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Purpose: Ovarian cancer patients with HR proficiency (HRP) have had limited benefits from PARP inhibitor treatment, highlighting the need for improved therapeutic strategies. In this study, we developed a novel SIK2 inhibitor, SIC-19, and investigated its potential to enhance the sensitivity and expand the clinical utility of PARP inhibitors in ovarian cancer., Methods: The SIK2 protein was modeled using a Molecular Operating Environment (MOE), and the most favorable model was selected based on a GBVI/WSA dG scoring function. The Chembridge Compound Library was screened, and the top 20 candidate compounds were tested for their interaction with SIK2 and downstream substrates, AKT-pS473 and MYLK-pS343. SIC-19 emerged as the most promising drug candidate and was further evaluated using multiple assays., Results: SIC-19 exhibited selective and potent inhibition of SIK2, leading to its degradation through the ubiquitination pathway. The IC50 of SIC-19 correlated inversely with endogenous SIK2 expression in ovarian cancer cell lines. Treatment with SIC-19 significantly inhibited cancer cell growth and sensitized cells to PARP inhibitors in vitro, as well as in ovarian cancer organoids and xenograft models. Mechanistically, SIK2 knockdown and SIC-19 treatment reduced RAD50 phosphorylation at Ser635, prevented nuclear translocation of RAD50, disrupted nuclear filament assembly, and impaired DNA homologous recombination repair, ultimately inducing apoptosis. These findings highlight the crucial role of SIK2 in the DNA HR repair pathway and demonstrate the significant PARP inhibitor sensitization achieved by SIC-19 in ovarian cancer., Conclusions: SIC-19, a novel SIK2 inhibitor, effectively inhibits tumor cell growth in ovarian cancer by interfering with RAD50-mediated DNA HR repair. Furthermore, SIC-19 enhances the efficacy of PARP inhibitors, providing a promising therapeutic strategy to improve outcomes for ovarian cancer patients., Competing Interests: Declaration of Competing Interest The all authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The Normal Risk Ovarian Screening Study (NROSS) tested a two-stage screening strategy in postmenopausal women at conventional hereditary risk where significantly rising cancer antigen (CA)-125 prompted transvaginal sonography (TVS) and abnormal TVS prompted surgery to detect ovarian cancer., Methods: A total of 7,856 healthy postmenopausal women were screened annually for a total of 50,596 woman-years in a single-arm study (ClinicalTrials.gov identifier: NCT00539162). Serum CA125 was analyzed with the Risk of Ovarian Cancer Algorithm (ROCA) each year. If risk was unchanged and <1:2,000, women returned in a year. If risk increased above 1:500, TVS was undertaken immediately, and if risk was intermediate, CA125 was repeated in 3 months with a further increase in risk above 1:500 prompting referral for TVS. An average of 2% of participants were referred to TVS annually., Results: Thirty-four patients were referred for operations detecting 15 ovarian cancers and two borderline tumors with 12 in early stage (I-II). In addition, seven endometrial cancers were detected with six in stage I. As four ovarian cancers and two borderline tumors were diagnosed with a normal ROCA, the sensitivity for detecting ovarian and borderline cancer was 74% (17 of 23), and 70% of ROCA-detected cases (12 of 17) were in stage I-II. NROSS screening reduced late-stage (III-IV) disease by 34% compared with UKCTOCS controls and by 30% compared with US SEER values. The positive predictive value (PPV) was 50% (17 of 34) for detecting ovarian cancer and 74% (25 of 34) for any cancer, far exceeding the minimum acceptable study end point of 10% PPV., Conclusion: While the NROSS trial was not powered to detect reduced mortality, the high specificity, PPV, and marked stage shift support further development of this strategy.

Pancreatic ductal adenocarcinoma (PDAC) and low-grade ovarian cancer (LGSOC) are characterized by the prevalence of KRAS oncogene mutations. DIRAS3 is the first endogenous non-RAS protein that heterodimerizes with RAS, disrupts RAS clustering, blocks RAS signaling, and inhibits cancer cell growth. Here, we found that DIRAS3-mediated KRAS inhibition induces ROS-mediated apoptosis in PDAC and LGSOC cells with KRAS mutations, but not in cells with wild-type KRAS, by downregulating NFE2L2/Nrf2 transcription, reducing antioxidants, and inducing oxidative stress. DIRAS3 also induces cytoprotective macroautophagy/autophagy that may protect mutant KRAS cancer cells from oxidative stress, by inhibiting mutant KRAS, activating the STK11/LKB1-PRKAA/AMPK pathway, increasing lysosomal CDKN1B/p27 localization, and inducing autophagic gene expression. Treatment with chloroquine or the novel dimeric chloroquine analog DC661 significantly enhances DIRAS3-mediated inhibition of mutant KRAS tumor cell growth in vitro and in vivo. Taken together, our study demonstrates that DIRAS3 plays a critical role in regulating mutant KRAS-driven oncogenesis in PDAC and LGSOC. Abbreviations: AFR: autophagic flux reporter; ATG: autophagy related; CQ: chloroquine; DCFDA: 2'-7'-dichlorodihydrofluorescein diacetate; DIRAS3: DIRAS family GTPase 3; DOX: doxycycline; KRAS: KRAS proto-oncogene, LGSOC: low-grade serous ovarian cancer; MiT/TFE: microphthalmia family of transcription factors; NAC: N-acetylcysteine; PDAC: pancreatic ductal adenocarcinoma; ROS: reactive oxygen species; TFEB: transcription factor EB.

