Therapeutic Target For Ovarian Cancer Research Articles

LncRNA SNHG17 promotes proliferation and invasion of ovarian cancer cells through up-regulating FOXA1.

This study was designed to investigate the specific mechanism through which long non-coding RNA (lncRNA) SNHG17 promotes the proliferative capacity and invasiveness of ovarian tumor cells. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) detected the expressions of SNHG17 and FOXA1 in 30 pairs of ovarian cancer tissue specimens and corresponding adjacent ones. Meanwhile, in ovarian cancer cell lines (A2780, OVCAR3, SKOV3, CAOV3) and normal ovarian epithelial cell line (IOSE80), SNHG17 and FOXA1 mRNA levels were also examined. In in vitro experiment, si-SNHG17, si-FOXA1, and their corresponding negative controls were transfected into ovarian cancer cell lines, respectively. After that, Cell Counting Kit-8 (CCK-8) and plate cloning experiments were carried out to examine cell proliferation ability, while transwell assay was performed for cell invasiveness detection. Lastly, the interplay between SNHG17 and FOXA1 was further assessed via qRT-PCR and Western blot. qRT-PCR results indicated that SNHG17 expression was remarkably enhanced in ovarian cancer tissue samples compared with that in adjacent ones. In addition, ovarian cancer cells also contained higher expression of SNHG17 than the normal ovarian epithelial cells. However, down-regulating SNHG17 attenuated the cell proliferation and invasive ability. At the same time, compared with that in adjacent tissue samples, FOXA1 also showed a higher expression in ovarian cancer tissues, which was positively correlated with SNHG17. Silencing SNHG17 markedly downregulated FOXA1 expression at both mRNA and protein levels. Furthermore, downregulation of FOXA1 expression was found to be able to inhibit cell proliferation and invasion as well. LncRNA SNHG17 can promote ovarian tumor cell proliferative ability and invasiveness by upregulating FOXA1, and serve as a potential therapeutic target for ovarian cancer.

Clinical Significance of miR-575 in Ovarian Cancer and Its Effect on Ovarian Cancer Cells

Ovarian cancer (OC) is a common form of gynecological cancer that increases in incidence annually, seriously threatening the health and safety of patients. The critical role of miR-575 in multiple neoplastic diseases has already been well established. We suspected that miRNA-575 may be strongly associated with the occurrence of OC and may be the key to OC diagnosis and treatment in the future. To test this hypothesis, we selected 68 OC patients (research group, or RG) who were admitted to our hospital and 60 concurrent healthy controls (control group, or CG) as the study participants. Human OC cell lines A2780, OVCAR-3, and SK-OV-3, and human normal ovarian epithelial cells IOSE80 were used in this study. Nanoparticle-assisted polymerase chain reaction (Nano-PCR), was used to identify miRNA-575 expression and its diagnostic implications in OC. miRNA-575 expression in OC cells and its influence on OC cell biological behaviors were investigated, and the correlation between miR-575 and the phosphatase and tensin homolog (PTEN) was discussed. The results revealed a highly expressed miRNA-575 in OC. Detection of biological behaviors was conducted after transfection of miRNA-575 into OC cells. Cell proliferation and invasion in the mimics-miRNA-575 group was statistically enhanced compared to the negative control (NC) and ininhibition-miRNA-575 groups, and the apoptosis and apoptosis protein count significantly declined (P < 0.05). Subsequently, PTEN was observed to present a lower expression in OC cells. Online target gene analysis website (http://www.targetscan.org/vert_72/) identified the related sites that could bind miR-575 and PTEN. PTEN protein detection in the mimics-miR-575, ininhibition-miR-575, and NC groups showed that the PTEN protein expression in the mimics-miR-575 group was reduced in comparison with that of the other groups, while that for ininhibition-miR-575 was elevated compared to that of the NC group (P < 0.05). Therefore, we came to the conclusion that a high expression of miR-575 can bolster OC cell proliferation and invasion and inhibit apoptosis, while the impact of miR-575 on OC cells may be achieved via targeting the PTEN protein. miR-575 presents important clinical applications in OC diagnosis as an underlying therapeutic target of OC and is a major breakthrough toward future OC research.

