High-grade Serous Ovarian Cancer Cell Research Articles

Identifying and overcoming a mechanism of resistance to WEE1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells

Objective: As a result of TP53 gene mutation high grade serous ovarian cancer (HGSOC) is dependent on the G2 checkpoint for the repair of DNA damage and survival. The key role of WEE1 kinase at this checkpoint makes inhibition of WEE1 kinase in combination with DNA damaging agents an attractive therapeutic strategy for HGSOC. Our aim was to characterise resistance mechanisms to WEE1 inhibitor AZD1775 and identify ways to overcome resistance ready for use in the clinic. Methods: AZD1775-resistant HGSOC cell clones were isolated and western blotting, cell cycle analysis, growth assays, RNA-Seq and gene expression analysis were used to characterise resistance mechanisms and investigate a way to overcome resistance. Results: A resistance mechanism previously reported in small cell lung cancer did not operate in HGSOC. Instead, resistance resulted from different cell cycle control pathway changes that slow AZD1775-induced cell cycle progression and reduce accumulation of replication associated DNA damage. One major change was reduced levels of CDK1, the substrate for WEE1 kinase inhibition; another was increased levels of PKMYT1, which can also inhibit CDK1. Increased expression of TGFβ signalling to slow cell cycle progression occurred in resistant clones. A TGFβR1 inhibitor overcame resistance in a clone with the highest TGFβR1 receptor expression. Conclusions: Although overexpression of the membrane glycoprotein MDR1 is a common mechanism of drug resistance, it was not involved in our HGSOC cells. Instead AZD1775 resistance resulted from cell cycle control pathway changes that combine to slow AZD1775-induced cell cycle progression and so reduce accumulation of replication-associated DNA damage.

Functional analysis of the 1p34.3 risk locus implicates GNL2 in high-grade serous ovarian cancer

The high-grade serous ovarian cancer (HGSOC) risk locus at chromosome 1p34.3 resides within a frequently amplified genomic region signifying the presence of an oncogene. Here, we integrate in silico variant-to-function analysis with functional studies to characterize the oncogenic potential of candidate genes in the 1p34.3 locus. Fine mapping of genome-wide association statistics identified candidate causal SNPs local to H3K27ac-demarcated enhancer regions that exhibit allele-specific binding for CTCF in HGSOC and normal fallopian tube secretory epithelium cells (FTSECs). SNP risk associations colocalized with eQTL for six genes (DNALI1, GNL2, RSPO1, SNIP1, MEAF6, and LINC01137) that are more highly expressed in carriers of the risk allele, and three (DNALI1, GNL2, and RSPO1) were upregulated in HGSOC compared to normal ovarian surface epithelium cells and/or FTSECs. Increased expression of GNL2 and MEAF6 was associated with shorter survival in HGSOC with 1p34.3 amplifications. Despite its activation of β-catenin signaling, RSPO1 overexpression exerted no effects on proliferation or colony formation in our study of ovarian cancer and FTSECs. Instead, GNL2, MEAF6, and SNIP1 silencing impaired in vitro ovarian cancer cell growth. Additionally, GNL2 silencing diminished xenograft tumor formation, whereas overexpression stimulated proliferation and colony formation in FTSECs. GNL2 influences 60S ribosomal subunit maturation and global protein synthesis in ovarian cancer and FTSECs, providing a potential mechanism of how GNL2 upregulation might promote ovarian cancer development and mediate genetic susceptibility of HGSOC.

