Chemoresistant Ovarian Cancer Cells Research Articles

Retro-inversion follicle-stimulating hormone peptide-modified nanoparticles for delivery of PDK2 shRNA against chemoresistant ovarian cancer by switching glycolysis to oxidative phosphorylation

BackgroundMost ovarian cancers are diagnosed at advanced stages characterized by abdominal dissemination and frequently exhibit chemoresistance. Pyruvate dehydrogenase kinase 2 (PDK2) regulates the switch between glycolysis and oxidative phosphorylation and contributes to tumor progression and chemoresistance. Here, we investigated the effects of PDK2 blockade on metabolic reprogramming and cisplatin sensitivity and evaluated the in vivo antitumor effects of PDK2 shRNA in chemoresistant ovarian cancer using retro-inverso follicle-stimulating hormone peptide-modified nanoparticle as carriers.MethodsThe expression of PDK2 was detected by immunohistochemistry, Western blot and real-time PCR. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry. Cell migration was detected by Transwell assay. Seahorse Analyzer was used to evaluate metabolic changes. The cisplatin-resistant ovarian cancer cells A2780cp were used to establish the mouse model of peritoneal metastatic ovarian cancer.ResultsA higher expression level of PDK2 was observed in chemoresistant ovarian cancer tissues and cell lines and was associated with shorter progression-free survival. PDK2 knockdown inhibited proliferation and migration and promoted apoptosis of both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Cisplatin sensitivity was increased even in cisplatin-resistant ovarian cancer cells. Mechanistically, PDK2 knockdown resulted in an increased oxygen consumption rate and decreased extracellular acidification rate, along with reduced lactate production, increased PDHC activity and increased levels of electron transport chain complexes III and V. The metabolism switched from glycolysis to oxidative phosphorylation. Finally, to specifically and effectively deliver PDK2 shRNA in vivo, we formulated a targeted delivery system containing retro-inverso follicle-stimulating hormone peptide as a targeting moiety and polyethylene glycol–polyethylenimine copolymers as carriers. The nanoparticle complex significantly suppressed tumor growth and peritoneal metastasis of cisplatin-resistant ovarian cancer without obvious toxicities.ConclusionsOur findings showed the link between metabolic reprogramming and chemoresistance in ovarian cancer and provided an effective targeting strategy for switching metabolic pathways in cancer therapy.

Identification of key genes and pathways related to cancer-associated fibroblasts in chemoresistance of ovarian cancer cells based on GEO and TCGA databases

BackgroundStudies have revealed the implications of cancer-associated fibroblasts (CAFs) in tumor progression, metastasis, and treatment resistance. Here, in silico analyses were performed to reveal the key genes and pathways by which CAFs affected chemoresistance in ovarian cancer.MethodsCandidate genes were obtained from the intersected differentially expressed genes in ovarian cancer, ovarian cancer chemoresistance, and ovarian CAF-related microarrays and chemoresistance-related genes from GeneCards databases. Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Set Enrichment Analysis were employed to identify the pathways engaged in ovarian cancer chemoresistance and ovarian CAF-related pathways. The top genes with high Degree in the protein-protein interaction network were intersected with the top genes enriched in the key pathways, followed by correlation analyses between key genes and chemotherapeutic response. The expression profiles of key genes were obtained from Human Protein Atlas database and TCGA-ovarian cancer data.Resultsp53, cell cycle, PI3K-Akt, and MAPK pathways were the key pathways related to the implication of CAFs in ovarian cancer chemoresistance. 276 candidate genes differentially expressed in CAFs were associated with ovarian cancer chemoresistance. MYC, IGF1, HRAS, CCND1, AKT1, RAC1, KDR, FGF2, FAS, and EGFR were enriched in the key chemoresistance-related ways. Furthermore, MYC, EGFR, CCND1 exhibited close association with chemotherapeutic response to platinum and showed a high expression in ovarian cancer tissues and platinum-resistant ovarian cancer cells.ConclusionThe study suggests the key genes (MYC, EGFR, and CCND1) and pathways (p53, cell cycle, PI3K-Akt, and MAPK) responsible for the effect of CAFs on ovarian cancer chemoresistance.

