High-grade Serous Ovarian Cancer Cell Research Articles

The role and impact of alternative polyadenylation and miRNA regulation on the expression of the multidrug resistance-associated protein 1 (MRP-1/ABCC1) in epithelial ovarian cancer

The ATP-binding cassette transporter (ABCC1) is associated with poor survival and chemotherapy drug resistance in high grade serous ovarian cancer (HGSOC). The mechanisms driving ABCC1 expression are poorly understood. Alternative polyadenylation (APA) can give rise to ABCC1 mRNAs which differ only in the length of their 3′untranslated regions (3′UTRs) in a process known as 3′UTR-APA. Like other ABC transporters, shortening of the 3′UTR of ABCC1 through 3′UTR-APA would eliminate microRNA binding sites found within the longer 3′UTRs, hence eliminating miRNA regulation and altering gene expression. We found that the HGSOC cell lines Caov-3 and Ovcar-3 express higher levels of ABCC1 protein than normal cells. APA of ABCC1 occurs in all three cell lines resulting in mRNAs with both short and long 3′UTRs. In Ovcar-3, mRNAs with shorter 3′UTRs dominate resulting in a six-fold increase in protein expression. We were able to show that miR-185-5p and miR-326 both target the ABCC1 3′UTR. Hence, 3′UTR-APA should be considered as an important regulator of ABCC1 expression in HGSOC. Both HGSOC cell lines are cisplatin resistant, and we used erastin to induce ferroptosis, an alternative form of cell death. We showed that we could induce ferroptosis and sensitize the cisplatin resistant cells to cisplatin by using erastin. Knocking down ABCC1 resulted in decreased cell viability, but did not contribute to erastin induced ferroptosis.

Targeting RAS-ERK pathway alterations with MEK inhibitors to improve chemosensitivity in high grade serous ovarian cancers

ObjectiveAssess if MEK inhibitor blockade of RAS-ERK pathway adaptive response in high grade serous ovarian cancers (HGSOC) improves platinum sensitivity. MethodsThree HGSOC cell lines and three patient derived organoid (PDOs) samples from ascites of platinum resistant HGSOC patients were collected. Cell lines and PDOs were exposed to carboplatin and MEK inhibitors cobimetinib or trametinib. Cytotoxic effects of MEK inhibitors alone or combined with carboplatin were established. Western blots demonstrated RAS-ERK pathway blockage after MEK inhibitor treatment. RNA sequencing assessed gene expression after MEK inhibitor treatment. Cell line NF1 gene knockdown was performed with corresponding chemosensitivity levels. ResultsHigh carboplatin IC50 levels indicated platinum resistance in cell lines and PDOs. Cobimetinib induced cytotoxicity in cell lines and PDOs, while trametinib was less effective. Western blot confirmed MEK-ERK pathway blockage at minimal concentrations of MEK inhibitors in cell lines and PDOs. Phosphorylated-ERK levels of untreated cells indicated higher levels of RAS-ERK pathway activation in OVSAHO and OVCAR7 compared to OVCAR3. OVSAHO harbors a NF1 mutation and had highest levels of RAS-ERK activation. Cotreatment with carboplatin and MEK inhibitors showed varying synergistic cytotoxic effects at different combinations. Synergistic effect was most prominent in the OVSAHO carboplatin and cobimetinib combination. RNA sequencing identified downregulation of c-MYC and FOXM1 gene expression after MEK inhibitor treatment. NF1 gene knockdown showed an acquired increased IC50 compared to parental cells. ConclusionMEK inhibitors block RAS-ERK pathways in platinum resistant HGSOC cells and PDOs. MEK inhibitors with carboplatin have select synergistic effects which may indicate a strategy to improve platinum sensitivity.