Abstract 1285: Functional analysis of ZFHX4 as a novel therapeutic target in ovarian cancer

Abstract [Introduction] Ovarian cancer is one of the most lethal gynecologic malignancies. More than half of the patients are diagnosed in advanced stages of disease where the prognosis is poor due to the inevitable acquired resistance to platinum agents. Although maintenance therapy with PARP inhibitors or bevacizumab has been proven to improve patient survival, platinum sensitivity is still the most important prognostic factor. Thus, novel therapeutic strategies effective for platinum-resistant cases or to overcome platinum-resistance are required. Zinc Finger Homeobox 4 (ZFHX4) has been known as one of the major transcription regulators in nerve and muscle differentiation. Although not fully elucidated, recent researches including our genome-wide exploration indicate ZFHX4 is also involved in carcinogenesis and cancer progression. The objective of this study was to investigate the potential of ZFHX4 as a novel therapeutic target in ovarian cancer by analyzing clinical database and cell model experiments. [Methods] Correlations between ZFHX4 mRNA expression and overall survival in 61 ovarian cancer cases treated at our institute and in the cases registered in TCGA database respectively were assessed by log-lank test. RNAi knockdown was introduced to several ovarian cancer cells to investigate the influence of ZFHX4 on cancer cell proliferation. Scratch assay was performed to assess the effect of RNAi knockdown on cell migration ability. Cells were also treated with cisplatin after 72 hours of siRNA transfection to assess the influence of ZHFX4 on platinum sensitivity. [Results] Both in our clinical dataset and TCGA data, the overall survival was significantly poor in the group in which ZFHX4 is highly expressed. In cell model assay, ZFHX4 knockdown significantly suppressed ovarian cancer cell proliferation. Decreased motility was observed in the cells treated with siRNA targeting ZFHX4 by scratch assay. Furthermore, ZFHX4 knockdown increased cisplatin sensitivity in ovarian cancer cells. [Conclusions] Altogether, our results suggest ZFHX4 functions to promote cancer cell proliferation, migration and platinum-resistance. Interruption of ZFHX4 function independently or in combination with platinum agents has a potential to bring benefit for the treatment of ovarian cancer. Citation Format: Yoshiko Oyama, Shogo Shigeta, Keita Tsuji, Hideki Tokunaga, Muneaki Shimada, Nobuo Yaegashi. Functional analysis of ZFHX4 as a novel therapeutic target in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1285.

Abstract 3052: Targeting AXL in PIK3R2-amplified ovarian cancer

Abstract The p85 regulatory subunit and its associated p110 catalytic subunit are components of the class IA PI3K that mediates the downstream signal of receptor tyrosine kinases. PIK3R2 (encoding p85β, one of the p85 isoforms) is commonly amplified in ovarian cancer patients. Importantly, high PIK3R2 expression significantly correlated with worse patient survival. Here, we showed that p85β promoted tumorigenic properties of ovarian cancer cells. The protein expression of a receptor tyrosine kinase AXL was positively regulated by p85β. AXL is a member of the TAM receptor family. The regulation was specific to AXL because the other two members (Tyro3 and MERTK) were not altered by p85β. Drug sensitivity assays suggested that inhibition of AXL reduced oncogenic activities of p85β in vitro and in vivo. Together, we propose p85β as a potential therapeutic target in ovarian cancer and therapeutic blockade of AXL signaling may be an effective strategy for ovarian cancer with PIK3R2 amplification. Citation Format: Ling Rao, Victor C. Mak, Yuan Zhou, Dong Zhang, Xinran Li, Yiling Lu, Gordon B. Mills, Lydia W. Cheung. Targeting AXL in PIK3R2-amplified ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3052.

