Abstract Background: Human papillomavirus (HPV) infection is the primary cause of cervical carcinoma. Variations in HPV prevalence, tumor genomic alterations, and survival outcomes across population groups remain incompletely characterized. Understanding the interplay of viral and host tumor biology is essential to optimize prevention and treatment in cervical cancer. Methods: High-risk HPV prevalence was evaluated in non-Hispanic White (NHW), non-Hispanic Black (NHB), and Hispanic (HSP) females aged 18-59 years using the National Health and Nutrition Examination Survey (NHANES, 2005-2016). Age-adjusted incidence of cervical cancer was evaluated using data from 21 regions in SEER (2004-2020). Somatic driver mutations were analyzed in tumors from patients with cervical cancer using the Genomics Evidence Neoplasia Information Exchange (GENIE v16.0). Overall survival was examined using Kaplan-Meier and Cox regression models among stage I-IV cervical cancer cases in the National Cancer Database (NCDB, 2004-2020). Results: Among 11,033 NHANES participants, high-risk HPV prevalence was similar in White and Hispanic women but higher in Black women (2.1-fold higher odds of any HPV infection; 1.5-fold higher odds of HPV16/18). Fig 1A shows the higher distribution of high-risk HPV genotypes individually and concurrent high-risk infections in NHB patients compared with either HSP or NHW patients. Data from SEER shows that age-adjusted incidence of cervical cancer was higher in both HSP and NHB patients compared with NHW patients. Rates declined in all groups from 2004-2000 (Fig 1B). Fig 1C displays somatic mutations in tumors from 595 cervical cancer patients in GENIE. BRD4, ERBB3, MTOR, and GRM3 mutations were more frequent in NHB than NHW patients. ERBB3, CIC, PIK3R1, and PI3K pathway alterations were enriched in NHB compared with HSP patients, whereas SMARCA4, CIC, KDM5C, and RANBP2 mutations were more common in White than Hispanic patients. In 117,170 NCDB cases, 5-year survival was 76% for HSP, 69% for NHW, and 60% for NHB patients (Fig 1D, p<0.0001). Compared with HSP women, mortality risk was 40% worse for NHW and 91% worse for NHB patients (p<0.001). Conclusions: Differences in HPV infection pattern are linked with cervical cancer incidence, somatic mutations, and survival, reflecting biologic and clinical heterogeneity across populations. Integrating viral genotyping with incidence rates and tumor molecular profiling may inform precision prevention, risk assessment, and personalized treatment to improve outcomes for all patients. Translational Relevance: This study links viral, incidence, genomic and survival data across multiple national datasets to reveal population-level variations in cervical cancer. These insights support efforts to enhance HPV vaccination, early detection, and individualized prevention, screening and therapeutic decision-making. Citation Format: S. Ahmed Hussain, Chunqiao Tian, Christopher Tarney, Thomas Beltran, Pouya Javadian, Ryan McLaughlin, Paulette Mhawech-Fauceglia, Doris M. Benbrook, Sean Cronin, Zachary Kopelman, Colin Sitler, Leslie M. Randall, John Chan, Daniel Kapp, Chad A. Hamilton, Charles A. Leath, Christina Washington, Kathleen Moore, Kristen Bunch, Nicholas Bateman, Thomas P. Conrads, G. Larry Maxwell, Kathleen M. Darcy. Population-based patterns of human papillomavirus infections and cervical cancer incidence, driver mutations and overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4114.