Function of m5C RNA methyltransferase NOP2 in high-grade serous ovarian cancer

ABSTRACT RNA methyltransferase nucleolar protein p120 (NOP2), commonly referred to as NOP2/Sun RNA methyltransferase family member 1 (NSUN1), is involved in cell proliferation and is highly expressed in various cancers. However, its role in high-grade serous ovarian cancer (HGSOC) remains unclear. Our study investigated the expression of NOP2 in HGSOC tissues and normal fimbria tissues, and found that NOP2 was significantly upregulated in HGSOC tissues. Our experiments showed that NOP2 overexpression promoted cell proliferation in vivo and in vitro and increased the migration and invasion ability of HGSOC cells in vitro. Furthermore, we identified Rap guanine nucleotide exchange factor 4 (RAPGEF4) as a potential downstream target of NOP2 in HGSOC. Finally, our findings suggest that the regulation of NOP2 and RAPGEF4 may depend on m5C methylation levels.

134P Doxorubicin and olaparib (OLA) synergism in high-grade serous ovarian (HGOC) and triple-negative breast cancer (TNBC) cell lines with olaparib-resistance

134P Doxorubicin and olaparib (OLA) synergism in high-grade serous ovarian (HGOC) and triple-negative breast cancer (TNBC) cell lines with olaparib-resistance

EXPRESSION OF PROGRAMMED CELL DEATH PROTEIN 1 (PD-1) AND ITS LIGAND PD-L1 IN HIGH-GRADE SEROUS OVARIAN CANCER

Recently, the immunotherapeutic treatment of ovarian cancer is of much concern. This is due to the discovery of the signaling pathway of the PD-1/PD-L1 immune checkpoints, which are capable of controlling autoimmune response severity and duration.
 The aim of the study is to determine the dependence of PD-1/PD-L1 expression on the proliferative activity of high-grade serous ovarian cancer cells.
 Materials and Methods. The study was performed at Central Research Laboratory, Pacific State Medical University, Ministry of Health of the Russian Federation. The material was collected at Primorsky Regional Oncological Dispensary, 2016–2021. The pathological and anatomical samples were analyzed by immunohistochemistry using p53, Ki-67, PD-L1, and Anti-Hu CD279 (PD-1). The main group included 74 patients with high-grade serous ovarian cancer, the comparative group consisted of 26 patients with benign ovarian tumors.
 Results. PD-1/PD-L1 expression was observed in tumor tissues in ovarian cancer. There was also an increase in the positive reaction to Ki-67 and p53 according to grading and staging of cancer.
 Conclusion. Despite the claimed immunomodulatory function, high PD-1/PD-L1 levels are favorable prognostic indicators in ovarian cancer. The obtained data show that PD-1/PD-L1 molecules are biologically significant regulators of the immune response in high-grade serous ovarian cancer, which is an argument in favor of immune checkpoint inhibitors. Thus, PD-L1 in the immune cells infiltrating the tumor is a favorable prognostic factor. A statistically significant direct correlation between Ki-67 and PD-L1 proves a direct dependence of PD-L1 expression on proliferative activity of the ovarian tumor. A statistically significant inverse correlation between p53 and PD-1 shows the dependence of PD-1 expression on apoptotic changes in the ovarian tumor.

SLFN11 is a BRCA Independent Biomarker for the Response to Platinum-Based Chemotherapy in High-Grade Serous Ovarian Cancer and Clear Cell Ovarian Carcinoma.

BRCA1/2 mutations are robust biomarkers for platinum-based chemotherapy in epithelial ovarian cancers. However, BRCA1/2 mutations in clear cell ovarian carcinoma (CCC) are less frequent compared with high-grade serous ovarian cancer (HGSC). The discovery of biomarkers that can be applied to CCC is an unmet need in chemotherapy. Schlafen 11 (SLFN11) has attracted attention as a novel sensitizer for DNA-damaging agents including platinum. In this study, we investigated the utility of SLFN11 in HGSC and CCC for platinum-based chemotherapy. SLFN11 expression was analyzed retrospectively by IHC across 326 ovarian cancer samples. The clinicopathologic significance of SLFN11 expression was analyzed across 57 advanced HGSC as a discovery set, 96 advanced HGSC as a validation set, and 57 advanced CCC cases, all of whom received platinum-based chemotherapy. BRCA1/2 mutation was analyzed using targeted-gene sequencing. In the HGSC cohort, the SLFN11-positive and BRCA mutation group showed significantly longer whereas the SLFN11-negative and BRCA wild-type group showed significantly shorter progression-free survival and overall survival. Moreover, SLFN11-positive HGSC shrunk significantly better than SLFN11-negative HGSC after neoadjuvant chemotherapy. Comparable results were obtained with CCC but without consideration of BRCA1/2 mutation due to a small population. Multivariate analysis identified SLFN11 as an independent factor for better survival in HGSC and CCC. The SLFN11-dependent sensitivity to platinum and PARP inhibitors were validated with genetically modified non-HGSC ovarian cancer cell lines. Our study reveals that SLFN11 predicts platinum sensitivity in HGSC and CCC independently of BRCA1/2 mutation status, indicating that SLFN11 assessment can guide treatment selection in HGSC and CCC.

