Specific targeting of the pseudogene RPSAP52 reduces ovarian tumor growth in patient-derived orthoxenograft models.

Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. We previously identified bone marrow stromal antigen 2 (BST2) as an endometrial cancer antigen using iTRAQ-based quantitative proteomic technology focused on cell surface membrane proteins, and also demonstrated the usefulness of an anti-BST2 monoclonal antibody (mAb) for endometrial cancer. In this study, we aimed to identify a new ovarian cancer antigen. We also aimed to develop a novel monoclonal antibody (mAb) and evaluate its preclinical efficacy against ovarian cancer. To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. As the new therapeutic target for ovarian cancer, we identified lipolysis-stimulated lipoprotein receptor (LSR) which had one of the largest significant differences in protein level between normal ovarian epithelial and ovarian cancer cell lines. Immunohistochemical analysis showed that the overall survival of ovarian serous carcinoma patients with high LSR expression was significantly shorter than those with low LSR (p = 0.0293). We newly developed anti-LSR mAb and investigated its preclinical efficacy. Anti-LSR mAb showed significant in vivo inhibition of tumor growth against a xenograft model of hLSR-positive ovarian cancer in an antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) independent manner (p = 0.0001). And anti-LSR mAb also induced G0/G1 cell cycle arrest by regulation of MEK and p44/42 MAPK activities and expression levels of cell cycle related proteins in vitro. Furthermore, anti-hLSR mAb, which crossreacts with mouse LSR, had little detectable toxicity in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition in ADCC and CDC independent manner in vivo. Anti-human LSR mAb also inhibited LSR function and showed direct tumor growth inhibition inducing G0/G1 cell cycle arrest in vitro. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Satoshi Nakagawa, Akiko Morimoto, Minoru Fujimoto, Takuhei Yokoyama, Yusuke Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Eiichi Morii, Tadashi Kimura, Tetsuji Naka. Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2015-4382

The United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) recently reported a reduction in the average overall mortality among ovarian cancer patients screened with an annual sequential, multimodal strategy that tracked biomarker CA125 over time, where increasing serum CA125 levels prompted ultrasound. However, multiple cases were documented wherein serum CA125 levels were rising, but ultrasound screens were normal, thus delaying surgical intervention. A significant factor which could contribute to false negatives is that many aggressive ovarian cancers are believed to arise from epithelial cells on the fimbriae of the fallopian tubes, which are not readily imaged. Moreover, because only a fraction of metastatic tumors may reach a sonographically-detectable size before they metastasize, annual screening with ultrasound may fail to detect a large fraction of early-stage ovarian cancers. The ability to detect ovarian carcinomas before they metastasize is critical and future efforts towards improving screening should focus on identifying unique features specific to aggressive, early-stage tumors, as well as improving imaging sensitivity to allow for detection of tubal lesions. Implementation of a three-stage multimodal screening strategy in which a third modality is employed in cases where the first-line blood-based assay is positive and the second-line ultrasound exam is negative may also prove fruitful in detecting early-stage cases missed by ultrasound.

Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Objective:Ovarian cancer is the most lethal gynecological malignancy. Five-year survival of advanced ovarian cancer patients remains less than 50% and the mortality rate has not changed in recent years. This study is aimed to screen for novel ovarian cancer therapeutic targets using quantitative proteomic approach and to develop antibody-based medicine targeting novel ovarian cancer antigens. Method:Exhaustive quantitative proteome analysis focused on cell surface membrane proteins were performed by isobaric tags for relative and absolute quantitation(iTRAQ) using a normal ovarian surface epithelial cell line and 7 ovarian cancer cell lines. By this approach, ovarian cancer-specific membrane proteins were identified. We confirmed the expression of lipolysis-stimulated lipoprotein receptor (LSR) using immunohistochemical staining, western blotting method, and flow cytometry. By modified MTT assay, cell growth inhibition was performed in LSR-knock down cells compared with control cells. Cell adhesion assay was also performed in vitro. Anti-LSR monoclonal antibody (anti-LSR mAb) was generated to evaluate the efficacy of the LSR targeted antibody therapy in vivo. For subcutaneous xenograft experiments, ovarian cancer cell line (RMG-1) was injected subcutaneously into the CB17/SCID mice. Mice were then randomly divided into two groups and administrated intraperitoneally anti-LSR mAb or control IgG antibody twice a week for 6 times. In addition, luciferase transfected ovarian cancer cell line (A2780-luc) was implanted intraperitoneally into the CB17/SCID mice. Mice were administrated anti-LSR mAb or control IgG antibody intraperitoneally twice a week for 5 times. Result:By iTRAQ analysis, we identified 1685 proteins and one of the ovarian cancer candidates protein, LSR was identified. We confirmed the expression of LSR in 8 out of 11 ovarian cancer cell lines and all of ovarian cancer clinical specimens. We performed proliferation assay using siRNA against ovarian cancer cell lines. When expression of LSR was suppressed, a tumor growth was significantly inhibited at day 4(p<0.01). In adhesion assay, we observed significant inhibition of cell adhesion in LSR knockdown cells. These results suggested that LSR is related to cell proliferation and adhesion. A significant anti-tumour effect with 78% was observed in the anti-LSR mAb antibody administrated group compared with control IgG antibody administrated group after 25 days (p <0.001). Furthermore, in intraperitoneal xenograft model, anti-LSR antibody administration group significantly decreased signal intensities compared with the control antibody administration group at day 22 after implantation. Conclusion:We showed that the LSR is related to cell proliferation and adhesion in vitro in ovarian cancer cells and proved the antitumor effect of anti-LSR mAb. These results suggested that LSR can be a new therapeutic target of ovarian cancer. Citation Format: Satoko Matsuzaki, Kosuke Hiramatsu, Satoshi Serada, Satoshi nakagawa, Shinya Matsuzaki, Yutaka Ueda, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4998.

