Shogo Shigeta

Ceramide limits phosphatidylinositol-3-kinase C2β-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2β (PI3KC2β) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2β-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2β and affect its compartmentalization, thereby suppressing PI3KC2β activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2β knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2β in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2β-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Inhibition of plasminogen activator inhibitor-1 is a potential therapeutic strategy in ovarian cancer

Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.

Transferrin facilitates the formation of DNA double-strand breaks via transferrin receptor 1: the possible involvement of transferrin in carcinogenesis of high-grade serous ovarian cancer.

Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Assessing the effect of guideline introduction on clinical practice and outcome in patients with endometrial cancer in Japan: a project of the Japan Society of Gynecologic Oncology (JSGO) guideline evaluation committee

Objective The Japan Society of Gynecologic Oncology (JSGO) published the first practice guideline for endometrial cancer in 2006. The JSGO guideline evaluation committee assessed the effect of this guideline introduction on clinical practice and patient outcome using data provided by the Japan Society of Obstetrics and Gynecology (JSOG) cancer registration system. Methods Data of patients with endometrial cancer registered between 2000 and 2012 were analyzed, and epidemiological and clinical trends were assessed. The influence of guideline introduction on survival was determined by analyzing data of patients registered between 2004 and 2009 using competing risk model. Results In total, 65,241 cases of endometrial cancer were registered. Total number of patients registered each year increased about 3 times in the analyzed period, and the proportion of older patients with type II endometrial cancer rapidly increased. The frequency of lymphadenectomy had decreased not only among the low-recurrence risk group but also among the intermediate- or high-recurrence risk group. Adjuvant therapy was integrated into chemotherapy (p<0.001). Overall survival did not significantly differ before and after the guideline introduction (hazard ratio [HR]=0.891; p=0.160). Additional analyses revealed patients receiving adjuvant chemotherapy showed better prognosis than those receiving adjuvant radiation therapy when limited to stage I or II (HR= 0.598; p=0.003). Conclusion It was suggested that guideline introduction influenced the management of endometrial cancer at several aspects. Better organized information and continuous evaluation are necessary to understand the causal relationship between the guideline and patient outcome.

Leptomeningeal metastasis from gynecologic cancers diagnosed by brain MRI

Leptomeningeal metastasis (LM) is rarely observed in gynecologic cancers. As gadolinium-enhanced magnetic resonance imaging (Gd-MRI) is highly effective for diagnosing LM, the aim of this study is to describe the clinical behaviors and outcomes of LM patients who were diagnosed by Gd-MRI. After securing institutional review board approvals, we retrospectively reviewed patient records. Eight patients were found to have LM from gynecological malignancies. Primary tumors included three ovarian cancers, one tubal cancer, one peritoneal cancer, two endometrial cancers, and one cervical cancer. Gd-MRI of the brain and the spine is indicated as the high-priority inspection for the diagnosis of this devastating complication.

Novel cooperative pathway of c-Myc and Furin, a pro-protein convertase, in cell proliferation as a therapeutic target in ovarian cancers

c-Myc is a master regulator of various oncogenic functions in many types of human cancers. However, direct c-Myc-targeted therapy has not been successful in the clinic. Here, we explored a novel therapeutic target, which shows synthetic lethality in c-Myc-driven ovarian cancers, and examined the molecular mechanism of the synthetic lethal interaction. By high throughput siRNA screening with a library of 6,550 genes, Furin, a pro-protein convertase, was identified as the top hit gene. Furin inhibition by siRNA or a Furin inhibitor significantly suppressed cell proliferation in high c-Myc-expressing ovarian cancer cells compared with low c-Myc-expressing cells. Conversely, Furin overexpression in the presence of high c-Myc significantly promoted cell proliferation compared with only c-Myc or Furin overexpression. Notch1, one of the Furin substrates, was upregulated by c-Myc, and Notch1 cleaved by Furin increased cell proliferation of high c-Myc-expressing ovarian cancer cells. Notch1 was involved in the cooperative pathway of c-Myc and Furin in cell proliferation. In clinical ovarian cancer specimens, co-expression of c-Myc and Furin correlated with poor survival. In conclusion, we found that c-Myc cooperates with Furin to promote cell proliferation. Furin may be a promising therapeutic target in c-Myc-driven ovarian cancer.

Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel

Introduction Uterine serous carcinoma (USC) is more aggressive than other subtypes of endometrial carcinoma and is associated with a poor prognosis. We analyzed the metabolomic profile of USC with acquired resistance to paclitaxel. Results Glutathione (GSH) concentration in PTX-1 cells was higher than in USPC-1 cells. In addition, GSH concentration in the USPC-1 cells increased after treatment with paclitaxel but was unchanged in PTX-1 cells. Glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P) concentrations in PTX-1 cells were higher than those in USPC-1 cells. G6P concentration in the USPC-1 cells was unchanged after treatment with paclitaxel, while it decreased in PTX-1 cells. Conclusion Our results indicate that increased GSH and glucose metabolism may be related to acquiring resistance to paclitaxel in USC and thus may be targets for anti-USC therapy. Materials and Methods We compared metabolic profiles and reactions to paclitaxel in both a wild type USC cell line (USPC-1) and PTX-1, a cell line derived from USPC-1 which acquired paclitaxel resistance, using a capillary electrophoresis CE-MS/MS system.

Ceramide nanoliposomes as a MLKL-dependent, necroptosis-inducing, chemotherapeutic reagent in ovarian cancer

Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therapy is now being tested in dose-escalating phase I clinical trials as a cancer treatment. Multiple nanoscale delivery systems for ceramide have been proposed to overcome the inherent toxicities, poor pharmacokinetics, and difficult biophysics associated with ceramide. Using the ceramide nanoliposomes (CNL), we now investigate the therapeutic efficacy and signaling mechanisms of this nanoscale delivery platform in refractory ovarian cancer. Treatment of ovarian cancer cells with CNL decreased the number of living cells through necroptosis but not apoptosis. Mechanistically, dying SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like (MLKL) as evidenced by oligomerization and relocalization to the blebbing membranes, showing necroptotic characteristics. Knockdown of MLKL, but not its upstream protein kinases such as receptor-interacting protein kinases, with siRNA significantly abolished CNL-induced cell death. Monomeric MLKL protein expression inversely correlated with the IC50 values of CNL in distinct ovarian cancer cell lines, suggesting MLKL as a possible determinant for CNL-induced cell death. Finally, systemic CNL administration suppressed metastatic growth in an ovarian cancer cell xenograft model. Taken together, these results suggest that MLKL is a novel pronecroptotic target for ceramide in ovarian cancer models. Mol Cancer Ther; 17(1); 50–59. ©2017 AACR.

Abstract NTOC-091: INVOLVEMENT OF TIE–1 TYROSINE KINASE RECEPTOR IN CHEMO–RESISTANCE: POTENTIAL OF TIE1 AS A NOVEL THERAPEUTIC TARGET

