Chemoresistant Ovarian Cancer Cell Lines Research Articles

Dual Inhibition of SYK and EGFR Overcomes Chemoresistance by Inhibiting CDC6 and Blocking DNA Replication.

Targeting multiple signaling pathways has been proposed as a strategy to overcome resistance to single-pathway inhibition in cancer therapy. A previous study in epithelial ovarian cancers identified hyperactivity of spleen tyrosine kinase (SYK) and epidermal growth factor receptor (EGFR), which mutually phosphorylate and activate each other. Given the potential for pharmacologic inhibition of both kinases with clinically available agents, this study aimed to assess the antitumor efficacy of both pharmacologic and genetic SYK and EGFR co-inhibition using a multifaceted approach to analyze the global phosphoproteome and chemoresistant ovarian cancer cell lines, patient-derived organoids, and xenograft models. Dual inhibition of SYK and EGFR in chemoresistant ovarian cancer cells elicited a highly synergistic antitumor effect. Notably, the combined inhibition strategy activated the DNA damage response, induced G1 cell cycle arrest, and promoted apoptosis. The phosphoproteomic analysis revealed that perturbation of SYK and EGFR signaling induced a significant reduction in both phosphorylated and total protein levels of cell division cycle 6 (CDC6), a crucial initiator of DNA replication. Together, this study offers preclinical evidence supporting dual inhibition of SYK and EGFR as a promising treatment for chemoresistant ovarian cancer that disrupts DNA synthesis by impairing formation of the prereplication complex. These findings warrant further clinical investigation to explore the potential of this combination therapy in overcoming drug resistance and improving patient outcomes.

Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer

ObjectiveOvarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. MethodsThe human EOC cell lines A2780, HeyA8, SKOV3ip1, A2780-CP20, HeyA8-MDR, and SKOV3-TR were treated with BNZ-111, and cell proliferation, apoptosis, and cell cycle were assessed. ResultsIt demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating β-3 tubulin expression to suppress microtubule dynamics. ConclusionBNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology.

Abstract 7139: Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer

Abstract Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. With improved pharmacokinetic parameters, BNZ-111 effectively disrupts tubulin polymerization through hydrogen bonds and hydrophobic interactions. It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating β-3 tubulin expression to suppress microtubule dynamics. This study suggests that BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology Citation Format: Jeong-Won Lee, Kwangho Lee, Byumseok Koh, Yoo-Young Lee, Duk-Soo Bae. Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7139.

Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

BackgroundAmong the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion.MethodsWestern blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively.ResultsWe observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity.ConclusionsDysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

The potential benefits of dinitrophenol combination with chemotherapy in the treatment of ovarian cancer.

2,4-dinitrophenol (DNP), an uncoupling mitochondrial agent, has been identified as a source of oxidative stress and linked to the pathogenesis of ovarian cancer. In this study, we determine the cytotoxic effect of DNP alone or in combination with chemotherapies in ovarian cancer cells. We utilized human ovarian cancer cell lines SKOV-3 and MDAH-2774 with their chemoresistant counterparts. Cancer stem cells (CSCs) were isolated from SKOV-3 utilizing magnetic-activated cell sorting technique for CD44+/CD117+ cells. Human normal primary ovarian epithelial (NOEC) and HOSEpiC cell lines were used as a control. Cells were treated with and without chemotherapy (taxotere 0.3 µM or cisplatin 50 µM), with or without increasing doses of DNP (0.125, 0.25, or 0.5 mM) for 24 hours followed by evaluation of cell viability and IC50 utilizing MTT assay. For determination of synergism, Fa-combination Index plots were created using the CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). All data were run in triplicates and analyzed by t-test. DNP treatment of ovarian cancer and chemoresistant ovarian cancer cell lines as well as CSCs resulted in decreased cell viability in a dose dependent manner with no effect on normal cells. Combination of DNP with chemotherapy synergistically enhances cytotoxicity of chemotherapeutics in all ovarian cancer cells as compared to chemotherapy alone. Our data indicates the potential of the addition of DNP to the arsenal of drugs available to treat ovarian cancer, whether alone or in combination with chemotherapies. The synergistic effects of DNP in reducing the required amount of chemotherapy, is critical for the alleviation of harmful side effects.

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.

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.

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.

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.

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.