Abstract 2391: Alterations in metformin mediated metabolism in chemoresistant ovarian cancer cells

Abstract Ovarian cancer (OVCA) is the fifth leading cause of death in post-menopausal women in comparison to all other gynecologic malignancies. Late detection of OVCA is common as screening is done mostly in women over 63. This delay in screening coupled with poor prognosis presents the need for novel treatment measures. Repurposing of Metformin (Met), a biguanide used to increase insulin responses in the body, has been identified as a promising approach for therapeutic treatment in OVCA patients. The purpose of this study is to isolate metabolic pathways used by Met to alter OVCA onset. Preliminary results show that Met inhibits cell proliferation and clonogenic growth in both HeyA8 and HeyA8MDR which are drug-sensitive and drug-resistant cell lines, respectively. In combination with carboplatin, Met treatment enhanced chemosensitivity in both cell lines. In addition, lipidomic analysis was done to monitor the impact of lipid metabolites on OVCA cells. Our analysis showed differences in neutral lipid composition in drug-sensitive and resistant cells which indicates the role of various lipid species play in mediating drug-resistance. Furthermore, Met treatment triggered anti-cancerous pathways through syntheses of lipid metabolites. This data indicates that cell proliferation, along with clonogenic growth are inhibited dose dependently by Met treatment in drug-resistant and drug-sensitive cell lines. Chemosensitivity is enhanced by Met treatment in combination with carboplatin in HeyA8 and HeyA8MDR cell lines. Together, the data suggests that autophagy and lipid biogenesis can be altered through metabolic pathways by Met treatment. Through the use of repurposed Metformin, this approach shows promising results of a treatment regimen and potential prevention of OVCA. Citation Format: Brenita Colette Jenkins, Debarshi Roy, Kristal Maner-Smith, Viji Shridhar. Alterations in metformin mediated metabolism in chemoresistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2391.

Abstract 906: Targeting CD166 to overcome platinum resistance in ovarian cancer

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids by OC cells disseminated in the peritoneal fluid. Within a spheroid, OC cells undergo an epithelial-to-mesenchymal transition (EMT), which involves changes in the expression of cell adhesion molecules (CAMs) and acquisition of cancer stem cell (CSC) characteristics, therefore protecting OC cells during chemotherapy. Cluster of differentiation 166 (CD166) is one of the CAMs involved in tumor progression and its modulation impacts cell-cell adhesion, leading to impaired ability of tumor cells to metastasize. However, limited number of studies are available on the functional role of CD166 in the initiation, progression, and chemoresistance of OC. Our goal was to investigate the contribution of CD166 in supporting chemoresistance and the tumorigenic OCSC phenotype, thus proposing this molecule as a potential new therapeutic target in OC. Methods: We compared CD166 expression in primary OC cells grown as spheroids and monolayers and in chemoresistant versus sensitive OC cell lines by q-PCR and Western Blot (WB). CD166 blockade by shRNA knockdown (KD) or anti-CD166 inhibitory antibody (clone AZN-L50) was evaluated on spheroid assay and colony formation. Combinatorial carboplatin and AZN-L50 treatment was assessed on spheroid assays and by WB of pro-apoptotic proteins. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the CD166 promoter. Results: OC cells grown as spheroids showed a significant increase in CD166 expression compared to monolayers. Our results also demonstrated an increase in CD166 expression levels in chemoresistant OC cell lines when compared to the chemosensitive counterpart. A comparison of normal adjacent ovarian epithelium, localized, and metastatic OC tissues on a multitissue array revealed that CD166 expression coincided with an advanced promigratory phenotype (n=88, P<0.001), correlating with poor outcome. CD166 blockade by either shRNA-mediated KD or AZN-L50 treatment in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. The combination of carboplatin and CD166 blockade increased cleaved-PARP and -caspase-3 levels compared to single agent alone, indicating sustained DNA damage. Mechanistically, β-catenin-KD decreased CD166 expression levels and β-catenin coimmunoprecipitated with the CD166 promoter, suggesting that CD166 is a direct β-catenin target. Conclusions: CD166 is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that inhibition of CD166 along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Rula Atwani, Mayuri Prasad, George E Sandusky, Salvatore Condello. Targeting CD166 to overcome platinum resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 906.

Abstract 5500: A PTP4A3 inhibitor reduces ovarian cancer dissemination and alters STAT3 and ERK activation

