Patient-Derived Exosomes as siRNA Carriers in Ovarian Cancer Treatment.

RNA interference is a powerful gene-silencing tool with potential clinical applications. However, its therapeutic use is challenging because suitable carriers are unavailable. Exosomes are stable small endogenous vesicles that can transport functional molecules to target cells, making them ideal small interfering RNA (siRNA) carriers. Herein, we elucidated the therapeutic potential of patient-derived exosomes as an siRNA carrier for ovarian cancer (OC) treatment. The exosomes were extracted from the culture medium of primary fibroblasts collected from the omentum of patients with OC during surgery. MET proto-oncogene, receptor tyrosine kinase (MET) was selected for gene silencing, c-Met siRNAs were synthesized and loaded into the exosomes (Met-siExosomes) via electroporation, and the treatment effect of the Met-siExosomes was assessed in vitro and in vivo. The Met-siExosomes downregulated the c-Met protein levels and inhibited OC cell proliferation, migration, and invasion. In xenograft experiments using SKOV3-13 and ES-2 cells, Met-siExosomes were selectively extracted from peritoneally disseminated tumors. Intraperitoneal treatment suppressed the c-Met downstream targets in cancer cells and prolonged mouse survival. The synthesized siRNAs were successfully and selectively delivered via the exosomes to intraperitoneally disseminated tumors. As patients with OC routinely undergo omentectomy and abundant fibroblasts can be easily collected from the omentum, patient-derived exosomes may represent a promising therapeutic siRNA carrier to treat OC.

Spatial proteo-transcriptomic profiling reveals the molecular landscape of borderline ovarian tumors and their invasive progression.

Serous borderline tumors (SBT) are epithelial neoplastic lesions of the ovaries that commonly have a good prognosis. In 10-15% of cases, however, SBT will recur as low-grade serous cancer (LGSC), which is deeply invasive and responds poorly to current standard chemotherapy1,2,3. While genetic alterations suggest a common origin, the transition from SBT to LGSC remains poorly understood4. Here, we integrate spatial proteomics5 with spatial transcriptomics to elucidate the evolution from SBT to LGSC and its corresponding metastasis at the molecular level in both the stroma and the tumor. We show that the transition of SBT to LGSC occurs in the epithelial compartment through an intermediary stage with micropapillary features (SBT-MP), which involves a gradual increase in MAPK signaling. A distinct subset of proteins and transcripts was associated with the transition to invasive tumor growth, including the neuronal splicing factor NOVA2, which was limited to expression in LGSC and its corresponding metastasis. An integrative pathway analysis exposed aberrant molecular signaling of tumor cells supported by alterations in angiogenesis and inflammation in the tumor microenvironment. Integration of spatial transcriptomics and proteomics followed by knockdown of the most altered genes or pharmaceutical inhibition of the most relevant targets confirmed their functional significance in regulating key features of invasiveness. Combining cell-type resolved spatial proteomics and transcriptomics allowed us to elucidate the sequence of tumorigenesis from SBT to LGSC. The approach presented here is a blueprint to systematically elucidate mechanisms of tumorigenesis and find novel treatment strategies.

Decoding evolutionary trajectories of ovarian cancer metastasis.

The genetic changes during high-grade serous ovarian cancer metastasis have largely remained a mystery. Lahtinen et al. show that ovarian cancer metastasizes along three different evolutionary states that have distinct mutations and signaling pathways, potentially allowing the identification of targeted treatments.

Continuous Administration of Anti-VEGFA Antibody Upregulates PAI-1 Secretion from Ovarian Cancer Cells via miR-143-3p Downregulation.