The Effect of Olaparib and Bortezomib Combination Treatment on Ovarian Cancer Cell Lines

Objective: Ovarian cancer (OC) is the deadliest gynecologic malignancy and has a poor survival rate due to late diagnosis and chemoresistance development. In the standard treatment of OC, platinum-based chemotherapeutics are used. However, following several rounds of chemotherapy, these drugs’ efficacy eventually becomes limited due to the development of chemoresistance in most patients who previously responded to this treatment. Therefore, overcoming chemoresistance in the treatment of OC is of high importance. In this study, we investigated the effect of combinatorial inhibition of poly(ADPribose) polymerase (PARP) and proteasome by olaparib and bortezomib on chemosensitive and chemoresistant OC cell lines. Materials and Methods: We used sulphorhodamine B assay to screen cell viability following drug treatments alone or in combination, and used the cytotoxicity data to model the effect of drugs on cell death in R programming environment. In addition to olaparib and bortezomib, we performed cytotoxicity screenings where we applied cisplatin to OC cells. We also carried out flow cytometry analysis to quantify apoptotic cells following treatments. Results: We showed that combination treatment was more effective on chemosensitive OC cell lines when cisplatin was not used. In the presence of cisplatin, olaparib and bortezomib combination treatment resulted in higher cytotoxicity in chemoresistant OC lines compared to chemosensitive OC cell lines. Combinatorial inhibition of PARP and proteasome led to a higher number of apoptotic cells in OV2008 chemosensitive cell line compared to drugs alone. Conclusion: Our data shows that olaparib and bortezomib combination treatment might show promise in vivo in the treatment of OC. Also, the efficacy of this combination treatment might be dependent on OC cells’ chemosensitivity profiles.

Binding mode of Pyridoclax to myeloid cell leukemia-1 (Mcl-1) revealed by nuclear magnetic resonance spectroscopy, docking and molecular dynamics approaches

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family proteins. Its amplification is one of the most frequent genetic aberrations found in human cancers. Pyridoclax, a promising BH3 mimetic inhibitor, interacts directly with Mcl-1 and induces massive apoptosis at a concentration of 15 µM in combination with anti-Bcl-xL strategies in chemo-resistant ovarian cancer cell lines. In this study, a combined experimental and theoretical approach was used to investigate the binding mode of Pyridoclax to Mcl-1. The representative poses generated from dynamics simulations compared with NMR data revealed: (i) Pyridoclax bound to P1 and P2 pockets of Mcl-1 BH3 binding groove through its styryl and methyl groups establishing mainly hydrophobic contacts, (ii) one of the ending pyridines interacts through electrostatic interaction with K234 side chain, a negatively charged residue present only in this position in Mcl-1. Communicated by Ramaswamy H. Sarma

Analysis of Differentially Expressed MicroRNAs and Circulating Tumor Cells as Predictive Biomarkers of Platinum Chemoresistance in Primary Ovarian Carcinomas: A Prospective Study

Lesson Learned.Circulating tumor cells, microRNA markers, or other biomarkers merit examination as part of correlative scientific analyses in prospective clinical trials.Background.Platinum chemotherapy resistance occurs in approximately 25% of patients with ovarian carcinoma; however, no biomarkers of ovarian carcinoma chemoresistance have been validated. We performed a prospective trial designed to identify tumor‐based predictive biomarkers of platinum resistance.Methods.Tumor specimens were collected from 29 women with newly diagnosed histopathologically proven primary ovarian carcinoma. Of these, 23 women had specimens accessible for assessment and outcome data available regarding chemosensitive versus chemoresistance status via review of the medical record. Tumor slices were stained with antibodies against two microRNAs (miRNAs 29b and 199a) differentially expressed in chemoresistant ovarian cancer cell lines. Additionally, blood samples obtained at the time of diagnosis were analyzed for the presence of circulating tumor cells (CTCs).Results.The average age of the patients was 64 years, and 82.6% had high‐grade epithelial carcinomas. The baseline median CA‐125 was 464 (range 32–2,782). No statistically significant differences were observed in miR29b or 199a expression in platinum‐resistant/refractory versus platinum‐sensitive tumors. Furthermore, the presence of CTCs was not found to be statistically significantly predictive of eventual platinum resistance.Conclusion.Our analysis showed no differences in miR29b and 199a expression, and differences in baseline CTCs in women with newly diagnosed ovarian tumors were not statistically significant.

MUL1 E3 ligase regulates the antitumor effects of metformin in chemoresistant ovarian cancer cells via AKT degradation.

Chemoresistance is one of most critical clinical problems encountered when treating patients with ovarian cancer, due to the fact that the disease is usually diagnosed at advanced stages. Metformin is used as a first‑line drug for the treatment of type2 diabetes; however, drug repositioning studies have revealed its antitumor effects, mainly mediated through AMP‑activated protein kinase(AMPK) activation and AKT/mammalian target of rapamycin(mTOR) pathway inhibition in various types of cancer, including drug‑resistant cancer cells. The current study revealed that the novel antitumor mechanism of metformin is mediated by regulation of mitochondrialE3 ubiquitin protein ligase1(MUL1) expression that negatively regulates AKT. The results demonstrated that metformin decreased the expression of AKT protein levels via MUL1E3 ligase. In addition, metformin increased both mRNA and protein levels of MUL1 and promoted degradation of AKT in a proteasome‑dependent manner. Silencing MUL1 expression suppressed the metformin‑mediated AKT degradation and its downstream effects. Cell cycle analysis and a clonogenic assay demonstrated that knockdown of MUL1 significantly diminished the antitumor effects of metformin. Together, these data indicate that MUL1 regulates metformin‑mediated AKT degradation and the antitumor effects of metformin in chemoresistant ovarian cancer cell lines.

