Autophagy In Ovarian Cancer Cells Research Articles

Repressing IRS1/2 by NT157 inhibits the malignant behaviors of ovarian cancer through inactivating PI3K/AKT/mTOR pathway and inducing autophagy.

Insulin receptor substrate 1 and 2 (IRS1/2) have been found involved in many cancers development and their inhibitors exert significant tumor-suppressive effects. Here, we tried to explore the function of NT157, an IGF1R-IRS1/2 inhibitor, in ovarian cancer. We treated ovarian cancer cells with varying doses of NT157. The MTT assay was employed to evaluate cell proliferation and colony formation assay was used for detecting colony-forming ability. TUNEL assay was adopted to test cell apoptosis. Cell invasion was checked by the Transwell assay. The expression of apoptosis-related proteins, autophagy markers, IRS1/2, and PI3K/AKT/mTOR pathway was compared by Western blot, immunofluorescence, or qRT-PCR. As indicated by the data, NT157 abated the viability, proliferation, and induced autophagy of ovarian cancer cells. Overexpressing IRS1/2 attenuated the tumor-suppressive effect of NT157 and heightened the PI3K/AKT/mTOR pathway activation. Inhibition of the PI3K/AKT/mTOR pathway enhanced the tumor-suppressive effect of NT157 and facilitated NT157-mediated autophagy. However, the autophagy inhibitor 3-MA partly reversed NT-157-mediated antitumor effects. In conclusion, this study disclosed that NT157 suppressed the malignant phenotypes of ovarian cancer cells by inducing autophagy and hampering the expression of IRS1/2 and PI3K/AKT/mTOR pathway.

Brucea javanica Oil Emulsion Promotes Autophagy in Ovarian Cancer Cells Through the miR-8485/LAMTOR3/mTOR/ATG13 Signaling Axis.

Background: Ovarian cancer is a common malignant tumor of the female reproductive tract, with the highest mortality rate. At present, no effective approaches to improve the survival rate exist. B. javanica Oil Emulsion (BJOE), an extract from B. javanica (L.) Merr. [Simaroubaceae], exhibits antitumor effects and can increase the sensitivity of radiotherapy and chemotherapy in many types of cancers. MiR-8485, a discovered miRNA, has been shown to be involved in the occurrence and development of tumors. The purpose of this study was to investigate the effect of BJOE on the regulation of mammalian rapamycin target protein (mTOR) autophagy signal pathway and related autophagy factors on ovarian cancer cells through miR-8485. Methods: The main chemical constituents of BJOE were determined by UHPLC-MS/MS. Detection of miR-8485 expression in ovarian cancer cells treated with BJOE by quantitative reverse transcription polymerase chain reaction (qRT-PCR). CCK8 experiment and flow cytometry were used to observe the effects of BJOE and overexpression of miR-8485 on cell proliferation and apoptosis. Then, monodansylcadaverine (MDC) fluorescence staining was used to observe the changes of autophagy vesicles before and after the effect of BJOE and overexpressed miR-8485 on cancer cells. Next, the binding sites between miR8485 and mammalian rapamycin target protein activator 3 (LAMTOR3) were detected by double luciferase reporter assay. Furthermore, qRT-PCR and Western blot experiments were used to explore the changes of autophagy-related factors LAMTOR3, mTOR and autophagy-related 13 (ATG13), and microtubule associated protein 1 light chain 3 beta (LC3-Ⅱ) after BJOE and overexpression of miR-8485, in addition to autophagy inhibitor (3-MA) for rescue experiment verification. Results: The qRT-PCR results showed that the expression of miR-8485 increased after BJOE treatment in the SKOV3 cell. The CCK8 assay and flow cytometry analysis revealed that both BJOE and miR-8485 overexpression inhibited the proliferation and promoted the apoptosis of the SKOV3 cell. MDC fluorescence staining showed that BJOE and miR-8485 overexpression led to a significant increase in autophagy vesicles in the SKOV3 cell. Double luciferase reporter assay confirmed the existence of binding sites between miR8485 and LAMTOR3. The results of qRT-PCR and Western blot showed that BJOE and overexpressed miR-8485 downregulated the expression of LAMTOR3 and mTOR and up-regulated the expression of ATG13 and LC3-Ⅱ. Conclusion: 1) MiR-8485 may be the key factor of BJOE in promoting autophagy and apoptosis and inhibiting cell proliferation of ovarian cancer cells; 2) BJOE may play an antitumor role by regulating LAMTOR3/mTOR/ATG13 signaling axis through miR-8485 to promote autophagy in ovarian cancer cells.