Abstract 5044: Integrin-linked kinase gene knockdown results in decreased growth of ovarian cancer xenograft models

Abstract Integrin-linked kinase (ILK) is overexpressed in ovarian cancer cells. In the present study, we sought to assess the effect of ILK silencing on ovarian cancer cell proliferation using shRNA. Our lab previously demonstrated that phosphorylation of ILK acts as a proliferative signal in a variety of ovarian cancer cell lines. Cytotoxicity of ILK knockdown using shRNA was analyzed in vitro using SKOV3 cell lines. Transfected cells were incubated in Incucyte®, which allowed for simultaneous assessment of viable cells at the end of the incubation period using CellTiter glo assay. Knockdown of ILK using shRNA significantly inhibited the proliferation of SKOV3 cells. SKOV3 cells were then infected with lentivirus expressing ILK shRNA to obtain stable knockdown of ILK, which was confirmed with Western blot. Both parental and ILK shRNA infected cells were injected subcutaneously in NOD SCID Gamma immunocompromised mice. Tumor growth was assessed by sequential tumor measurements and final weights. Xenograft data indicated significantly decreased tumor volume over multiple time points in the transfected cells (p <0.0001) (Figure 1). Average final volume for the ILK-shRNA group (n = 15) and control group (n = 13) was 660mm3 (SD = 366.7, range 271 - 1667 mm3) and 1165 mm3 (SD = 668.7, range 506 - 2664 mm3), respectively. Similarly, average tumor weight was less for the ILK-shRNA group (404.4mg [range 226.1 - 801mg]) relative to control (584.3mg [range 293.2 - 1033.4mg]). Our findings demonstrate that knockdown of ILK in ovarian cancer cell lines results in decreased cell proliferation, as well as decreased tumor growth in xenograft models. This supports the role for further investigation into ILK as a potential therapeutic target in ovarian cancer. Days From Tumor Implantation1214161921232628313438VectorAverage Tumor Volume (mm^3)142.4151.6203274.7379.3482.1671.4741.2845.11083.81164.6Standard Error26.983.430.835.759.682112.3110.7133.9172.2185.5ILK-shRNAAverage Tumor Volume (mm^3)45.9107.282.4113150.9177263.4345.2397.3568.8659.9Standard Error6.39.99.915.225.822.841.558.455.19994.7 Citation Format: Tiffany Redfern, Benjamin Wilson, Michael Ulm, Adam ElNaggar, Suriyan Ponnusamy, Yinan Wang, Sarah Asemota, Ramesh Narayanan. Integrin-linked kinase gene knockdown results in decreased growth of ovarian cancer xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5044.

Tumor Microenvironment in Ovarian Cancer: Function and Therapeutic Strategy.

Ovarian cancer is one of the leading causes of death in patients with gynecological malignancy. Despite optimal cytoreductive surgery and platinum-based chemotherapy, ovarian cancer disseminates and relapses frequently, with poor prognosis. Hence, it is urgent to find new targeted therapies for ovarian cancer. Recently, the tumor microenvironment has been reported to play a vital role in the tumorigenesis of ovarian cancer, especially with discoveries from genome-, transcriptome- and proteome-wide studies; thus tumor microenvironment may present potential therapeutic target for ovarian cancer. Here, we review the interactions between the tumor microenvironment and ovarian cancer and various therapies targeting the tumor environment.

CircKIF4A sponges miR-127 to promote ovarian cancer progression.

Ovarian cancer is a major gynecologic cancer and common cause of gynecologic cancer death worldwide. However, the molecular mechanisms of ovarian cancer progression are still unclear. circular RNAs (circRNAs) are recently reported to be involved in cancer progression regulation but the potential functions of circRNAs in ovarian cancer remains unknown. In this study, we explored the expression of circKIF4A in ovarian cancer tissues. Then, a series of experiments were conducted to investigate how circKIF4A functioned in ovarian cancer in vitro and in vivo. The results revealed that circKIF4A was highly expressed in ovarian cancer tissues. Knockdown of circKIF4A suppressed cell proliferation and migration in ovarian cancer. Subsequent mechanism study revealed that circKIF4A acted as a competitive endogenous RNA (ceRNA) to promoted ovarian cancer progression by sponging miR-127 and upregulated the expression of Junctional adhesion molecule 3 (JAM3). Therefore, circKIF4A could be a novel biomarker and therapeutic target for ovarian cancer.

Long non‑coding RNA NEAT1 modifies cell proliferation, colony formation, apoptosis, migration and invasion via the miR‑4500/BZW1 axis in ovarian cancer.