Background: Multiple antigens, autoantibodies (AAb), and antigen-autoantibody (Ag-AAb) complexes were compared for their ability to complement CA125 for early detection of ovarian cancer., Methods: Twenty six biomarkers were measured in a single panel of sera from women with early stage (I-II) ovarian cancers (n = 64), late stage (III-IV) ovarian cancers (186), benign pelvic masses (200) and from healthy controls (502), and then split randomly (50:50) into a training set to identify the most promising classifier and a validation set to compare its performance to CA125 alone., Results: Eight biomarkers detected ≥ 8% of early stage cases at 98% specificity. A four-biomarker panel including CA125, HE4, HE4 Ag-AAb and osteopontin detected 75% of early stage cancers in the validation set from among healthy controls compared to 62% with CA125 alone (p = 0.003) at 98% specificity. The same panel increased sensitivity for distinguishing early-stage ovarian cancers from benign pelvic masses by 25% (p = 0.0004) at 95% specificity. From 21 autoantibody candidates, 3 AAb (anti-p53, anti-CTAG1 and annt-Il-8) detected 22% of early stage ovarian cancers, potentially lengthening lead time prior to diagnosis., Conclusion: A four biomarker panel achieved greater sensitivity at the same specificity for early detection of ovarian cancer than CA125 alone., (© 2024. The Author(s).)

Purpose: To assess the contributions of circulating metabolites for improving upon the performance of the risk of ovarian malignancy algorithm (ROMA) for risk prediction of ovarian cancer among women with ovarian cysts., Experimental Design: Metabolomic profiling was performed on an initial set of sera from 101 serous and nonserous ovarian cancer cases and 134 individuals with benign pelvic masses (BPM). Using a deep learning model, a panel consisting of seven cancer-related metabolites [diacetylspermine, diacetylspermidine, N-(3-acetamidopropyl)pyrrolidin-2-one, N-acetylneuraminate, N-acetyl-mannosamine, N-acetyl-lactosamine, and hydroxyisobutyric acid] was developed for distinguishing early-stage ovarian cancer from BPM. The performance of the metabolite panel was evaluated in an independent set of sera from 118 ovarian cancer cases and 56 subjects with BPM. The contributions of the panel for improving upon the performance of ROMA were further assessed., Results: A 7-marker metabolite panel (7MetP) developed in the training set yielded an AUC of 0.86 [95% confidence interval (CI): 0.76-0.95] for early-stage ovarian cancer in the independent test set. The 7MetP+ROMA model had an AUC of 0.93 (95% CI: 0.84-0.98) for early-stage ovarian cancer in the test set, which was improved compared with ROMA alone [0.91 (95% CI: 0.84-0.98); likelihood ratio test P: 0.03]. In the entire specimen set, the combined 7MetP+ROMA model yielded a higher positive predictive value (0.68 vs. 0.52; one-sided P < 0.001) with improved specificity (0.89 vs. 0.78; one-sided P < 0.001) for early-stage ovarian cancer compared with ROMA alone., Conclusions: A blood-based metabolite panel was developed that demonstrates independent predictive ability and complements ROMA for distinguishing early-stage ovarian cancer from benign disease to better inform clinical decision making., (©2022 American Association for Cancer Research.)

Importance: Despite similar histologic appearance among high-grade serous ovarian cancers (HGSOCs), clinical observations suggest vast differences in gross appearance. There is currently no systematic framework by which to classify HGSOCs according to their gross morphologic characteristics., Objective: To develop and characterize a gross morphologic classification system for HGSOC., Design, Setting, and Participants: This cohort study included patients with suspected advanced-stage ovarian cancer who presented between April 1, 2013, and August 5, 2016, to the University of Texas MD Anderson Cancer Center, a large referral center. Patients underwent laparoscopic assessment of disease burden before treatment and received a histopathologic diagnosis of HGSOC. Researchers assigning morphologic subtype and performing molecular analyses were blinded to clinical outcomes. Data analysis was performed between April 2020 and November 2021., Exposures: Gross tumor morphologic characteristics., Main Outcomes and Measures: Clinical outcomes and multiomic profiles of representative tumor samples of type I or type II morphologic subtypes were compared., Results: Of 112 women (mean [SD] age 62.7 [9.7] years) included in the study, most patients (84% [94]) exhibited a predominant morphologic subtype and many (63% [71]) had a uniform morphologic subtype at all involved sites. Compared with those with uniform type I morphologic subtype, patients with uniform type II morphologic subtype were more likely to have a favorable Fagotti score (83% [19 of 23] vs 46% [22 of 48]; P = .004) and thus to be triaged to primary tumor reductive surgery. Similarly, patients with uniform type II morphologic subtype also had significantly higher mean (SD) estimated blood loss (639 [559; 95% CI, 391-887] mL vs 415 [527; 95% CI, 253-577] mL; P = .006) and longer mean (SD) operative time (408 [130; 95% CI, 350-466] minutes vs 333 [113; 95% CI, 298-367] minutes; P = .03) during tumor reductive surgery. Type I tumors had enrichment of epithelial-mesenchymal transition (false discovery rate [FDR] q-value, 3.10 × 10-24), hypoxia (FDR q-value, 1.52 × 10-5), and angiogenesis pathways (FDR q-value, 2.11 × 10-2), whereas type II tumors had enrichment of pathways related to MYC signaling (FDR q-value, 2.04 × 10-9) and cell cycle progression (FDR q-value, 1.10 × 10-5) by integrated proteomic and transcriptomic analysis. Abundances of metabolites and lipids also differed between the 2 morphologic subtypes., Conclusions and Relevance: This study identified 2 novel, gross morphologic subtypes of HGSOC, each with unique clinical features and molecular signatures. The findings may have implications for triaging patients to surgery or chemotherapy, identifying outcomes, and developing tailored therapeutic strategies.

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Salt-inducible kinase 2 (SIK2; also known as serine/threonine-protein kinase SIK2) is overexpressed in several cancers and has been implicated in cancer progression. However, the mechanisms by which SIK2 regulates cancer cell motility, migration and metastasis in ovarian cancer have not been fully discovered. Here, we identify that SIK2 promotes ovarian cancer cell motility, migration and metastasis in vitro and in vivo. Mechanistically, SIK2 regulated cancer cell motility and migration by myosin light chain kinase, smooth muscle (MYLK)-meditated phosphorylation of myosin light chain 2 (MYL2). SIK2 directly phosphorylated MYLK at Ser343 and activated its downstream effector MYL2, promoting ovarian cancer cell motility and metastasis. In addition, we found that adipocytes induced SIK2 phosphorylation at Ser358 and MYLK phosphorylation at Ser343, enhancing ovarian cancer cell motility. Moreover, SIK2 protein expression was positively correlated with the expression of MYLK-pS343 in ovarian cancer cell lines and tissues. The co-expression of SIK2 and MYLK-pS343 was associated with reduced median overall survival in human ovarian cancer samples. Taken together, SIK2 positively regulates ovarian cancer motility, migration and metastasis, suggesting that SIK2 is a potential candidate for ovarian cancer treatment., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7-associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.