Abstract The objective of this study was twofold: (1) to examine the pharmacological effects of a potent, allosteric, small molecule inhibitor (KVX-053 or 7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione) of the oncogenic protein tyrosine phosphatase PTP4A3, which is overexpressed in human ovarian cancer and is associated with poor patient prognosis, and (2) to define alterations in ovarian cancer cells with acquired resistance to this PTP4A3 phosphatase inhibitor. PTP4A3 promotes cell migration, survival, metabolism, angiogenesis, and tumor formation, which are mediated at least partly by PTP4A3’s ability to function as a positive signal transducer capable of activating STAT3 and ERK1/2. We found KVX-053 inhibited the migration and, at higher concentrations, the viability of chemosensitive and chemoresistant human ovarian cancer cell lines in vitro. Using an established ovarian cancer metastasis prevention model in which taxane-resistant SKOV3TRip2 cells were injected i.p. followed by four treatments with 20 mg/kg KVX-053, we observed a median survival of 68 days compared to the median survival of 58 days in the vehicle treated mice. We also observed a statistically significant reduction in the number of tumor implantation sites with KVX-053 treatment. Since drug resistance frequently occurs with targeted cancer therapies, we next generated A2780 and OVCAR4 ovarian cancer cells that were 32- and 4-fold resistant to KVX-053, respectively. This was accomplished by using stepwise increased compound exposure over a 3-4 month period to probe acquired cellular resistance changes caused by KVX-053. The resulting KVX-053-resistant cells had similar in vitro growth rates with the wildtype cells and retained resistance to KVX-053 for at least one month when grown in the absence of the small molecule inhibitor. They were not cross-resistant to paclitaxel, cisplatin or teniposide but were cross-resistant to chemical analogs of KVX-053. The KVX-053-resistant A2780 cells exhibited a 95% decrease in the activated form of STAT3, Y705 phospho-STAT3, and a 57% decrease in the phosphorylation of a second activation site S727 without any marked differences in the total STAT3 levels. In contrast, phospho-ERK1/2 and total ERK1/2 were elevated in the ovarian cancer cells with stable resistance to KVX-053. These results further confirm the in vivo activity of KVX-053 against ovarian cancer and suggest that chronic KVX-053 exposure disrupts the PTP4A3/STAT3 and PTP4A3/ERK cellular signaling pathways. Citation Format: John S. Lazo, Kelly N. Isbell, Danielle C. Llaneza, Ettore J. Rastelli, Charles N. Landen, Peter Wipf, Elizabeth R. Sharlow. A PTP4A3 inhibitor reduces ovarian cancer dissemination and alters STAT3 and ERK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5500.

Ascitic fluid shear stress in concert with hepatocyte growth factor drive stemness and chemoresistance of ovarian cancer cells via the c-Met-PI3K/Akt-miR-199a-3p signaling pathway.

Overcoming drug resistance is an inevitable challenge to the success of cancer treatment. Recently, in ovarian cancer, a highly chemoresistant tumor, we demonstrated an important role of shear stress in stem-like phenotype and chemoresistance using a three-dimensional microfluidic device, which most closely mimics tumor behavior. Here, we examined a new mechanosensitive microRNA—miR-199a-3p. Unlike most key microRNA biogenesis in static conditions, we found that Dicer, Drosha, and Exportin 5 were not involved in regulating miR-199a-3p under ascitic fluid shear stress (0.02 dynes/cm2). We further showed that hepatocyte growth factor (HGF), but not other ascitic cytokines/growth factors such as epidermal growth factor and tumor necrosis factor α or hypoxia, could transcriptionally downregulate miR-199a-3p through its primary transcript miR-199a-1 and not miR-199a-2. Shear stress in the presence of HGF resulted in a concerted effect via a specific c-Met/PI3K/Akt signaling axis through a positive feedback loop, thereby driving cancer stemness and drug resistance. We also showed that miR-199a-3p expression was inversely correlated with enhanced drug resistance properties in chemoresistant ovarian cancer lines. Patients with low miR-199a-3p expression were more resistant to platinum with a significantly poor prognosis. miR-199a-3p mimic significantly suppressed ovarian tumor metastasis and its co-targeting in combination with cisplatin or paclitaxel further decreased the peritoneal dissemination of ovarian cancer in mice. These findings unravel how biophysical and biochemical cues regulate miR-199a-3p and is important in chemoresistance. miR-199a-3p mimics may serve as a novel targeted therapy for effective chemosensitization.

MSLN Correlates With Immune Infiltration and Chemoresistance as a Prognostic Biomarker in Ovarian Cancer.

Mesothelin (MSLN) is a glycoprotein with various expression degrees in different tumors including mesothelioma, ovarian cancer, pancreatic cancer, etc. MSLN is considered to play an important role in cell survival, proliferation, and tumor progression. Although the expression of MSLN in tumors makes it a potential therapeutic target, its mechanism of action is still unclear, especially its correlation with immune cells infiltration in the tumor microenvironment has not been investigated. In this study, we detected the overexpression of MSLN in ovarian cancer using database analysis and tissue-array staining. We further evaluated the diagnostic value of MSLN and found it was associated with poor overall survival in ovarian cancer. In addition, the high expression of MSLN was significantly related to the immune-related genes and chemoresistant genes. We confirmed the overexpression of MSLN in the chemoresistant ovarian cancer cell lines. Our research suggests that MSLN participates in a variety of pathways related to the suppression of immune activation and promotion of chemoresistance, leading to a poor prognosis in ovarian cancer.