Ovarian cancer (OC) is a frequently occurring malignant tumor in women. Increasing evidence has indicated that long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) participates in OC pathogenesis. Thus, the aim of the present study was to explore the function of NEAT1 during OC progression. The expression levels of NEAT1, microRNA (miR)-4500 and basic leucine zipper and W2 domain-containing protein 1 (BZW1) were assessed via reverse transcription-quantitative PCR and western blotting. Furthermore, cell proliferation, colony formation, apoptosis, migration and invasion were assessed using Cell-Counting Kit 8, colony formation, flow cytometry and Transwell assays, respectively. Cell glycolysis was analyzed using an XF96 metabolic flux analyzer, and the relationship between miR-4500 and NEAT1 or BZW1 was verified via dual-luciferase reporter and RNA binding protein immunoprecipitation assays. miR-4500 expression levels were low, whereas NEAT1 expression levels were high in OC tissues and cell lines compared with control tissues and cell lines. Moreover, the results indicated that NEAT1 was a sponge of miR-4500, which directly targeted BZW1. NEAT1 knockdown induced OC cell apoptosis, and inhibited OC cell proliferation, colony formation, migration, invasion and glycolysis. miR-4500 inhibitor reversed NEAT1 knockdown-mediated effects. Similarly, miR-4500 mimic-mediated effects on cell functions were reversed by BZW1 overexpression. In addition, the results indicated that BZW1 expression was regulated by NEAT1 and miR-4500. Collectively, the present study suggested that NEAT1 modulated cell proliferation, colony formation, apoptosis, migration, invasion and glycolysis via the miR-4500/BZW1 axis in OC; therefore, NEAT1 may serve as a therapeutic target for OC.

Role of M2-like macrophages in the progression of ovarian cancer

In this study, we noninvasively assessed whether M2-like macrophages accelerate the progression of ovarian cancer by performing molecular imaging of ovarian cancer cells expressing enhanced firefly luciferase (Effluc) in living mice. First, murine ovarian cancer ID8 cells expressing Effluc (ID8/Effluc cells) were established by retroviral infection. Subsequently, macrophages were isolated from the peritoneal exudate of mice injected with thioglycollate medium and differentiated into M2-like macrophages by adding interleukin 4. To characterize these M2-like macrophages, F4/80 and cluster of differentiation 206 expression levels were determined. Then, the M2-like macrophages were co-cultured with the ID8/Effluc cells and bioluminescence imaging (BLI) of signals from the ID8/Effluc cells was completed. Additionally, migration and wound healing were assessed to evaluate the effects of conditioned medium (CM) from M2-like macrophages on ID8/Effluc cell motility. In the in vivo study, mice were first given either liposome–phosphate-buffered saline or liposome-clodronate (lipo-clodronate). After 24 h, ID8/Effluc cells were intraperitoneally injected into the mice and BLI was completed at the designed time points. Next, histological analysis was conducted to characterize the infiltrated tumor. Flow cytometric analysis revealed high levels of CD206 expression in the differentiated M2-like macrophages. Meanwhile, ID8/Effluc cells co-cultured with these M2-like macrophages proliferated rapidly in an M2-like macrophage, number-dependent manner. The migration of the ID8/Effluc cells was also increased by the application of CM from M2-like macrophages. In vivo BLI revealed that the growth rate of intraperitoneally injected ovarian cancer cells was inhibited following macrophage depletion by treatment with lipo-clodronate. M2-like macrophages accelerated the progression of ovarian cancer, suggesting they are a new therapeutic target for ovarian cancer and that ovarian cancer could be managed by altering the nature of communication between ovarian cancer and macrophages.

Multi-kinase targeted therapy as a promising treatment strategy for ovarian tumors expressing sfRon receptor.