Proteomic Analysis Reveals Low-Dose PARP Inhibitor-Induced Differential Protein Expression in BRCA1-Mutated High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer diagnosed in patients worldwide. Patients with BRCA1/2-mutated HGSOC have benefited from targeted treatments such as poly(ADP-ribose) polymerase inhibitors (PARPi). Despite the initial success of PARPi-based ovarian cancer treatment regimens, approximately 70% of patients with ovarian cancer relapse and the 5-year survival rate remains at 30%. PARPi exhibit variable treatment efficacy and toxicity profiles. Furthermore, the off-target effects of PARP inhibition have not yet been fully elucidated, warranting further study of these classes of molecules in the context of HGSOC treatment. Highly reproducible quantitative mass spectrometry-based proteomic workflows have been developed for the analysis of tumor tissues and cell lines. To detect the off-target effects of PARP inhibition, we conducted a quantitative mass spectrometry-based proteomic analysis of a BRCA1-mutated HGSOC cell line treated with low doses of two PARPi, niraparib and rucaparib. Our goal was to identify PARPi-induced protein signaling pathway alterations toward a more comprehensive elucidation of the mechanism of action of PARPi beyond the DNA damage response pathway. A significant enrichment of nuclear and nucleoplasm proteins that are involved in protein binding was observed in the rucaparib-treated cells. Shared upregulated proteins between niraparib and rucaparib treatment demonstrated RNA II pol promoter-associated pathway enrichment in transcription regulation. Pathway enrichment analyses also revealed off-target effects in the Golgi apparatus and the ER. The results from our mass spectrometry-based proteomic analysis highlights notable off-target effects produced by low-dose treatment of BRCA1-mutated HGSOC cells treated with rucaparib or niraparib.

Nelfinavir Induces Cytotoxicity towards High-Grade Serous Ovarian Cancer Cells, Involving Induction of the Unfolded Protein Response, Modulation of Protein Synthesis, DNA Damage, Lysosomal Impairment, and Potentiation of Toxicity Caused by Proteasome Inhibition.

Simple SummaryHigh-grade serous ovarian cancer (HGSOC) accounts for 70% of all ovarian-cancer-related deaths. Mainstay treatment with platinum-based drugs following surgery results in favorable outcomes in the majority of patients; however, in >80% of cases, the disease relapses with eventual drug resistance. As such, urgent development of improved alternative therapies is necessary for HGSOC patients with lower life expectancy. Rapid repurposing of market available drugs for cancer therapy is a cost-effective alternative to bypass the decade-long traditional drug development pipeline. Among potential drug-repurposing candidates, nelfinavir (NFV)—an anti-infective agent to treat acquired immunodeficiency syndrome (AIDS)—has shown anti-cancer effects against diverse cancers; however, its remedial benefits against HGSOC are unknown. In this study, we explored how NFV targets HGSOC cells obtained from patients at platinum-sensitive and -resistant stages. We observed beneficial efficacy elicited by NFV against HGSOC in both disease conditions through multiple mechanistic avenues, suggesting positive drug-repurposing prospects.High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction in the HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction in clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho-eIF2α, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile.

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells.

Background: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. Methods: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). Results: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. Conclusions: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs’ structure might lead to new treatment options against ovarian cancer.

The Influence of Angiotensin Peptides on Survival and Motility of Human High-Grade Serous Ovarian Cancer Cells in Serum Starvation Conditions.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent and malignant form of ovarian cancer. A local renin–angiotensin system (RAS) has been found in the ovary, and changes in selected components of this system were observed in pathological states and also in ovarian cancer. In the present study, we examined the effect of three peptides, Ang-(1-7), Ang-(1-9) and Ang-(3-7), on proliferation and motility of the OVPA8 cell line, a new well-defined and preclinical model of HGSOC. We confirmed the presence of mRNA for all angiotensin receptors in the tested cells. Furthermore, our findings indicate that all tested angiotensin peptides increased the metabolic serum in the medium by activation of cell defense mechanisms such as nuclear factor kappaB signaling pathway andapoptosis. Moreover, tested angiotensin peptides intensified serum starvation-induced cell cycle arrest at the G0/G1 phase. In the case of Ang-(3-7), a significant decrease in the number of Ki67 positive cells (Ki67+) and reduced percentage of activated ERK1/2 levels in ovarian cancer cells were additionally reported. The angiotensin-induced effect of the accumulation of cells in the G0/G1 phase was not observed in OVPA8 cells growing on the medium with 10% FBS. Moreover, in the case of Ang-(3-7), the tendency was quite the opposite. Ang-(1-7) but not Ang-(1-9) or Ang-(3-7) increased the mobility of reluctant-to-migrate OVAP8 cells cultured in the serum-free medium. In any cases, the changes in the expression of VIM and HIF1A gene, associated with epithelial–mesenchymal transition (EMT), were not observed. In conclusion, we speculate that the adaptation to starvation in nutrient-deprived tumors can be modulated by peptides from the renin–angiotensin system. The influence of angiotensin peptides on cancer cells is highly dependent on the availability of growth factors and nutrients.

