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. siRNA 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.

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.

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 IC 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. .

The Japanese government began a human papillomavirus (HPV) vaccination program for girls aged 12‐16 years in 2010 but withdrew its recommendation in 2013 because of potential adverse effects, leading to drastically reduced vaccination uptake. To evaluate population‐level effects of HPV vaccination, women younger than 40 years of age newly diagnosed with cervical intraepithelial neoplasia grade 1‐3 (CIN1‐3), adenocarcinoma in situ (AIS), or invasive cervical cancer (ICC) have been registered at 21 participating institutes each year since 2012. A total of 7709 women were registered during 2012‐2017, of which 5045 were HPV genotyped. Declining trends in prevalence of vaccine types HPV16 and HPV18 during a 6‐year period were observed in CIN1 (50.0% to 0.0%, Ptrend < .0001) and CIN2‐3/AIS (83.3% to 45.0%, Ptrend = .07) only among women younger than 25 years of age. Overall, HPV vaccination reduced the proportion of HPV16/18‐attributable CIN2‐3/AIS from 47.7% to 33.0% (P = .003): from 43.5% to 12.5% as routine vaccination (P = .08) and from 47.8% to 36.7% as catch‐up vaccination (P = .04). The HPV16/18 prevalence in CIN2‐3/AIS cases was significantly reduced among female individuals who received their first vaccination at age 20 years or younger (P = .02). We could not evaluate vaccination effects on ICC owing to low incidence of ICC among women aged less than 25 years. We found HPV vaccination to be effective in protecting against HPV16/18‐positive CIN/AIS in Japan; however, our data did not support catch‐up vaccination for women older than 20 years. Older adolescents who skipped routine vaccination due to the government’s suspension of its vaccine recommendation could benefit from receiving catch‐up vaccination before age 20 years.

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.