Ascites from ovarian cancer patients are increasingly recognized as a valuable biofluid for cancer research, as its protein composition reflects the disease state and may reveal biomarkers of treatment sensitivity and response. However, the detection of low-abundance proteins is hindered by the presence of highly abundant proteins such as albumin. In this study, we evaluated five protein preparation methods for their effectiveness in depleting high-abundance or enriching low-abundance proteins in ovarian cancer ascites. The Norgen (Nor), Minutes (Min), and Perchloric acid (PerCA) methods were based on abundant protein depletion, while the Urine (Uri) and Nanomics (Nano) kits focused on low-abundance protein enrichment. Processed samples were analyzed using label-free quantitative bottom-up proteomics by LC-MS/MS, followed by a bioinformatics assessment. Compared with undepleted ascites (UnD), Min, Nor, Nano, and PerCA increased protein identifications, whereas Uri produced profiles similar to those of UnD. Notably, PerCA and Nano enabled the identification of distinct protein subsets associated with cancer-related pathways, including immune responses and autophagy. PerCA enriched transmembrane and secreted immunomodulatory glycoproteins, whereas Nano enrichment primarily captured secreted, nuclear, and cytoplasmic soluble proteins. Overall, our results show that both high-abundance protein depletion and low-abundance enrichment improve ascites proteome coverage, each offering distinct advantages in identifying biologically relevant low-abundance proteins.

Chemotherapy is a common treatment for women with ovarian cancer, and many women treated with chemotherapy experience cognitive dysfunction. However, few studies have investigated the mechanisms of chemotherapy-related cognitive impairment in women with ovarian cancer. The goal of this study was to assess the relationships among neurovascular coupling, iron levels and cognitive functions in women with ovarian cancer after first-line chemotherapy. Simultaneous fNIRS and EEG data were collected in women diagnosed with advanced stage ovarian cancer at baseline (N = 13) and after 3-9 rounds of chemotherapy (N = 8). The attentional network task (ANT) was administered for neurocognitive evaluation. Blood iron levels were measured using standard clinical assays. In parallel, similar concurrent fNIRS-EEG data were acquired in a group of 10 healthy participants in a test-retest design. Cognitive performance declined, as indicated by decreases in ANT sub scores after chemotherapy. Blood iron biomarkers indexing oxygen transport also declined and were related to decreases in the ANT sub scores. Both fNIRS and EEG data responses were shown to have excellent reliability in the healthy subjects, while cancer patients showed significant decreases in oxygenated hemoglobin response in fNIRS, despite no changes in EEG responses after chemotherapy. A significant dose relationship was also found in the changes of fNIRS responses. Our data indicate that chemotherapy produced cognitive deficits and decreases in the oxygenated hemoglobin response and that these may be related to reduced oxygen transport capacity. Results also suggest the utility of using fNIRS-EEG to monitor the progression of cognitive impairment and characterize those mechanisms.

Mortalin is an essential chaperone for the import of nuclear-encoded proteins into mitochondria and is elevated in ovarian cancer in association with poor patient prognosis. The investigational new drug, SHetA2, interacts with mortalin releasing its client proteins. In this study, interactions of SHetA2 moieties and mortalin substrate binding domain (SBD) amino acids are demonstrated by surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) to occur at low micromolar SHetA2 concentrations that selectively kill cancer cells over noncancerous cells. In both ovarian cancer and noncancerous cells SHetA2 reduces: mitochondria import of mortalin, degradation of mortalin's mitochondrial localization sequence (MLS), mortalin/inositol 1,4,5-trisphosphate receptors complexes and oxidative phosphorylation. In cancer cells only, SHetA2 reduces calcium levels, mitochondrial length and fusion proteins, while inducing autophagy and PTEN-induced kinase 1 (PINK1)/PARKIN-mediated mitophagy. Noncancerous cells exhibit increased mitochondrial branch length in response to SHetA2 and a low level of inducible autophagy that is resistant to SHetA2. Inhibition of autophagosome-lysosome fusion reduces, or increases, SHetA2 cytotoxicity in ovarian cancer or noncancerous cells, respectively. SHetA2 inhibits mortalin and growth, and induces mitophagy in ovarian cancer xenografts and increases survival post-surgical tumor removal. In conclusion, SHetA2 binds directly to mortalin's SBD and causes distinct responses in ovarian cancer and noncancerous cells.