Although bevacizumab (BEV) plays a key role in ovarian cancer treatment, BEV resistance is often observed in clinical settings. This study aimed to identify the genes responsible for BEV resistance. C57BL/6 mice inoculated with ID-8 murine ovarian cancer cells were treated with anti-VEGFA antibody or IgG (control) twice weekly for 4 weeks. The mice were sacrificed, then, RNA was extracted from the disseminated tumors. qRT-PCR assays were performed to identify angiogenesis-related genes and miRNAs that were altered by anti-VEGFA treatment. SERPINE1/PAI-1 was found to be upregulated during BEV treatment. Therefore, we focused on miRNAs to elucidate the mechanism underlying the upregulation of PAI-1 during BEV treatment. Kaplan-Meier plotter analysis revealed that higher expression levels of SERPINE1/PAI-1 were associated with poor prognoses among BEV-treated patients, suggesting that SERPINE1/PAI may be involved in the acquisition of BEV resistance. miRNA microarray analysis followed by in silico and functional assays revealed that miR-143-3p targeted SERPINE1 and negatively regulated PAI-1 expression. The transfection of miR-143-3p suppressed PAI-1 secretion from ovarian cancer cells and inhibited in vitro angiogenesis in HUVECs. Next, miR-143-3p-overexpressing ES2 cells were intraperitoneally injected into BALB/c nude mice. ES2-miR-143-3p cells downregulated PAI-1 production, attenuated angiogenesis, and significantly inhibited intraperitoneal tumor growth following treatment with anti-VEGFA antibody. Continuous anti-VEGFA treatment downregulated miR-143-3p expression, which upregulated PAI-1 and activated an alternative angiogenic pathway in ovarian cancer. In conclusion, the substitution of this miRNA during BEV treatment may help overcome BEV resistance, and this may be used as a novel treatment strategy in clinical settings. IMPLICATIONS: Continuous administration of VEGFA antibody upregulates SERPINE1/PAI-1 expression via the downregulation of miR-143-3p, which contributes to acquiring bevacizumab resistance in ovarian cancer.

Clinical trial assessing the safety of edoxaban with concomitant chemotherapy in patients with gynecological cancer-associated thrombosis (EGCAT study).

BACKGROUND: Gynecological cancer is one of the highest risk factors for cancer-associated thrombosis (CAT). Although low-molecular-weight heparin (LMWH) is recommended as an anticoagulant for treating CAT, recent studies have shown that direct oral anticoagulants (DOACs) are an acceptable alternative. Patients with cancer require a series of chemotherapies concomitantly with DOAC administration; however, the extent to which these drugs influence DOAC blood concentrations is unknown. In this study, we measured the plasma concentration of edoxaban during chemotherapy for gynecological cancers to determine its safety. METHODS: Patients histologically diagnosed with ovarian or uterine corpus cancer and CAT were recruited after primary surgery and before the initiation of postoperative adjuvant chemotherapy, including paclitaxel. Patients were administered edoxaban (30 or 60 mg) orally for CAT. The plasma concentrations of edoxaban and active factor Xa were determined and their percentage change before and after chemotherapy was calculated. Additionally, blood coagulation tests were analyzed. RESULTS: Sixteen patients with gynecological cancer (12 with ovarian cancer and 4 with uterine corpus cancer) were enrolled. Among these, 15 samples were collected one day after chemotherapy initiation. During chemotherapy, the trough concentration of edoxaban changed from 17.6 ± 10.6 to 20.0 ± 15.6 ng/ml, and the mean percentage change in edoxaban concentration was 14.5%. Therefore, the trough concentrations of edoxaban, which represent excretion capacity, were not significantly increased by chemotherapy with paclitaxel. The area under the plasma edoxaban concentration-time curve and the active factor Xa concentration were also unaffected. CONCLUSION: Patients with CAT and ovarian or uterine corpus cancer administered edoxaban orally showed no significant increase in the trough concentration of edoxaban while undergoing chemotherapy. This suggests the safety of edoxaban use during the treatment of gynecological cancers. TRIAL REGISTRATION: EGCAT study; Japan Registry of Clinical Trials, jRCTs051190024.

Exosomal CD47 Plays an Essential Role in Immune Evasion in Ovarian Cancer.