Nanoparticles of Metal-Organic Cages Overcoming Drug Resistance in Ovarian Cancer.

A long-standing challenge in the treatment of ovarian cancer is drug resistance to standard platinum-based chemotherapy. Recently, increasing attention has been drawn to the use of self-assembled metal-organic complexes as novel therapeutics for cancer treatment. However, high hydrophobicity that is often associated with these structures lowers their solubility and hinders their clinical translation. In this article, we present a proof-of-concept study of using nanoprecipitation to formulate the hydrophobic metal-organic cages and facilitate their use in treating chemoresistant ovarian cancer. The Pt6L4 Cage 1 is an octahedral cage formed by self-assembly of six 1,10-phenanthroline-Pt(II) centers and four 2,4,6-tris(4-pyridyl)-1,3,5-triazine ligands (L). Cage 1 is able to trigger DNA damage and exhibits promising in vitro potency against a panel of human ovarian cancer cell lines. However, due to the large portion of aromatic components, this cage structure has very limited solubility in cell culture media (<20μM). Notably, upon nanoformulation by using fluorescein (2) and a pegylated anionic polymer (3), the concentration of Cage 1 can reach up to 0.4 mM. Production of the nanoparticles of metal-organic cages (nMOC) is driven by the formation of the 1:1 host-guest complex of 1 and 2 in aqueous solution, which then form nanoprecipitation in presence of poly glutamic acid-b-poly ethylene glycol (3). The resulted nMOC are about 100 nm in diameter, and they serve as a delivery platform that slowly releases the therapeutic content. The use of fluorescein facilitates monitoring cell entry of nMOC and drug release using flow cytometry. Finally, comparing to cisplatin, the nMOC exhibit comparable in vitro efficacy against a panel of human cancer cell lines, and notably, it shows a much lower resistance factor against chemoresistant ovarian cancer cell lines.

Mechanism of Cepharanthine Cytotoxicity in Human Ovarian Cancer Cells.

Cepharanthine (CEP), a medicinal product derived from Stephania cephalantha Hayata, possesses a potent cytotoxicity against several types of cancers. Recently, we have found that CEP could efficiently inhibit the growth of mutated p53 colon cancer cells, which are often resistant to commonly used chemotherapeutic agents. In this study, we evaluated the cytotoxic effect and the underlying mechanisms of CEP on both chemosensitive CaOV-3 and chemoresistant OVCAR-3 ovarian cancer cell lines. The present study demonstrated that CEP significantly inhibited the growth of CaOV-3 and OVCAR-3 cells in a time- and concentration-dependent manner. CEP arrested CaOV-3 and OVCAR-3 cells in the G1 phase and S phase of cell cycle, respectively. Western blot analysis demonstrated that CEP markedly increased the expression of p21Waf1 protein and decreased the expression of cyclins A and D proteins in both CaOV-3 and OVCAR-3 cells. Additionally, CEP triggered apoptotic cell death in OVCAR-3 cells. Taken together, the above results suggest that CEP is a promising anticancer drug for ovarian cancer.

Abstract 3996: Pyridoclax and its derivatives from oligopyridine family directly inhibit Mcl-1 and exert potent antitumor effects on ovarian cancer in vitro and in vivo