Early detection of ovarian cancer remains an important unmet medical need. Effective screening could reduce mortality by 10%-30%. Used individually, neither serum CA125 nor transvaginal sonography (TVS) is sufficiently sensitive or specific. Two-stage strategies have proven more effective, where a significant rise above a woman's baseline CA125 prompts TVS and an abnormal sonogram prompts surgery. Two major screening trials have documented that this strategy has adequate specificity, but sensitivity for early-stage (I-II) disease must improve to have a greater impact on mortality. To improve the first stage, different panels of protein biomarkers have detected cases missed by CA125. Autoantibodies against TP53 have detected 20% of early-stage ovarian cancers 8 months before elevation of CA125 and 22 months before clinical diagnosis. Panels of autoantibodies and antigen-autoantibody complexes are being evaluated with the goal of detecting >90% of early-stage ovarian cancers, alone or in combination with CA125, while maintaining 98% specificity in control subjects. Other biomarkers, including micro-RNAs, ctDNA, methylated DNA, and combinations of ctDNA alterations, are being tested to provide an optimal first-stage test. New technologies are also being developed with greater sensitivity than TVS to image small volumes of tumor. See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible.", (©2020 American Association for Cancer Research.)

Background: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease., Methods: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2., Results: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling., Conclusions: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered., (© 2020 American Cancer Society.)

Background: Early detection of ovarian cancer could significantly improve patient outcomes. Cancer antigen 125 (CA 125) is elevated in sera from approximately 60% of patients with early-stage (I/II) disease. Sensitivity might be improved through the combination of CA 125 with other biomarkers. Among potential biomarkers, antigen-autoantibody (Ag-AAb) complexes have received relatively little attention., Methods: Luminex-based immunoassays were used to measure human epididymis protein 4 (HE4), anti-HE4 autoantibody, and HE4 Ag-AAb complexes in sera from patients with early- (n = 73) and late-stage ovarian cancers (n = 49) at the time of diagnosis and from asymptomatic women with (n = 15) or without ovarian cancer (n = 212) enrolled in the Normal Risk Ovarian Cancer Screening Study., Results: At 98% specificity for healthy, asymptomatic women, 7% of patients with early-stage (I/II) ovarian cancer and 4% of patients with late-stage (III/IV) disease had elevated levels of HE4 autoantibody, whereas elevated levels of HE4 Ag-AAb complexes were detected in sera from 38% of early-stage cases and 31% of late-stage cases. Complementarity was observed in receiver operating characteristic (ROC) curves between HE4 Ag-AAb complexes and CA 125 levels in early-stage ovarian cancer (P < .001). CA 125 detected 63% of cases, and a combination of CA 125 and HE4 Ag-AAb complexes detected 81%. Complementarity was also observed in ROC curves for an independent validation set with 69 early-stage patients (P = .039). HE4 Ag-AAb complexes were detected in serial preclinical serum samples from women destined to develop ovarian cancer: they correlated with CA 125 but did not provide a lead time., Conclusions: HE4 Ag-AAb complexes could complement CA 125 in detecting a higher fraction of early-stage ovarian cancers., (© 2019 American Cancer Society.)

Background: Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors., Methods: Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line., Results: Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy., Conclusions: PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer., (© 2019 American Cancer Society.)

Purpose: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 ( PFKFB2 ) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo ., Results: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wt TP53 ). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of si PFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cells with wt TP53 , decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma., Conclusions: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wt TP53 ., (©2019 American Association for Cancer Research.)

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease., (© 2019 American Cancer Society.)

Background: Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells., Methods: Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models., Results: Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P < .05) and curing a percentage of mice., Conclusions: Treatment with a combination of anti-VEGF, anti-IL-8, and anti-IGF receptor antibodies prevented the outgrowth of dormant cells and prolonged survival in a preclinical model., (© 2019 American Cancer Society.)

Background: This systematic review will assess the effectiveness of advanced nursing care (ANC) on depression in patients with ovarian cancer (OC)., Methods: We will identify any relevant randomized controlled trial from Cochrane Library, MEDLINE, Embase, Web of Science, Springer, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure from their inceptions to March 5, 2019. The primary outcome includes depression. The secondary outcomes consist of anxiety, quality of life, and adverse events. Data that meets all the eligibility criteria will be extracted, pooled, and analyzed by using RevMan 5.3 software. Methodological quality for each eligible study will be assessed by using Cochrane risk of bias tool., Results: This study will analyze depression, anxiety, quality of life, and adverse events of ANC on depression in patients with OC., Conclusion: The findings of this study will provide the latest evidence for the effectiveness and adverse events of ANC on depression in patients with OC., Ethics and Dissemination: No ethic approval is required for this study, because all the data will be extracted from previous published studies. The results of this study will be presented at conference or will be published at a peer-reviewed journal., Prospero Registration Number: PROSPERO CRD42019126374.

