Exosomal miR-4516 derived from ovarian cancer stem cells enhanced cisplatin tolerance in ovarian cancer by inhibiting GAS7.

Ovarian cancer (OC) is a devastating disease for women, with chemotherapy resistance taking the lead. Cisplatin has been the first-line therapy for OC for a long time. However, the resistance of OC to cisplatin is an important impediment to its efficacy. Mounting studies showed that ovarian cancer stem cells (OCSCs) affected chemotherapy resistance by secreting exosomes. MicroRNAs (miRNAs) play important roles in exosomes secreted by OCSCs. Here, through the analysis of GEO database (GSE107155) combined with RT-qPCR of OC-related cells/clinical tissues, it was found that hsa-miR-4516 (miR-4516) was significantly up-regulated in OCSCs. Then, OCSCs-derived exosomes were isolated and identified, and it was observed the influence of exosomes on the chemoresistance in SKOV3/cisplatin (SKOV3/DDP) cells. These results manifested that OCSCs-mediated exosomes facilitated the chemoresistance of SKOV3/DDP cells by delivering miR-4516 into them. Growth arrest-specific 7 (GAS7), a downstream target of miR-4516, was determined by bioinformatics prediction combined with molecular biological detection. Next, we up-regulated GAS7 expression and discovered that the promotion of chemoresistance in SKOV3/DDP cells by OCSCs-derived exosomes was significantly impaired. Finally, the mice tumor model of SKOV3/DDP cells was built to estimate the effect of GAS7 over-expression on OC growth. The results showed that GAS7 inhibited the chemoresistance of OC in vivo. In conclusion, our experiments suggested that OCSCs-derived exosomes enhanced OC cisplatin resistance by suppressing GAS7 through the delivery of miR-4516. This study provides a possible target for the treatment of OC DDP resistance.

Advances in ovarian tumor stem cells and therapy.

Ovarian cancer is considered the most lethal among all gynecological malignancies due to its early metastatic dissemination, extensive spread, and malignant ascites. The current standard of care for advanced ovarian cancer involves a combination of cytoreductive surgery and chemotherapy utilizing platinum-based and taxane-based agents. Although initial treatment yields clinical remission in 70-80% of patients, the majority eventually develop treatment resistance and tumor recurrence. A growing body of evidence indicates the existence of cancer stem cells within diverse solid tumors, including ovarian cancer, which function as a subpopulation to propel tumor growth and disease advancement by means of drug resistance, recurrence, and metastasis. The presence of ovarian cancer stem cells is widely considered to be a significant contributor to the unfavorable clinical outcomes observed in patients with ovarian cancer, as they play a crucial role in mediating chemotherapy resistance, recurrence, and metastasis. Ovarian cancer stem cells possess the capacity to reassemble within the entirety of the tumor following conventional treatment, thereby instigating the recurrence of ovarian cancer and inducing resistance to treatment. Consequently, the creation of therapeutic approaches aimed at eliminating ovarian cancer stem cells holds great potential for the management of ovarian cancer. These cells are regarded as one of the most auspicious targets and mechanisms for the treatment of ovarian cancer. There is a pressing need for a comprehensive comprehension of the fundamental mechanisms of ovarian cancer's recurrence, metastasis, and drug resistance, alongside the development of effective strategies to overcome chemoresistance, metastasis, and recurrence. The implementation of cancer stem cell therapies may potentially augment the tumor cells' sensitivity to existing chemotherapy protocols, thereby mitigating the risks of tumor metastasis and recurrence, and ultimately improving the survival rates of ovarian cancer patients.

Active DNA Demethylase, TET1, Increases Oxidative Phosphorylation and Sensitizes Ovarian Cancer Stem Cells to Mitochondrial Complex I Inhibitor.