Investigation of role of CpG methylation in some epithelial mesenchymal transition gene in a chemoresistant ovarian cancer cell line.

Ovarian cancer is one of the lethal gynecologic cancers. Chemoresistance is an essential reason for treatment failure and high mortality. Emerging evidence connects epithelial-mesenchymal transition (EMT) like changes and acquisition of chemoresistance in cancers. Including EMT, DNA methylation influences cellular processes. Here, EMT-like changes were investigated in cisplatin-resistant A2780 ovarian cancer cells (A2780cis), wherein role of DNA methylation in some EMT genes regulations was studied. Cell viability assay was carried out to test the sensitivity of A2780, and A2780cis human cancer cell lines to cisplatin. Differential mRNA expression of EMT markers using qPCR was conducted to investigate EMT like changes. CpG methylation role in gene expression regulation was investigated by 5-azacytidine (5-aza) treatment. DNA methylation changes in EMT genes were identified using Methylscreen assay between A2780 and A2780cis cells. In order to evaluate if DNA methylation changes are causally underlying EMT, treatment with 5-aza followed by Cisplatin was done on A2780cis cells. Accordingly, morphological changes were studied under the microscope, whereas EMT marker’s gene expression changes were investigated using qPCR. In this respect, A2780cis cell line has maintained its cisplatin tolerance ability and exhibits phenotypic changes congruent with EMT. Methylscreen assay and qPCR study have revealed DNA hypermethylation in promoters of epithelial adhesion molecules CDH1 and EPCAM in A2780cis compared to the cisplatin-sensitive parental cells. These changes were concomitant with gene expression down-regulation. DNA hypomethylation associated with transcription up-regulation of the mesenchymal marker TWIST2 was observed in the resistant cells. Azacytidine treatment confirmed DNA methylation role in regulating gene expression of CDH1, EPCAM and TWIST2 genes. A2780cis cell line undergoes EMT like changes, and EMT genes are regulated by DNA methylation. To that end, a better understanding of the molecular alterations that correlate with chemoresistance may lead to therapeutic benefits such as chemosensitivity restoration.

Contribution of stress granules and spliceosomal components to the formation of chemoresistance in ovarian cancer cells

The emergence of chemoresistance in cancer cells is one of the main reasons for the high mortality from oncological diseases. We have shown that signaling molecules from dying cancer cells contribute to a more aggressive therapy‐resistant phenotype. However, the molecular mechanisms underlying this effect remain elusive.Here we aimed to find out which proteins secreted by dying tumor cells get into recipient cells and to determine the molecular cascades triggered in response to incubation with therapy‐induced secretomes. We modified the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) technology. A heavy‐labeled primary culture of ovarian cancer cells was treated or untreated with cisplatin for 48 h. Then using ultrafiltration cartridges, a fraction of extracellular vesicles (EV) were isolated from culture media. The obtained EV were resuspended in a medium containing Medium‐labeled amino acids and added to the recipient chemonaive unlabeled tumor cells for 24 h. During incubation, recipient cells absorbed EV with Heavy‐labeled proteins from donor cells and synthesized new proteins with Medium label. Then, we performed proteomic analysis of recipient cells. In total, we identified 4224 proteins. Interestingly, dying tumor cells secrete and recipient cells absorb a large number of spliceosomal and ribosomal proteins along with translation initiation complex. These proteins are often presented in stress granules. In response to EV from dying tumor cells, recipient cells upregulated the translation of genes responsible for the regulation of the cell cycle and oxidative phosphorylation.To our knowledge, this is the first report about the secretion of stress granules and spliceosomal components into the extracellular space and their internalization in other cells. To confirm these data we performed transfection of donor cells with constructs encoding the fluorescent proteins TIA1 (protein of stress granules) and SRSF4 (protein of the spliceosome). We showed that both proteins are re‐located together from the nucleus into the cytoplasm under the cisplatin treatment. Then, we demostrated the absorption of fluorescent‐labeled proteins TIA1 and SRSF4 by recipient cells after incubation with therapy‐induced EV. Our findings demonstrate that in response to cisplatin treatment tumor cells secrete spliceosomal components into the extracellular space (presumably as a part of stress granules), then these components are absorbed by recipient chemonaive cells and trigger a restructuring of metabolism and cell cycle.