Purpose: Most patients with ovarian cancer receive paclitaxel chemotherapy, but less than half respond. Pre-treatment microtubule stability correlates with paclitaxel response in ovarian cancer cell lines. Microtubule stability can be increased by depletion of individual kinases. As microtubule stability can be regulated by phosphorylation of microtubule-associated proteins (MAPs), we reasoned that depletion of pairs of kinases that regulate phosphorylation of MAPs could induce microtubule stabilization and paclitaxel sensitization. Experimental Design: Fourteen kinases known to regulate paclitaxel sensitivity were depleted individually in 12 well-characterized ovarian cancer cell lines before measuring proliferation in the presence or absence of paclitaxel. Similar studies were performed by depleting all possible pairs of kinases in six ovarian cancer cell lines. Pairs that enhanced paclitaxel sensitivity across multiple cell lines were studied in depth in cell culture and in two xenograft models. Results: Transfection of siRNA against 10 of the 14 kinases enhanced paclitaxel sensitivity in at least six of 12 cell lines. Dual knockdown of IKBKB/STK39 or EDN2/TBK1 enhanced paclitaxel sensitivity more than silencing single kinases. Sequential knockdown was superior to concurrent knockdown. Dual silencing of IKBKB/STK39 or EDN2/TBK1 stabilized microtubules by inhibiting phosphorylation of p38 and MAP4, inducing apoptosis and blocking cell cycle more effectively than silencing individual kinases. Knockdown of IKBKB/STK39 or EDN2/TBK1 enhanced paclitaxel sensitivity in two ovarian xenograft models. Conclusions: Sequential knockdown of dual kinases increased microtubule stability by decreasing p38-mediated phosphorylation of MAP4 and enhanced response to paclitaxel in ovarian cancer cell lines and xenografts, suggesting a strategy to improve primary therapy. Clin Cancer Res; 24(20); 5072-84. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Among the 3 GTPases in the DIRAS family, DIRAS3/ARHI is the best characterized. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26-kDa GTPase that shares 60% homology to RAS and RAP. DIRAS3 is downregulated in many tumor types, including ovarian cancer, where re-expression inhibits cancer cell growth, reduces motility, promotes tumor dormancy and induces macroautophagy/autophagy. Previously, we demonstrated that DIRAS3 is required for autophagy in human cells. Diras3 has been lost from the mouse genome during evolutionary re-arrangement, but murine cells can still undergo autophagy. We have tested whether DIRAS1 and DIRAS2, which are homologs found in both human and murine cells, could serve as surrogates to DIRAS3 in the murine genome affecting autophagy and cancer cell growth. Similar to DIRAS3, these 2 GTPases share 40-50% homology to RAS and RAP, but differ from DIRAS3 primarily in the lengths of their N-terminal extensions. We found that DIRAS1 and DIRAS2 are downregulated in ovarian cancer and are associated with decreased disease-free and overall survival. Re-expression of these genes suppressed growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death. Mechanistically, DIRAS1 and DIRAS2 induce and regulate autophagy by inhibition of the AKT1-MTOR and RAS-MAPK signaling pathways and modulating nuclear localization of the autophagy-related transcription factors FOXO3/FOXO3A and TFEB. Taken together, these data suggest that DIRAS1 and DIRAS2 likely serve as surrogates in the murine genome for DIRAS3, and may function as a backup system to fine-tune autophagy in humans.

Purpose: The TP53 tumor-suppressor gene is mutated in >95% of high-grade serous ovarian cancers. Detecting an autologous antibody response to TP53 that might improve early detection. Experimental Design: An immunoassay was developed to measure TP53 autoantibody in sera from 378 cases of invasive epithelial ovarian cancer and 944 age-matched healthy controls from the United States, Australia, and the United Kingdom. Serial preclinical samples from cases and controls were also assayed from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Results: Using a cutoff value of 78 U/mL to achieve a specificity of 97.4%, TP53 autoantibody was elevated in 30% of 50 cases from MD Anderson, 21.3% of 108 cases from the Australian Ovarian Cancer Study, and 21% of 220 cases from the UKCTOCS. Among 164 cases with rising CA125 detected with the UKCTOCS risk of ovarian cancer algorithm (ROCA), 20.7% had elevated TP53 autoantibody. In cases missed by the ROCA, 16% of cases had elevated TP53 autoantibody. Of the 34 ovarian cancer cases detected with the ROCA, TP53 autoantibody titers were elevated 11.0 months before CA125. In the 9 cases missed by the ROCA, TP53 autoantibody was elevated 22.9 months before cancer diagnosis. Similar sensitivity was obtained using assays with specific mutant and wild-type TP53. Conclusions: TP53 autoantibody levels provide a biomarker with clinically significant lead time over elevation of CA125 or an elevated ROCA value. Quantitative assessment of autoantibodies in combination with CA125 holds promise for earlier detection of invasive epithelial ovarian cancer. Clin Cancer Res; 23(19); 5912-22. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Introduction: Despite advances in surgery and chemotherapy for ovarian cancer, 70% of women still succumb to the disease. Biomarkers have contributed to the management of ovarian cancer by monitoring response to treatment, detecting recurrence, distinguishing benign from malignant pelvic masses and attempting to detect disease at an earlier stage. Areas covered: This review focuses on recent advances in biomarkers and imaging for management of ovarian cancer with particular emphasis on early detection. Relevant literature has been reviewed and analyzed. Expert commentary: Rising or persistent CA125 blood levels provide a highly specific biomarker for epithelial ovarian cancer, but not an optimally sensitive biomarker. Addition of HE4, CA 72.4, anti-TP53 autoantibodies and other biomarkers can increase sensitivity for detecting early stage or recurrent disease. Detecting disease recurrence will become more important as more effective therapy is developed. Early detection will require the development not only of biomarker panels, but also of more sensitive and specific imaging strategies. Effective biomarker strategies are already available for distinguishing benign from malignant pelvic masses, but their use in identifying and referring patients with probable ovarian cancer to gynecologic oncologists for cytoreductive operations must be encouraged.

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer. Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear-centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling. Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC 50 of 0.8 to 2.6 μmol/L, where the IC 50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = -0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip ( P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression. Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945-54. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Background: Autophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown., Methods: We employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging., Results: Induction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [ 18 F]-FDG uptake, and significantly altered choline metabolism., Conclusions: ARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors.