Increased expression of IDO1 is associated with improved survival and increased number of TILs in patients with high-grade serous ovarian cancer

BackgroundThe enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a crucial role in regulating the immune system's response to tumors, but its exact role in cancer, especially in high-grade serous ovarian cancer (HGSOC), remains controversial. We aimed to investigate the prognostic impact of IDO1 expression and its correlation with tumor-infiltrating lymphocytes (TILs) in HGSOC. MethodsImmunohistochemical (IHC) staining and bioimage analysis using the QuPath software were employed to assess IDO1 protein expression in a well-characterized cohort of 507 patients with primary HGSOC. Statistical evaluation was performed using SPSS, and in silico validation considering IDO1 mRNA expression in bulk and single-cell gene expression datasets was conducted. Additionally, IDO1 expression in interferon-gamma (IFNG) stimulated HGSOC cell lines was analyzed. ResultsOur findings revealed that IDO1 protein and mRNA expression serve as positive prognostic markers for overall survival (OS) and progression-free survival (PFS) in HGSOC. High IDO1 expression was associated with a significant improvement in OS by 21 months (p < 0.001) and PFS by 6 months (p = 0.016). Notably, elevated IDO1 expression correlated with an increased number of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ TILs (p < 0.001). Furthermore, high IDO1 mRNA expression and protein level were found to be associated with enhanced responsiveness to pro-inflammatory cytokines, particularly IFNG. ConclusionsOur study provides evidence that IDO1 expression serves as a positive prognostic marker in HGSOC and is associated with an increased number of CD3+, CD4+ and CD8+ TILs. Understanding the intricate relationship between IDO1, TILs, and the tumor microenvironment may hold the key to improving outcomes in HGSOC.

Efficacy and safety of glycosphingolipid SSEA-4 targeting CAR-T cells in an ovarian carcinoma model.

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized SSEA-4 cell-surface glycolipid as a target for CAR-T cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed pre-clinically in vitro and in vivo for anti-tumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4 positive cells in all the tested cancer cell lines whereas SSEA-4 negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific anti-tumor response at all the CAR-T cell doses used. At high T cell doses, CAR-T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR-T cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR-T therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.

Evaluating Alkaline Phosphatase-Instructed Self-Assembly of d-Peptides for Selectively Inhibiting Ovarian Cancer Cells.

Cancer is a major public health concern requiring novel treatment approaches. Enzyme-instructed self-assembly (EISA) provides a unique approach for selectively inhibiting cancer cells. However, the structure and activity correlation of EISA remains to be explored. This study investigates new EISA substrates of alkaline phosphatase (ALP) to hinder ovarian cancer cells. Analogues 2-8 were synthesized by modifying the amino acid residues of a potent EISA substrate 1 that effectively inhibits the growth of OVSAHO, a high-grade serous ovarian cancer (HGSOC) cell line. The efficacy of 2-8 against OVSAHO was assessed, along with the combination of substrate 1 with clinically used drugs. The results reveal that substrate 1 displays the highest cytotoxicity against OVSAHO cells, with an IC50 of around 8 μM. However, there was limited synergism observed between substrate 1 and the tested clinically used drugs. These findings indicate that EISA likely operates through a distinct mechanism that necessitates further elucidation.