Importance:Three-year pelvic recurrence rate in women with low-risk cervical carcinoma was not inferior following simple hysterectomy (SH) vs modified radical hysterectomy (MRH) or radical hysterectomy (RH) in the Simple Hysterectomy and Pelvic Node Assessment randomized clinical trial, but the survival analysis of the trial was underpowered. Objective:To evaluate long-term survival in low-risk cervical carcinoma following SH vs MRH or RH. Design, Setting, and Participants:This cohort study included women undergoing SH, MRH or RH in US Commission on Cancer-accredited facilities participating in the National Cancer Database who received a diagnosis between January 2010 and December 2017 of International Federation of Gynecology and Obstetrics 2009 stage IA2 or IB1 squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma of the cervix (≤2 cm) and clinically negative lymph nodes. Exposure:SH, MRH, or RH following diagnosis of stage IA2 or IB1 squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma of the cervix. Main Outcomes and Measures:Survival was the primary end point, evaluated with and without propensity score balancing. Survival rates, survival distributions, adjusted hazard ratio (aHR) of death, and restricted mean survival times (RMST) were analyzed as of September 2024. Two multivariable models were fitted. Model 1 included the hysterectomy type and 9 baseline factors (age, comorbidity score, race and ethnicity, insurance status, treatment facility, stage, histologic subtype, tumor grade, and surgical approach). Model 2 included the model 1 variables plus 4 additional clinical factors (surgical margin, LVSI, pathologic LN metastasis, and adjuvant treatment). Results:This cohort study evaluated 2636 women (mean [SD] age, 45.4 [11.4] years; median [IQR] follow-up, 85 [64-110] months), including 982 with SH, 300 with MRH, 927 with traditional RH, and 427 with unspecified MRH or RH. Survival was similar following SH vs MRH or RH (7 year survival rate, 93.9%; 95% CI, 91.9%-95.4% vs 95.3%; 95% CI, 94.0%-96.3%%; P = .07) and SH vs MRH vs RH (7 year survival rate, 93.9%; 95% CI, 91.9%-95.4% vs 94.2%; 95% CI, 90.1%-96.7% vs 95.4%; 95% CI, 93.6%-96.6%; P = .15). Risk of death following either SH vs MRH or RH, SH vs RH, or MRH vs RH remained similar after adjusting for baseline covariates alone or baseline covariates plus clinical factors. Survival remained similar within subsets by age, comorbidity score, race and ethnicity, facility type, stage, histologic subtype, tumor grade, surgical approach, and year of diagnosis. Adjusted survival remained similar in patients with SH vs MRH or RH after propensity score balancing for baseline covariates (aHR, 1.19; 95% CI, 0.86-1.65; P = .31) with similar 3-year (98.3%; 95% CI, 97.2%-99.0% vs 97.6%; 95% CI, 96.6%-98.2%), 5-year (95.9%; 95% CI, 94.3%-97.1% vs 96.5%; 95% CI, 95.5%-97.3%), 7-year (94.5%; 95% CI, 92.5%-95.9% vs 95.1%; 95% CI, 93.7%-96.1%), and 10-year (89.8%; 95% CI, 86.3%-92.5% vs 91.7%; 95% CI, 89.4%-93.4%) survival rates. Sensitivity analysis for patients who received a diagnosis between 2010 and 2013 documented similar 10-year RMST following SH vs MRH or RH, SH vs RH, SH vs MRH, and MRH vs RH. Conclusions and Relevance:In this cohort study, long-term survival was similar following SH vs MRH or RH, supporting the use of SH in select patients with low-risk early-stage cervical carcinoma.