Ovarian cancer is largely diagnosed at advanced stages upon detection of multiple peritoneal dissemination, resulting in poor outcomes. CD47 is overexpressed in tumors, facilitates tumor immune evasion, and is located on exosomes. We aimed to investigate the role of exosomal CD47 in ovarian cancer progression. Prognostic significance of CD47 expression in ovarian cancer was examined using a public database including 1,435 patients and validated with 26 patients at our institution. CD47 expression was associated with poor progression-free survival and inversely correlated with macrophage infiltration in ovarian cancer tissues. Exosomes were collected from ovarian cancer cell lines, and CD47 expression on exosomes was confirmed via flow cytometry. Inhibition of exosome secretion with GW4869 and exosome uptake with 5-(N-ethyl-N-isopropyl)-amiloride inhibited the surface CD47 expression on ovarian cancer cells and promoted phagocytosis by macrophages. RAB27A (a key regulator of exosome release) knockdown inhibited exosome secretion and led to CD47 downregulation in ovarian cancer cells. In a xenograft mouse model, suppression of the release of tumor-derived exosomes by GW4869 or RAB27A knockdown suppressed tumor progression and enhanced M1 macrophage phagocytosis in cancer tissues. Collectively, CD47 expression was correlated with poor prognoses in patients with ovarian cancer, suggesting the importance of immune evasion. CD47 was expressed on exosomes and the inhibition of exosome secretion and/or uptake enhanced cancer cell phagocytosis by macrophages, and thus, suppressed peritoneal dissemination. This suggests the potential of a novel immune checkpoint therapeutic agent that focuses on exosomes. IMPLICATIONS: Mechanistic insight from the current study suggests that exosomal CD47 may be an advantageous therapeutic target in ovarian cancer.

Paclitaxel exposure downregulates miR-522 expression and its downregulation induces paclitaxel resistance in ovarian cancer cells.

Paclitaxel resistance is a critical challenge in ovarian cancer treatment. This study aimed to identify microRNAs (miRNAs) that modulate paclitaxel resistance for use as potential therapeutic targets in such settings. Paclitaxel-resistant cell lines were established using two ovarian cancer cell lines: SKOV3ip1 and HeyA8. The evaluation of miRNA polymerase chain reaction (PCR) arrays indicated that the expression of miR-522-3p was downregulated in paclitaxel-resistant cells. The restoration of miR-522-3p sensitized the resistant cells to paclitaxel, and its downregulation desensitized the parental cells. Using PCR arrays, we focused on E2F2, with the luciferase reporter assay revealing that it was a direct target for miR-522-3p. The paclitaxel-resistant cells showed stronger E2F2 expression than the parental cells, while E2F2 inhibition sensitized the resistant cells to paclitaxel. Forced E2F2 expression in the parental cells led to the acquisition of paclitaxel resistance, while miR-522-3p inhibited E2F2 expression and was associated with retinoblastoma protein phosphorylation attenuation, which resulted in G0/G1 arrest. The effects of miR-522-3p and E2F2 in ovarian cancer were examined using public databases, revealing that low miR-522-3p expression and high E2F2 expression were associated with significantly poorer overall survival. In conclusion, miR-522-3p attenuated the degree of paclitaxel resistance in vitro through the downregulation of E2F2; miR-522-3p supplementation may be a therapeutic target for paclitaxel-resistant ovarian cancer.

Exploring the potential of engineered exosomes as delivery systems for tumor-suppressor microRNA replacement therapy in ovarian cancer.

MicroRNA (miRNA) plays a pivotal role in cancer biology. Therefore, tumor suppressor (TS) miRNAs are an attractive target for cancer therapy. However, clinical trials have failed due to the difficulties in miRNA delivery, warranting the development of a novel drug delivery system (DDS). Exosomes are stable in circulation and selectively picked up by cancer cells, indicating that they can serve as a miRNA carrier. The aim of this study was to explore the possibility of exosomes as a carrier for miRNA replacement therapy for ovarian cancer (OC). First, exosomes were purified from primary-cultured omental fibroblasts of OC patients. miR-199a-3p was selected as a TS miRNA, and the synthesized miR-199a-3p was loaded into exosomes by electroporation. Treatment with miR199a-3p-loaded-exosomes (miR-199a-3p-Exo) drastically increased miR-199a-3p expression level in OC cell lines (CaOV3; 8592-, SKOV3; 67188-, and OVCAR3; 2280-fold). miR-199a-3p-Exo suppressed c-Met expression, a direct target of miR-199a-3p, and thereby inhibited cell proliferation and invasion. In a xenograft study, miR-199a-3p-Exo also drastically inhibited peritoneal dissemination in OC mice model, and diminished c-Met expression, ERK phosphorylation, and MMP2 expression in tumors. These results suggest that miRNA replacement therapy using exosomes shows promise for treatment of OC. Given that omental fibroblasts can be obtained from most OC patients, patient-derived exosomes can be utilized as a DDS for future molecular-targeted therapies.