FAK inhibitor PF-562271 inhibits the migration and proliferation of high-grade serous ovarian cancer cells through FAK and FAK mediated cell cycle arrest.

Focal adhesion kinase (FAK) is a promising therapeutic target for various cancers and its inhibitor development is in full swing. PF-562271 is a classic FAK inhibitor that has shown promising preclinical data and has been found to exhibit an anti-migration effect on some cancer cells. However, its anticancer effect on high-grade serous ovarian cancer (HGSOC) has not been reported. In this study, we evaluated the anti-migration and anti-proliferation effects of PF-562271 against HGSOC SKOV3 and A2780 cells, as well as the underlying mechanism. The results demonstrated that FAK was overexpressed in clinical HGSOC tissues and was positively correlated with the pathological progression of HGSOC. Moreover, HGSOC patients with high FAK expression levels exhibited low survival rates. PF-562271 treatment significantly inhibited the cell adhesion and migration of SKOV3 and A2780 cells by inhibiting p-FAK expression and decreasing the FA surface area. Additionally, PF-562271 treatment inhibited colony formation and induced cell senescence through G1 phase cell cycle arrest mediated DNA replication inhibition. Taken together, the findings demonstrated that FAK inhibitor PF-562271 significantly inhibits HGSOC cell adhesion, migration, and proliferation process through FAK and/or FAK mediated cell cycle arrest, and suggested that PF-562271 could serve as a potential oncotherapeutic agent for HGSOC targeting treatment.

Characteristics of Extracellular Vesicles from a High-Grade Serous Ovarian Cancer Cell Line Derived from a Platinum-Resistant Patient as a Potential Tool for Aiding the Prediction of Responses to Chemotherapy.

Platinum-resistant high-grade serous ovarian cancer (HGSOC) is invariably a fatal disease. A central goal of ovarian cancer research is therefore to develop new strategies to overcome platinum resistance. Treatment is thus moving towards personalized therapy. However, validated molecular biomarkers that predict patients' risk of developing platinum resistance are still lacking. Extracellular vesicles (EVs) are promising candidate biomarkers. EpCAM-specific EVs are largely unexplored biomarkers for predicting chemoresistance. Using transmission electron microscopy, nanoparticle tracking analysis and flow cytometry, we compared the characteristics of EVs released from a cell line derived from a clinically confirmed cisplatin-resistant patient (OAW28) and EVs released from two cell lines from tumors sensitive to platinum-based chemotherapy (PEO1 and OAW42). We demonstrated that EVs released from the HGSOC cell line of chemoresistant patients exhibited greater size heterogeneity, a larger proportion of medium/large (>200 nm) Evs and a higher number of released EpCAM-positive EVs of different sizes, although the expression of EpCAM was predominant in EVs larger than 400 nm. We also found a strong positive correlation between the concentration of EpCAM-positive EVs and the expression of cellular EpCAM. These results may contribute to the prediction of platinum resistance in the future, although they should first be validated in clinical samples.

CDK2 regulates collapsed replication fork repair in CCNE1-amplified ovarian cancer cells via homologous recombination.

CCNE1 amplification is a common alteration in high-grade serous ovarian cancer and occurs in 15-20% of these tumors. These amplifications are mutually exclusive with homologous recombination deficiency, and, as they have intact homologous recombination, are intrinsically resistant to poly (ADP-ribose) polymerase inhibitors or chemotherapy agents. Understanding the molecular mechanisms that lead to this mutual exclusivity may reveal therapeutic vulnerabilities that could be leveraged in the clinic in this still underserved patient population. Here, we demonstrate that CCNE1-amplified high-grade serous ovarian cancer cells rely on homologous recombination to repair collapsed replication forks. Cyclin-dependent kinase 2, the canonical partner of cyclin E1, uniquely regulates homologous recombination in this genetic context, and as such cyclin-dependent kinase 2 inhibition synergizes with DNA damaging agents in vitro and in vivo. We demonstrate that combining a selective cyclin-dependent kinase 2 inhibitor with a DNA damaging agent could be a powerful tool in the clinic for high-grade serous ovarian cancer.