Background: Ovarian cancer (OC) is the most common cause of death among gynecologic cancers. A key reason for the high lethality of OC is that early detection of OC is uncommon, and the majority of OC patients have advanced stage disease at diagnosis. Current non-invasive tests do not adequately distinguish benign from malignant adnexal masses. Diagnostic tests that reliably distinguish early stage OC from benign conditions may lead to earlier diagnosis of OC and improved survival. Methods: We designed a cohort study of plasma biomarkers in ovarian cancer patients. Specimens were analyzed from 100 patients with advanced OC (AJCC Stage III and IV), 50 patients with early stage OC (Stage I and II), and 50 patients with benign surgical conditions from the Mayo Ovarian SPORE Biospecimens Core. Presurgical plasma samples were assayed for multiple toll-like receptor agonists, cytokines, and vascular growth factors by ELISA and electrochemiluminescence. Biomarkers that were reliably detected in plasma were analyzed for association with OC. Differences in plasma biomarker levels between benign, early, and advanced OC patient groups were assessed using plate-adjusted logistic regression models. Results: Out of 23 biomarkers tested, 7 were excluded due to unreliable plasma detection. Of the remaining 16 biomarkers, 6_including interferon gamma (IFNγ), interleukin 6 (IL-6), IL-8, IL-10, tumor necrosis factor alpha (TNFα), and placental growth factor (PlGF)_were univariately associated with OC (all p<0.005), and one_IL-6_was associated with early stage OC (p < 0.0001). Heat shock protein 90kDa beta member 1 (HSP90B1, gp96) was associated with OC and early stage OC with borderline statistical significance (p = 0.039 and p = 0.026, respectively). However, when adjusted for cancer antigen 125 (CA-125), only HSP90B1 independently predicted OC (p = 0.008), as well as early stage OC (p = 0.014). Conclusions: The plasma cytokines IFNγ, IL-6, IL-8, IL-10, TNFα, and PlGF are associated with OC. However, after adjusting for CA-125, only HSP90B1 independently predicts OC, including early stage OC. These data warrant further investigation to determine whether measuring plasma HSP90B1 can aid in patient evaluation. Citation Format: Matthew S. Block, Matthew J. Maurer, Krista Goergen, Kimberly R. Kalli, Courtney L. Erskine, Marshall D. Behrens, Keith L. Knutson. Plasma heat shock protein 90kDa beta member 1 levels predict both early stage and advanced stage ovarian cancer independently from cancer antigen 125 in patients with an indeterminate adnexal mass. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1873. doi:10.1158/1538-7445.AM2014-1873

Globally, the numbers of ovarian cancer are about 225,500 new cases and 140,200 deaths per year. In 2013, about 22,240 new cases of ovarian cancer will be diagnosed and 15,500 women will die of ovarian cancer in the USA. Early-stage ovarian cancer (International Federation of Gynecology and Obstetrics [FIGO] stages I to IIA) is approximately 30 % of patients with ovarian cancer; the disease is restricted to the true pelvis when it is diagnosed. Palpation of an adnexal mass during a pelvic examination is essential for diagnosis of ovarian tumor and cancer. Performing pelvic high resolution ultrasound examination is a useful noninvasive diagnostic test. In addition, measurement of cancer antigen 125 (CA-125) is important as it was found to be elevated in greater than 80 % of patients with epithelial ovarian cancer (EOC). The standard primary treatment for epithelial ovarian cancer involves pelvic and para-aortic lymphadenectomy, followed by adjuvant chemotherapy consisting of carboplatin and paclitaxel. Systematic lymphadenectomy resection and pathological examination of retroperitoneal lymph nodes have clinical significance for accurate assessment of staging and prognosis. This procedure may involve resection of para-aortic lesions, and therefore it may increase surgical morbidity, such as increased operation time, increased blood loss, lymph cyst formation, and edema in the leg. The surgical approach for EOC could be through laparotomy, laparoscopy, or robot-assisted laparoscopy, which has the advantage of being safe, better visualization of the abdominal and pelvis organs, minimal blood loss, and short hospital stay. Following surgery, patients with early ovarian cancer—FIGO stages I to IIA, except stage IA, grade 1—benefit from three to six cycles of platinum-based chemotherapy, in terms of both overall survival. Fertility-sparing surgery (FSS) for women of childbearing age with early-stage ovarian cancer is a desirable alternative to more radical approach. In addition, preservation of the adnexa and uterus is recommended in patients with non-epithelial tumors and epithelial borderline ovarian cancer.