Pathophysiological Role and Potential Therapeutic Exploitation of Exosomes in Ovarian Cancer.

Exosomes are extracellular vesicles involved in several biological and pathological molecules and can carry many bioactive materials to target cells. They work as important mediators of cell-cell communication and play essential roles in many diseases, especially in cancer. Ovarian cancer is one of the most common gynecological malignancies. Most patients are diagnosed at advanced stages involving widespread peritoneal dissemination, resulting in poor prognosis. Emerging evidence has shown that exosomes play vital roles throughout the progression of ovarian cancer. Moreover, the development of engineered exosome-based therapeutic applications- including drug delivery systems, biomolecular targets and immune therapy-has increased drastically. Herein, we review the functional features of exosomes in ovarian cancer progression and the therapeutic application potential of exosomes as novel cancer treatments.

Clinical effects of switching from minodronate to denosumab treatment in patients with postmenopausal osteoporosis: a retrospective study.

BACKGROUND: Denosumab is a major treatment option for patients with postmenopausal osteoporosis; however, the evidence for its use is lacking. Therefore, in this 24-month retrospective study, we aimed to evaluate the effects of switching from minodronate (MIN) to denosumab in these patients. METHODS: Patients with postmenopausal osteoporosis either switched from MIN to denosumab (Group 1; n = 32) or continued MIN treatment (Group 2; n = 24). Bone mineral density (BMD) of the lumbar spine (L2-L4) and femoral neck was assessed at baseline and every 6 months for 24 months. Serum bone-specific alkaline phosphatase (BAP) and N-terminal telopeptide were measured at baseline, 12 months, and 24 months. RESULTS: Twenty-nine of the 32 patients (90.6%) in group 1 and all patients (24/24) in group 2 completed the 24-month follow-up. Switching from MIN to denosumab (Group 1) significantly increased lumbar BMD at 12, 18, and 24 months (6.1, 7.4, and 9.6%, respectively) and femoral neck BMD at 12, 18, and 24 months (2.8, 3.2, and 3.4%, respectively), whereas MIN continuous treatment (Group 2) showed no significant difference from baseline. Switching therapy also showed a significant decrease in serum BAP from baseline to 12 and 24 months (- 19.3 and - 26.5%, respectively) and serum NTX from baseline to 12 months (- 13.1%), whereas continuous MIN treatment failed to show any significant differences from baseline. CONCLUSION: Switching from MIN to denosumab in patients with postmenopausal osteoporosis showed clinical benefits with regard to BMD and bone turnover markers in comparison with continuous MIN treatment. It may therefore be a valid treatment option in the clinical setting.

Role of the Exosome in Ovarian Cancer Progression and Its Potential as a Therapeutic Target.

Peritoneal dissemination is a distinct form of metastasis in ovarian cancer that precedes hematogenic or lymphatic metastasis. Exosomes are extracellular vesicles of 30-150 nm in diameter secreted by different cell types and internalized by target cells. There is emerging evidence that exosomes facilitate the peritoneal dissemination of ovarian cancer by mediating intercellular communication between cancer cells and the tumor microenvironment through the transfer of nucleic acids, proteins, and lipids. Furthermore, therapeutic applications of exosomes as drug cargo delivery are attracting research interest because exosomes are stabilized in circulation. This review highlights the functions of exosomes in each process of the peritoneal dissemination of ovarian cancer and discusses their potential for cancer therapeutics.

Direct oral anticoagulants are effective and safe for the treatment of venous thromboembolism associated with gynecological cancers.