Abstract Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. Although the patients are initially quite sensitive to the taxane and platinum-based first-line chemotherapy, most of them relapse and develop chemoresistance. Defects in apoptosis regulation in ovarian cancer allow the cancer cells to evade cell death and contribute to chemoresistance. Mcl-1 is an anti-apoptotic member of the Bcl-2 proteins family and its amplifıcation is one of the most frequent genetic aberrations found in human cancers. Its expression is at the origin of the acquired resistance to chemotherapy and to Bcl-2 and Bcl-xL inhibitors. In ovarian cancers, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to prevent cancer cells from undergoing apoptotic cell death. Their concomitant inhibitions lead to massive apoptosis even in absence of chemotherapy. Moreover, in some cases, Mcl-1 inhibition is itself able to lead to apoptosis. If clinically relevant pharmacologic inhibition of Bcl-xL is available using ABT-263, selective direct inhibition of Mcl-1 remains problematic. In this context, our teams have designed and synthesized small compounds based on a pyridyl scaffold, named oligopyridines, which potentially target the Mcl-1 hydrophobic binding pocket. We demonstrated that the lead of the first generation of oligopyridines, named Pyridoclax, interacts directly with Mcl-1, releases its pro-apoptotic partners Bim and Bak and induces massive apoptosis at 25 µM concentration in combination with anti-Bcl-xL strategies in chemoresistant ovarian cancer cell lines (Gloaguen et al., J Med Chem 2015). In the present study, we investigated the antitumor activity of Pyridoclax hydrochloride in three subcutaneous xenograft models derived from the injection of chemoresistant ovarian cancer cell lines. Different routes of Pyridoclax hydrochloride administration were tested and its antitumor effect was analyzed at different doses as single agent or in combination with ABT-263. This study highlighted an effective antitumor activity of 20mg/kg of Pyridoclax administered intravenously as single agent in two of three xenograft models without side effects. In order to improve its biological activity, we evaluated the cytotoxic effects of a second generation of oligopyridines derived from the Pyridoclax. This allowed us to identify the MR31367, one of the most potent oligopyridines that shows a stronger pro-apoptotic activity in association with to Bcl-xL-targeting strategies in ovarian cancer cell lines. Further characterization showed that this derivative binds Mcl-1 and release Bim and Bak from it, leading to Bak-mediated apoptosis. Overall, these results open up interesting perspectives for the clinical use of Mcl-1 inhibitors as single agent or in combination with anticancer drugs to improve the clinical management of ovarian cancers. Citation Format: Siham Hedir, Louis-Bastien Weiswald, Marcella De Giorgi, Jade Fogha, Martina De Pascale, Emilie Brotin, Bogdan Marekha, Peggy Suzanne, Fabien Gautier, Philippe Juin, Laetitia Ligat, Frédéric Lopez, Rémi Legay, Ronan Bureau, Sylvain Rault, Jana Sopkova-de Oliveira Santos, Anne-Sophie Voisin-Chiret, Laurent Poulain. Pyridoclax and its derivatives from oligopyridine family directly inhibit Mcl-1 and exert potent antitumor effects on ovarian cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3996.

Abstract 1921: The sensitivity of ovarian cancer cells to the HDAC inhibitor belinostat is improved by inhibiting Bcl-xL or Mcl-1 anti-apoptotic proteins

Abstract The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancers, which remain the leading cause of death from gynecologic cancer. Our previous studies have shown that the anti-apoptotic proteins Bcl-xL and Mcl-1 cooperate to protect resistant ovarian cancer cells from apoptosis as their concomitant inhibition results in massive apoptotic cell death. Moreover, their BH3-only pro-apoptotic partners Bim and Puma are crucial actors in the induced cell death. This suggests that ovarian cancer cell apoptosis can be triggered by inhibiting Bcl-xL and Mcl-1 and/or by promoting their BH3-only partners, especially Bim and Puma. The expression of these Bcl-2 family proteins has been reported to be modulated by HDAC (histone deacetylases) inhibitors in various cancer models. The objective of the present study was to evaluate the efficacy of the FDA-approved pan-HDAC inhibitor belinostat (i) to reduce the [Bcl-xL and Mcl-1] / BH3-only proteins ratio and (ii) to trigger apoptosis in chemoresistant ovarian cancer cell lines, alone or in combination with other pharmacological molecules that modulate these Bcl-2 family proteins. HDAC inhibition by belinostat led to a G2/M blockade and consequently reduced cell proliferation. Moreover, belinostat used at high concentrations (from 1000 to 2000 nM) efficiently triggered apoptosis in chemoresistant ovarian cancer cells. This cytotoxic effect was associated with both an up-regulation of Bim and Puma protein expression and an inhibition of Bcl-xL protein expression. Used at sub-toxic concentrations, belinostat also up-regulated Bim and Puma proteins but failed to down-regulate Bcl-xL and Mcl-1 protein expression. We therefore investigated whether Bcl-xL or Mcl-1 inhibition could sensitize ovarian cancer cells to sub-toxic concentrations of belinostat. Interestingly, the combination of belinostat with the BH3-mimetic ABT-737, that inhibits Bcl-xL, induced massive apoptosis. Moreover, Mcl-1 direct or indirect inhibition was also very efficient to sensitize ovarian cancer cells to belinostat. These results suggest that belinostat, alone or in association with pharmacological molecules that inhibit Mcl-1 or Bcl-xL, represents a promising approach for the treatment of chemoresistant ovarian cancers. Citation Format: Charlène Duboc, Edwige Abeilard, Anne Sophie Voisin-Chiret, Pascal Gauduchon, Laurent Poulain, Marie Villedieu. The sensitivity of ovarian cancer cells to the HDAC inhibitor belinostat is improved by inhibiting Bcl-xL or Mcl-1 anti-apoptotic proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1921.

Correction: Niclosamide and its analogs are potent inhibitors of Wnt/β-catenin, mTOR and STAT3 signaling in ovarian cancer.

[This corrects the article DOI: 10.18632/oncotarget.13466.].