Icariside II suppresses the tumorigenesis and development of ovarian cancer by regulating miR-144-3p/IGF2R axis.

Ovarian cancer is one of the three major gynecological malignancies. It has been reported that Icariside II was able to block the occurrence and development of ovarian cancer. However, the detailed mechanism by which Icariside II regulates the development of ovarian cancer is widely unknown. EdU staining and transwell assays were applied to detect the proliferation, migration, and invasion of ovarian cancer cells. Next, the relationship between miR-144-3p and IGF2R was verified by the dual-luciferase reporter assay. Moreover, in vivo animal model was constructed to verify the effect of Icariside II on the development of ovarian cancer. Icariside II notably inhibited the proliferation, migration, and invasion and induced the apoptosis of ovarian cancer cells. Additionally, Icariside II markedly increased the level of miR-144-3p in ovarian cancer cells. Moreover, IGF2R was targeted by miR-144-3p directly. Icariside II significantly decreased the expression of IGF2R and the phosphorylation level of AKT and mTOR in ovarian cancer cells, which were partially reversed by miR-144-3p inhibitor. Meanwhile, Icariside II remarkably promoted the autophagy of ovarian cancer cells, as confirmed by the increased expression of Beclin-1 and ATG-5 and decreased expression of p62; however, co-treatment with miR-144-3p inhibitor notably decreased autophagy. Furthermore, the result of animal study suggested Icariside II notably inhibited ovarian tumor growth as well. Collectively, Icariside II could suppress the tumorigenesis and development of ovarian cancer by promoting autophagy via miR-144-3p/IGF2R axis. These results may be beneficial for future studies on the use of Icariside II to treat ovarian cancer.

Long non-coding RNA HOXA11-AS knockout inhibits proliferation and overcomes drug resistance in ovarian cancer

ABSTRACT In ovarian carcinogenesis and progression, long non-coding RNAs (lncRNAs) have been shown to have a role, although the underlying processes remain a mystery. By modulating the degree of autophagy in ovarian cancer cells, we sought to learn more about the function lncRNA HOXA11-AS plays in the development of ovarian cancer. The expression of HOXA11-AS in ovarian normal cells and ovarian cancer cell lines was measured using R package and qRT-PCR. Ovarian cancer cells expressed HOXA11-AS substantially higher than normal cells, while cisplatin-resistant cells expressed HOXA11-AS significantly higher than ovarian cancer cells. Next, we studied the prognostic data of HOXA11-AS in ovarian cancer in the Tissue Cancer Genome Atlas (TCGA). In the next step, lentiviral transfection of ovarian cancer cells A2780, OVCAR3, and A2780/DDP (cisplatin-resistant) were performed, and HOXA11-AS knockdown was found to significantly inhibit cell viability, migration, and invasion of A2780 and OVCAR3 cells, and promote apoptosis by CCK-8 assay, transwell assay, cell cycle, and apoptosis assay, and promoted the sensitivity of A2780/DDP cells to cisplatin. It has been shown by the western blot test that HOXA11-AS knockdown increases the amount of cellular autophagy in cells. In contrast, adding the autophagy inhibitor 3-methyladenine (3-MA) to HOXA11-AS cells knocked down in vivo reduced its anti-tumor properties. As a whole, this study found that HOXA11-AS knockdown increased the expression of autophagy-related proteins and improved cisplatin sensitivity, decreased ovarian cancer cell proliferation, and promoted cell apoptosis. This study provides new insights into the role of HOXA11-AS in ovarian cancer regulation.

Resveratrol Contrasts IL-6 Pro-Growth Effects and Promotes Autophagy-Mediated Cancer Cell Dormancy in 3D Ovarian Cancer: Role of miR-1305 and of Its Target ARH-I.