Abstract P205: Novel 1,1-diarylethylene compounds degrade FOXM1 and selectively and potently reduce survival of high-grade serous ovarian cancer cells

Abstract Background: Forkhead box M1 (FOXM1) is a master transcriptional regulator and multifunctional oncoprotein that is widely overexpressed in ovarian cancer and associated with poor prognosis. Due to its promotion of numerous oncogenic phenotypes in ovarian cancer, FOXM1 has emerged as an attractive therapeutic target for ovarian cancer treatment. Recently, we reported that 1,1-diarylethylene compounds, NB-55, NB-73, and NB-115 (NB compounds), potently inhibit FOXM1 in breast cancer cells and mediate anti-cancer effects. Given the high molecular similarity between triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC), we characterized this new class of FOXM1 inhibitors in HGSC cells in the present study and compared their effects to other reported FOXM1 inhibitors. Methods: We conducted dose-response curves of NB compounds and other FOXM1 inhibitors in HGSC cells (CAOV3 and OVCAR4) and normal control (FT282 C11) cells. Western blot analyses probed for FOXM1, p-FOXM1, CCNB1, FOXA1, and FOXO3A in HGSC cells treated with NB compounds. We used the proteosome inhibitor MG132 to investigate the mechanism of FOXM1 inhibition by NB-73 and NB-115. We investigated FOXM1 transcriptional activity, colony formation, cell cycle, and apoptosis in HGSC cells treated with NB-73 and NB-115. Results: Dose-response curves in HGSC cells revealed IC50 values in the low nM for monensin; mid-nM for NB-73 and NB-115; high nM for thiostrepton; and low µM for NB-55, FDI-6, and n-phenylphenanthren-9-amine. RCM-1 did not affect HGSC cell proliferation. Dose-response curves in normal control fallopian tube epithelial (FTE) cells revealed IC50 values of high nM for thiostrepton and low µM for NB compounds and FDI-6. HGSC cells treated with NB compounds demonstrated decreased levels of FOXM1 and p-FOXM1 but not FOXA1 and FOXO3A. NB-73 and NB-115 decreased FOXM1 promoter activity and gene target expression but were no longer able to decrease FOXM1 levels when combined with MG132 in HGSC cells. Clonogenic assays of HGSC cells treated with NB-73 and NB-115 revealed decreased colony formation at low nM concentrations. HGSC cells treated with NB-73 and NB-115 have higher cell populations in G2/M and undergo higher rates of apoptosis compared to non-treated HGSC cells. Conclusions: NB-73 and NB-115 promote proteasomal degradation of FOXM1 and are more potent FOXM1 inhibitors of cell proliferation than previously characterized FOXM1 inhibitors in HGSC cells. NB-73 and NB-115 potently reduce HGSC survival and selectively inhibit HGSC cells as compared to normal control FTE cells. Our data encourage further assessment of NB compounds as a novel therapeutic strategy in the vast majority of HGSC that overexpress FOXM1. Support: NIH T32CA009476(CL), research grants 083(BSK) and 084(JAK) from the Breast Cancer Research Foundation, the Landfield Cancer Research Fund(BSK), NIH R03CA224339(ARK), The Marsha Rivkin Center(ARK), The Betty J. and Charles D. McKinsey Ovarian Cancer Research Fund(ARK), and The Fred & Pamela Buffett Cancer Center (NCI P30CA036727)(ARK). Citation Format: Cassie Liu, Catalina Muñoz-Trujillo, John A. Katzenellenbogen, Benita S. Katzenellenbogen, Adam R. Karpf. Novel 1,1-diarylethylene compounds degrade FOXM1 and selectively and potently reduce survival of high-grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P205.