Ten-eleven translocation 1 (TET1) is a methylcytosine dioxygenase involved in active DNA demethylation. In our previous study, we demonstrated that TET1 reprogrammed the ovarian cancer epigenome, increased stem properties, and activated various regulatory networks, including metabolic networks. However, the role of TET1 in cancer metabolism remains poorly understood. Herein, we uncovered a demethylated metabolic gene network, especially oxidative phosphorylation (OXPHOS). Contrary to the concept of the Warburg effect in cancer cells, TET1 increased energy production mainly using OXPHOS rather than using glycolysis. Notably, TET1 increased the mitochondrial mass and DNA copy number. TET1 also activated mitochondrial biogenesis genes and adenosine triphosphate production. However, the reactive oxygen species levels were surprisingly decreased. In addition, TET1 increased the basal and maximal respiratory capacities. In an analysis of tricarboxylic acid cycle metabolites, TET1 increased the levels of α-ketoglutarate, which is a coenzyme of TET1 dioxygenase and may provide a positive feedback loop to modify the epigenomic landscape. TET1 also increased the mitochondrial complex I activity. Moreover, the mitochondrial complex I inhibitor, which had synergistic effects with the casein kinase 2 inhibitor, affected ovarian cancer growth. Altogether, TET1-reprogrammed ovarian cancer stem cells shifted the energy source to OXPHOS, which suggested that metabolic intervention might be a novel strategy for ovarian cancer treatment.

Novel players in the development of chemoresistance in ovarian cancer: ovarian cancer stem cells, non-coding RNA and nuclear receptors.

Ovarian cancer (OC) ranks as the fifth leading factor for female mortality globally, with a substantial burden of new cases and mortality recorded annually. Survival rates vary significantly based on the stage of diagnosis, with advanced stages posing significant challenges to treatment. OC is primarily categorized as epithelial, constituting approximately 90% of cases, and correct staging is essential for tailored treatment. The debulking followed by chemotherapy is the prevailing treatment, involving platinum-based drugs in combination with taxanes. However, the efficacy of chemotherapy is hindered by the development of chemoresistance, both acquired during treatment (acquired chemoresistance) and intrinsic to the patient (intrinsic chemoresistance). The emergence of chemoresistance leads to increased mortality rates, with many advanced patients experiencing disease relapse shortly after initial treatment. This review delves into the multifactorial nature of chemoresistance in OC, addressing mechanisms involving transport systems, apoptosis, DNA repair, and ovarian cancer stem cells (OCSCs). While previous research has identified genes associated with these mechanisms, the regulatory roles of non-coding RNA (ncRNA) and nuclear receptors in modulating gene expression to confer chemoresistance have remained poorly understood and underexplored. This comprehensive review aims to shed light on the genes linked to different chemoresistance mechanisms in OC and their intricate regulation by ncRNA and nuclear receptors. Specifically, we examine how these molecular players influence the chemoresistance mechanism. By exploring the interplay between these factors and gene expression regulation, this review seeks to provide a comprehensive mechanism driving chemoresistance in OC.

Advances in the role of GPX3 in ovarian cancer (Review).

Ovarian cancer (OC) is the 5th most common malignancy in women, and the leading cause of death from gynecologic malignancies. Owing to tumor heterogeneity, lack of reliable early diagnostic methods and high incidence of chemotherapy resistance, the 5­year survival rate of patients with advanced OC remains low despite considerable advances in detection and therapeutic approaches. Therefore, identifying novel therapeutic targets to improve the prognosis of patients with OC is crucial. The expression of glutathione peroxidase 3 (GPX3) plays a crucial role in the growth, proliferation and differentiation of various malignant tumors. In OC, GPX3 is the only antioxidant enzyme the high expression of which is negatively correlated with the overall survival of patients. GPX3 may affect lipid metabolism in tumor stem cells by influencing redox homeostasis in the tumor microenvironment. The maintenance of stemness in OC stem cells (OCSCs) is strongly associated with poor prognosis and recurrence in patients. The aim of the present study was to review the role of GPX3 in OC and investigate the potential factors and effects of GPX3 on OCSCs. The findings of the current study offer novel potential targets for drug therapy in OC, enhance the theoretical foundation of OC drug therapy and provide valuable references for clinical treatment.

Anisomycin inhibits the activity of human ovarian cancer stem cells via regulating antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling.