OBJECTIVES: Platinum-resistance is one of the most challenging difficulties in the treatment of ovarian cancer patients. To overcome this problem, we have explored a target molecule which can conquer platinum-resistance of ovarian cancer cells utilizing a functional genomics approach. MATERIALS AND METHODS: High-throughput functional siRNA screening was designed to target 6550 genes in cisplatin-resistant A2780 CP ovarian cancer cells. Cell viability was assessed by luminescent cell viability assay. After identifying a candidate molecule, cisplatin uptake was determined by atomic absorption spectrometry. DNA damages were determined by the western blotting and immunofluorescent staining using γH2AX antibodies. RESULTS: Through a functional screening, receptor tyrosine kinase TIE 1 was identified as a top candidate gene, of which inhibition give rise to enhancement of cisplatin sensitivity in ovarian cancer cells. Conversely, over-expression of TIE 1 gene significantly decreased susceptibility to cisplatin-induced cell death without affecting cisplatin uptake. DNA damages induced by cisplatin was significantly suppressed in TIE 1 over-expressed cells, raising novel potential mechanisms of TIE 1 in nucleotide excision repair system that removes chemicals adduct to DNA. In addition, over-expression of TIE 1 increased the expression of XPC, which is responsible for nucleotide excision repair. CONCLUSION: We have identified TIE 1 as a molecular target to overcome platinum-resistance in ovarian cancer cells. TIE 1 contribute platinum- resistance in ovarian cancer cells by promoting XPC-dependent DNA repairing system. Citation Format: Masumi Ishibashi, Masafumi Toyoshima, Mahy Egiz, Xuewei Zhang, Junko Minato, Shogo Shigeta, Kazuyuki Kitatani, Nobuo Yaegashi. INVOLVEMENT OF TIE–1 TYROSINE KINASE RECEPTOR IN CHEMO–RESISTANCE: POTENTIAL OF TIE1 AS A NOVEL THERAPEUTIC TARGET [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-091.

Abstract A13: Transferrin facilitates the formation of DNA-double strand breaks via transferrin receptor 1 in fallopian tube epithelial cells.

Objectives: High-grade serous ovarian cancer (HGSOC) is now believed to arise from fallopian tube epithelium (FTE), and p53 signatures are indicated to be the early oncogenic change in HGSOC. Although accumulation of DNA-double strand breaks (DNA-DSBs) are frequently observed in p53 signatures, the mechanism of DNA-DSBs formation in FTE has not been revealed yet. Hydroxyl radicals, which strongly induce DNA-DSBs, are the most active reactive oxygen species (ROS) in a living organism, and hydroxyl radicals are produced in a Fenton reaction catalyzed by free iron ions. Focusing on transferrin, which is a transporter of iron ion and exists abundantly in the follicular fluid or retrograde menstrual blood, we inspected the role of transferrin and transferrin receptor family in DNA-DSBs formation at FTE. Materials and methods: The expressions of transferrin receptor 1(TfR1) and 2 (TfR2) in human FTE were assessed by immunohistochemistry. Immortalized fallopian tube secretory epithelial cells (kindly provided by Dr. Ronny Drapkin, Dana-Farber Cancer institute) and A2780 ovarian cancer cells were cultured with holo-transferrin or vehicle, and the extent of DNA-DSBs was compared. γH2AX was adopted as a marker of DNA-DSBs. ROS were also measured to verify whether transferrin promotes a Fenton reaction. The involvement of TfR1 and TfR2 were assessed with siRNA knockdown strategy. Further, the ex vivo study were performed using murine fallopian tubes. Results: In immunohistochemistry, both TfR1 and TfR2 were ubiquitously positive in human FTE. Transferrin administration significantly increased the γH2AX expression in these cells and led ROS formation. In addition, transferrin treatment also amplified hydrogen peroxide-inducing γH2AX expression. TfR1 knockdown cancelled the uptake of transferrin, subsequent γH2AX expression and ROS formation but TfR2 knockdown didn9t. Also we confirmed that transferrin treatment facilitated γH2AX formation in murine FTE ex vivo. Conclusion: We identified transferrin-TfR1 axis facilitates DNA-DSBs by promoting a Fenton reaction. It is possible that FTE exposed to the extracellular transferrin highly concentrated in the follicular fluid or retrograde menstrual blood is deeply involved with the carcinogenesis of HGSOC. Citation Format: Shogo Shigeta, Masafumi Toyoshima, Kazuyuki Kitatani, Masumi Ishibashi, Toshinori Usui, Nobuo Yaegashi. Transferrin facilitates the formation of DNA-double strand breaks via transferrin receptor 1 in fallopian tube epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A13.