The combination of APR-246 and carboplatin in olaparib-resistant high grade serous ovarian cancer (HGSOC) and triple negative breast cancer (TNBC) cell lines.

e17504 Background: PARP inhibitors (PARPi), as Olaparib (OLA), have changed the therapeutic landscape in HGSOC and TNBC. However, most patients will develop resistance to PARPi. Although resistance mechanisms are poorly understood, it is suggested that BRCA mutations reversions or cell cycle alterations could be involved. APR-246 is a new targeted agent that restores p53 in TP53-mutated cells. The aim of our study was to assess the potential synergism of the combination of carboplatin (carbo) plus APR-246 in both OLA-sensitive and OLA-resistant HGSOC and TNBC cell lines. Methods: We selected two HGSOC (PEO1-S and Kuramochi-S) and one TNBC TP53 mutant cell lines (MDA-MB-231-S). Resistance to OLA was generated by pulse doses with IC75 OLA for 24 hours and recovered for two weeks during a period of 6 months (PEO1-R) and to increased concentrations of OLA for 72 hours during a period of 1 year (Kuramochi-R, MDA-MB-231-R). Next-generation sequencing was performed in order to assess mutations. IC50 of APR-246 and Carbo and the combination of both (combo) were calculated for Kura-S/R, PEO1-S/R and 231-S/R cell lines. Synergism between both drugs was assessed with Chou-Talalay method for combination index (CI) at Fa50. DNA damage was assessed by gamma-H2AX expression by western-blot (WB). Results: 231-S harboured a TP53 mutation (mut) (missense R280K) and BRCA wildtype (wt), in PEO1-S BRCA2 mut p.Y1655* and TP53 mut was detectable. Kuramochi-S was TP53 mut (missense D816Y) and BRCA2 mut. BRCA and p53 mutation remained unchanged in the resistant lines generated.The mutation profile of each line and the IC50 of APR-246, carbo and combo are shown in the table. IC50 of carboplatin is superior in all 3 resistant cell lines when compared to their sensitive parental line, suggesting a potential cross-resistance to platinum in OLA-resistance. The combination of carbo and APR-246 is synergistic even in OLA-resistant cell lines, in fact in those cross-resistant lines values of synergism by Chou-Talalay are improved by the combo suggesting a potential role of this combination in PARPi resistant cell lines. WB showed an increased DNA damage with the combo when compared to either single agent, with an increase in H2AX in all lines either sensitive or resistant. Conclusions: The combination of APR246 and carboplatin is synergistic in all 3 studied cell lines but also in the OLA resistant ones. The combo increased its synergistic effect suggesting a potential role of combo in OLA-resistant tumors. [Table: see text]

MYC amplification segment-size mutual exclusivity analysis with other oncogenic events in a pan-cancer sequencing cohort.