Deactivation of STAT3 and ERK and Activation of p38 in Ovarian Cancer Cells Treated with a Small Molecule Inhibitor of PTP4A3 Phosphatase

The Protein Tyrosine Phosphatase of Regenerating Liver‐3, also known as PRL‐3 or PTP4A3, is among the most oncogenic known protein tyrosine phosphatases, promoting cell migration, survival, metabolism, angiogenesis, and tumor formation. These diverse cancer‐associated phenotypic actions are thought to be mediated at least in part by the unique ability of PTP4A3 to function as a positive signal transducer capable of activating STAT3 and ERK1/2 and deactivating p38 kinase. PTP4A3 is significantly upregulated in a majority of human ovarian cancers and is associated with a poor patient prognosis. Our hypothesis is that PTP4A3 phosphatase inhibitors can be effective targeted treatments for ovarian cancer. We previously described a potent, allosteric, small molecule PTP4A3 inhibitor, 7‐imino‐2‐phenylthieno[3,2‐c]pyridine‐4,6(5H,7H)‐dione, which is also known as KVX‐053 or JMS‐053. KVX‐053 shows no significant in vitro inhibition against a panel of protein kinases or protein tyrosine phosphatases other than PTP4A1‐3. KVX‐053 is cytotoxic to chemosensitive and chemoresistant human ovarian cancer cell lines in vitro and has in vivo antitumor activity. The goal of the current study was to examine the impact of a PTP4A3 phosphatase inhibitor on human ovarian cancer STAT3, ERK1/2, and p38 activation. We observed a concentration‐dependent decrease in the activated form of STAT3, namely, Y705 phospho‐STAT3, after a 4 h exposure of A2780 ovarian cancer cells to KVX‐053 in vitro. A 4 h exposure to a concentration of KVX‐053 that reduced A2780 cell viability by 25% (EC25), namely 157 nM, decreased Y705 phospho‐STAT3 levels by 46%. An EC50 (470 nM) or EC90 (4.23 μM) concentration decreased Y705 phospho‐STAT3 levels by 61% and 87%, respectively. STAT3 levels remained unchanged except for a modest decrease with 4.23 μM of KVX‐053. ERK1/2 phosphorylation was decreased 64% with a 4 h treatment of an EC90 concentration of KVX‐053 but not with the EC25 or EC50 concentrations. In contrast, KVX‐053 caused a concentration‐dependent increase in p38 kinase phosphorylation and activation. These results suggest that acute KVX‐053 exposure disrupts the PTP4A3/STAT3, PTP4A3/ERK, and PTP4A3/p38 homeostatic loops, providing additional information about the role of PTP4A3 in cancer cell signaling and survival.

Comparing the Secretomes of Chemorefractory and Chemoresistant Ovarian Cancer Cell Populations.

Simple SummaryEpithelial ovarian cancer (EOC) is a gynecological disease that is complicated to treat due to its heterogenous nature and because many women develop resistance to various therapeutic strategies. Tumor recurrence can be examined as a two-pronged approach: resistance developed after multiple exposures to frontline anticancer drugs or resistance developed in response to poor microenvironmental conditions, such as hypoxia. Although there are numerous ways to confer chemoresistance, studies have shown that chemoresistant EOC cells release unique secretome profiles that include cytokines, growth factors, and extracellular vesicles (EVs). These secreted factors activate intracellular pathways that contribute to chemoresistance. Secreted EVs transfer biomaterials (including proteins, RNAs, and microRNAs) to other cells, which is critical in cell–cell communication; thus, changes in EV content, in particular exosome miRNAs, have been used to project EOC prognosis. This review examines the feedback loop where chemoresistant EOC cells release unique secretome profiles that confer chemoresistance in normal bystander cells and cancer cells.High-grade serous ovarian cancer (HGSOC) constitutes the majority of all ovarian cancer cases and has staggering rates of both refractory and recurrent disease. While most patients respond to the initial treatment with paclitaxel and platinum-based drugs, up to 25% do not, and of the remaining that do, 75% experience disease recurrence within the subsequent two years. Intrinsic resistance in refractory cases is driven by environmental stressors like tumor hypoxia which alter the tumor microenvironment to promote cancer progression and resistance to anticancer drugs. Recurrent disease describes the acquisition of chemoresistance whereby cancer cells survive the initial exposure to chemotherapy and develop adaptations to enhance their chances of surviving subsequent treatments. Of the environmental stressors cancer cells endure, exposure to hypoxia has been identified as a potent trigger and priming agent for the development of chemoresistance. Both in the presence of the stress of hypoxia or the therapeutic stress of chemotherapy, cancer cells manage to cope and develop adaptations which prime populations to survive in future stress. One adaptation is the modification in the secretome. Chemoresistance is associated with translational reprogramming for increased protein synthesis, ribosome biogenesis, and vesicle trafficking. This leads to increased production of soluble proteins and extracellular vesicles (EVs) involved in autocrine and paracrine signaling processes. Numerous studies have demonstrated that these factors are largely altered between the secretomes of chemosensitive and chemoresistant patients. Such factors include cytokines, growth factors, EVs, and EV-encapsulated microRNAs (miRNAs), which serve to induce invasive molecular, biophysical, and chemoresistant phenotypes in neighboring normal and cancer cells. This review examines the modifications in the secretome of distinct chemoresistant ovarian cancer cell populations and specific secreted factors, which may serve as candidate biomarkers for aggressive and chemoresistant cancers.