Simple SummaryTumor dormancy is the period during which patients are asymptomatic before recurrence and it is difficult to target pharmacologically. Inflammatory cytokines present in the tumor microenvironment likely contribute to this event. We show that IL-6 inhibits autophagy in ovarian cancer cells via miRNAs downregulation of ARH-I, an effect contrasted by the nutraceutical resveratrol (RV). In detail, RV keeps the cancer cells in an autophagy-mediated dormant-like state contrasting the IL-6 pro-growth activity. Additionally, we show that ARH-I (DIRAS3) is a bona fide target of miR-1305, a novel oncomiRNA upregulated by IL-6 and downregulated by RV. Our findings have a translational impact since we observed that patients with high DIRAS3 expression have a better outcome, and this correlates with MIR1305 downregulation. Overall, maintaining a permanent cell dormancy by the chronic administration of RV might be considered a therapeutic option to prevent the “awakening” of cancer cells in response to a permissive microenvironment.Tumor dormancy is the extended period during which patients are asymptomatic before recurrence, and it represents a difficult phenomenon to target pharmacologically. The relapse of tumors, for instance arising from the interruption of dormant metastases, is frequently observed in ovarian cancer patients and determines poor survival. Inflammatory cytokines present in the tumor microenvironment likely contribute to such events. Cancer cell dormancy and autophagy are interconnected at the molecular level through ARH-I (DIRAS3) and BECLIN-1, two tumor suppressors often dysregulated in ovarian cancers. IL-6 disrupts autophagy in ovarian cancer cells via miRNAs downregulation of ARH-I, an effect contrasted by the nutraceutical protein restriction mimetic resveratrol (RV). By using three ovarian cancer cell lines with different genetic background in 2D and 3D models, the latter mimicking the growth of peritoneal metastases, we show that RV keeps the cancer cells in a dormant-like quiescent state contrasting the IL-6 growth-promoting activity. Mechanistically, this effect is mediated by BECLIN-1-dependent autophagy and relies on the availability of ARH-I. We also show that ARH-I (DIRAS3) is a bona fide target of miR-1305, a novel oncomiRNA upregulated by IL-6 and downregulated by RV. Clinically relevant, bioinformatic analysis of a transcriptomic database showed that the high expression of DIRAS3 and MAP1LC3B mRNAs together with that of CDKN1A, directing a cellular dormant phenotype, predicts better overall survival in ovarian cancer patients, and this correlates with MIR1305 downregulation. The possibility of maintaining a permanent cell dormancy in ovarian cancer by the chronic administration of RV should be considered as a therapeutic option to prevent the “awakening” of cancer cells in response to a permissive microenvironment, thus limiting the risk of tumor relapse and metastasis.

MicroRNA miR-30a inhibits cisplatin resistance in ovarian cancer cells through autophagy

ABSTRACT We study whether microRNA miR-30a inhibits the autophagy through transforming growth factor (TGF)-β/Smad4 to generate cisplatin (DDP) resistance in ovarian cancer cells. The expression of miR-30a, Smad4, and TGF-β was detected in the serum of ovarian cancer patients and DDP-resistant cell lines (A2780) by quantitative real-time polymerase chain reaction (qRT-PCR). Computational search and western blotting were used to demonstrate the downstream target of miR-30a in ovarian cancer cells. Cell viability was measured with CCK8 assay. Apoptosis and autophagy of ovarian cancer cells were analyzed by flow cytometry and transmission electron microscopy, and the expressions of Beclin1 and LC3II protein were detected by western blotting. Expression of miR-30a was significantly decreased, while expressions of TGF-β and Smad4 mRNA were increased in serum of ovarian cancer patients after DDP chemotherapy as well as in DDP-resistant cells. Activation of autophagy contributed to DDP-resistance cells. Moreover, Bioinformatics software predicted Smad4 to be a target of miR-30a. Overexpression of miR-30a decreased the expression of Smad4 and TGF-β. Additionally, miR-30a-overexpressing inhibited DDP-induce autophagy and promoted DDP-resistant cell apoptosis. In conclusion, miR-30a mediates DDP resistance in ovarian cancer by inhibiting autophagy via the TGF-β/Smad4 pathway.

Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.