3073 Background: MYC gene alterations resulting in increased copy number are recurrent oncogenic alterations across human malignancies. Presently no clear data exists to aid in interpreting MYC amplification characteristics for identifying MYC-dependent tumors. Methods: Analysis was conducted on a center-wide cohort of 68,920 tumor-normal matched samples from pediatric and adult patients sequenced with an FDA-authorized targeted sequencing panel (MSK-IMPACT). To ascertain tumor content, ploidy and allelic specific gene copy number, tumor-normal samples were analyzed with FACETS. Samples with tumor purity &lt;20% were excluded. MYC amplification was defined as total copy number (TCN) minus ploidy ≥ 4. All samples were queried for presence of concurrent oncogenic or likely-oncogenic alterations based on OncoKb annotation. Co-occurrence (Co) and mutual-exclusivity (Me) analysis between MYC amplification according to segment size and other oncogenic alterations was performed with DISCOVER (false discovery rate threshold of 0.01) for each tumor type. Results: After removing low-quality samples (n=19,010/68,920), 49,910 samples were included in the analysis. 3,219 (6.4%) samples harbored MYC amplification, with segment size ranging from less than 100Kb to more than 100Mb, with 38% samples displaying focal amplifications shorter than 10 Mb and 62% broad amplifications longer than 10 Mb. TCN was inversely correlated with segment size (R= -0.34, p=2.2e-16). Triple-negative breast cancer (13.3%), hepatocellular carcinoma (8.7%), neuroendocrine tumors of the rectum (8.3%), high-grade serous ovarian cancer (7.6%) and large cell neuroendocrine carcinomas of the lung (6.7%) had the highest prevalence of MYC amplification without concurrent potentially driver oncogene mutations, amplifications or fusions. CoMe analysis demonstrated mutual exclusivity of MYC gene amplification regardless of segment size with most major tumor-type specific oncogenic events. All MYC amplifications were mutually exclusive with MET splice variants and mutations (q&lt; 0.001) and KRAS mutations (q&lt; 0.001) in lung adenocarcinoma (LUAD), FGFR3 mutations (q&lt; 0.001) and fusions (q&lt; 0.001) in bladder cancer, PTEN mutations (q&lt; 0.001) in hormone receptor-positive breast cancer, KRAS mutations (q&lt; 0.001) in colorectal cancer and CDK4 amplifications (q&lt; 0.001) in sarcomas. Broad MYC amplifications were mutually exclusive with RET, ROS1 and ALK fusions in LUAD, while focal ones co-occurred with RET (q=0.007) and ALK fusions (q&lt; 0.001) or secondary mutations (q&lt; 0.001). Conclusions: This dataset defines a subgroup of MYC amplified cancers without co-occurring drivers and further subdivided by segment size, providing information that may help better identify patients for trials of novel MYC-directed therapies.

AXL pathway as a target to overcome resistance to PARP inhibitors in high-grade serous ovarian (HGSOC) and triple-negative breast cancer (TNBC) cell lines.

e17502 Background: PARP inhibitors (PARPi) as olaparib (OLA) have become the new standard in maintenance treatment in high grade serous ovarian carcinoma (HGSOC) and triple negative breast cancer (TNBC). Thus there is an important need to identify the mechanisms for PARPi resistance and the strategies to overcome it. Our aim is to intervene in vitro AXL, a receptor tyrosine kinase, linking its function to the OLA-resistance mechanism and assess its suitability as a therapeutical target to overcome PARPi-resistance. Methods: Three cell lines, 2 from HGSOC (PEO1-S and Kuramochi-S) and 1 from TNBC (MDA-MB-231-S) were selected, isogenic resistant pairs generated in vitro by pulsed exposure to OLA (PEO1-R) or by exposure to growing concentrations of OLA (Kuramochi-R and MDA-MB-231-R) were obtained. Resistance was confirm by the comparison of IC50 of OLA in -S vs -R lines. AXL expression was assessed by western blot (WB). Cells were treated with OLA before and after the downregulation of AXL expression by a specific siRNA. Cell viability was assessed by MTT assay and DNA damage by the evaluation of markers by western blot (WB). Results: AXL expression was maintained upon OLA treatment in all 3 resistant cell lines. However, AXL expression was reduced upon OLA exposure in 3 sensible parental cell lines. AXL silencing (Si) lead to a significantly decrease of OLA IC50 in all 3 sensitive and resistant cell lines. For sensitive cell lines, IC50 of OLA was in 1) PEO1S with control (scramble) was 5.79 mM vs 2.24 mM after silencing with siRNA, p &lt; 0.0001; 2) Kuramochi-S scramble OLA IC50 was 481.8 mM vs 199.7 mM with siRNA, p &lt; 0.0001 and 3) MDA-MB-231-S with control of scramble the OLA IC50 was 61.62 mM vs 26.21 mM with siRNA, p = 0.001. For resistant cell lines, 4) PEO1R control with scramble 91.49 mM vs siRNA 45.72 mM, p &lt; 0.0001; 5) Kuramochi-R scramble OLA IC50 579.8 mM vs 219.8 mM with siRNA, p = 0.0008 and 6) 231-R scramble OLA IC50 68.22 mM vs 9.247 mM with siRNA, p &lt; 0.0001. These results suggest that AXL downregulation increases sensitivity to OLA in both sensitive and resistant cell lines. Histone γ-H2AX was analysed pre and post silencing and both increased when treating with OLA, especially when AXL was downregulated in alll three resistant cell lines. Conclusions: This in vitro study suggest a potential role of AXL in OLA resistance and AXL downregulation migh represent a potential mechanism to overcome resistance to OLA in ovarian and TNBC cell lines.