The Fibrillin-1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells.

BackgroundChemotherapy resistance is a primary reason of ovarian cancer therapy failure; hence it is important to investigate the underlying mechanisms of chemotherapy resistance and develop novel potential therapeutic targets.MethodsRNA sequencing of cisplatin‐resistant and ‐sensitive (chemoresistant and chemosensitive, respectively) ovarian cancer organoids was performed, followed by detection of the expression level of fibrillin‐1 (FBN1) in organoids and clinical specimens of ovarian cancer. Subsequently, glucose metabolism, angiogenesis, and chemosensitivity were analyzed in structural glycoprotein FBN1‐knockout cisplatin‐resistant ovarian cancer organoids and cell lines. To gain insights into the specific functions and mechanisms of action of FBN1 in ovarian cancer, immunoprecipitation, silver nitrate staining, mass spectrometry, immunofluorescence, Western blotting, and Fӧrster resonance energy transfer‐fluorescence lifetime imaging analyses were performed, followed by in vivo assays using vertebrate model systems of nude mice and zebrafish.ResultsFBN1 expression was significantly enhanced in cisplatin‐resistant ovarian cancer organoids and tissues, indicating that FBN1 might be a key factor in chemoresistance of ovarian cancer. We also discovered that FBN1 sustained the energy stress and induced angiogenesis in vitro and in vivo, which promoted the cisplatin‐resistance of ovarian cancer. Knockout of FBN1 combined with treatment of the antiangiogenic drug apatinib improved the cisplatin‐sensitivity of ovarian cancer cells. Mechanistically, FBN1 mediated the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) at the Tyr1054 residue, which activated its downstream focal adhesion kinase (FAK)/protein kinase B (PKB or AKT) pathway, induced the phosphorylation of signal transducer and activator of transcription 2 (STAT2) at the tyrosine residue 690 (Tyr690), promoted the nuclear translocation of STAT2, and ultimately altered the expression of genes associated with STAT2‐mediated angiogenesis and glycolysis.ConclusionsThe FBN1/VEGFR2/STAT2 signaling axis may induce chemoresistance of ovarian cancer cells by participating in the process of glycolysis and angiogenesis. The present study suggested a novel FBN1‐targeted therapy and/or combination of FBN1 inhibition and antiangiogenic drug for treating ovarian cancer.

A phase II study of Navitoclax (ABT-263) as single agent in women heavily pretreated for recurrent epithelial ovarian cancer: The MONAVI – GINECO study

BackgroundThere are limited treatment options for ovarian cancer patients with early relapse after platinum chemotherapy. In preclinical studies, we previously demonstrated the promising activity of ABT-737, a Bcl-2/Bcl-xL anti-apoptotic protein inhibitor, in chemo-resistant ovarian cancer cells and tumors, suggesting its potential activity in platinum-resistant patients. MethodsWe conducted a prospective multicenter single-arm phase II study to assess the efficacy of Navitoclax (orally available ABT-737 analogue) monotherapy in 46 heavily pretreated (2–12 lines, median = 4) patients with high-grade serous platinum-resistant ovarian tumors. Navitoclax was administered at the daily dose of 150 mg during a lead-in period (7–14 days) and then increased to 250 mg daily in the absence of dose-limiting thrombocytopenia (<G3). Progression-free survival (PFS) based on RECIST v1.1 criteria was the primary endpoint. Analysis of efficacy according to the expression of Bcl-2 family proteins in tumor biopsies was also planned. ResultsThe 3-month PFS was 22.7% [95%CI: 13.2–39.2], median PFS was 1.64 months [95%CI: 1.58–2.30]. There were 16 (35.6%, 95%CI: 22.3–51.3) overall responses (RECIST v1.1): 1 partial response and 15 stable diseases. No correlation between the expression of Bim, Mcl-1 and P-ERK with clinical response was found in this study.Thrombocytopenia was the major side-effect (G3/4: n = 12; 26%), leading to pursue at the daily dose of 150 mg in 8 patients and to discontinue treatment in 3 patients. Neither significant bleeding nor toxic death were observed. ConclusionsNavitoclax monotherapy had poor activity that was not correlated with the expression of Bim, Mcl-1 and P-ERK, without unacceptable toxicity.Trial Registration: Clinicaltrials.gov identifier: NCT02591095

Chromatin softening and nuclear envelope breaching potentiates killing of chemoresistant cancer cells

The nuclei of metastatic cancer cells are more pliable because of reduced lamin A/C expression and decreased chromatin condensation, facilitating effective invasion through tissue interstices. Taking advantage of the fact that nuclei are already softer in chemoresistant metastatic ovarian cancer cells, we strategize to further compromising the nucleus integrity by treating cancer cells with low-dose drug paclitaxel (PTX) and fostamatinib. PTX is a commonly used chemotherapeutic drug for many cancer types, and fostamatinib is a recently approved adjuvant by FDA to treat ovarian cancer.