Objectives: Longitudinal multimarker combinations have the potential to improve sensitivity while maintaining the high specificity required for the early detection of ovarian cancer. The use of multiple markers to improve sensitivity over cancer antigen 125 (CA125) in longitudinal algorithms for early ovarian cancer detection requires the selection of markers with optimal discriminatory power and low longitudinal variance relative to disease-initiated changes. Our objective was to identify a multimarker panel suitable for ovarian cancer, where each individual marker has its own baseline, permitting longitudinal algorithm development., Materials and Methods: In this retrospective study, we measured CA125, human epididymis protein 4 (HE4), matrix metalloproteinase-7 (MMP-7), CA72-4, CA19-9, CA15-3, carcinoembryonic antigen, and soluble vascular cell adhesion molecule (sVCAM) concentrations using immunoassays in pretreatment sera from 142 stage I ovarian cancer cases and 5 annual samples each from 217 healthy controls. After random division into training and validation sets, all possible biomarker combinations were explored exhaustively using linear classifiers to identify the panel with the greatest sensitivity for stage I disease at a high specificity of 98%. To evaluate longitudinal performance of the individual markers, the within-person over time and the between-person coefficient of variation (CV) were estimated. Hierarchical modeling across women of log-concentrations enabled the borrowing of information across subjects to moderate variance estimates given the small number of observations per subject., Results: The 4-marker panel comprising CA125, HE4, MMP-7, and CA72-4 performed with the highest sensitivity (83.2%) at 98% specificity. The within-person CVs were lower for CA125, HE4, MMP-7, and CA72-4 (15%, 25%, 25%, and 21%, respectively) compared with their corresponding between-person CV (49%, 20%, 35%, and 84%, respectively) indicating baselines in healthy volunteers. After simple log-transformations, the within-volunteer variation across volunteers was modeled with a normal distribution permitting parsimonious hierarchical modeling., Conclusions: The multiplex panel chosen is suitable for the early detection of ovarian cancer and the individual markers have their own baseline permitting longitudinal algorithm development.

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

NDN is a maternally imprinted gene consistently expressed in normal ovarian epithelium, is dramatically downregulated in the majority of ovarian cancers. Little or no NDN expression could be detected in 73% of 351 epithelial ovarian cancers. NDN was also downregulated in 10 ovarian cancer cell lines with total loss in 6 of 10. Re-expression of NDN decreased Bcl-2 levels and induced apoptosis, which significantly inhibited ovarian cancer cell growth in cell culture and in xenografts. In addition, re-expression of NDN inhibited cell migration by decreasing actin stress fiber and focal adhesion complex formation through deactivation of Src, FAK and RhoA. Loss of NDN expression in ovarian cancers could be attributed to LOH in 28% of 18 informative cases and to hypermethylation of CpG sites 1 and 2 of NDN promoter in 23% and 30% of 43 ovarian cancers, respectively. Promoter hypermethylation was also found in 5 of 10 ovarian cancer cell lines. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored NDN expression in 4 of 7 cell lines with enhanced promoter methylation levels. These observations support the conclusion that NDN is an imprinted tumor suppressor gene which affects cancer cell motility, invasion and growth and that its loss of function in ovarian cancer can be caused by both genetic and epigenetic mechanisms.

Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

microRNAs (miRNAs/miRs) belong to a class of small noncoding RNAs that can negatively regulate messenger RNA (mRNA) expression of target genes. miRNAs are involved in multiple aspects of ovarian cancer cell dysfunction and the phenotype of ovarian cancer cells can be modified by targeting miRNA expression. miRNA profiling has detected a number of candidate miRNAs with the potential to regulate many important biologic functions in ovarian cancer, but their role still needs to be clarified, given the remarkable heterogeneity among ovarian cancers and the context-dependent role of miRNAs. This review summarizes the data collected from The Cancer Genome Atlas (TCGA) and several other genome-wide projects to identify dysregulated miRNAs in ovarian cancers. Copy number variations (CNVs), epigenetic alterations, and oncogenic mutations are also discussed that affect miRNA levels in ovarian disease. Emphasis is given to the role of particular miRNAs in altering expression of genes in human ovarian cancers with the potential to provide diagnostic, prognostic, and therapeutic targets. Particular attention has been given to TP53, BRCA1/2, CA125 (MUC16), HE4 (WFDC2), and imprinted genes such as ARHI (DIRAS3). A better understanding of the abnormalities in miRNA expression and downstream transcriptional and biologic consequences will provide leads for more effective biomarkers and translational approaches in the management of ovarian cancer., (©2014 American Association for Cancer Research.)

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P<0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21-23% of primary ovarian cancers but in 81-84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P<0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.

The initiation of angiogenesis can mark the transition from tumor dormancy to active growth and recurrence. Mechanisms that regulate recurrence in human cancers are poorly understood, in part because of the absence of relevant models. The induction of ARHI (DIRAS3) induces dormancy and autophagy in human ovarian cancer xenografts but produces autophagic cell death in culture. The addition of VEGF to cultures maintains the viability of dormant autophagic cancer cells, thereby permitting active growth when ARHI is downregulated, which mimics the "recurrence" of growth in xenografts. Two inducible ovarian cancer cell lines, SKOv3-ARHI and Hey-ARHI, were used. The expression level of angiogenesis factors was evaluated by real-time PCR, immunohistochemistry, immunocytochemistry and western blot; their epigenetic regulation was measured by bisulfite sequencing and chromatin immunoprecipitation. Six of the 15 angiogenesis factors were upregulated in dormant cancer cells (tissue inhibitor of metalloproteinases-3, TIMP3; thrombospondin-1, TSP1; angiopoietin-1; angiopoietin-2; angiopoietin-4; E-cadherin, CDH1). We found that TIMP3 and CDH1 expression was regulated epigenetically and was related inversely to the DNA methylation of their promoters in cell cultures and in xenografts. Increased H3K9 acetylation was associated with higher TIMP3 expression in dormant SKOv3-ARHI cells, while decreased H3K27me3 resulted in the upregulation of TIMP3 in dormant Hey-ARHI cells. Elevated CDH1 expression during dormancy was associated with an increase in both H3K4me3 and H3K9Ac in two cell lines. CpG demethylating agents and/or histone deacetylase inhibitors inhibited the re-growth of dormant cancer cells, which was associated with the re-expression of anti-angiogenic genes. The expression of the anti-angiogenic genes TIMP3 and CDH1 is elevated during dormancy and is reduced during the transition to active growth by changes in DNA methylation and histone modification.