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models.

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

Combination ATR (ceralasertib) and PARP (olaparib) Inhibitor (CAPRI) trial in acquired PARP-inhibitor-resistant homologous recombination deficient ovarian cancer.

Addition of ataxia telangiectasia and Rad3-related kinase inhibitors (ATRi) to poly-ADP ribose polymerase inhibitors (PARPi) overcomes PARPi-resistance in high grade serous ovarian cancer (HGSOC) cell and mouse models. We present the results of an investigator-initiated study of combination PARPi(olaparib) and ATRi(ceralasertib) in patients with acquired PARPi-resistant HGSOC. Eligible patients had recurrent, platinum-sensitive BRCA1/2 mutated or homologous recombination (HR) deficient HGSOC and clinically benefited from PARPi (response by imaging/CA-125 or duration of maintenance therapy; >12 months 1st-line or >6 months ≥2nd-line) before progression. No intervening chemotherapy was permitted. Patients received olaparib 300mg twice daily and ceralasertib 160mg daily on days 1-7 of a 28-day cycle. Primary objectives were safety and objective response rate (ORR). Thirteen patients enrolled were evaluable for safety and 12 for efficacy. 62%(n=8) had germline BRCA1/2 mutations, 23% (n=3) somatic BRCA1/2 mutations, and 15%(n=2) HR-deficient tumors. Prior PARPi indication was treatment for recurrence (54%, n=7), 2nd line-maintenance (38%, n=5), and frontline treatment with carboplatin/paclitaxel (8%, n=1). There were 6 partial responses yielding an ORR of 50% (95% CI:0.15, 0.72). Median treatment duration was 8 cycles (range 4-23+). Grade(G) 3/4 toxicities were 38%(n=5); 15%(n=2) G3 anemia, 23%(n=3) G3 thrombocytopenia, 8% (n=1) G4 neutropenia. Four patients required dose-reductions. No patient discontinued treatment due to toxicity. Combination olaparib and ceralasertib is tolerable and shows activity in HR-deficient platinum-sensitive recurrent HGSOC that benefited and then progressed with PARPi as the penultimate regimen. These data suggest that ceralasertib re-sensitizes PARPi resistant HGSOCs to olaparib, warranting further investigation.

Exploring the Control of PARP1 Levels in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a leading cause of mortality from gynecologic malignancies worldwide. Although a transformative improvement has been shown with the introduction of PARP (poly(ADP-ribose) polymerase) inhibitors, the emergence of resistance to these drugs represents a therapeutic challenge. Hence, expanding our understanding of mechanisms behind the control of PARP1 expression can provide strategic guidance for the translation of novel therapeutic strategies. The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors critically involved in the regulation of important cellular functions. Notably, we recently demonstrated that, in cervical cancer cells, STAT1 controls PARP1 levels through multiple mechanisms, possibly involving also STAT3. Here, we tested the hypothesis that a similar mechanism might be operative in HGSOC. To this end, the impact of STAT1/STAT3 modulation on PARP1 expression was assessed in established and primary HGSOC cells, and molecular biology studies proved that STAT1 might act at both transcriptional and post-transcriptional levels to modulate the PARP1 level. Notably, bioinformatics analysis of TCGA databases demonstrated that increased STAT1 mRNA expression levels are associated with a favorable prognosis and with response to chemotherapy in HGSOC patients. Our findings suggest an alternative strategy for targeting HGSOC cells based on their dependency on PARP1.