Cancer of the ovary is the leading cause of death for women with gynecologic malignancies. Over 90% of ovarian cancers derive from malignant transformation of the ovarian surface epithelium (OSE), from which cells disseminate into the peritoneal cavity, invade locally or spread via lymphatics,

<div>Abstract<p>Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes, we characterized transforming growth factor β/BMP receptor and Smad expression in immortalized ovarian surface epithelial cells and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signaling in ovarian cancer. Using small interfering RNA, ligand trap, inhibitor, and ligand stimulation approaches, we show that BMP9 acts as a proliferative factor for immortalized ovarian surface epithelial cells and ovarian cancer cell lines, signaling predominantly through an ALK2/Smad1/Smad4 pathway rather than through ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signaling that is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that ∼25% of epithelial ovarian cancers express BMP9, whereas normal human ovarian surface epithelial specimens do not. Our data indicate that BMP9 signaling through ALK2 may be a novel therapeutic target in ovarian cancer. [Cancer Res 2009;69(24):9254–62]</p></div>

<div>Abstract<p>Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes, we characterized transforming growth factor β/BMP receptor and Smad expression in immortalized ovarian surface epithelial cells and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signaling in ovarian cancer. Using small interfering RNA, ligand trap, inhibitor, and ligand stimulation approaches, we show that BMP9 acts as a proliferative factor for immortalized ovarian surface epithelial cells and ovarian cancer cell lines, signaling predominantly through an ALK2/Smad1/Smad4 pathway rather than through ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signaling that is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that ∼25% of epithelial ovarian cancers express BMP9, whereas normal human ovarian surface epithelial specimens do not. Our data indicate that BMP9 signaling through ALK2 may be a novel therapeutic target in ovarian cancer. [Cancer Res 2009;69(24):9254–62]</p></div>

Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes, we characterized transforming growth factor beta/BMP receptor and Smad expression in immortalized ovarian surface epithelial cells and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signaling in ovarian cancer. Using small interfering RNA, ligand trap, inhibitor, and ligand stimulation approaches, we show that BMP9 acts as a proliferative factor for immortalized ovarian surface epithelial cells and ovarian cancer cell lines, signaling predominantly through an ALK2/Smad1/Smad4 pathway rather than through ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signaling that is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that approximately 25% of epithelial ovarian cancers express BMP9, whereas normal human ovarian surface epithelial specimens do not. Our data indicate that BMP9 signaling through ALK2 may be a novel therapeutic target in ovarian cancer.

The aims of this study were to investigate the hypoxia-inducible factor 1alpha (HIF-1alpha) protein inhibition and tumor growth by a molecular target of rapamycin inhibitor, rapamycin, in xenogeneic transplant model of ovarian cancer and to study the correlation of apoptosis with HIF-1alpha and vascular endothelial growth factor (VEGF) expression. Four groups of female nude mice were inoculated subcutaneous with SKOV-3 cells and treated with vehicle, rapamycin, paclitaxel, or rapamycin plus paclitaxel. The expressions of HIF-1alpha and VEGF and microvessel density (MVD) were assessed by immunohistochemistry. While messenger RNA (mRNA) expression of Glut1, bcl-2, and VEGF was studied by reverse transcription-polymerase chain reaction, and apoptosis of tumor cells was determined by terminal deoxynucleotidyl biotin-dUTP nick end labeling (TUNEL). The HIF-1alpha was expressed in epithelial ovarian cancer. There was a significant correlation between HIF-1alpha protein expression and VEGF or MVD. Tumor burden treated with rapamycin alone, rapamycin plus paclitaxel, and paclitaxel alone was reduced (47.91%, 51.03%, and 31.75%, respectively) compared with controls. The expression of HIF-1alpha was inhibited, and apoptotic index of tumor cell increased in rapamycin and rapamycin plus paclitaxel group. HIF-1alpha may upregulate VEGF expression both in mRNA and protein level. There is a positive correlation between HIF-1alpha and MVD. Rapamycin inhibits expression of HIF-1alpha and suppresses ovarian tumor growth. Our data suggested that a combination of HIF-1alpha inhibitor and chemotherapy could provide an effective approach for inhibiting tumor growth in ovarian cancer.