Simple SummaryOvarian cancer (OC) is the most common cause of cancer-related deaths among women worldwide, and its incidence has been increasing and has continued to prove resistant to a variety of therapeutics. This observation is principally disturbing given the amount of money invested in identifying novel therapies for this disease. A comparatively rapid and economical pipeline for identification of novel drugs is drug repurposing. We reported earlier that the antimalarial drug Quinacrine (QC) also has anticancer activity and here we discovered that QC significantly upregulates cathepsin L (CTSL) and promoting autophagic flux in ovarian cancer. QC-induced CTSL activation promotes lysosomal membrane permeability resulting in active CTSL release into the cytosol, which promotes Bid cleavage, mitochondrial membrane permeability, cytochrome-c release and cell death in both in-vitro and in-vivo models. Therefore, QC is a promising candidate for OC treatment.We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

MKNK2 enhances chemoresistance of ovarian cancer by suppressing autophagy via miR-125b

Chemoresistance is a major cause for high mortality and poor survival in patients with ovarian cancer. Changes of cellular autophagy is associated with tumor cell chemoresistance. MAP kinase interacting serine/threonine kinase 2 (MKNK2) belongs to the protein kinase superfamily mediating cell cycle, apoptosis and angiogenesis. However, its effects on chemoresistance during ovarian cancer development remain unclear. In this study, we found that MKNK2 expression levels were markedly up-regulated in chemoresistant ovarian cancer cells compared with the sensitive cells. In addition, significantly increased expression of MKNK2 was detected in clinical ovarian cancer tissues, particularly in tumor samples from patients with drug resistance, and high MKNK2 expression is closely associated with poor prognosis. Our in vitro experiments subsequently showed that MKNK2 knockdown markedly reduced the proliferation of chemoresistant ovarian cancer cells, which was confirmed in SKOV3/DDP xenograft mouse models. Importantly, MKNK2 knockdown considerably induced autophagy in ovarian cancer cells with drug resistance, which was involved in the suppression of cell proliferation. Of note, we showed that miR-125b directly targeted MKNK2, and a negative correlation was observed between the expression of them in clinical tumor tissues. MKNK2 silence also increased miR-125b expression levels in drug-resistant ovarian cancer cells. Intriguingly, MKNK2 knockdown-suppressed cell proliferation and -induced autophagy were almost abrogated by miR-125b inhibition in chemoresistant ovarian cancer cells. Together, these findings demonstrated that MNKN2 is responsible for chemoresistance in ovarian cancer through modulating autophagy by targeting miR-125b, which may be a promising therapeutic target to develop strategies against ovarian cancer with drug resistance.

ROS-Mediated Apoptosis and Autophagy in Ovarian Cancer Cells Treated with Peanut-Shaped Gold Nanoparticles.

BackgroundEven with considerable improvement in treatment of epithelial ovarian cancer achieved in recent years, an increasing chemotherapy resistance and disease 5-year relapse is recorded for a majority part of patients that encourages the search for better therapeutic options. Gold nanoparticles (Au NPs) due to plethora of unique physiochemical features are thoroughly tested as drug delivery, radiosensitizers, as well as photothermal and photodynamic therapy agents. Importantly, due to highly controlled synthesis, it is possible to obtain nanomaterials with directed size and shape.MethodsIn this work, we developed novel elongated-type gold nanoparticles in the shape of nanopeanuts (AuP NPs) and investigated their cytotoxic potential against ovarian cancer cells SKOV-3 using colorimetric and fluorimetric methods, Western blot, flow cytometry, and fluorescence microscopy.ResultsPeanut-shaped gold nanoparticles showed high anti-cancer activity in vitro against SKOV-3 cells at doses of 1–5 ng/mL upon 72 hours treatment. We demonstrate that AuP NPs decrease the viability and proliferation capability of ovarian cancer cells by triggering cell apoptosis and autophagy, as evidenced by flow cytometry and Western blot analyses. The overproduction of reactive oxygen species (ROS) was noted to be a critical mediator of AuP NPs-mediated cell death.ConclusionThese data indicate that gold nanopeanuts might be developed as nanotherapeutics against ovarian cancer.

Saponins Extracted from Tea (Camellia Sinensis) Flowers Induces Autophagy in Ovarian Cancer Cells.