OBJECTIVE: To retrospectively review the efficacy and safety of novel direct oral anticoagulants (DOACs) and compare the results with those of vitamin K antagonists (VKA) when used in clinical practice to treat venous thromboembolism (VTE) because there is insufficient evidence regarding its use in patients with gynecological cancers. METHODS: A study was conducted of patients diagnosed with gynecological cancers at Osaka University Hospital between January 2010 and December 2017. The medical records of those who suffered from deep venous thrombosis (DVT) and/or pulmonary embolism (PE) were retrospectively reviewed. RESULTS: Among the 1698 cases of gynecological cancers, 107 (6.3%) cases were diagnosed as having VTE. A total of 34 (31.8%) patients presented DVT plus PE and 73 (68.2%) patients had DVT alone. Fifty-four cases were treated with DOACs and 53 with VKA. Although 3 of the 53 patients (5.7%) in the VKA group developed recurrent VTE, only 1 (1.9%) patient in the DOAC group showed clinically relevant bleeding from a tumor penetrating the rectum. DOACs were non-inferior to VKA with respect to the composite outcome, including recurrent venous thrombosis and relevant bleeding (hazard ratio 0.31, 95% confidence interval 0.03-3.12, P=0.363). CONCLUSION: DOACs can be effectively and safely used in VTE patients with gynecological cancers.

Downregulation of miR-194-5p induces paclitaxel resistance in ovarian cancer cells by altering MDM2 expression.

Paclitaxel is a first-line drug for treating epithelial ovarian cancer (EOC). However, prognosis for patients with advanced stage cancer remains poor due to primary or acquired drug resistance. Therefore, overcoming chemoresistance is one of the greatest challenges in treating EOC. In this study, we identified microRNAs (miRNA) that regulate paclitaxel resistance and tested their potential utility as therapeutic targets. Paclitaxel-resistant cell lines were established using two EOC cell lines: SKVO3ip1 and HeyA8. miRNA PCR arrays showed that miR-194-5p was downregulated in paclitaxel-resistant cells. Forced expression of miR-194-5p resensitized resistant cells to paclitaxel. Conversely, miR-194-5p inhibition induced paclitaxel resistance in parental cells. In silico analysis and luciferase reporter assay revealed that MDM2 is a direct target of miR-194-5p. MDM2 was upregulated in paclitaxel resistant cells compared with parental cells. MDM2 inhibition also resensitized resistant cells to paclitaxel and forced MDM2 induced paclitaxel resistance in parental cells. miR-194-5p induced p21 upregulation and G1 phase arrest in resistant cells by downregulating MDM2. Furthermore, a public database showed that high MDM2 expression was associated with a shorter progression-free survival in EOC patients treated with paclitaxel. Collectively, our results show that restoring miR-194-5p expression resensitizes EOCs to paclitaxel, and this may be exploited as a therapeutic option.

Exosomal miR-1290 is a potential biomarker of high-grade serous ovarian carcinoma and can discriminate patients from those with malignancies of other histological types.

BACKGROUND: microRNAs (miRNAs) stably exist in circulating blood encapsulated in extracellular vesicles such as exosomes; therefore, serum miRNAs have the potential to serve as novel cancer biomarkers. New diagnostic markers to detect high grade serous ovarian cancer (HGSOC) are urgently needed. The aim of this study was to identify miRNAs specific to HGSOC and analyze whether serum miRNA can discriminate HGSOC patients from healthy controls or patients with ovarian malignancies of other histological types. METHODS: Exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray analysis revealed several elevated miRNAs specific to HGSOC. Among these, we focused on miR-1290. Sera from 70 ovarian cancer patients and 13 healthy controls were gathered and its expression levels detected by quantitative real-time polymerase chain reaction. RESULTS: In HGSOC patients, serum miR-1290 was significantly overexpressed compared to in healthy controls (3.52 fold; P = 0.03), unlike in patients with ovarian cancers of other histological types. The relative expression of miR-1290 was higher in advanced stages of HGSOC than in early stages (4.23 vs. 1.58; P = 0.23). Its expression significantly decreased after operation (5.87 to 1.17; P < 0.01), indicating that this miRNA reflects tumor burden. A receiver operating characteristic curve analysis showed that at the cut-off of 1.20, the sensitivity and specificity were 63% and 85% respectively for discriminating patients with HGSOC (area under the curve [AUC] = 0.71) from healthy controls, and at the cut-off of 1.55, the sensitivity and specificity were 47% and 85% respectively for discriminating patients with HGSOC (AUC = 0.76) from those with malignancies of other histological types. CONCLUSIONS: Serum miR-1290 is significantly elevated in patients with HGSOC and can be used to discriminate these patients from those with malignancies of other histological types; it is a new potential diagnostic biomarker for HGSOC.