The sfRon kinase is an important therapeutic target in ovarian cancer that contributes to prominent tumor growth and disease progression. We reasoned that a multi-kinase inhibition of sfRon pathway might be an effective strategy to achieve a sustained anti-tumor response, while simultaneously preventing treatment resistance. We performed a detailed dissection of sfRon signaling in vitro and demonstrated that S6K1 is a key component of a multi-kinase targeting strategy in sfRon expressing ovarian tumors. We selected AD80 compound that targets several kinases within sfRon pathway including AKT and S6K1, and compared its efficacy with inhibitors that selectively target either sfRon or PI3 kinase. Using human ovarian xenografts and clinically relevant patient-derived xenografts (PDXs), we demonstrated that in vivo treatment with single agent AD80 shows superior efficacy to a standard-care chemotherapy (cisplatin/paclitaxel), or to the direct inhibition of sfRon kinase by BMS777607. Our findings indicate that ovarian tumors expressing sfRon are most effectively treated with multi-kinase inhibitors simultaneously targeting AKT and S6K1, such as AD80, which results in long-term anti-tumor response and prevents metastasis development.

Deubiquitinase USP35 restrains STING-mediated interferon signaling in ovarian cancer.

Ovarian cancer is the most lethal malignant tumor of female reproductive system. It is well-known that induction of STING-mediated type I interferons can enhance the resultant antitumor activity. However, STING pathway is usually inactivated in cancer cells at multiple levels. Here, we identified deubiquitinase USP35 is upregulated in ovarian cancer tissues. High level of USP35 was correlated with diminished CD8+ T cell infiltration and poor prognosis in ovarian cancer patients. Mechanistically, we found that silencing USP35 reinforces the activation of STING-TBK1-IRF3 pathway and promotes the expression of type I interferons. Our data further showed that USP35 can directly deubiquitinate and inactivate STING. Interestingly, activation of STING promotes its binding to USP35 in a STING phosphorylation-dependent manner. Functionally, we found that knockdown of USP35 sensitizes ovarian cancer cells to the DNA-damage chemotherapeutic drug cisplatin. Overall, our study indicates that upregulation of USP35 may be a mechanism of the restricted STING activity in cancer cells, and highlights the significance of USP35 as a potential therapeutic target for ovarian cancer.

SLC7A2 serves as a potential biomarker and therapeutic target for ovarian cancer.

The solute carrier (SLC) family is the largest group of membrane transporters, but their functions in ovarian cancer (OV) remain unclear. We analyzed SLC family members with amino acids-transporting functions in OV. The mRNA expression levels and prognostic values of SLCs in OV were analyzed in the Gene Expression Profiling Interactive Analysis and Kaplan–Meier Plotter database. Solute carrier family 7 member 2 (SLC7A2), which showed differential expression and the most significant prognostic value, was selected for further analyses. The cBioPortal database, Gene Set Enrichment Analysis and Weighted Correlation Network Analysis were used to explore the potential functions and molecular mechanisms of SLC7A2 in OV. Validations in our own samples and in Gene Expression Omnibus datasets were conducted. Functional validation in OV cell lines was carried out. In total, 73 SLC family members were analyzed. Seven members were upregulated while 11 members were downregulated in OV and 15 members were protective factors for prognosis while 12 members were risk factors. SLC7A2 was downregulated in OV, and it was positively associated with prognosis. Knockdown of SLC7A2 promoted viability, invasion and migration of OV cells. These SLC family members and in particular SLC7A2 represented novel biomarkers for diagnosis and treatment for OV.

Abstract B70: Rac1 as a therapeutic target in ovarian cancer

Abstract Rac1 is a high-value therapeutic target for cancer based on its tumor-promoting activities, yet clinical applications targeting Rac1 are in their infancy. High expression and hyperactivation of Rac1 in ovarian cancer, along with our identification of R-ketorolac as a novel Rac1 and Cdc42 selective inhibitor with translational potential, prompt us to test the hypothesis that targeting Rac1 has therapeutic utility for ovarian cancer. Ascites tumor cell samples from ovarian cancer patients in a prospective study receiving racemic ketorolac for clinically indicated use in pain relief were previously reported to show time-dependent reduction of Rac1 and Cdc42 activities post-treatment. New RNA seq data of these patient samples reveals significant downregulation of genes involved in endocytosis, regulation of actin cytoskeleton, ER protein processing, TNF and NOD signaling. Conversely, the identified downregulated genes were overexpressed and associated with worse survival in ovarian cancer patients analyzed through The Cancer Genome Atlas (TCGA). Among the downregulated genes in the NOD pathway are chemokines and proinflammatory cytokines. Follow-up cytokine panels from patients confirm that racemic ketorolac treatment reduces the levels of immunosuppressive cytokines IL-6, IL-10, and RANTES in ascites fluids. Together, these data indicate there may be a benefit to the anti-inflammatory activity of the S- enantiomer, as well as the GTPase inhibitory activity of the R-enantiomer of ketorolac for ovarian cancer treatment. Citation Format: Melanie Rivera, Martha M. Grimes, Dayna Dominguez, S. Ray Kenney, Yuna Guo, Elsa Romero, Kathryn J. Brayer, Yan Guo, Scott A. Ness, Sarah F. Adams, Carolyn Muller, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 as a therapeutic target in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B70.