Renal microvascular injury occurs in most patients with hypertension-induced nephropathy (HN). We have shown that overexpression of adaptor protein p66Shc is implicated in the loss of renal microvascular reactivity in hypertensive rats. Since sulfur heteroarotinoid A2 (SHetA2) modulates p66Shc, we tested whether SHetA2 would restore renal microvascular reactivity and mitigate kidney injury in a rat HN model. Dahl salt sensitive (SS) and p66Shc knockout (p66Shc-KO) rats were used in a well-established rat model of HN, characterized by severe renal vascular dysfunction. SHetA2 was either added acutely to isolated rat afferent arterioles or chronically administrated to rats during HN development. The ability of SHetA2 treatment to restore afferent arteriolar contraction in response to increased perfusion pressure or ATP was evaluated using the perfused juxtamedullary nephron preparation. The progression of renal damage was evaluated by measuring urinary protein excretion and conducting analysis of glomerular injury. Comparison of renal microvascular responses to perfusion pressure in p66Shc-KO rats and parental SS rats, in the presence and absence of acute preincubation with SHetA2, revealed a dose-dependent ability of SHetA2 to restore renal microvascular reactivity in SS rats with little effect upon p66Shc knockouts. Moreover, chronic treatment with SHetA2 prevented loss of renal microvascular responses and decline in renal function. SHetA2 was more potent and effective in males compared with females. Targeting p66Shc with SHetA2 diminishes renal damage and restores renal afferent arteriolar reactivity caused by hypertension. These results justify further translation of these findings to develop SHetA2 for prevention and treatment of hypertension-induced kidney damage.NEW & NOTEWORTHY Acute preincubation with modulator of p66Shc signaling sulfur heteroarotinoid A2 (SHetA2) revealed dose-dependent ability of SHetA2 to restore renal microvascular reactivity in rats with hypertension-induced nephropathy. Moreover, chronic treatment with SHetA2 prevented loss of renal microvascular responses and decline in renal function. Thus, targeting p66Shc with SHetA2 diminishes renal damage and restores renal afferent arteriolar reactivity caused by hypertension.

Ovarian cancer is one of the most lethal gynecological malignancies and is often associated with fluid build-up in the peritoneal cavity, known as ascites. Nearly one-third of patients with ovarian cancer present with ascites at the time of initial diagnosis, and more frequently with recurrent ovarian cancer. Ascites is a uniquely valuable tool for research, as it is representative of both the tumor and its microenvironment. Ascites is composed of cells (single cells and multicellular aggregates) and acellular components that contribute to the development of peritoneal metastasis and chemoresistance. Ascites is an underutilized resource that provides an opportunity to improve our understanding of ovarian cancer biology, identify novel drug targets, assess drug responses, and identify diagnostic and/or prognostic biomarkers. This review summarizes the current understanding of ovarian cancer ascites with a focus on, (1) etiology, (2) cytopathological and molecular characterization, (3) the role its cellular and acellular components play in shaping the tumor microenvironment, and (4) its application in translational research for drug development (organoids and patient-derived ascites xenografts) and biomarker discovery. Lastly, options for the treatment of malignant ascites, along with future opportunities to use ascites as a translational research tool to improve our understanding of ovarian cancer biology and to develop new therapeutic strategies, are discussed.

Background: The literature on the relationships among blood iron levels, cognitive performance, and brain iron levels specific to women at the menopausal transition is ambiguous at best. The need to better understand these potential relationships in women for whom monthly blood loss (and thus iron loss) is ceasing is highlighted by iron’s accumulation in brain tissue over time, thought to be a factor in the development of neurodegenerative disease. Methods: Non-anemic women who were either low in iron or had normal iron levels for their age and race/ethnicity provided blood samples, underwent MRI scans to estimate brain iron levels, and performed a set of cognitive tasks with concurrent EEG. Results: Cognitive performance and brain dynamics were positively related to iron levels, including measures associated with oxygen transport. There were no relationships between any of the blood measures of iron and brain iron. Conclusions: Higher iron status was associated with better cognitive performance in a sample of women who were neither iron deficient nor anemic, without there being any indication that higher levels of systemic iron were related to higher levels of brain iron. Consequently, addressing low iron levels at the menopausal transition may be a candidate approach for alleviating the “brain fog” commonly experienced at menopause.

