 h incubation with HB and after 3 h incubation with HMME. Within a certain range, the higher the concentration, the stronger the fluorescence became and at 40 µg/ml, the intracellular photosensitizer had reached saturation. Based on these results PDT was applied to SKOV3 cells. All the cells were killed when the photosensizer dose reached 40 µg/ml. CONCLUSION: PDT is an effective therapy for ovarian cancer cells.