Tea flower saponins (TFS) possess effective anticancer properties. The diversity and complexity of TFS increases the difficulty of their extraction and purification from tea flowers. Here, multiple methods including solvent extraction, microporous resin separation and preparative HPLC separation were used to obtain TFS with a yield of 0.34%. Furthermore, we revealed that TFS induced autophagy—as evidenced by an increase in MDC-positive cell populations and mCherry-LC3B-labeled autolysosomes and an upregulation of LC3II protein levels. 3-MA reversed the decrease in cell viability induced by TFS, showing that TFS induced autophagic cell death. TFS-induced autophagy was not dependent on the Akt/mTOR/p70S6K signaling pathway. TFS-induced autophagy in OVCAR-3 cells was accompanied by ERK pathway activation and reactive oxygen species (ROS) generation. This paper is the first report of TFS-mediated autophagy of ovarian cancer cells. These results provide new insights for future studies of the anti-cancer effects of TFS.

LncRNA GAS8-AS1 Inhibits Ovarian Cancer Progression Through Activating Beclin1-Mediated Autophagy.

BackgroundEarly detection and diagnosis of ovarian cancer (OC) is complicated due to the concealment of the ovarian anatomical position and the lack of clinical manifestations and specific indicators of early OC. Therefore, it is urgent to study the pathogenesis of OC, especially the molecular mechanism.ResultsLncRNA GAS8-AS1 was decreased in OC tissues and cell lines, and high expression of GAS8-AS1 indicated a higher 5-year survival rate of OC patients. Overexpression of GAS8-AS1 suppressed growth of OC cells, while deletion of GAS8-AS1 promoted the progression of OC cells. Further data indicated GAS8-AS1 activated autophagy in OC cells. Functional experiments showed that 3-MA removed the inhibitory effect of GAS8-AS1 in OC cells. On the contrary, Rapamycin reversed the promoting effect of GAS8-AS1 in OC cells. Furthermore, GAS8-AS1 bound with Beclin1 and promoted its expression, and silencing of Beclin1 reversed the inhibitory role of GAS8-AS1 in OC progression. In vivo tumorigenesis assay showed GAS8-AS1 suppressed OC progression and activated Beclin1 mediated autophagy.ConclusionOur study suggested GAS8-AS1 inhibited OC progression by activating autophagy via binding with Beclin1, and GAS8-AS1 might be a potential therapeutic target for OC clinical treatment.

Potential relationship between Sirt3 and autophagy in ovarian cancer.

Sirtuin 3 (Sirt3) is an important member of the sirtuin protein family. It is a deacetylase that was previously reported to modulate the level of reactive oxygen species (ROS) production and limit the extent of oxidative damage in cellular components. As an important member of the class III type of histone deacetylases, Sirt3 has also been documented to mediate nuclear gene expression, metabolic control, neuroprotection, cell cycle and proliferation. In ovarian cancer (OC), Sirt3 has been reported to regulate cellular metabolism, apoptosis and autophagy. Sirt3 can regulate autophagy through a variety of different molecular signaling pathways, including the p62, 5′AMP-activated protein kinase and mitochondrial ROS-superoxide dismutase pathways. However, autophagy downstream of Sirt3 and its association with OC remains poorly understood. In the present review, the known characteristics of Sirt3 and autophagy were outlined, and their potential functional roles were discussed. Following a comprehensive analysis of the current literature, Sirt3 and autophagy may either serve positive or negative roles in the regulation of OC. Therefore, it is important to identify the appropriate expression level of Sirt3 to control the activation of autophagy in OC cells. This strategy may prove to be a novel therapeutic method to reduce the mortality of patients with OC. Finally, potential research directions into the association between Sirt3 and other signaling pathways were provided.

Abstract 3717: LncRNA RNF157-AS1 inhibits autophagy in ovarian cancer cells by downregulating ARHI: The different role in proliferation and drug resistance