Hedgehog-Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1.

BackgroundCisplatin (DDP) resistance remains a key challenge in improving the clinical outcome of patients with ovarian cancer (OC). Gli2 overexpression can lead to DDP resistance in OC cells, but the specific underlying regulatory mechanism remains unclear. The membrane transporter encoding gene MDR1 positively regulates chemotherapy resistance in various cancer types. We evaluated MDR1 as a potential Gli2 downstream target and the contribution of the Gli2/MDR1 axis in promoting DDP resistance in OC cells.MethodsTo generate drug-resistant SKOV3/DDP cells, SKOV3 cells were grown for six months under continuous induction wherein the DDP concentration was steadily increased. Gli2 expression in OC cells with varying DDP sensitivities was detected using western blot. Cell counting kit-8 assays were used to assess the DDP sensitivity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells and reversal of DDP resistance in SKOV3/DDP and A2780/DDP cells. Cell proliferation was analyzed using 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays. The transcriptional regulation of MDR1 by Gli2 was determined using luciferase reporter assays. Finally, xenograft OC tumors were generated in nude mice, which were then treated with intraperitoneal DDP or phosphate-buffered saline (PBS) injections to investigate if Gli2 affected DDP resistance in OC in vivo.ResultsDDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive OC cells. Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression, whereas it increased DNA damage, thereby sensitizing OC cells to DDP. Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor (GANT61) in SKOV3/DDP and A2780/DDP cells. In cells stably overexpressing Gli2, treatment with gradient concentrations of verapamil, an MDR1 inhibitor, significantly inhibited MDR1 expression. Our findings indicate that downregulation of MDR1 expression may reverse OC cell resistance to DDP. Moreover, dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2, with Gli2 positively regulating MDR1 expression. Finally, subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating OC drug resistance.ConclusionsWe identified a mechanism by which Hedgehog-Gli signaling regulates OC chemoresistance by modulating MDR1 expression. Hence, Gli2 and MDR1 are potential biomarkers and therapeutic targets in patients with chemoresistant OC.

Curcumin and resveratrol inhibit chemoresistance in cisplatin-resistant epithelial ovarian cancer cells via targeting P13K pathway.

The activation of the PI3K/AKT/mTOR pathway has been proved to be associated with survival as well as proliferation of various tumour cells in multiple cancer types, including epithelial ovarian cancer (EOC). Moreover, the activation of the PI3K/AKT/mTOR pathway is the key mechanism responsible for higher invasiveness and migratory capacities of ovarian cancer cells. Furthermore, PI3K is crucial for activation of the PI3K/AKT/mTOR pathway; therefore, its inhibition might be an effective strategy against cancer. The combination approach is now an established strategy against cancer. So, the present study evaluated molecular mechanics behind the synergistic effects of curcumin and resveratrol along with cisplatin treatment on inhibition of the PI3K pathway in ovarian cancer cells. The present study confirmed significant inhibition of the PI3K/AKT/mTOR pathway as observed by Matrigel invasion assay, Western blot expression of important molecular markers and apoptotic markers. The present study concludes that the combination of curcumin and resveratrol significantly sensitized the EOC cells to cisplatin treatment, thereby inhibiting chemoresistance in ovarian cancer cells by significant inhibition of the PI3K/AKT/mTOR pathway.

Effects of trans-(±)-kusunokinin on chemosensitive and chemoresistant ovarian cancer cells.

Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC50 value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.

Curcumin and resveratrol inhibit chemoresistance in cisplatin-resistant epithelial ovarian cancer cells via targeting P13K pathway.

The activation of the PI3K/AKT/mTOR pathway has been proved to be associated with survival as well as proliferation of various tumour cells in multiple cancer types, including epithelial ovarian cancer (EOC). Moreover, the activation of the PI3K/AKT/mTOR pathway is the key mechanism responsible for higher invasiveness and migratory capacities of ovarian cancer cells. Furthermore, PI3K is crucial for activation of the PI3K/AKT/mTOR pathway; therefore, its inhibition might be an effective strategy against cancer. The combination approach is now an established strategy against cancer. So, the present study evaluated molecular mechanics behind the synergistic effects of curcumin and resveratrol along with cisplatin treatment on inhibition of the PI3K pathway in ovarian cancer cells. The present study confirmed significant inhibition of the PI3K/AKT/mTOR pathway as observed by Matrigel invasion assay, Western blot expression of important molecular markers and apoptotic markers. The present study concludes that the combination of curcumin and resveratrol significantly sensitized the EOC cells to cisplatin treatment, thereby inhibiting chemoresistance in ovarian cancer cells by significant inhibition of the PI3K/AKT/mTOR pathway.