Ovarian cancer presents therapeutic challenges, particularly in advanced stages, where malignant ascites—a buildup of fluid in the abdominal cavity—complicates treatment. Within this ascitic environment, clusters of cancer cells, known as spheroids, form, proliferate, and implant upon various organs, contributing to the metastatic nature of ovarian cancer. As they limit drug penetration and have a hypoxic environment that fosters angiogenesis, spheroids exhibit significant chemoresistance, amplified by the inflammatory and tumor-promoting microenvironment (Peng). Therefore, to reveal the potential mechanisms of chemoresistance in ovarian cancer, directly targeting these spheroids is crucial to develop effective therapies that could help reduce the high mortality rate associated with ovarian cancer. ShetA2 (Sulfur Heteroarotinoid A2) is a novel, promising anticancer drug that exhibits unique potential by inducing apoptosis in cancer cells and disrupting mitochondrial pathways without harming healthy cells (Benbrook). Currently in phase 1 clinical trials, it is vital to understand its efficacy and optimize its therapeutic potential. However, conventional quantification methods for SHetA2 primarily rely on plasma analysis in animal models or human samples, providing only indirect measures and failing to replicate microenvironmental factors in the tumor’s 3D structure that inhibit drug access to spheroids (Sharma et al.). Consequently, a direct approach to measure drug concentration in single spheroids is necessary to understand the pharmacodynamics and pharmacokinetics of anticancer drugs (Peng). To address this gap, we developed a highly sensitive nano-flow liquid chromatography-mass spectrometry (nanoLC-MS) method for quantification of SHetA2 in patient-derived spheroids treated ex vivo with cell free-ascites. First, spheroids are washed and lysed. Second, an isotopically labeled compound d4-SHetA2 with known concentration is added to the lysate as the internal standard. Third, a C18 ZipTip is used to clean and desalt the sample to remove competing ions that could reduce detection sensitivity. Lastly, the prepared samples are analyzed using flow ultra-high-performance liquid chromatography (UHPLC) coupled with a high-resolution Thermo Orbitrap mass spectrometer. This integrated approach will provide highly sensitive and accurate quantification of SHetA2 at low concentrations, offering insights into its distribution, penetration, and activity within single patient-derived spheroids. Furthermore, by enabling a more precise and clinically relevant analysis of SHetA2’s efficacy in targeting spheroids, this approach will help establish robust criteria for dose selection and scheduling intervals in clinical applications. Annelise Huynh, Amit Singh, Zongkai Peng, Doris M. Benbrook, Anthony W. Burgett, Zhibo Yang. Quantification of SHetA2 in patient-derived ovarian cancer spheroids to study drug uptake and resistance in a physiologically relevant model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5756.

High grade serous ovarian and endometrial cancers possess TP53 missense mutations that result in loss of the central tumor suppressive functions of wild type p53 (WT p53). In normal unstressed cells, p53 protein is regulated through ubiquitin-tagging and shuttling to the 26S proteasome by the E3 ubiquitin ligase MDM2 (Murine Double Minute 2). In cancer cells with mutant forms of p53, MDM2 is not adequately expressed in such cells to perform its regulatory role, thus leading to mutant p53 aggregates. Understanding mechanisms to target mutant p53 proteins for degradation in cancers has tremendous therapeutic potential. Recent discoveries in p53 refolding agents to reinstate WT p53 functionality for the treatment of gynecologic malignancies have shown promise. Treatment of cancer cells harboring p53 mutations with p53 reactivators HO-3867 and our novel curcumin analogue AKT-100 displayed an upregulated expression of heat shock protein 70 (HSP70) family member genes, as well as the autophagy-inducing co-chaperone BAG3 (Bcl-2-associated athanogene 3) by RNA sequencing analysis. Western blot analysis showed a decrease in p53 protein expression after treatment with the curcumin analogues, while MDM2 levels were unchanged, indicating an alternative mechanism of mutant p53 degradation. We hypothesize that the BAG3-HSP70 interaction varies with different mutations in p53 as well as the various HSP70 family members involved and can be therapeutically normalized to restore WT p53 regulation. Using our cell models of high grade serous ovarian and endometrial cancer, we have determined through co-immunoprecipitation and mass spectrometry that p53 binds HSPA6 and BAG3 upon treatment only with AKT-100 in KLE (p53 R175H) and COV362 (p53 Y220C) cells. Furthermore, co-treatment of curcumin analogue AKT-100 plus HSP inhibitor SHetA2 (sulfur heteroarotinoid A2, NSC 726189) highly increased the expression of BAG3, HSPA1A, HSPA6 and ubiquitination while decreasing mutant p53 expression in KLE cells. Confocal microscopy in KLE cells also displayed a shift of mutant p53 and HSPA6 from nuclear to mostly cytoplasmic upon treatment with AKT-100. Co-treatment with AKT-100 and SHetA2 highly increased HSPA6 expression throughout the cell, while p53 returned to the nucleus to perform normal apoptotic functions based on CyQuant proliferation data. Our data indicates a novel mechanistic insight into therapeutically intervening to re-establish normal p53 levels and regulation to treat women with advanced, mutant p53 gynecologic malignancies. Geneva L. Williams, Jamie L. Padilla, Lane E. Smith, Jun-yong Choe, Doris M. Benbrook, Kimberly K. Leslie. Disruption of aggregated oncogenic mutant p53 as a novel therapeutic strategy for gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4327.