Ovarian cancer is the fifth most common cancer among women worldwide. Detection of metastasis of ovarian cancer is crucial for diagnosis and prolongs the life of patients. This study focused on whether SNAI1 overexpression relates to invasion of ovarian cancer in vitro and in vivo. Invasion, colony formation and wound healing assays and flow cytometric analysis were performed to test the invasion and proliferation of SKOV3 ovarian cancer cells after transfection. The effect of SNAI1 on ovarian cancer in vivo was validated using a murine xenograft model. In vitro, SNAI1 upregulation led to an increased percent of CD133+ SKOV3 cells and promoted SKOV3 cell invasion and proliferation. In vivo, the SNAI1 overexpression group showed the highest rate of tumor growth compared with SNAI2 and the control group (60 and 50%, respectively). Our results show that SNAI1 expression induces an increase in the number of CD133+ cells, a change important for the epithelial to mesenchymal transition and the proliferation in ovarian cancer. It is suggested that SNAI1 may serve as a novel target for ovarian cancer prediction and therapy.

Background: Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up-regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re-expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts., Methods: Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5-aza-20-deoxycytidine [DAC] or 5-azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamicacid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re-expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate-binding Di-RAS-like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model., Results: The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re-expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC-induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo., Conclusions: A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re-expression of imprinted tumor suppressor genes., (Cancer © 2011 American Cancer Society.)

Background: Dasatinib, an inhibitor of Src/Abl family kinases, can inhibit tumor growth of several solid tumors. However, the effect and mechanism of action of dasatinib in human ovarian cancer cells remains unknown., Methods: Dasatinib-induced autophagy was determined by acridine orange staining, punctate localization of GFP-LC3, LC3 protein blotting, and electron microscopy. Significance of beclin 1, AKT, and Bcl-2 in dasatinib-induced autophagy and growth inhibition was assayed by small interfering RNA (siRNA) silencing and/or overexpression of the gene of interest., Results: Dasatinib inhibited cell growth by inducing little apoptosis, but substantial autophagy in SKOv3 and HEY ovarian cancer cells. In vivo studies showed dasatinib inhibited tumor growth and induced both autophagy and apoptosis in a HEY xenograft model. Knockdown of beclin 1 and Atg12 expression with their respective siRNAs diminished dasatinib-induced autophagy, whereas knockdown of p27Kip1 with specific siRNAs did not. Small hairpin RNA knockdown of beclin 1 expression reduced dasatinib-induced autophagy and growth inhibition. Dasatinib reduced the phosphorylation of AKT, mTOR, p70S6K, and S6 kinase expression. Constitutive expression of AKT1 and AKT2 inhibited dasatinib-induced autophagy in both HEY and SKOv3 cells. Dasatinib also reduced Bcl-2 expression and activity. Overexpression of Bcl-2 partially prevented dasatinib-induced autophagy., Conclusions: Dasatinib induces autophagic cell death in ovarian cancer that partially depends on beclin 1, AKT, and Bcl-2. These results may have implications for clinical use of dasatinib., (Copyright © 2010 American Cancer Society.)

Regulators of mitosis have been successfully targeted to enhance response to taxane chemotherapy. Here, we show that the salt inducible kinase 2 (SIK2) localizes at the centrosome, plays a key role in the initiation of mitosis, and regulates the localization of the centrosome linker protein, C-Nap1, through S2392 phosphorylation. Interference with the known SIK2 inhibitor PKA induced SIK2-dependent centrosome splitting in interphase while SIK2 depletion blocked centrosome separation in mitosis, sensitizing ovarian cancers to paclitaxel in culture and in xenografts. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher expression of SIK2 significantly correlated with poor survival in patients with high-grade serous ovarian cancers. We believe these data identify SIK2 as a plausible target for therapy in ovarian cancers., (2010 Elsevier Inc. All rights reserved.)

Objective: To study the clinicopathologic features of granulocytic sarcoma., Methods: The clinical and pathologic findings of 38 cases of granulocytic sarcoma were retrospectively analyzed. Immunohistochemical study was performed and the literature was reviewed., Results: The age of patients ranged from 2 to 77 years (mean = 43.3 years). The male-to-female ratio was 1.5:1. Major clinical presentations included superficial lymph node enlargement and painful soft tissue mass. Follow-up data were available in 18 patients; and 14 of them died of tumor-related diseases. The average duration of survival of the patients was 16.9 months. Histologically, the tumor cells were relatively uniform in appearance and small to medium in size. The cytoplasm was scanty and pale in color. The nuclei were round or focally irregular, with fine chromatin and inconspicuous nucleoli. Mitosis figures were readily identified. Scattered immature eosinophilic myelocytes were seen. Immunohistochemical study showed that the tumor cells in all cases expressed MPO and CD43. Most cases were also positive for CD68, lysozyme, CD99 and TdT. The staining for CD3, CD20, CD79a, pan-cytokeratin and PLAP were negative., Conclusions: Granulocytic sarcoma is a known histologic mimicker of non-Hodgkin lymphoma, Ewing sarcoma/PNET and embryonal rhabdomyosarcoma. Detailed morphologic examination, when coupled with immunohistochemical study, is useful in arriving at a correct diagnosis.

Currently, an effective gene therapy strategy, which not only retains cancer-specific expression but also limits toxicity, has yet to be developed for ovarian cancer. Mounting reports over the years have shown that human telomerase activity is significantly elevated in cancer cells compared with normal cells. In this study, we evaluated the human telomerase reverse transcriptase (hTERT; T) promoter and showed that it can direct target gene expression preferentially in ovarian cancer cells. However, its promoter (T) activity is much lower than that of cytomegalovirus (CMV), a commonly used nonspecific promoter. To overcome this problem, we have integrated the T promoter into our recently developed VP16-Gal4-WPRE integrated systemic amplifier (VISA) system and dramatically enhanced transgene expression. In addition, to further develop this cancer-specific promoter gene expression system into an applicable therapeutic vector, we expressed E1A (an adenoviral type 5 transcription factor that possesses anticancer properties) through this novel VISA platform. We showed that the T-VISA system specifically targeted the expression of E1A to ovarian cancer cells at a level greater than or comparable with the commonly used CMV promoter, yet remained nearly silent in normal cells, thus making this a suitable gene therapy construct. By using this cancer-specific promoter that limits target gene expression in normal cells/tissues, potential toxicity induced by the CMV promoter would be prevented. More importantly, we showed significant antitumor activity with much less toxicity in animal models through i.v. delivery of T-VISA-E1A:liposomal nanoparticles, suggesting a promising role of T-VISA-E1A for ovarian cancer treatment under a gene therapy setting.