Downregulation of long noncoding RNA TUSC7 promoted cell growth, invasion and migration through sponging with miR-616-5p/GSK3β pathway in ovarian cancer.

Long non-coding RNAs (lncRNAs) can serve as prognostic markers for cancer patients, including ovarian cancer. The objective of this study was to explore the potential functions and mechanisms of lncRNA-TUSC7 in ovarian cancer. RT-PCR or Western blot (WB) was performed to detect expressions of TUSC7, miR-616-5p and GSK3β in ovarian cancer tissues, adjacent normal tissues and ovarian cancer cell lines. Correlation analysis was performed to analyze the correlations between TUSC7 and miR-616-5p, miR-616-5p and GSK3β, TUSC7 and GSK3β. Furthermore, Kaplan-Meier survival analysis was used to analyze overall survival (OS) of patients with TUSC7 low and high expression. Besides, CCK-8 assay was carried out to measure cell proliferation ability and transwell assay was used to measure cell invasion and migration abilities. In addition, WB was performed to measure protein levels in ovarian cancer tissues and ovarian cancer cell lines. Finally, Luciferase reporter assay was performed to verify the binding sites between TUSC7 and miR-616-5p, miR-616-5p and GSK3β. In this study, we first found that TUSC7 was significantly reduced in ovarian cancer tissues, which was associated with advanced stage and poor diagnosis for ovarian cancer patients. MiR-616-5p was increased in ovarian cancer tissues and cancer cell lines, which was negatively correlated with TUSC7 and GSK3β. GSK3β was found to be reduced in ovarian cancer tissues, which was positively correlated with TUSC7. Of note, we found that TUSC7 overexpression inhibited cell proliferation, invasion and migration in SKOV3 cells. Moreover, protein expressions of Cyclin D1, N-cadherin, Vimentin, MMP-9 and MMP-2 were also repressed. Notably, Luciferase reporter assay proved that TUSC7 could directly sponge with miR-616-5p, which could directly bind with GSK3β, a tumor suppressor, regulating the β-catenin signaling. Finally, we proved that TUSC7 regulated cell proliferation, invasion and migration via miR-616-5p/GSK3β signaling pathway in SKOV3 cells. According to the results, our study revealed that TUSC7 was repressed in patients with ovarian cancer, which might be used as a prognostic factor for ovarian cancer patients. Furthermore, we first discovered that the reduced TUSC7 promoted cell proliferation, invasion and migration of ovarian cancer via miR-616-5p/GSK3β/β-catenin pathway, which might provide a novel promising therapeutic target for ovarian cancer.

Angiogenesis and vasculogenic mimicry as therapeutic targets in ovarian cancer

Tumor angiogenesis is an essential process for growth and metastasis of cancer cells as it supplies tumors with oxygen and nutrients. During tumor angiogenesis, many pro-angiogenic factors are secreted by tumor cells to induce their own vascularization via activation of pre-existing host endothelium. However, accumulating evidence suggests that vasculogenic mimicry (VM) is a key alternative mechanism for tumor vascularization when tumors are faced with insufficient supply of oxygen and nutrients. VM is a tumor vascularization mechanism in which tumors create a blood supply system, in contrast to tumor angiogenesis mechanisms that depend on pre-existing host endothelium. VM is closely associated with tumor progression and poor prognosis in many cancers. Therefore, inhibition of VM may be a promising therapeutic strategy and may overcome the limitations of anti-angiogenesis therapy for cancer patients. In this review, we provide an overview of the current anti-angiogenic therapies for ovarian cancer and the current state of knowledge regarding the links between microRNAs and the VM process, with a focus on the mechanism that regulates associated signaling pathways in ovarian cancer. Moreover, we discuss the potential for VM as a therapeutic strategy against ovarian cancer.