BACKGROUND: Ovarian cancer stem cells (OCSCs) are the main cause of relapse and drug resistance in patients with ovarian cancer. Anisomycin has been shown to be an effective antitumor agent, but its mechanism of action in ovarian cancer remains elusive. METHODS: CD44+/CD133+ human OCSCs were isolated from human ovarian cancer tissues. OCSCs were interfered with using anisomycin and specific small-interfering RNA (siRNA). Microarray assay, MTT, in vivo tumorigenic experiments, transwell assay, cell cycle assay, colony formation assay, angiogenesis assay, and hematoxylin and eosin staining were used to detect the mechanism of anisomycin with respect to inhibiting the activity of OCSCs. Expression of the NCBP2-AS2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) pathway was examined using western blotting, a quantitative real-time PCR (RT-qPCR) and immunofluorescence staining. Bioinformatics analysis was used for predictive analysis of NCBP2-AS2 expression in urogenital tumors. RESULTS: Microarray analysis showed that treatment with anisomycin significantly decreased the expression of antisense RNA NCBP2-AS2 in OCSCs. In vitro cellular experiments showed that interfering with endogenous antisense RNA NCBP2-AS2 using siRNA distinctly inhibited the proliferation, migration and angiogenesis of OCSCs, whereas in vivo animal experiments revealed decreased tumorigenesis in nude mice. Moreover, the results of RT-qPCR and western blotting demonstrated that both anisomycin treatment and NCBP2-AS2 silencing led to significant reductions in the mRNA and protein expression levels of NCBP2-AS2, MEK, ERK and STAT3. From a bioinformatic point of view, antisense RNA NCBP2-AS2 exhibited significantly differential expression between urogenital tumors and normal controls, and a similar expression pattern was found in the genes NCBP2, RPL35A, DNAJC19 and ECE2, which have similarity to NCBP2-AS2. CONCLUSIONS: Anisomycin suppresses the in vivo and in vitro activity of human OCSCs by downregulating the antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling pathway, whereas the antisense RNA NCBP2-AS2 and genes with similarity have the potential to serve as markers for clinical diagnosis and prognosis of urogenital tumors.

miR-134-3p driven by anisomycin impairs ovarian cancer stem cell activity through inhibiting GPR137 expression.

Background: Ovarian cancer recurrence and metastasis are predominantly attributed to ovarian cancer stem cells; however, the mechanism by which anisomycin regulates human ovarian cancer stem cells (HuOCSCs) remains unclear. Methods: cDNA microArray was used to screen microRNAs (miRNAs) targeted by anisomycin, and RT-qPCR validated the miRNA targets. TargetScan database, GO enrichment analysis, and RT-qPCR, accompanied by a fluorescent reporter system, were employed to verify the miRNA target genes. In vitro experimental cell proliferation inhibition assay, flow cytometry, Transwell, angiogenesis assay, and in vivo transplantation tumor assay were implemented to assess the ability of the overexpressed miRNAs to hinder HuOCSC activity. Western blot, RT-qPCR, and immunofluorescence were applied to measure the transcriptional and protein-level expression of the miRNA target genes and their related genes. Bioinformatic analysis predicted and deciphered the role of the miRNA target genes and related genes in the development and prognosis of ovarian cancer. Results: The expression levels of multiple DLK1-DIO3 imprinted microRNA cluster members were altered by anisomycin, among which miR-134-3p expression was most significantly elevated. miR-134-3p overexpression significantly suppressed HuOCSC activity. The screening and validation of target genes uncovered that miR-134-3p was able to markedly suppress GPR137 expression. Additionally, miR-134-3p regulated the cytoskeleton, migration-related protein in the NDEL1/DYNEIN/TUBA1A axis through targeting GPR137. Bioinformatics prediction unveiled a close association of GPR137, NDEL1, DYNC1H1, and TUBA1A with ovarian cancer development and prognosis. Conclusions: The activity of HuOCSCs may be compromised by anisomycin through the regulation of miR-134-3p, which inhibits the GPR137/NDEL1/DYNEIN/TUBA1A axis.

"DEPHENCE" system-a novel regimen of therapy that is urgently needed in the high-grade serous ovarian cancer-a focus on anti-cancer stem cell and anti-tumor microenvironment targeted therapies.

Ovarian cancer, especially high-grade serous type, is the most lethal gynecological malignancy. The lack of screening programs and the scarcity of symptomatology result in the late diagnosis in about 75% of affected women. Despite very demanding and aggressive surgical treatment, multiple-line chemotherapy regimens and both approved and clinically tested targeted therapies, the overall survival of patients is still unsatisfactory and disappointing. Research studies have recently brought some more understanding of the molecular diversity of the ovarian cancer, its unique intraperitoneal biology, the role of cancer stem cells, and the complexity of tumor microenvironment. There is a growing body of evidence that individualization of the treatment adjusted to the molecular and biochemical signature of the tumor as well as to the medical status of the patient should replace or supplement the foregoing therapy. In this review, we have proposed the principles of the novel regimen of the therapy that we called the "DEPHENCE" system, and we have extensively discussed the results of the studies focused on the ovarian cancer stem cells, other components of cancer metastatic niche, and, finally, clinical trials targeting these two environments. Through this, we have tried to present the evolving landscape of treatment options and put flesh on the experimental approach to attack the high-grade serous ovarian cancer multidirectionally, corresponding to the "DEPHENCE" system postulates.