Downregulating vaccinia-related kinase 1 by luteolin suppresses ovarian cancer cell proliferation by activating the p53 signaling pathway

ObjectivesOvarian cancer constitutes one of the most common causes of cancer-related deaths, and preventing chemotherapy resistance and recurrence in patients with ovarian cancer remains a challenge. Herein, we aimed to identify the effect of luteolin, a novel therapeutic agent targeting vaccinia-related kinase 1 (VRK1), on high-grade serous ovarian cancer (HGSOC). MethodsPhosphokinase array, RNA sequencing, and cell cycle and apoptosis assays were conducted to determine the underlying mechanism of the effect of luteolin on HGSOC cells. The anticancer effects of oral and intraperitoneal luteolin administration were assessed in patient-derived xenograft models via several methods, including the assessment of tumor size and immunohistochemistry of phospho-p53, phosphor-HistoneH3 and cleaved caspase 3. ResultsLuteolin reduced HGSOC cell proliferation and increased apoptosis and cell cycle arrest at G2/M. Compared with controls, several genes were dysregulated in luteolin-treated cells, and luteolin activated the p53 signaling pathway. The human phosphokinase array revealed distinct p53 upregulation in luteolin-treated cells, as confirmed by p53 phosphorylation at ser15 and ser46 using western blot analysis. In patient-derived xenograft models, oral or intraperitoneal luteolin administration substantially suppressed tumor growth. Moreover, combination treatment involving luteolin and cisplatin inhibited tumor cell proliferation, especially in cisplatin-resistant HGSOC cell lines. ConclusionsLuteolin demonstrated considerable anticancer effect on HGSOC cells, reduced VRK1 expression, and activated the p53 signaling pathway, thereby inducing apoptosis and cell cycle arrest in G2/M and inhibiting cell proliferation. Furthermore, luteolin exhibited a synergistic effect with cisplatin both in vivo and in vitro. Thus, luteolin can be considered a promising cotreatment option for HGSOC.

Abstract 2551: CASC4 drives anoikis resistance in high grade serous ovarian cancer through recycling of EGFR

Abstract Introduction: Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian cancer (HGSOC) subtype accounts for almost 60% of ovarian cancers and is the deadliest. HGSOC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. Survival of tumor cells in the peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). CRISPR/Cas9 and transcriptomic screens identified the Golgi protein CASC4 (GOLM2, H63) as a “driver” of anoikis resistance. As CASC4 is highly uncharacterized in literature, we sought to determine how CASC4 confers anoikis resistance to HGSOC cells. Methods: Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse clinical outcomes, such as worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we used HGSOC cell lines PEO1, CaOV3, and OVCAR3 with shRNA-mediated CASC4 knockdowns (CASC4 KD), cultured in forced suspension. Results: Culturing cells in suspension, to recapitulate the peritoneal fluid environment in vitro, showed that CASC4 KD hampers cell proliferation and colony formation ability, and increases apoptosis. Additionally, a Reverse Phase Protein Assay (RPPA) showed that CASC4 KD results in significant changes in proteins known to be localized to the cell surface or secreted, including decreased protein levels of the receptor tyrosine kinase (RTK) Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways. A paralog of CASC4, GOLM1, is known to drive hepatocellular carcinoma through various mechanisms, such as the recycling of internalized RTKs, including EGFR. Indeed, our experiments showed that CASC4 KD in ovarian cancer cells leads to decreased EGFR levels and decreased EGFR recycling. Conclusion: Knocking down CASC4 results in decreased survivability in suspension, decreased EGFR levels at the plasma membrane, and decreased recycling of EGFR. Further elucidating mechanisms of CASC4-dependent anoikis resistance could lead to the development of novel therapeutic approaches, such as inhibitory peptides, and may assist in developing newer biomarkers for predicting ovarian cancer malignancy. Citation Format: Jaidev Bapat, Lindsay W. Brubaker, Zachary L. Watson, Tomomi M. Yamamoto, Benjamin G. Bitler. CASC4 drives anoikis resistance in high grade serous ovarian cancer through recycling of EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2551.