ETV5-mediated upregulation of lncRNA CTBP1-DT as a ceRNA facilitates HGSOC progression by regulating miR-188-5p/MAP3K3 axis

High-grade serous ovarian cancer (HGSOC) is a common and lethal cancer of the female reproductive system. Long non-coding RNAs (lncRNAs) are aberrantly expressed in various cancers and play crucial roles in tumour progression. However, their function and molecular mechanism in HGSOC remain largely unknown. Based on public databases and bioinformatics analyses, the overexpression of lncRNA CTBP1-DT in HGSOC tissues was detected and validated in a cohort of HGSOC tissues. High expression of lncRNA CTBP1-DT was associated with poor prognosis and was an independent risk factor for survival. Overexpression of lncRNA CTBP1-DT promoted malignant biological behaviour of HGSOC cells, whereas its depletion induced growth arrest of HGSOC cells by vitro and in vivo assays. Mechanistically, lncRNA CTBP1-DT could competitively bind to miR-188-5p to protect MAP3K3 from degradation. Moreover, our results revealed that ETV5 could specifically interact with the promoter of lncRNA CTBP1-DT and activate its transcription. Collectively, these results reveal a novel ETV5/lncRNA CTBP1-DT/miR-188-5p/MAP3K3 pathway for HGSOC progression and suggest that lncRNA CTBP1-DT might be a potential biomarker and therapeutic target for HGSOC.

Multiomic analysis identifies CPT1A as a potential therapeutic target in platinum-refractory, high-grade serous ovarian cancer

SummaryResistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line).

Repurposing Ceritinib Induces DNA Damage and Enhances PARP Inhibitor Responses in High-Grade Serous Ovarian Carcinoma.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Biological and prognostic value of ETV5 in high-grade serous ovarian cancer

BackgroundETS transcription factors are known to act as either positive or negative regulators of the expression of genes involved in various biological processes. It was reported that ETS variant transcription factor 5 (ETV5), a key member of the ETS family, mainly plays a role as an potential oncogene in various malignant tumors. However, the role and mechanism of ETV5 in high-grade serous ovarian cancer (HGSOC) have not been elucidated.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect ETV5 messenger ribonucleic acid (mRNA) expression in 87 HGSOC tissues and 35 normal fallopian tube tissues. Western blotting and qRT-PCR were used to detect the protein and mRNA expression of ETV5 in six ovarian cancer (OC) and human embryonic cell lines. Knockdown or overexpression of ETV5 in HGSOC cell lines, Cell Counting Kit-8, colony formation, and transwell assays were used to detect HGSOC cell proliferation, invasion, and migration capabilities. The chi-square test was used to analyze the clinicopathological characteristics of HGSOC patients. Survival analysis was performed using the Kaplan-Meier method, and the log-rank test was used to analyze the correlation between ETV5 expression and HGSOC patient prognosis. Univariate and multivariate analyses using the Cox regression model were conducted to determine the independent significance of relevant clinical covariates.ResultsBioinformatic analysis demonstrated that ETV5 expression was significantly upregulated in OC (p < 0.05). qRT-PCR showed that ETV5 was significantly overexpressed in HGSOC tissues than in fallopian tube tissues (p < 0.05). qRT-PCR and western blotting assays demonstrated that ETV5 was relatively highly expressed in OC cell lines. ETV5 overexpression was positively associated with poor survival in HGSOC patients, therefore making it a high-risk factor for HGSOC progression. Furthermore, ETV5 promoted the proliferation, migration, and invasion capabilities of HGSOC cells.ConclusionETV5 has a carcinogenic effect in HGSOC and can be used as a clinically effective biomarker to determine the prognosis of HGSOC patients.

Molecular response to PARP1 inhibition in ovarian cancer cells as determined by mass spectrometry based proteomics

BackgroundPoly (ADP)-ribose polymerase (PARP) inhibitors have entered routine clinical practice for the treatment of high-grade serous ovarian cancer (HGSOC), yet the molecular mechanisms underlying treatment response to PARP1 inhibition (PARP1i) are not fully understood.MethodsHere, we used unbiased mass spectrometry based proteomics with data-driven protein network analysis to systematically characterize how HGSOC cells respond to PARP1i treatment.ResultsWe found that PARP1i leads to pronounced proteomic changes in a diverse set of cellular processes in HGSOC cancer cells, consistent with transcript changes in an independent perturbation dataset. We interpret decreases in the levels of the pro-proliferative transcription factors SP1 and β-catenin and in growth factor signaling as reflecting the anti-proliferative effect of PARP1i; and the strong activation of pro-survival processes NF-κB signaling and lipid metabolism as PARPi-induced adaptive resistance mechanisms. Based on these observations, we nominate several protein targets for therapeutic inhibition in combination with PARP1i. When tested experimentally, the combination of PARPi with an inhibitor of fatty acid synthase (TVB-2640) has a 3-fold synergistic effect and is therefore of particular pre-clinical interest.ConclusionOur study improves the current understanding of PARP1 function, highlights the potential that the anti-tumor efficacy of PARP1i may not only rely on DNA damage repair mechanisms and informs on the rational design of PARP1i combination therapies in ovarian cancer.