MiR-454 suppresses the proliferation and invasion of ovarian cancer by targeting E2F6

BackgroundThe aberrant expression of microRNA-454 (miR-454) has been confirmed to be involved in the development of cancers. However, the functional role of miR-454 in the progression of ovarian cancer remains unclear.MethodsThe expression of miR-454 in ovarian cancer cells and serum of ovarian cancer patients was detected by RT-PCR. CCK8, colony formation, transwell, and flow cytometry assays were conducted to assess the effects of miR-454 on ovarian cancer cell proliferation, migration, invasion, and apoptosis, respectively. Dual-luciferase reporter assay was used to confirm the targeting relationship between miR-454 and E2F6. The expression pattern of E2F6 in ovarian cancer tissues was detected using immunohistochemistry (IHC) assay. The relative expression of related proteins was examined using western blot analysis.ResultsmiR-454 was markedly down-regulated by hypoxia in ovarian cancer cells. Compared with normal samples, the expression of miR-454 was up-regulated in the serum of ovarian cancer patients, and correlated with the clinicopathological stages of ovarian cancer. Next, we found that miR-454 overexpression inhibited the proliferation, migration and invasion of OVCAR3 and SKOV3 cells, as well as promoted apoptosis. In addition, the Akt/mTOR and Wnt/β-catenin signaling pathway were inhibited by miR-454 in ovarian cancer cells. Mechanically, bioinformatic analysis and dual-luciferase reporter assay confirmed that E2F6 was a direct target of miR-454 and negatively regulated by miR-454 in ovarian cancer cells. Moreover, IHC analysis showed that E2F6 was highly expressed in ovarian cancer tissues. Finally, we found that the increasing cell proliferation and migration triggered by E2F6 overexpression were abolished by miR-454 overexpression.ConclusionTaken together, these results highlight the role of miR-454 as a tumor suppressor in ovarian cancer cells by targeting E2F6, indicating that miR-454 may be a potential diagnostic biomarker and therapeutic target for ovarian cancer.

The Novel Zinc Finger Protein 587B Gene, ZNF587B, Regulates Cell Proliferation and Metastasis in Ovarian Cancer Cells in vivo and in vitro.

BackgroundThe zinc finger protein 587B (ZNF587B) is a novel cisplatin-sensitive gene that was identified in our previous research by using a genome-scale CRISPR-Cas9 knockout library in ovarian cancer (OC) cell lines. ZNF587B belongs to the C2H2-type zinc finger protein (ZFP) family. Many ZFP protein could inhibit tumor development and malignancy. However, the function of ZNF587B remains unknown.MethodsQuantitative PCR (qPCR) was utilized to compare ZNF587B mRNA expression levels in OC and normal ovarian cell lines. The small interfering RNA (siRNA) and full-length ZNF587B eukaryotic expression plasmid were constructed and transfected into OC cells later. Colony formation, 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell assay, and xenograft experiment were conducted to evaluate the effect of ZNF587B on OC cells.ResultsZNF587B was downregulated by approximately 43% and 17% in the OC cell lines SKOV3 and A2780, respectively, compared with that in the normal ovarian cell line IOSE80. Overexpression of ZNF587B reduced cell proliferation, colony formation, migration, and invasion, which could be reversed by knockdown of ZNF587B via siRNA. Xenograft experiments also confirmed that ZNF587B could suppress tumor growth. Survival data of OC patients in the SurvExpress database showed that with respect to overall survival, low-risk patients grouped by the prognostic index had a higher expression of ZNF587B and a better prognosis than high-risk group (HR = 1.77, 95% CI: 0.55–0.70, p = 0.023). Moreover, overexpression of ZNF587B promoted OC cells apoptosis when pretreated with cisplatin.ConclusionZNF587B is a novel potential tumor suppressor of OC and may be a therapeutic target for OC.

RHPN1-AS1 promotes cell proliferation and migration via miR-665/Akt3 in ovarian cancer.