Short peptide domains of the Wnt inhibitor sFRP4 target ovarian cancer stem cells by neutralizing the Wnt ß-catenin pathway, disrupting the interaction between ß-catenin and CD24 and suppressing autophagy.

AIMS: One of the hallmarks of cancer stem cells (CSC) is hyperactive Wnt ß-catenin signaling due to the decreased presence of Wnt antagonists such as secreted frizzled related protein 4 (SFRP4). Cysteine-rich domain (CRD) and netrin-like domain (NLD) are the two functional domains of SFRP4 having anti-tumor properties. In this study, we have explored the effectiveness of short micropeptides SC-301 (from CRD) and SC-401 (from NLD) on CSC properties, EMT, apoptosis and autophagy in ovarian CSCs enriched from PA-1 and SKOV-3 cell lines. MAIN METHODS: Gene expression analysis, Western blot and immunocytochemistry were performed on ovarian CSCs to evaluate the inhibitory potential of micropeptides to various CSC associated oncogenic properties. Co-immunoprecipitation was performed to detect the binding of CD24 to ß-catenin protein complex. CYTO-ID Autophagy Detection Kit 2.0 was used to monitor autophagic flux in peptide treated CSCs. KEY FINDINGS: It is clearly seen that the micropeptides derived from both the domains inhibit Wnt pathway, initiate apoptosis, inhibit migration and chemosensitize CSCs. Specifically, CD24, a defining marker of ovarian CSC was suppressed by peptide treatment. Notably, interaction between CD24 and ß-catenin was disrupted upon peptide treatment. SFRP4 peptide treatment also suppressed the autophagic process which is crucial for CSC survival. SIGNIFICANCE: The study demonstrated that although both peptides have inhibitory effects, SC-401 was emphatically more effective in targeting CSC properties and down regulating the Wnt ß-catenin machinery.

Anisomycin has a potential toxicity of promoting cuproptosis in human ovarian cancer stem cells by attenuating YY1/lipoic acid pathway activation.

Ovarian cancer is a highly malignant gynecologic tumor that seriously endangers women's health. We previously demonstrated that anisomycin significantly inhibited the activity of ovarian cancer stem cells (OCSCs) in vitro and in vivo. In the present study, anisomycin treatment of OCSCs significantly reduced ATP and T-GSH content; and increased pyruvate, LPO, and MDA. Anisomycin also significantly inhibited the proliferation of OCSCs in vitro, and its effect was similar to that of elesclomol and buthionine sulfoximine (BSO), suggesting that it has the potential to promote cuproptosis of OCSCs. Our subsequent cDNA microarray analysis results showed that anisomycin significantly reduced the transcriptional levels of genes that protect copper metabolism and cuproptosis, including the PDH complex, metallothionein, lipoid acid pathway, and FeS cluster proteins. Bioinformatics analysis revealed that four core factors (lipoic acid pathway FDX1, DLD, DLAT, PDH), and transcription factor YY1 were highly expressed in ovarian cancer tissues and were significantly correlated with an unfavorable prognosis. Further analysis depicted multiple YY1-recognized motif basic sites as existing in the promoters of the above four factors. In addition, the expression levels of YY1 in the tissue samples from ovarian cancer patients were significantly positively correlated with the expression levels of FDX1, DLD, DLAT, PDHB, and other genes. Finally, the analysis of the peripheral blood exosome database disclosed that the contents of the four key factors of YY1 and the lipoic acid pathway in the peripheral blood exosomes of patients with ovarian cancer were significantly elevated relative to those of normal healthy individuals. Therefore, our molecular biology experiments combined with bioinformatics analysis results suggest that the direct target of anisomycin-induced cuproptosis in ovarian cancer stem cells is probably a YY1 transcription factor. By inhibiting the expression and activity of YY1, anisomycin could not activate the transcriptional activity of the core genes of the lipoic acid pathway (i.e.,FDX1, DLD, DLAT, and PDHB), and induced the accumulation of cytotoxic substances, eventually leading to potential cuproptosis in ovarian cancer stem cells.