Cancer antigen 125 (CA125) is an antigen that is elevated in the serum of women with ovarian carcinoma, but can also be detected in serum from healthy women. CA125 is expressed in 80% of human ovarian cancers, as well as in normal adult endometrium, lung, and amnion. The gene encoding human CA125 was identified as MUCIN16 (MUC16). A database search identified the orthologous mouse gene, Muc16. Reverse transcription-polymerase chain reaction and RNA in situ hybridization detected Muc16 transcripts in the surface epithelia of the upper respiratory tract, the mesothelia lining body cavities and the internal organs, as well as male and female reproductive organs, and the amnion. Antibodies raised against human MUC16 do not recognize mouse MUC16. Therefore, a rabbit anti-mouse polyclonal antibody against recombinant mouse MUC16 was generated. Immunohistochemistry using this anti-mouse MUC16 antibody revealed expression in the luminal epithelia of the trachea, the epithelia of the secretory glands in the oral cavity, the surface of the olfactory epithelia, as well as mesothelial cells lining body cavities (i.e., pleural, peritoneal, and pelvic cavities), and male and female reproductive organs. In addition, MUC16 protein was detected in other cell types, such as the surface epithelia of the cochlear duct and chief cells of the stomach, suggesting multiple roles for MUC16. In mouse serous epithelial ovarian cancer, MUC16 protein was detected at the apical surface of well-differentiated tumors, but not poorly differentiated tumors. These findings document the presence of MUC16 in murine ovarian cancer and in normal tissues and provide a foundation for future functional studies.

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

Background: Imprinted tumor suppressor genes may be particularly important in the pathogenesis of ovarian cancer. Two imprinted genes, paternally expressed 3 (PEG3) and aplasia Ras homologue member I (ARHI), are the most frequently down-regulated in ovarian cancers on gene expression arrays., Methods: PEG3 and ARHI expression levels were evaluated with real-time reverse-transcriptase polymerase chain reaction (PCR) analysis. Promoter methylation was measured by pyrosequencing, and loss of heterozygosity (LOH) was detected by PCR-LOH assays., Results: PEG3 was down-regulated in 75% and ARHI was down-regulated in 88% of 40 ovarian cancers. ARHI CpG islands I and II were hypermethylated in 13 of 42 ovarian cancers (31%) and in 5 of 42 ovarian cancers (12%), respectively, and hypermethylation was associated with reduced ARHI expression in all 18 samples of ovarian cancer with CpG island hypermethylation. PEG3 was hypermethylated in 11 of 42 ovarian cancers (26%), and PEG3 expression was down-regulated in 10 of those 11 cancers. LOH was detected in 8 of 35 informative cases for ARHI (23%) and in 5 of 25 informative cases for PEG3 (20%). PEG3 and ARHI expression was highly correlated in human ovarian cancers (correlation coefficient [R]=0.69; P< .0001). PEG3 and ARHI also were methylated concordantly in ovarian cancers (R=0.36; P= .019). Re-expression of PEG3, similar to that of ARHI, markedly inhibited ovarian cancer growth. ARHI and PEG3 expression could be restored by treatment with 5-aza-2'-deoxycytidine and trichostatin A, consistent with the importance of promoter methylation and histone acetylation in regulating expression of both genes., Conclusions: Loss of expression of the growth-inhibitory imprinted genes ARHI and PEG3 through promoter methylation, LOH, and other mechanisms may stimulate clonogenic growth and contribute to the pathogenesis of a majority of ovarian cancers.

Objectives: Early detection of ovarian cancer should improve overall survival. Multiple serum markers have been evaluated as possible tests to detect early stage disease, but few urine markers have been studied. Mesothelin has been detected in serum from patients with ovarian cancer, but has not been previously reported in urine., Methods: Mesothelin was assayed in the serum and in the urine from 28 patients with early stage (I/II) invasive epithelial ovarian cancers, 111 with advanced stage (III/IV) invasive disease and 19 with tumors of low malignant potential. Marker values have been compared to those in healthy controls and 115 patients with benign pelvic masses. Thresholds were set to include 95% of mesothelin values for 127 sera and 89 urines from healthy women. Urine values were considered: (1) as assayed; (2) normalized using the ratio of serum to urine creatinine; and (3) normalized using the Cockroft-Gault formula for glomerular filtration rate (GFR). Urines were also assayed for human chorionic gonadotropin (hCG) free beta subunit and beta subunit core fragment and similarly normalized., Results: Optimal sensitivity for early stage disease was obtained when urine mesothelin was normalized using GFR. A greater fraction of patients with early stage disease was detected with the mesothelin urine assay (42%) than with the serum assay (12%). Similarly, 75% of patients with advanced ovarian cancer had elevated mesothelin in urine compared to 48% in serum. Serum and urine levels of mesothelin correlated for early (p=0.02) and late (p<0.001) disease. Urine mesothelin exhibited greater sensitivity for early stage ovarian cancer than did hCG free beta subunit or beta subunit core fragment and complementarity was not observed., Conclusion: Urine mesothelin deserves further evaluation as a biomarker for detection of early stage ovarian cancer in combination with other urinary markers.