Recent efforts have revealed that long non-coding RNAs exert crucial roles in cancer initiation and progression. RHPN1-AS1 is a 2030 bp transcript from human chromosome 8q24, and involved in tumorigenesis in uveal melanoma and non-small cell lung cancer, but it remains unknown in ovarian cancer. This study focused on the role of RHPN1-AS1 in ovarian cancer and found that RHPN1-AS1 was up-regulated in ovarian cancer tissues and cell lines. Overexpression of RHPN1-AS1 promoted ovarian cancer cell proliferation, migration, and invasion. Mechanistically, overexpression of RHPN1-AS1 decreased the expression of miR-665 and subsequently promoted the expression of Akt3 at posttranscriptional level. Taken together, RHPN1-AS1 positively regulated the expression of Akt3 through sponging miR-665, and exerted an oncogenic role in ovarian cancer progression, and indicates that RHPN1-AS1 may be a potential therapeutic target in ovarian cancer.

Elucidating resistance mechanism to PARP inhibitors for the development of novel therapeutic approaches in high-grade serous ovarian cancer.

6078 Background: PARP inhibitors (PARPi) have been established as a targeted therapeutic approach not only in patients with high-grade serous ovarian cancer (HGSOC) that have genetic loss of function of BRCA1/2-associated DNA repair. However, treatment efficacy varies and neither BRCA mutation, nor homolog recombination deficiency (HRD) status seem to be optimal predictors. Moreover, mechanisms of treatment resistance are poorly understood and novel approaches are urgently required. Methods: Here we created gene expression data of HGSOC patients (n = 52) before PARPi treatment to elucidate key signaling pathways of resistance to increase their efficacy in combinatorial therapeutic strategies. We performed a comprehensive bioinformatics analysis of the differentially expressed genes between the 25% extreme responders (n = 26; 13 each group), including gene set enrichment analysis (GSEA) and causal inference analysis with the CARNIVAL pipeline to elucidate the underlying molecular and regulatory mechanisms governing treatment efficacy and resistance. Results: In accordance with recent publications, we found higher levels of MYC activity in non-responders and deregulation of the Wnt/ß-catenin signaling pathway resulting in PARPi treatment resistance. The pathway enrichment analysis also revealed specific pathways especially PDGFR, FGFR, PI3K/mTOR and MAPK signaling pathway associated with resistant phenotype. Furthermore, we have identified key kinases, particularly JAK1/2 and SRC that might mediate resistance to PARP inhibition. In addition, differential gene expression analysis revealed folate receptor 1 (FOLR1) to be significantly higher expressed in non-responders (logFC = 2.66; p < 0.0026) with the potential as a serum-based biomarker not only for ovarian cancer, as it correlates closely with CA125, but also PARPi treatment efficacy. Conclusions: In conclusion, these findings define a network of pathways, that are crucial to mediate mechanism of PARPi resistance and identified key signaling kinases as therapeutic targets in ovarian cancer.

Circulating Tumor Cells Characterization Revealed TIMP1 as a Potential Therapeutic Target in Ovarian Cancer.

Background: Recent studies showed a relevant role of hematogenous spread in ovarian cancer and the interest of circulating tumor cells (CTCs) monitoring as a prognosis marker. The aim of the present study was the characterization of CTCs from ovarian cancer patients, paying special attention to cell plasticity characteristics to better understand the biology of these cells. Methods: CTCs isolation was carried out in 38 patients with advanced high-grade serous ovarian cancer using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize these cells. Results: Epithelial CTCs were found in 21% of patients, being their presence higher in patients with extraperitoneal metastasis. Importantly, this population was characterized by the expression of epithelial markers as MUC1 and CK19, but also by genes associated with mesenchymal and more malignant features as TIMP1, CXCR4 and the stem markers CD24 and CD44. In addition, we evidenced the relevance of TIMP1 expression to promote tumor proliferation, suggesting its interest as a therapeutic target. Conclusions: Overall, we evidenced the utility of the molecular characterization of EpCAM+ CTCs from advanced ovarian cancer patients to identify biomarkers with potential applicability for disseminated disease detection and as therapeutic targets such as TIMP1.