Abstract Ovarian cancer(OC) is the commonest cause of female cancer death. Long noncoding RNAs (lncRNAs) has been shown to have regulatory roles in the procession of many cancers, including OC. Here, in our previous study, we reported that lncRNA RNF157-AS1 was overexpression in OC tissues, which is confirmed by GSE144077 and TCGA matched GTEx normal data. Further experiments revealed that RNF157-AS1 knockdown significantly repressed the proliferation both in vitro and in vivo and overexpressed RNF157-AS1 promoted the proliferation of OC cells. However, the higher expression of RNF157-AS1 predicted a better prognosis for OC patients in TCGA database. RNA-seq analysis revealed that ARHI was significant overexpressed after RNF157-AS1 silencing and it is negative correlated with RNF157-AS1 in TCGA and matched GTEx normal data. ARHI, a autophagy-related gene, could induced autophagy in OC cells. Because of 70% of OC patients were diagnosed with advanced tumors and need to chemotherapy. Overexpression of RNF157-AS1 could inhibit autophagy-associated cell death and promote rapid proliferation of OC cells, while overexpressed RNF157-AS1 decrease autophagy and induced cells apoptosis under adverse environment. To test our hypothesis, CCK8 assay suggested overexpression of RNF157-AS1 significantly decreased the chemoresistance to cisplatin (DDP) and silencing of RNF157-AS1 increased the DDP-resistance of OC cells, which are confirmed by clone formation assay and apoptosis experiments. Western blot assay also suggested that RNF157-AS1 silencing promote ARHI, LC3II/I and beclin1 expression. To our knowledge, this is the first report revealed that the different roles of RNF157-AS1 in OC cells' proliferation and chemoresistance and our study also can provide guidance for clinical treatment of OC patients. Citation Format: Sujuan Xu, Pengfei Xu. LncRNA RNF157-AS1 inhibits autophagy in ovarian cancer cells by downregulating ARHI: The different role in proliferation and drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3717.

Abstract 4083: Crizotinib improves the therapeutic efficacy of olaparib in ovarian cancer

Abstract Despite improvements in treatment, ovarian cancer still proves lethal in >60% of cases. One of the most important factors contributing to poor patient outcomes is the ability of dormant ovarian cancer cells to survive following primary surgery and chemotherapy. Poly (ADP) ribose polymerase inhibitors (PARPi) are promising agents for treating both primary and recurrent ovarian cancers. However, acquired drug resistance to PARPi decreases the clinical efficacy of these agents. We have previously found that olaparib induces autophagy in ovarian cancer and the addition of drugs that inhibit autophagy, such as chloroquine, can overcome drug resistance and enhance the anti-cancer activity of PARP inhibitors in ovarian cancer cells, cell line-derived xenograft and patient-derived xenograft models. Building upon this work, our group has sought drugs that are selectively toxic for autophagic ovarian cancer cells and that might enhance sensitivity to PARP inhibitors. In a previous study, our laboratory had found that knockdown of the anaplastic lymphoma kinase (ALK) significantly reduced survival of ovarian cancer cells that were undergoing autophagy. The FDA-approved ALK inhibitor, crizotinib, exhibited significantly greater toxicity for autophagic ovarian cancer cells in culture and cured a fraction of mice with dormant, autophagic ovarian cancer xenografts. As olaparib induces autophagy in ovarian cancer cells and crizotinib selectively targets autophagic cancer cells, we 1) explored the vulnerability of autophagic cells to the combination treatment of olaparib and crizotinib both in vitro and in vivo; and 2) the mechanism by which the combination of olaparib and crizotinib regulates cell proliferation and programmed cell death. Enhancement of olaparib activity was observed in 4 ovarian cancer cell lines (OVCAR8, OVCAR3, HEY and SKOV3). Moreover, a combination of olaparib and crizotinib proved more effective than either single agent in a xenograft model of the OVCAR8 ovarian cancer cell line. Mechanistically, a combination of olaparib and crizotinib induces DNA damage and accumulation of γ-H2AX. A combination of olaparib and crizotinib increased reactive oxygen species, while decreasing the phosphorylation of AKT. This decrease in p-AKT was associated with a decrease in p-mTOR, all of which contributed to an increase in autophagy, documented by western blot analysis, LC3 puncta and electron microscopy. Decreased p-AKT also correlated with a decrease in its downstream effector, survivin, leading to apoptosis. Together, our data suggest that crizotinib can enhance the therapeutic efficacy of olaparib in ovarian cancer by modulating autophagy and apoptosis. Moreover, we have identified a unique combination of therapy with olaparib and crizotinib that could improve the therapeutic efficacy of olaparib and outcomes for ovarian cancer patients. Citation Format: Janice M. Santiago-O'Farrill, Alicia M. Blessing, Sarah E. Becker, Zhen Lu, Robert C. Bast. Crizotinib improves the therapeutic efficacy of olaparib in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4083.