Epithelial ovarian cancer is neither a common nor a rare disease. In the United States, the prevalence of ovarian cancer in postmenopausal women (1 in 2,500) significantly affects strategies for prevention and detection. If chemoprevention for ovarian cancer were provided to all women over the age of 50, side effects would have to be minimal in order to achieve an acceptable ratio of benefit to risk. This ratio might be improved by identifying subsets of individuals at increased risk or by bundling prevention of ovarian cancer with treatment for other more prevalent conditions. Approximately 10% of ovarian cancers are familial and relate to mutations of BRCA1, BRCA2, and mismatch repair genes. More subtle genetic factors are being sought in women with apparently sporadic disease. Use of oral contraceptive agents for as long as 5 years decreases the risk of ovarian cancer in later life by 50%. In one study, fenretinide (4-HPR) delayed development of ovarian cancer in women at increased risk of developing breast and ovarian cancer. Accrual to confirmatory studies has been prohibitively slow and prophylactic oophorectomy is recommended for women at increased genetic risk. Vaccines may have a role for prevention of several different cancers. Breast and ovarian cancers express mucins that could serve as targets for vaccines to prevent both cancers. Early detection of ovarian cancer requires a strategy with high sensitivity (> 75% for stage I disease) and very high specificity (> 99.6%) to achieve a positive predictive value of 10%. Transvaginal sonography (TVS) has achieved these values in some studies, but is limited by the cost of annual screening in a general population. Two-stage strategies that incorporate both serum markers and TVS promise to be more cost-effective. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risks for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the United Kingdom that will critically test the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125. Recent candidates include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s) and soluble EGF receptor. Proteomic approaches have been used to define a distinctive pattern of peaks on mass spectroscopy or to identify a limited number of critical markers that can be assayed by more conventional methods. Several groups are placing known markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

Purpose: ARHI expression is lost or markedly down-regulated in the majority of ovarian cancers. The mechanism by which ARHI is down-regulated in ovarian cancers is still not clear. Our previous reports indicated that ARHI promoter activity was reduced in ovarian cancer cells, due in part to the effects of negative regulatory transcription factor(s)., Experimental Design and Results: We now show that E2F1 and E2F4, but not E2F2, E2F3, or E2F5, bind to the ARHI promoter and repress its activity in ovarian cancer cells. Consistent with this observation, immunochemical staining of cell lines and of 364 samples of ovarian cancer tissue show that the expression of E2F1 and E2F4 proteins is much higher in ovarian cancer cells than in normal ovarian epithelial cells, and that increased expression of E2Fs was negatively correlated with ARHI expression (P < 0.05). Mutation of the putative E2F binding site in the ARHI promoter reversed this inhibitory effect and significantly increased ARHI promoter activity. In addition to the effects of transcriptional regulation, ARHI mRNA also exhibited a significantly reduced half-life in ovarian cancer cells when compared with that in normal ovarian epithelial cells (P < 0.01), suggesting posttranscriptional regulation of ARHI expression. ARHI mRNA contains AU-rich elements (ARE) in the 3'-untranslated region. We have found that these AREs interact with HuR, an ARE-binding protein that stabilizes bound mRNAs, possibly contributing to the rapid turnover of ARHI mRNA. Finally, reduced HuR ARE binding activity was observed in ovarian cancer cells when compared with normal ovarian surface epithelium., Conclusions: Taken together, our data suggest that ARHI expression is regulated at both the transcriptional and the posttranscriptional levels, contributing to the dramatic decrease in ARHI expression in ovarian cancers.

ARHI is a maternally imprinted tumor suppressor gene that is downregulated in 60% of ovarian and breast cancers. Loss of ARHI expression is associated with tumor progression in breast cancer and decreased disease-free survival in ovarian cancer. ARHI encodes a 26-kDa protein with 55-62% homology to Ras and Rap. In contrast to Ras, ARHI inhibits growth, motility, and invasion. ARHI contains a unique 34 amino-acid extension at its N-terminus and differs from Ras in residues critical for GTPase activity and for its putative effector function. Deletion of ARHI's unique N-terminal extension markedly reduces its inhibitory effect on cell growth. The gene maps to chromosome 1p31 at a site of LOH in 40% of ovarian and breast cancers. Mutations have not been detected, but the remaining allele is silenced by methylation in approximately 10-15 % of cases. In the remaining cancers, ARHI is downregulated by transcriptional mechanisms that involve E2F1 and E2F4, as well as by the loss of RNA binding proteins that decrease the half-life of ARHI mRNA. Transgenic expression of human ARHI in mice produces small stature, induces ovarian atrophy, and prevents postpartum milk production. Reexpression of ARHI in cancer cells inhibits signaling through Ras/Map and PI3 kinase, upregulates P21(WAF1/CIP1), downregulates cyclin D1, induces JNK, and inhibits signaling through STAT3. Marked overexpression of ARHI with a dual adenoviral vector induces caspase-independent, calpain-dependent apoptosis. When ARHI is expressed from a doxycycline-inducible promoter at more physiological levels, autophagy is induced, rather than apoptosis. Growth of ovarian and breast cancer xenografts is reversibly suppressed by ARHI, but expression of the NTD mutant produced only a limited inhibitory effect on growth of xenografts.

A Ras homologue member I (ARHI) is a novel imprinted tumor suppressor gene whose expression is frequently lost in breast and ovarian cancers. This small GTP-binding protein is a member of the Ras superfamily with significant homology to both Ras and Rap. Unlike the Ras oncogene, however, ARHI inhibits tumor cell growth. To elucidate the mechanisms by which ARHI inhibits cancer growth, we screened a human breast epithelial cell cDNA library using a yeast two-hybrid system for ARHI-interacting proteins. ARHI was found to interact with signal transducers and activators of transcription (STAT) 3, a latent transcription factor that transduces signals from the cell surface to the nucleus and activates gene transcription. STAT3 is frequently phosphorylated and activated in breast and ovarian cancers, where cytokines and growth factors up-regulate STAT3 and stimulate proliferation. The ARHI-STAT3 interaction was confirmed by coimmunoprecipitation in mammalian cells and shown to be specific for STAT3 but not STAT1 or STAT5a. When ARHI and STAT3 were coexpressed in SKOv3 cells, ARHI formed a complex with STAT3 in the cytoplasm and prevented interleukin-6-induced STAT3 accumulation in the nucleus. ARHI markedly reduced STAT3 binding to DNA and STAT3-dependent promoter activity while only moderately affecting STAT3 phosphorylation. Deletion of the NH2 terminus of ARHI significantly compromised its inhibitory activity, suggesting that this unique NH2-terminal extension contributes to ARHI's inhibition of STAT3-mediated transcriptional activity. Thus, the physical association between STAT3 and ARHI as well as the functional inhibition of STAT3 transcriptional activity by ARHI suggests a novel mechanism through which a putative tumor suppressor gene can inhibit STAT3 activity in breast and ovarian cancers.