Translational Physiology of Anti-Müllerian Hormone: Clinical Applications in Female Fertility Preservation and Cancer Treatment.

BackgroundWhilst the ability of AMH to induce the regression of the Müllerian ducts in the male fetus is well appreciated, AMH has additional biological actions in relation to steroid biosynthesis and ovarian follicle dynamics. An understanding of the physiology of AMH illuminates the potential therapeutic utility of AMH to protect the ovarian reserve during chemotherapy and in the treatment of female malignancies. The translation of the biological actions of AMH into clinical applications is an emerging focus of research, with promising preliminary results.Objective and RationaleStudies indicate AMH restrains primordial follicle development, thus administration of AMH during chemotherapy may protect the ovarian reserve by preventing the mass activation of primordial follicles. As AMH induces regression of tissues expressing the AMH receptor (AMHRII), administration of AMH may inhibit growth of malignancies expressing AMHR II. This review evaluates the biological actions of AMH in females and appraises human clinical applications.Search MethodsA comprehensive search of the Medline and EMBASE databases seeking articles related to the physiological functions and therapeutic applications of AMH was conducted in July 2021. The search was limited to studies published in English.OutcomesAMH regulates primordial follicle recruitment and moderates sex steroid production through the inhibition of transcription of enzymes in the steroid biosynthetic pathway, primarily aromatase and 17α-hydroxylase/17,20-lyase. Preliminary data indicates that administration of AMH to mice during chemotherapy conveys a degree of protection to the ovarian reserve. Administration of AMH at the time of ovarian tissue grafting has the potential to restrain uncontrolled primordial follicle growth during revascularization. Numerous studies demonstrate AMH induced regression of AMHR II expressing malignancies. As this action occurs via a different mechanism to traditional chemotherapeutic agents, AMH has the capacity to inhibit proliferation of chemo-resistant ovarian cancer cells and cancer stem cells.Wider ImplicationsTo date, AMH has not been administered to humans. Data identified in this review suggests administration of AMH would be safe and well tolerated. Administration of AMH during chemotherapy may provide a synchronistic benefit to women with an AMHR II expressing malignancy, protecting the ovarian reserve whilst the cancer is treated by dual mechanisms.

Substrate Stiffness Modulates the Growth, Phenotype, and Chemoresistance of Ovarian Cancer Cells.

Mechanical factors in the tumor microenvironment play an important role in response to a variety of cellular activities in cancer cells. Here, we utilized polyacrylamide hydrogels with varying physical parameters simulating tumor and metastatic target tissues to investigate the effect of substrate stiffness on the growth, phenotype, and chemotherapeutic response of ovarian cancer cells (OCCs). We found that increasing the substrate stiffness promoted the proliferation of SKOV-3 cells, an OCC cell line. This proliferation coincided with the nuclear translocation of the oncogene Yes-associated protein. Additionally, we found that substrate softening promoted elements of epithelial-mesenchymal transition (EMT), including mesenchymal cell shape changes, increase in vimentin expression, and decrease in E-cadherin and β-catenin expression. Growing evidence demonstrates that apart from contributing to cancer initiation and progression, EMT can promote chemotherapy resistance in ovarian cancer cells. Furthermore, we evaluated tumor response to standard chemotherapeutic drugs (cisplatin and paclitaxel) and found antiproliferation effects to be directly proportional to the stiffness of the substrate. Nanomechanical studies based on atomic force microscopy (AFM) have revealed that chemosensitivity and chemoresistance are related to cellular mechanical properties. The results of cellular elastic modulus measurements determined by AFM demonstrated that Young’s modulus of SKOV-3 cells grown on soft substrates was less than that of cells grown on stiff substrates. Gene expression analysis of SKOV-3 cells showed that mRNA expression can be greatly affected by substrate stiffness. Finally, immunocytochemistry analyses revealed an increase in multidrug resistance proteins, namely, ATP binding cassette subfamily B member 1 and member 4 (ABCB1 and ABCB4), in the cells grown on the soft gel resulting in resistance to chemotherapeutic drugs. In conclusion, our study may help in identification of effective targets for cancer therapy and improve our understanding of the mechanisms of cancer progression and chemoresistance.