FTO accelerates ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy

ObjectiveFat mass and obesity-associated protein (FTO) is identified as a critical demethylase involved in various physiological processes. Despite efforts have been made to study the biological functions of FTO in certain cancers, the role of FTO in ovarian cancer is largely unknown. In this study, we sought to investigate the function of FTO on proliferation, apoptosis and autophagy of ovarian cancer cells. MethodsQuantitative real-time PCR was performed to detect FTO expression in ovarian tumor tissues and ovarian cancer cell lines OVCAR-3, SKOV-3, COC1, HO-8910 and A2780. SKOV-3 cells were constructed with FTO overexpression and A2780 cells were constructed with FTO knockdown. CCK-8 assay was used to examine cell viability and flow cytometry was used to detect cell apoptosis. Activity assay kits were applied to detect caspase-3 and caspase-9 levels. Western blot was performed to measure the expressions of FTO, PCNA, Bax, Bcl-2, LC3, ATG5, P62, p-AKT and AKT. Stable FTO-overexpression SKOV-3 cells or FTO-depletion A2780 cells were injected subcutaneously into male Balb/c-nu mice. Xenografted tumors were assayed by H&E staining. Immunohistochemistry was subjected to measure FTO and Ki67 expressions. ResultsFTO was up-regulated in ovarian tumor tissues compared with non-cancerous ovarian tissues. FTO overexpression markedly increased viability and autophagy function, but decreased apoptosis of ovarian cancer cells. In addition, FTO overexpression promoted AKT phosphorylation. In contrast, FTO silence showed the opposite effect. ConclusionFTO accelerated ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy.

Metformin Affects Paclitaxel Sensitivity of Ovarian Cancer Cells Through Autophagy Mediated by Long Noncoding RNASNHG7/miR-3127-5p Axis.

Background: Ovarian cancer is the public health issue worldwide. Paclitaxel is a first-line chemotherapy drug for ovarian cancer, but paclitaxel resistance weakens the therapeutic effect. Metformin (Met) improved the paclitaxel sensitivity in a mouse model of ovarian cancer. However, the mechanism of Met on paclitaxel sensitivity is still unclear in ovarian cancer. Materials and Methods: Cell viability, apoptosis, migration, and invasion were measured by Cell Counting Kit-8 (CCK8), flow cytometry, and transwell assays severally. The expression of long noncoding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) and microRNA-3127-5p (miR-3127-5p) were detected by real-time quantitative polymerase chain reaction. The protein levels of poly (ADP-ribose) polymerase, microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, and Beclin 1 were examined by Western blot assay. RNA immunoprecipitation assay detected the relationship between SNHG7 and miR-3127-5p. Then, the binding correlation between SNHG7 and miR-3127-5p was predicted by starBase and verified by the dual-luciferase reporter. The effects of Met and SNHG7 on tumor growth were tested in ovarian cancer mice model. Results: Met inhibited cell viability, migration, invasion, SNHG7 level, and autophagy and promoted apoptosis in paclitaxel-resistant ovarian cancer cells. Moreover, Met partly reversed SNHG7-mediated paclitaxel sensitivity and autophagy in ovarian cancer cells. SNHG7 directly bound to miR-3127-5p. Met abolished the promoting effect of SNHG7 overexpression on tumor growth and autophagy in vivo. Conclusion: The authors' findings indicated that Met expedited paclitaxel sensitivity by regulating SNHG7/miR-3127-5p-mediated autophagy in ovarian cancer cells.