The aims of this study were to investigate the hypoxia-inducible factor 1α (HIF-1α) protein inhibition and tumor growth by a molecular target of rapamycin inhibitor, rapamycin, in xenogeneic transplant model of ovarian cancer and to study the correlation of apoptosis with HIF-1α and vascular endothelial growth factor (VEGF) expression. Four groups of female nude mice were inoculated subcutaneous with SKOV-3 cells and treated with vehicle, rapamycin, paclitaxel, or rapamycin plus paclitaxel. The expressions of HIF-1α and VEGF and microvessel density (MVD) were assessed by immunohistochemistry. While messenger RNA (mRNA) expression of Glut1, bcl-2, and VEGF was studied by reverse transcription–polymerase chain reaction, and apoptosis of tumor cells was determined by terminal deoxynucleotidyl biotin-dUTP nick end labeling (TUNEL). The HIF-1α was expressed in epithelial ovarian cancer. There was a significant correlation between HIF-1α protein expression and VEGF or MVD. Tumor burden treated with rapamycin alone, rapamycin plus paclitaxel, and paclitaxel alone was reduced (47.91%, 51.03%, and 31.75%, respectively) compared with controls. The expression of HIF-1α was inhibited, and apoptotic index of tumor cell increased in rapamycin and rapamycin plus paclitaxel group. HIF-1α may upregulate VEGF expression both in mRNA and protein level. There is a positive correlation between HIF-1α and MVD. Rapamycin inhibits expression of HIF-1α and suppresses ovarian tumor growth. Our data suggested that a combination of HIF-1α inhibitor and chemotherapy could provide an effective approach for inhibiting tumor growth in ovarian cancer.

Background: Ovarian cancer is the most fatal gynaecologic disease in the western world. In 2010 in the United States, an estimated 21,880 women will develop ovarian cancer and an estimated 13,850 women will succumb to this disease. Current treatments are limited to surgery and chemotherapy, but the disease often recurs highlighting the need for novel cancer therapeutics. We are currently evaluating the efficacy of a novel therapeutic, GAP-107B8 (PharmaGap Inc, Ottawa) using in vitro and in vivo models. GAP-107B8 was designed as a protein kinase C (PKC) inhibitor. The PKC family of serine/threonine kinases are involved in cellular proliferation, differentiation, apoptosis and cell polarity. One PKC isoform, PKC iota, has recently been identified as a human oncogene and has been shown to be overexpressed in epithelial ovarian cancers and is thus a potential therapeutic target for ovarian cancer. Objectives: 1) To test the novel inhibitor GAP-107B8 on ovarian cancer cell lines to determine its effects on cell proliferation, cell cycle progression and apoptosis; and 2) To determine the therapeutic potential of GAP-107B8 in xenograft models of two different ovarian cancer cell lines. Methods: Three ovarian cancer cell lines were treated with three different concentrations of GAP-107B8 and screened using high throughput assays to measure the proliferation of cells in adherent cultures. Apoptosis was measured by TUNEL staining, PARP cleavage and cell cycle analysis. Tumor burden was assessed in mice receiving subcutaneous implants of two ovarian cancer cell lines, followed by daily intra-tumoral injections of GAP-107B8. Results: GAP-107B8 caused a significant reduction in cell proliferation in 3 ovarian cancer cell lines tested (86% to 95%; p<0.001), including a cell line resistant to standard chemotherapy. In vivo, intra-tumoral treatment with GAP-107B8 resulted in a 45% reduction in average tumor size in the A2780cp-derived tumours (n=6/group, p<0.01) and 75% in the HEY-derived tumors (n=3/group, p<0.001). Cell killing may be mediated by apoptosis based on the observation of TUNEL staining 30 hours after treatment of cells in vitro with GAP-107B8. Apoptosis was confirmed by flow cytometry and PARP cleavage. GAP-107B8 also inhibited progression of cells through the cell cycle by blocking or delaying progression of cells through G2/M into the G1 phase. Conclusion: The novel inhibitor GAP-107B8 displays good efficacy in vitro in suppressing the proliferation of ovarian cancer cell lines. Cytotoxicity may be manifested in perturbations of the cell cyle and induction of apoptosis. Finally, GAP-107B8 showed therapeutic efficacy in ovarian cancer xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2592. doi:10.1158/1538-7445.AM2011-2592

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

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.