Robert C. Bast

DIRAS3 Inhibits Ovarian Cancer Cell Growth by Blocking the Fibronectin-Mediated Integrin β1/FAK/AKT Signaling Pathway

Autophagy is a crucial cellular process responsible for sustaining homeostasis through the degradation and recycling of proteins and organelles, providing energy during amino acid starvation and hypoxia. In cancer, autophagy can either inhibit tumor growth or support cancer cell survival. Our previous studies have shown that re-expression of the tumor suppressor gene DIRAS3 inhibits growth of ovarian cancer cells, promotes autophagic cell death in vitro, and induces tumor dormancy in vivo. Growth factors and extracellular matrix (ECM) components can, however, inhibit DIRAS3-induced autophagic cell death. This study explores whether fibronectin (FN) can counteract the growth inhibition induced by DIRAS3 in ovarian cancer cells. FN was found to inhibit DIRAS3-induced autophagy and to partially rescue ovarian cancer cells from DIRAS3-induced cell death while reducing DIRAS3-induced inhibition of p-FAK and p-AKT. Inhibiting FAK with defactinib in ovarian cancer cells enhanced DIRAS3-induced autophagy and cell death. Re-expression of DIRAS3 and treatment with defactinib produced tumor regression in xenograft models. Our findings suggest that ECM components in the tumor microenvironment like FN enhance the activities of β1 integrin, FAK, and AKT to inhibit DIRAS3-induced autophagic cell death, thereby promoting ovarian cancer cell survival.

Crizotinib Enhances PARP Inhibitor Efficacy in Ovarian Cancer Cells and Xenograft Models by Inducing Autophagy

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) can encounter resistance through various mechanisms, limiting their effectiveness. Our recent research showed that PARPi alone can induce drug resistance by promoting autophagy. Moreover, our studies have revealed that anaplastic lymphoma kinase (ALK) plays a role in regulating the survival of ovarian cancer cells undergoing autophagy. Here, we explored whether the ALK-inhibitor crizotinib could enhance the efficacy of PARPi by targeting drug-induced autophagic ovarian cancer cell and xenograft models. Our investigation demonstrates that crizotinib enhances the anti-tumor activity of PARPi across multiple ovarian cancer cells. Combination therapy with crizotinib and olaparib reduced cell viability and clonogenic growth in two-olaparib resistant cell lines. More importantly, this effect was consistently observed in patient-derived organoids. Furthermore, combined treatment with crizotinib and olaparib led to tumor regression in human ovarian xenograft models. Mechanistically, the combination resulted in increased levels of reactive oxygen species (ROS), induced DNA damage, and decreased the phosphorylation of AKT, mTOR, and ULK-1, contributing to increased olaparib-induced autophagy and apoptosis. Notably, pharmacologic, or genetic inhibition or autophagy reduced the sensitivity of ovarian cancer cell lines to olaparib and crizotinib treatment, underscoring the role of autophagy in cell death. Blocking ROS mitigated olaparib/crizotinib-induced autophagy and cell death while restoring levels of phosphorylated AKT, mTOR and ULK-1. These findings suggest that crizotinib can improve the therapeutic efficacy of olaparib by enhancing autophagy. Implications: The combination of crizotinib and PARPi presents a promising strategy, that could provide a novel approach to enhance outcomes for patients with ovarian cancer.

Targeting progesterone signaling prevents metastatic ovarian cancer

SignificanceWhy women carrying a pathogenic germlineBRCA1mutation are predisposed to ovarian and breast cancer remains elusive. This study points to ovarian progesterone as a culprit. Generally,BRCA1-mutation carriers exhibit high yet individually varying levels of progesterone during the menstrual cycle. Although not allBRCA1-mutation carriers develop these cancers, all of them are advised to undergo prophylactic surgeries at a young age (under 40 y to 45 y) to prevent ovarian and breast cancer. Insights from robust in vivo findings in this study offer a novel concept: Targeting progesterone signaling with antiprogestins could be an effective nonsurgical prophylactic option for ovarian and breast cancer prevention for these high-risk women.

Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition

BackgroundPoor outcomes for patients with ovarian cancer relate to dormant, drug‐resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease.MethodsBy using unbiased small‐interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small‐molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP‐binding protein Di‐Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib‐induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short‐hairpin RNA knockdown of autophagic proteins, and small‐molecule inhibitors of STAT3 and BCL‐2.ResultsInduction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3‐expressing tumors treated with crizotinib had significantly decreased tumor burden and long‐term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy‐mediated apoptosis by decreasing phosphorylated STAT3 and BCL‐2 signaling.ConclusionsCrizotinib may eliminate dormant, autophagic, drug‐resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second‐look operations should be seriously considered.

Human epididymis protein 4 antigen‐autoantibody complexes complement cancer antigen 125 for detecting early‐stage ovarian cancer

BackgroundEarly detection of ovarian cancer could significantly improve patient outcomes. Cancer antigen 125 (CA 125) is elevated in sera from approximately 60% of patients with early‐stage (I/II) disease. Sensitivity might be improved through the combination of CA 125 with other biomarkers. Among potential biomarkers, antigen‐autoantibody (Ag‐AAb) complexes have received relatively little attention.MethodsLuminex‐based immunoassays were used to measure human epididymis protein 4 (HE4), anti‐HE4 autoantibody, and HE4 Ag‐AAb complexes in sera from patients with early‐ (n = 73) and late‐stage ovarian cancers (n = 49) at the time of diagnosis and from asymptomatic women with (n = 15) or without ovarian cancer (n = 212) enrolled in the Normal Risk Ovarian Cancer Screening Study.ResultsAt 98% specificity for healthy, asymptomatic women, 7% of patients with early‐stage (I/II) ovarian cancer and 4% of patients with late‐stage (III/IV) disease had elevated levels of HE4 autoantibody, whereas elevated levels of HE4 Ag‐AAb complexes were detected in sera from 38% of early‐stage cases and 31% of late‐stage cases. Complementarity was observed in receiver operating characteristic (ROC) curves between HE4 Ag‐AAb complexes and CA 125 levels in early‐stage ovarian cancer (P < .001). CA 125 detected 63% of cases, and a combination of CA 125 and HE4 Ag‐AAb complexes detected 81%. Complementarity was also observed in ROC curves for an independent validation set with 69 early‐stage patients (P = .039). HE4 Ag‐AAb complexes were detected in serial preclinical serum samples from women destined to develop ovarian cancer: they correlated with CA 125 but did not provide a lead time.ConclusionsHE4 Ag‐AAb complexes could complement CA 125 in detecting a higher fraction of early‐stage ovarian cancers.

Poly(adenosine diphosphate ribose) polymerase inhibitors induce autophagy‐mediated drug resistance in ovarian cancer cells, xenografts, and patient‐derived xenograft models

BackgroundPoly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors.MethodsInduction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient‐derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line.ResultsFour PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ‐H2AX. Inhibition of autophagy also increased ROS and γ‐H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy.ConclusionsPARP inhibitor–induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild‐type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer.

A Blood-Based Metabolite Panel for Distinguishing Ovarian Cancer from Benign Pelvic Masses

Abstract Purpose: To assess the contributions of circulating metabolites for improving upon the performance of the risk of ovarian malignancy algorithm (ROMA) for risk prediction of ovarian cancer among women with ovarian cysts. Experimental Design: Metabolomic profiling was performed on an initial set of sera from 101 serous and nonserous ovarian cancer cases and 134 individuals with benign pelvic masses (BPM). Using a deep learning model, a panel consisting of seven cancer-related metabolites [diacetylspermine, diacetylspermidine, N-(3-acetamidopropyl)pyrrolidin-2-one, N-acetylneuraminate, N-acetyl-mannosamine, N-acetyl-lactosamine, and hydroxyisobutyric acid] was developed for distinguishing early-stage ovarian cancer from BPM. The performance of the metabolite panel was evaluated in an independent set of sera from 118 ovarian cancer cases and 56 subjects with BPM. The contributions of the panel for improving upon the performance of ROMA were further assessed. Results: A 7-marker metabolite panel (7MetP) developed in the training set yielded an AUC of 0.86 [95% confidence interval (CI): 0.76–0.95] for early-stage ovarian cancer in the independent test set. The 7MetP+ROMA model had an AUC of 0.93 (95% CI: 0.84–0.98) for early-stage ovarian cancer in the test set, which was improved compared with ROMA alone [0.91 (95% CI: 0.84–0.98); likelihood ratio test P: 0.03]. In the entire specimen set, the combined 7MetP+ROMA model yielded a higher positive predictive value (0.68 vs. 0.52; one-sided P < 0.001) with improved specificity (0.89 vs. 0.78; one-sided P < 0.001) for early-stage ovarian cancer compared with ROMA alone. Conclusions: A blood-based metabolite panel was developed that demonstrates independent predictive ability and complements ROMA for distinguishing early-stage ovarian cancer from benign disease to better inform clinical decision making.