Primary high-grade serous ovarian cancer cells are sensitive to senescence induced by carboplatin and paclitaxel in vitro

BackgroundVarious types of normal and cancer cells undergo senescence in response to carboplatin and paclitaxel, which are considered the gold standard treatments in ovarian cancer management. Surprisingly, the effect of these drugs on ovarian cancer cell senescence remained unknown.MethodsThe experiments were conducted on primary high-grade serous ovarian cancer cells. Molecular markers of senescence were evaluated using cytochemistry and immunofluorescence. Cell cycle distribution was analyzed using flow cytometry. Expression of cyclins and signaling pathways was tested using western blot. Telomere length and telomerase activity were measured using qPCR, and the colocalization of telomeres with DNA damage foci using immuno-FISH. Oxidative stress-related parameters were quantified using appropriate fluorescence probes. Production of cancerogenic agents was analyzed using qPCR and ELISA.ResultsCarboplatin applied with paclitaxel induces senescence of ovarian cancer cells in vitro. This activity was reflected by permanent G2/M growth arrest, a high fraction of cells expressing senescence biomarkers (SA-β-Gal and γ-H2A.X), upregulated expression of p16, p21, and p53 cell cycle inhibitors, and decreased expression of cyclin B1. Neither telomere length nor telomerase activity changed in the senescent cells, and the majority of DNA damage was localized outside telomeres. Moreover, drug-treated cancer cells exhibited increased production of STAT3 protein, overproduced superoxide and peroxides, and increased mitochondrial mass. They were also characterized by upregulated ANG1, CCL11, IL-6, PDGF-D, TIMP-3, TSP-1, and TGF-β1 at the mRNA and/or protein level.ConclusionsOur findings imply that conventional chemotherapy may elicit senescence in ovarian cancer cells, which may translate to the development of a cancer-promoting phenotype, despite the inability of these cells to divide.

Identification of Specific Cell Subpopulations and Marker Genes in Ovarian Cancer Using Single-Cell RNA Sequencing.

Objective Ovarian cancer is the deadliest gynaecological cancer globally. In our study, we aimed to analyze specific cell subpopulations and marker genes among ovarian cancer cells by single-cell RNA sequencing (RNA-seq). Methods Single-cell RNA-seq data of 66 high-grade serous ovarian cancer cells were employed from the Gene Expression Omnibus (GEO). Using the Seurat package, we performed quality control to remove cells with low quality. After normalization, we detected highly variable genes across the single cells. Then, principal component analysis (PCA) and cell clustering were performed. The marker genes in different cell clusters were detected. A total of 568 ovarian cancer samples and 8 normal ovarian samples were obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes were identified according to ∣log2fold change (FC) | >1 and adjusted p value <0.05. To explore potential biological processes and pathways, functional enrichment analyses were performed. Furthermore, survival analyses of differentially expressed marker genes were performed. Results After normalization, 6000 highly variable genes were identified across the single cells. The cells were divided into 3 cell populations, including G1, G2M, and S cell cycles. A total of 1,124 differentially expressed genes were identified in ovarian cancer samples. These differentially expressed genes were enriched in several pathways associated with cancer, such as metabolic pathways, pathways in cancer, and PI3K-Akt signaling pathway. Furthermore, marker genes, STAT1, ANP32E, GPRC5A, and EGFL6, were highly expressed in ovarian cancer, while PMP22, FBXO21, and CYB5R3 were lowly expressed in ovarian cancer. These marker genes were positively associated with prognosis of ovarian cancer. Conclusion Our findings revealed specific cell subpopulations and marker genes in ovarian cancer using single-cell RNA-seq, which provided a novel insight into the heterogeneity of ovarian cancer.