Background/ObjectivesPatients with ovarian cancer commonly experience metastases and recurrences, which contribute to high mortality. Our objective was to better understand ovarian cancer metastasis and identify candidate biomarkers and drug targets for predicting and preventing ovarian cancer recurrence.MethodsTranscripts of 770 cancer-associated genes were compared in cells collected from ascitic fluid versus resected tumors of an ES-2 orthotopic ovarian cancer mouse model. Associated cell types and pathways were explored with bioinformatics. FGF7 protein was measured using capillary-based immunoassays or ELISA in mouse and clinical specimens. Significances of differential gene expression and patient prognosis were determined by volcano plot and log-rank test, respectively.ResultsTumor transcriptomes exhibited higher endothelial cells, oxygenation, proteasome activity, and metabolism in comparison to ascites, but similar percentages of cancer-associated fibroblasts and immune cells. FGF7 mRNA was significantly higher in mouse tumors compared to ascites. FGF7 protein was significantly higher in tumors than in ascites in independent mouse models and clinical specimens. Serum FGF7 protein levels above the median of 25 patients with ovarian cancer were associated with worse progression-free and overall survival (p = 0.005 and 0.019, respectively) independent of patient and tumor characteristics.ConclusionsIn comparison to ascites, tumors exhibit different transcriptomic profiles that identify candidate biomarkers and drug targets for predicting and preventing recurrence. Among these, elevated tumoral FGF7 validated at the protein level and elevated serum FGF7 were significantly associated with worse patient survival. These results support further development of FGF7 receptor-targeted drugs and serum FGF7 to prevent and predict recurrence, respectively.

Although most advanced-stage ovarian cancers initially respond to platinum- and taxane-based chemotherapy, the majority of them will recur and eventually develop chemoresistance. Among all drug resistance mechanisms, reduced drug uptake in tumors is regarded as an important pathway acquired by drug-resistant cancer cells. For patients with ovarian cancer, chemoresistant cells can develop into multicellular spheroids and spread through ascite fluid that accumulates in their abdomen. These spheroids consist of 3D structures that are highly heterogeneous with different shapes, sizes, and compositions of cell types. Thus, studying drug uptake at the single spheroid level is important for understanding chemosensitivity and chemoresistance; however, drug-uptake studies in single spheroids have not been previously reported due to the lack of a suitable analytical technique. In this study, we cultured spheroids using the ovarian cancer cell line (OVCAR-8) and treated them using paclitaxel or OSW-1, a natural compound with anticancer properties. We then developed a method of quantifying drug uptake in single spheroids using LC/MS measurements and then normalized the drug amount in each spheroid to its size and total protein content. Our method can be used in translational studies of drug development, treatment, and prediction of drug efficacy prior to chemotherapy.