Autoantibodies, antigen-autoantibody complexes and antigens complement CA125 for early detection of ovarian cancer

Abstract Background Multiple antigens, autoantibodies (AAb), and antigen-autoantibody (Ag-AAb) complexes were compared for their ability to complement CA125 for early detection of ovarian cancer. Methods Twenty six biomarkers were measured in a single panel of sera from women with early stage (I-II) ovarian cancers (n = 64), late stage (III-IV) ovarian cancers (186), benign pelvic masses (200) and from healthy controls (502), and then split randomly (50:50) into a training set to identify the most promising classifier and a validation set to compare its performance to CA125 alone. Results Eight biomarkers detected ≥ 8% of early stage cases at 98% specificity. A four-biomarker panel including CA125, HE4, HE4 Ag-AAb and osteopontin detected 75% of early stage cancers in the validation set from among healthy controls compared to 62% with CA125 alone (p = 0.003) at 98% specificity. The same panel increased sensitivity for distinguishing early-stage ovarian cancers from benign pelvic masses by 25% (p = 0.0004) at 95% specificity. From 21 autoantibody candidates, 3 AAb (anti-p53, anti-CTAG1 and annt-Il-8) detected 22% of early stage ovarian cancers, potentially lengthening lead time prior to diagnosis. Conclusion A four biomarker panel achieved greater sensitivity at the same specificity for early detection of ovarian cancer than CA125 alone.

Normal Risk Ovarian Screening Study: 21-Year Update

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. PURPOSE The Normal Risk Ovarian Screening Study (NROSS) tested a two-stage screening strategy in postmenopausal women at conventional hereditary risk where significantly rising cancer antigen (CA)-125 prompted transvaginal sonography (TVS) and abnormal TVS prompted surgery to detect ovarian cancer. METHODS A total of 7,856 healthy postmenopausal women were screened annually for a total of 50,596 woman-years in a single-arm study (ClinicalTrials.gov identifier: NCT00539162 ). Serum CA125 was analyzed with the Risk of Ovarian Cancer Algorithm (ROCA) each year. If risk was unchanged and <1:2,000, women returned in a year. If risk increased above 1:500, TVS was undertaken immediately, and if risk was intermediate, CA125 was repeated in 3 months with a further increase in risk above 1:500 prompting referral for TVS. An average of 2% of participants were referred to TVS annually. RESULTS Thirty-four patients were referred for operations detecting 15 ovarian cancers and two borderline tumors with 12 in early stage (I-II). In addition, seven endometrial cancers were detected with six in stage I. As four ovarian cancers and two borderline tumors were diagnosed with a normal ROCA, the sensitivity for detecting ovarian and borderline cancer was 74% (17 of 23), and 70% of ROCA-detected cases (12 of 17) were in stage I-II. NROSS screening reduced late-stage (III-IV) disease by 34% compared with UKCTOCS controls and by 30% compared with US SEER values. The positive predictive value (PPV) was 50% (17 of 34) for detecting ovarian cancer and 74% (25 of 34) for any cancer, far exceeding the minimum acceptable study end point of 10% PPV. CONCLUSION While the NROSS trial was not powered to detect reduced mortality, the high specificity, PPV, and marked stage shift support further development of this strategy.