Targeting the DIO3 enzyme using first-in-class inhibitors effectively suppresses tumor growth: a new paradigm in ovarian cancer treatment.

The enzyme iodothyronine deiodinase type 3 (DIO3) contributes to cancer proliferation by inactivating the tumor-suppressive actions of thyroid hormone (T3). We recently established DIO3 involvement in the progression of high-grade serous ovarian cancer (HGSOC). Here we provide a link between high DIO3 expression and lower survival in patients, similar to common disease markers such as Ki67, PAX8, CA-125, and CCNE1. These observations suggest that DIO3 is a logical target for inhibition. Using a DIO3 mimic, we developed original DIO3 inhibitors that contain a core of dibromomaleic anhydride (DBRMD) as scaffold. Two compounds, PBENZ-DBRMD and ITYR-DBRMD, demonstrated attenuated cell counts, induction in apoptosis, and a reduction in cell proliferation in DIO3-positive HGSOC cells (OVCAR3 and KURAMOCHI), but not in DIO3-negative normal ovary cells (CHOK1) and OVCAR3 depleted for DIO3 or its substrate, T3. Potent tumor inhibition with a high safety profile was further established in HGSOC xenograft model, with no effect in DIO3-depleted tumors. The antitumor effects are mediated by downregulation in an array of pro-cancerous proteins, the majority of which known to be repressed by T3. To conclude, using small molecules that specifically target the DIO3 enzyme we present a new treatment paradigm for ovarian cancer and potentially other DIO3-dependent malignancies.

Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition

Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50–85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.

Quinacrine Induces Nucleolar Stress in Treatment-Refractory Ovarian Cancer Cell Lines.

Simple SummaryA high mortality rate in ovarian cancer imposes the need for improved therapy; most patients initially respond to systemic chemotherapy but later relapse with fatal treatment-refractory tumors. We previously evaluated repurposing the antimalarial agent quinacrine as an anticancer agent in chemoresistant ovarian cancer. Quinacrine has a long history of use in humans and we have demonstrated selectivity characteristics for reducing drug resistant tumors. In this study, we analyze different responses to quinacrine between drug resistant and sensitive cell lines to identify major pathways related to this selectivity. We confirm our results in notoriously drug resistant high-grade serous ovarian cancer cells and describe nucleostemin as a new quinacrine target related to ribosomal biogenesis and nucleolar stress. This study provides preclinical evidence that quinacrine may be effective against relapsed/refractory ovarian cancer.A considerable subset of gynecologic cancer patients experience disease recurrence or acquired resistance, which contributes to high mortality rates in ovarian cancer (OC). Our prior studies showed that quinacrine (QC), an antimalarial drug, enhanced chemotherapy sensitivity in treatment-refractory OC cells, including artificially generated chemoresistant and high-grade serous OC cells. In this study, we investigated QC-induced transcriptomic changes to uncover its cytotoxic mechanisms of action. Isogenic pairs of OC cells generated to be chemoresistant and their chemosensitive counterparts were treated with QC followed by RNA-seq analysis. Validation of selected expression results and database comparison analyses indicated the ribosomal biogenesis (RBG) pathway is inhibited by QC. RBG is commonly upregulated in cancer cells and is emerging as a drug target. We found that QC attenuates the in vitro and in vivo expression of nucleostemin (NS/GNL3), a nucleolar RBG and DNA repair protein, and the RPA194 catalytic subunit of Pol I that results in RBG inhibition and nucleolar stress. QC promotes the redistribution of fibrillarin in the form of extranuclear foci and nucleolar caps, an indicator of nucleolar stress conditions. In addition, we found that QC-induced downregulation of NS disrupted homologous recombination repair both by reducing NS protein levels and PARylation resulting in reduced RAD51 recruitment to DNA damage. Our data suggest that QC inhibits RBG and this inhibition promotes DNA damage by directly downregulating the NS–RAD51 interaction. Additionally, QC showed strong synergy with PARP inhibitors in OC cells. Overall, we found that QC downregulates the RBG pathway, induces nucleolar stress, supports the increase of DNA damage, and sensitizes cells to PARP inhibition, which supports new therapeutic stratagems for treatment-refractory OC. Our work offers support for targeting RBG in OC and determines NS to be a novel target for QC.