Increasing evidence is implicating roles for platelets in the development and progression of ovarian cancer, a highly lethal disease that can arise from the fallopian tubes, and has no current method of early detection or prevention. Thrombosis is a major cause of mortality of ovarian cancer patients suggesting that the cancer alters platelet behavior. The objective of this study was to develop a cell culture model of the pathological interactions of human platelets and ovarian cancer cells, using normal FT epithelial cells as a healthy control, and to test effects of the anti-platelet dihomo-gamma-linolenic acid (DGLA) in the model. Both healthy and cancer cells caused platelet aggregation, however platelets only affected spheroid formation by cancer cells and had no effect on healthy cell spheroid formation. When naturally-formed spheroids of epithelial cells were exposed to platelets in transwell inserts that did not allow direct interactions of the two cell types, platelets caused increased size of the spheroids formed by cancer cells, but not healthy cells. When cancer cell spheroids formed using magnetic nanoshuttle technology were put in direct physical contact with platelets, the platelets caused spheroid condensation. In ovarian cancer cells, DGLA promoted epithelial-to-mesenchymal (EMT) transition at doses as low as 100 μM, and inhibited metabolic viability and induced apoptosis at doses ≥150 μM. DGLA doses ≤150 μM used to avoid direct DGLA effects on cancer cells, had no effect on the pathological interactions of platelets and ovarian cancer cells in our models. These results demonstrate that the pathological interactions of platelets with ovarian cancer cells can be modeled in cell culture, and that DGLA has no effect on these interactions, suggesting that targeting platelets is a rational approach for reducing cancer aggressiveness and thrombosis risk in ovarian cancer patients, however DGLA is not an appropriate candidate for this strategy.

Abstract Introduction: Ovarian cancer (OvCa) is the most lethal gynecological malignancy. Although most OvCa patients initially respond to frontline platinum-based therapies with approximately 80% of patients experiencing remission, approximately 75% of those women experience a recurrence with only approximately 50% of patients alive 5 years following diagnosis. There is a critical need to develop novel therapeutic agents or methods to overcome chemoresistance and toxicities experienced with conventional chemotherapy regimens used for OvCa patients. Repurposing drugs is a promising strategy to safely and more rapidly evaluate potentially effective treatment regimens. Over the past two decades, Mebendazole (MBZ), an anti-parasitic drug, has gained much attention in oncology due to its favorable biosafety profile and potent anti-cancerous activity seen in several human malignancies. Therefore, we examined the efficiency and mechanism of MBZ against OvCa. Methods: The in vitro efficacy of MBZ was evaluated in cisplatin-resistant (CPR) and parent (WT/sensitive) OVCAR-8 cell lines as well as in ascites samples collected from chemo-resistant OvCa patients by using 2D and/or 3D cell viability assay and invasion assay. The in vivo efficacy of MBZ was assessed in orthotopic animal models, utilizing CPR-OVCAR8 spheroids, and patient-derived xenograft (PDX) animal models with specimens from chemo-resistant ovarian cancer patients. Molecular mechanisms were investigated through western blot and immunofluorescence analysis. Result: Our results confirmed that MBZ efficiently decreased cell viability, spheroid size, and the invasion ability of OvCa cell lines and ascites samples. OvCa cells treated with MBZ demonstrated downregulation of proliferation markers and upregulation of apoptosis markers indicating inhibition of cell proliferation and induction of apoptosis. Mechanistically, MBZ inhibited the Wnt/β-catenin signaling pathway by downregulating the expression and nuclear localization of β-catenin and epithelial-mesenchymal transition (EMT) markers. Preclinical studies showed that MBZ at 50 mg/kg/day dose for 28 days significantly reduced tumor growth in PDXs and orthotopic tumor models without any evidence of toxicity. Conclusion: Collectively, our study strongly supports the therapeutic potential of MBZ against OvCa particularly, which warrants further clinical studies. Citation Format: Rajani Rai, Dhanamjai Penta, Debasish Dey, Doris M Benbrook, Lin Wang, Magdalena Bieniasz, Lauren E Dockery. Efficacy and mechanism of mebendazole for ovarian cancer therapy [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 4671.