Corrigendum: SNORA72 activates the Notch1/c-Myc pathway to promote stemness transformation of ovarian cancer cells.

[This corrects the article DOI: 10.3389/fcell.2020.583087.].

Salinomycin-Loaded High-Density Lipoprotein Exerts Promising Anti-Ovarian Cancer Effects by Inhibiting Epithelial-Mesenchymal Transition.

Background: Effective treatments for ovarian cancer remain elusive, and survival rates have long been considered grim. Ovarian cancer stem cells (OCSCs) and epithelial-mesenchymal transition (EMT) are associated with cancer progression and metastasis, as well as drug resistance and eventual treatment failure. Salinomycin (Sal) has an extensive effect on a variety of cancer stem cells (CSCs); however, its poor water solubility and toxicity to healthy tissues at high doses limit further research into its potential as an anti-cancer drug. We proposed a therapeutic strategy by constructing a tumor-targeting carrier that mimics high-density lipoprotein (HDL) to synthesize salinomycin-loaded high-density lipoprotein (S-HDL). This strategy helps reduce the side effects of salinomycin, thereby improving its clinical benefits. Methods: OCSCs were isolated from ovarian cancer cells (OCCs) and the uptake of HDL nanoparticles was observed using laser confocal microscopes. After the cell viability analysis revealed the inhibitory effect of S-HDL on OCCs and OCSCs, the main biological processes influenced by S-HDL were predicted with a transcriptome sequencing analysis and verified in vitro and in vivo. Results: Cellular uptake analysis showed that the HDL delivery system was able to significantly enhance the uptake of Sal by OCCs, tentatively validating the targeting role of recombinant HDL, so that S-HDL could reduce the toxicity of Sal and increase its anti-ovarian cancer effects. Conversely, S-HDL could exert anti-ovarian cancer effects by inhibiting the proliferation of OCCs and OCSCs, promoting apoptosis, blocking EMT, and suppressing stemness and angiogenesis-related protein expression in vitro and in vivo. Conclusion: S-HDL had stronger anti-ovarian cancer effects than unencapsulated Sal. Thus, it may be a potential agent for ovarian cancer treatment in the future.

Anti-Cancer Activity of Cannabis sativa Phytocannabinoids: Molecular Mechanisms and Potential in the Fight against Ovarian Cancer and Stem Cells.

Ovarian cancer (OC) is the most lethal gynecological malignancy, with about 70% of cases diagnosed only at an advanced stage. Cannabis sativa, which produces more than 150 phytocannabinoids, is used worldwide to alleviate numerous symptoms associated with various medical conditions. Recently, studies across a range of cancer types have demonstrated that the phytocannabinoids Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) have anti-cancer activity in vitro and in vivo, but also the potential to increase other drugs' adverse effects. THC and CBD act via several different biological and signaling pathways, including receptor-dependent and receptor-independent pathways. However, very few studies have examined the effectiveness of cannabis compounds against OC. Moreover, little is known about the effectiveness of cannabis compounds against cancer stem cells (CSCs) in general and OC stem cells (OCSCs) in particular. CSCs have been implicated in tumor initiation, progression, and invasion, as well as tumor recurrence, metastasis, and drug resistance. Several hallmarks and concepts describe CSCs. OCSCs, too, are characterized by several markers and specific drug-resistance mechanisms. While there is no peer-reviewed information regarding the effect of cannabis and cannabis compounds on OCSC viability or development, cannabis compounds have been shown to affect genetic pathways and biological processes related to CSCs and OCSCs. Based on evidence from other cancer-type studies, the use of phytocannabinoid-based treatments to disrupt CSC homeostasis is suggested as a potential intervention to prevent chemotherapy resistance. The potential benefits of the combination of chemotherapy with phytocannabinoid treatment should be examined in ovarian cancer patients.

Differentially expression and function of circular RNAs in ovarian cancer stem cells.