Human omental adipose-derived mesenchymal stem cells enhance autophagy in ovarian carcinoma cells through the STAT3 signalling pathway

BackgroundOur previous study showed that human omental adipose-derived stem cells (ADSCs) promote ovarian cancer growth and metastasis. In this study, the role of autophagy in the ovarian cancer-promoting effects of omental ADSCs was further determined. MethodsThe growth and invasion of ovarian cancer cells were detected by CCK-8 and Transwell assays, respectively. The autophagy of ovarian cancer cells transfected with MRFP-GFP-LC3 adenoviral vectors was evaluated by confocal microscopy and western blot assay. Transfection of STAT3 siRNA was used to inhibit the expression of STAT3. ResultsOur results show that autophagy plays a vital role in ovarian cancer and is promoted by ADSCs. Specifically, we show that proliferation and invasion are correlated with autophagy induction by ADSCs in two ovarian cancer cell lines under hypoxic conditions. Mechanistically, ADSCs activate the STAT3 signalling pathway, thereby promoting autophagy. Knockdown of STAT3 expression using siRNA decreased hypoxia-induced autophagy and decreased the proliferation and metastasis of ovarian cancer cells. ConclusionTaken together, our data indicate that STAT3-mediated autophagy induced by ADSCs promotes ovarian cancer growth and metastasis.

Nobiletin Triggers Reactive Oxygen Species-Mediated Pyroptosis through Regulating Autophagy in Ovarian Cancer Cells.

Ovarian cancer is one of the most serious female malignancies worldwide. Despite intensive efforts being made to overcome ovarian cancer, there still remain limited optional treatments for this disease. Nobiletin, a prospective food-derived phytochemical extracted from citrus fruits, has recently been reported to suppress ovarian cancer cells, but the role of pyroptosis in ovarian carcinoma with nobiletin still remains unknown. In this study, we aim to explore the effect of nobiletin on ovarian carcinoma and further expound the underlying mechanisms of nobiletin-induced ovarian cancer cell death. Our results showed that nobiletin could significantly inhibit cell proliferation, induce DNA damage, and also lead to apoptosis by increasing the cleaved poly (ADP-ribose) polymerase (PARP) level of human ovarian cancer cells (HOCCs) in a dose-dependent manner. Moreover, we revealed that nobiletin decreased mitochondrial membrane potential and induced reactive oxygen species (ROS) generation and autophagy of HOCCs, contributing to gasdermin D-/gasdermin E-mediated pyroptosis. Taken together, nobiletin as a functional food ingredient represents a promising new anti-ovarian cancer candidate that could induce apoptosis and trigger ROS-mediated pyroptosis through regulating autophagy in ovarian cancer cells.

PD-L1 regulates tumorigenesis and autophagy of ovarian cancer by activating mTORC signaling.

PD-L1 is a well-known immune co-stimulatory molecule that regulates tumour cell escape from immunity by suppressing the immune response. However, the clinical significance of PD-L1 in the progression of ovarian cancer is unclear. Our study demonstrated that PD-L1 is up-regulated in ovarian tumour tissue compared with its expression level in adjacent normal tissue. Furthermore, we confirmed that PD-L1 increases the proliferation of cancer cells by activating the AKT-mTORC signalling pathway, which is also enhanced by the expression of S6K, the substrate of mTORC. In addition, PD-L1 promotes the autophagy of ovarian cancer cells by up-regulating the expression of BECN1, a crucial molecule involved in the regulation of autophagy. In conclusion, PD-L1 may provide a target for the development of a novel strategy for the treatment of ovarian cancer.

Inhibition LC3B can increase chemosensitivity of ovarian cancer cells

BackgroundOvarian cancer is often accompanied by the production of ascites, and patients with repeated ascites are associated with chemotherapy resistance. The previous study confirmed that the ovarian cancer patients who developed ascites after chemotherapy had elevated autophagy levels in the ascites and precipitated cells, which was positively correlated with MDR1 expression in the blood of patients.MethodsIn order to explore the correlation between autophagy and chemoresistant, we searched TCGA and GEO database to analyze the correlation between LC3B and MDR1, and identified the targeting miRNA of LC3B. It was verified by dual luciferase that miR-204 can target LC3B. The ovarian cancer cell line and the BALB/c nude mice tumor-bearing model were selected for in vitro and in vivo verification. In vitro studies confirmed that ovarian cancer cells were more sensitive to cisplatin by inhibiting LC3B.ResultsOverexpression of miR-204 reduced the expression of LC3B, Atg7, and MDR1, and promoted apoptosis. In vivo studies have also confirmed that reducing the level of autophagy in ovarian cancer cells increases the sensitivity to cisplatin.ConclusionsIt suggests that miR-204 can be used as a tumor suppressor gene and LC3B expression level can be used as a potential molecular marker to guide the diagnosis and treatment of patients with ovarian cancer.