Background: Patients with ovarian cancer have high platelet counts, which correlate with disease burden, incidence, and lethality of blood clots (thrombosis). We hypothesized that elevated aggregation is associated with both increased platelet number and altered behavior of platelets in patients with ovarian cancer. Methods: Healthy controls and patients with suspected or diagnosed ovarian cancer were evaluated for complete blood counts. To evaluate the effects of platelet count versus platelet behavior, equal platelet-rich plasma (PRP) volumes versus equal platelet numbers were used in platelet aggregation assays. Arachidonic acid, adenosine diphosphate, and collagen platelet agonists were used to induce aggregation. Volunteers were grouped into healthy controls (23), benign/borderline cases (7), and cancer cases (25 ovarian, 1 colorectal, and 2 endometrial). Results: The rate and amount of platelet aggregation were higher in patients compared to healthy controls regardless of whether the same platelet number or PRP volume was used. Compared to healthy controls, patients with untreated ovarian cancer exhibited high levels of platelet activation markers, P-selectin (27.06 vs. 31.06 ng/mL, p = 0.03), and beta-thromboglobulin (3.073 vs. 4.091 µg/mL, p = 0.02) in their plasma. The significance of the elevation and its correlations with platelet number or PRP volume varied depending on the agonist. Platelet (305.88 vs. 134.12, p < 0.0001) and white blood cell (8.459 vs. 5.395, p < 0.01) counts (×109/L) were elevated pre-chemotherapy and decreased post-chemotherapy, respectively. Conclusions: Elevated platelet aggregation is caused by both altered platelet number and behavior in patients with ovarian cancer. These results support the study of antiplatelet agents for thrombosis prevention in these patients.

Background: Platelets have been shown to promote ovarian cancer; however, the mechanism is poorly understood. Previously, we demonstrated that platelets reduce the size and increase the density of multi-cellular ovarian cancer spheroids in cell cultures. The objectives of this study were to determine if platelet inhibitors could counteract these effects, and to explore the mechanisms involved. Methods: FDA-approved platelet inhibitors were screened for their abilities to alter platelet effects on ovarian cancer spheroids. Mass spectrometry was used to identify proteins significantly altered in cancer cells upon exposure to platelets. The effects of platelets and/or liver x receptor agonists or antagonists on LXR activity were measured using ES-2 ovarian cancer cells transduced with an LXR-reporter vector. Results: Eptifibatide, a GPIIB-IIIA integrin inhibitor, and dipyridamole, an adenosine reuptake inhibitor, reduced and enhanced platelet effects on ovarian cancer spheroids, respectively. Proteomic studies identified the LXR/RXR and integrin pathways as mediators of platelet effects on ovarian cancer, and downstream effectors of eptifibatide. Conclusions: Integrin pathways and their downstream LXR/RXR effectors are implicated in how platelets alter ovarian cancer spheroid morphology. These results support studying eptifibatide and LXR/RXR agonists as candidate drugs for repurposing as therapeutic strategies to counteract platelet promotion of ovarian cancer.

Cervical cancer is predominantly driven by persistent infections with high-risk human papillomavirus and the continuous activity of its E6 and E7 oncoproteins. This study explored the role of heat shock proteins 70 kDa (HSP70s) in enhancing the function of these oncoproteins and examined the impact of SHetA2, an investigational new drug, on this interaction. We found that HSP70 specifically binds to E7, but not E6, protein and that SHetA2 disrupts this binding. This disruption led to a significant reduction in E6 and E7 mRNA and E7 protein levels, while effects on E6 protein levels were minimal. SHetA2 treatment also resulted in altered levels of cell cycle regulatory proteins, reduced cell cycle progression, and decreased metabolic viability in cervical cancer cell lines and xenograft models. These findings support the potential of SHetA2 to impair cervical cancer progression by targeting HSP70/E7 interactions, highlighting its promise as a therapeutic strategy for treating cervical cancer.