737P Phase II study of IV vinorelbine in relapsed platinum resistant or refractory C5 high grade serous primary peritoneal, fallopian tube or ovarian cancer (VIP)

The Australian Ovarian Cancer Study identified 4 distinct molecular subtypes of high-grade serous ovarian cancer (HGSOC). C5 subtype is linked to poor prognosis and stemness behavior. We have previously described dependency of C5 HGSOC on genes related to microtubule dynamics. In vitro and vivo studies have shown sensitivity of C5 subtype HGSOC cell lines and patient-derived xenografts to tubulin-depolymerizing vinca alkaloids such as vinorelbine.

18P In vitro analysis of the combination of APR-246 and carboplatin in triple negative breast cancer (TNBC) and high grade serous ovarian cancer (HGSOC) cell lines and its impact on Aurora kinase A (AURKA)-p53 pathway

AURKA is a protein that regulates mitotic spindle formation and p53 is involved in cell cycle regulation. p53 mutations and AURKA overexpression is a frequent alteration in both TNBC and HGSOC cell lines which lead to carboplatin sensitivity. APR-246 is a new targeted agent that modulates abnormal p53 in mutated cells. The aim of our study was to assess the effect of the combination of carboplatin (carbo) plus APR-246 in TNBC and HGSOC cell lines.

Phosphoproteomic response mapping to anti-angiogenic agents bevacizumab and cediranib in high-grade serous ovarian and endometrial cancer cell lines

<h3>Objectives:</h3> Antiangiogenics have become a cornerstone of treatment in advanced stage and recurrent high grade serous ovarian cancer. Both cediranib (ICON6) and bevacizumab (ICON7, GOG218) have demonstrated benefit as a maintenance treatment for ovarian cancer patients in clinical trials. Investigators have also explored their role in advanced stage and recurrent endometrial carcinoma (MITO-END 2, Rose 2017, Aghajanian 2011). There have been no head to head trials in gynecologic cancers comparing cediranib to bevacizumab, although in colorectal carcinoma, phase III data suggests equivalent efficacy (HORIZON III). We hypothesize that these drugs, although both categorized as anti-angiogenics, induce unique cellular responses and sought to explore the varying impact of each agent on cell signaling using established serous cell models. <h3>Methods:</h3> CAOV3 ovarian cancer cells and Hec50 endometrial cancer cells were exposed to 1µM bevacizumab or cediranib for 24 hours. Cell lysates were analyzed in duplicate using an antibody-based phosphoproteomic array of 875 phosphoproteins supplied by Kinexus Bioinformatics (Vancouver, BC). Priority signaling effects were defined for each drug and in each cell line as those with a significant (>75%) change from control with an acceptable sum of error (<75%). <h3>Results:</h3> Bevacizumab yielded 13 total leads, and cediranib yielded 9 in CAOV3 ovarian cancer cells (Table 1). Both agents significantly enhanced signaling events on the following epitopes: ABl, EZH2, HGK (2 sites), and IGF1R, indicating that these may be some of the core cellular effects of anti-angiogenic agents. Interestingly, all common leads induced by both agents in CAOV3 cells represent potential pro-oncogenic effects. In Hec50 cells (Table 1), both cediranib and bevacizumab treatment induced a unique set of phosphoproteins compared to the effects of these agents in CAOV3 cells, yet common findings were the phosphorylation of IGFR1 by bevacizumab and the activation of alternative MAP kinases in both cell lines. <h3>Conclusions:</h3> Angiogenesis plays a crucial role in tumor development and metastasis. Blocking angiogenesis using monoclonal antibodies such as bevacizumab or tyrosine kinase inhibitors such as cediranib is a strategy that is proving to be helpful in the care of patients with gynecologic malignancies. These molecular agents have both on-target, therapeutic effects but also may induce pathways of resistance. In these comprehensive phosphoproteomic mapping studies, we noted the activation of signaling pathways which were cell and drug specific. One consistent finding was the enhanced phosphorylation of IGFR1 and alternative MAP kinases that may portend eventual drug resistance.