BACKGROUND: Circular RNAs (circRNAs) are noncoding RNAs that regulate miRNA expression; however, their functions in cancer stem cells (CSCs) are not well known. METHODS: To determine the function of differentially expression of circRNAs associated with ovarian CSCs, circRNA profiling was conducted using a circRNA-based microarray on sphere-forming cells derived from A2780 and SKOV3 epithelial ovarian cancer cells termed A2780-SP and SKOV3-SP compared to monolayer cells such as A2780 and SKOV3 cells, respectively. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the biological functions of the circRNAs expressed in CSCs. RESULTS: The circRNA-based microarray data showed that 159 circRNAs were significantly upregulated (fold change > 1.5) and 55 circRNAs were downregulated in ovarian CSCs compared to monolayer cells. GO and KEGG enrichment analysis of differentially expressed circRNAs in ovarian CSCs showed that they were mainly involved in cell cycle, histone modification, cellular protein metabolic process, cell cycle, apoptotic signaling pathway, and ubiquitin-mediated proteolysis in ovarian cancer. In addition, the hsa-circRNA000963-miRNA-mRNA regulatory network was constructed based on potential target of miRNAs. These analyses involved that the biological function of the hsa-circRNA00096/miRNA/mRNA network was involved in signaling pathways regulating pluripotency of stem cells, PI3K-Akt signaling pathway, cell cycle, p53 signaling pathway, Wnt signaling pathway, calcium modulating pathway, and production of miRNAs involved in gene silencing by miRNA. CONCLUSIONS: Our data demonstrate the expression profiles of circRNAs in ovarian CSCs and suggest that circRNAs may be potential diagnostic and predictive biomarkers of ovarian cancer.

Plasma-activated medium inhibits cancer stem cell-like properties and exhibits a synergistic effect in combination with cisplatin in ovarian cancer.

Ovarian cancer stem-like cells (CSCs) have been implicated in tumor recurrence, metastasis, and drug resistance. Accumulating evidence has demonstrated the antitumor effect of plasma-activated medium (PAM) in various carcinomas, including ovarian cancer. Thus, PAM represents a novel onco-therapeutic strategy. However, its impact on ovarian CSCs is unclear. Here, we show that ovarian CSCs resistant to high-dose conventional chemotherapeutic agents used for ovarian cancer treatment exhibited dose-dependent sensitivity to PAM. In addition, PAM treatment reduced the expression of stem cell markers and sphere formation, along with the aldehyde dehydrogenase- or CD133-positive cell population. We further investigated the effect of PAM in combination with other chemotherapeutics on ovarian CSCs in vitro. PAM exhibited synergistic cytotoxicity with cisplatin (CDDP) but not with paclitaxel and doxorubicin. In a peritoneal metastasis xenograft model established via intraperitoneal spheroid injection, PAM intraperitoneal therapy significantly suppressed peritoneal carcinomatosis (tumor size and number), with a more significant decrease observed due to the combined effects of PAM and CDDP with no side effects. Taken together, our results indicate that PAM inhibits ovarian CSC traits and exhibits synergetic cytotoxicity with CDDP, demonstrating PAM as a promising intraparietal chemotherapy for enhancing antitumor efficacy and reducing side effects.

Cancer Stem Cells in Ovarian Cancer-A Source of Tumor Success and a Challenging Target for Novel Therapies.

Ovarian cancer is the most lethal neoplasm of the female genital organs. Despite indisputable progress in the treatment of ovarian cancer, the problems of chemo-resistance and recurrent disease are the main obstacles for successful therapy. One of the main reasons for this is the presence of a specific cell population of cancer stem cells. The aim of this review is to show the most contemporary knowledge concerning the biology of ovarian cancer stem cells (OCSCs) and their impact on chemo-resistance and prognosis in ovarian cancer patients, as well as to present the treatment options targeted exclusively on the OCSCs. The review presents data concerning the role of cancer stem cells in general and then concentrates on OCSCs. The surface and intracellular OCSCs markers and their meaning both for cancer biology and clinical prognosis, signaling pathways specifically activated in OCSCs, the genetic and epigenetic regulation of OCSCs function including the recent studies on the non-coding RNA regulation, cooperation between OCSCs and the tumor microenvironment (ovarian cancer niche) including very specific environment such as ascites fluid, the role of shear stress, autophagy and metabolic changes for the function of OCSCs, and finally mechanisms of OCSCs escape from immune surveillance, are described and discussed extensively. The possibilities of anti-OCSCs therapy both in experimental settings and in clinical trials are presented, including the recent II phase clinical trials and immunotherapy. OCSCs are a unique population of cancer cells showing a great plasticity, self-renewal potential and resistance against anti-cancer treatment. They are responsible for the progression and recurrence of the tumor. Several completed and ongoing clinical trials have tested different anti-OCSCs drugs which, however, have shown unsatisfactory efficacy in most cases. We propose a novel approach to ovarian cancer diagnosis and therapy.

Identification of microRNA expression profiles of CD44+ ovarian cancer stem cells.

PURPOSE: The aim of our study was to investigate microRNA (miRNA) expression profiles in CD44+ ovarian cancer stem cells (ovarian CSCs). METHODS: In this study, we enriched CD44+ ovarian CSCs using magnetic activated cell sorting (MACS). A combination of real-time quantitative PCR (qRT-PCR), western blot and sphere formation assays was used to demonstrate stem cell-like properties. RNA sequencing was used to detect the miRNA expression profiles in CD44+ ovarian CSCs. Transient transfection, qRT-PCR, western blot and sphere formation assays were further used to test the function of miR-181a-2-3p. RESULTS: We found that CD44+ ovarian CSCs showed enhanced sphere formation and expression of stemness-associated genes (NANOG, OCT4, SOX2) compared to ovarian cancer cells. The RNA sequencing results showed that the miRNA expression profiles of CD44+ ovarian CSCs were different from those of ovarian cancer cells. GO and KEGG pathway analyses indicated that these miRNAs regulate stem cell-like properties in CD44+ ovarian CSCs. In addition, miR-181a-2-3p negatively regulates the stem cell-like properties of CD44+ ovarian CSCs by targeting EGR1. CONCLUSION: Our data suggest that miRNAs play important roles in regulating the stem cell-like properties of CD44+ ovarian CSCs.

Loss of Krüppel-like factor 9 facilitates stemness in ovarian cancer ascites-derived multicellular spheroids via Notch1/slug signaling.

The ascites that develops in advanced OC, both at diagnosis and upon recurrence, is a rich source of multicellular spheroids/aggregates (MCSs/MCAs), which are the major seeds of tumor cell dissemination within the abdominal cavity. However, the molecular mechanism by which specific ascites-derived tumor cells survive and metastasize remains largely unknown. In this study, we elucidated cancer stem cell (CSC) properties of ascites-derived MCSs, concomitant with enhanced malignancy, induced EMT, and low KLF9 (Krüppel-like factor 9) expression, compared with PTCs. KLF9 was also downregulated in OC cell line-derived spheroids and the CD117+ CD44+ subpopulation in MCSs. Functional experiments demonstrated that KLF9 negatively modulated stem-like properties in OC cells. Mechanistic studies revealed that KLF9 reduced the transcriptional expression of Notch1 by directly binding to the Notch1 promoter, thereby inhibiting the function of slug in a CSL-dependent manner. Clinically, expression of KLF9 was associated with histological grade and loss of KLF9 predicts poor prognosis in OC.

Repositioning Trimebutine Maleate as a Cancer Treatment Targeting Ovarian Cancer Stem Cells.

The overall five-year survival rate for late-stage patients of ovarian cancer is below 29% due to disease recurrence and drug resistance. Cancer stem cells (CSCs) are known as a major contributor to drug resistance and recurrence. Accordingly, therapies targeting ovarian CSCs are needed to overcome the limitations of present treatments. This study evaluated the effect of trimebutine maleate (TM) targeting ovarian CSCs, using A2780-SP cells acquired by a sphere culture of A2780 epithelial ovarian cancer cells. TM is indicated as a gastrointestinal motility modulator and is known to as a peripheral opioid receptor agonist and a blocker for various channels. The GI50 of TM was approximately 0.4 µM in A2780-SP cells but over 100 µM in A2780 cells, demonstrating CSCs specific growth inhibition. TM induced G0/G1 arrest and increased the AV+/PI+ dead cell population in the A2780-SP samples. Furthermore, TM treatment significantly reduced tumor growth in A2780-SP xenograft mice. Voltage gated calcium channels (VGCC) and calcium-activated potassium channels (BKCa) were overexpressed on ovarian CSCs and targeted by TM; inhibition of both channels reduced A2780-SP cells viability. TM reduced stemness-related protein expression; this tendency was reproduced by the simultaneous inhibition of VGCC and BKCa compared to single channel inhibition. In addition, TM suppressed the Wnt/ß-catenin, Notch, and Hedgehog pathways which contribute to many CSCs characteristics. Specifically, further suppression of the Wnt/ß-catenin pathway by simultaneous inhibition of BKCa and VGCC is necessary for the effective and selective action of TM. Taken together, TM is a potential therapeutic drug for preventing ovarian cancer recurrence and drug resistance.