High-grade Serous Ovarian Cancer Cell Research Articles

A Unique Pattern of Mesothelial-Mesenchymal Transition Induced in the Normal Peritoneal Mesothelium by High-Grade Serous Ovarian Cancer.

The study was designed to establish whether high aggressiveness of high-grade serous ovarian cancer cells (HGSOCs), which display rapid growth, advanced stage at diagnosis and the highest mortality among all epithelial ovarian cancer histotypes, may be linked with a specific pattern of mesothelial-mesenchymal transition (MMT) elicited by these cells in normal peritoneal mesothelial cells (PMCs). Experiments were performed on primary PMCs, stable and primary ovarian cancer cells, tumors from patients with ovarian cancer, and laboratory animals. Results of in vitro and in vivo tests showed that MMT triggered by HGSOCs (primary cells and OVCAR-3 line) is far more pronounced than the process evoked by cells representing less aggressive ovarian cancer histotypes (A2780, SKOV-3). Mechanistically, HGSOCs induce MMT via Smad 2/3, ILK, TGF-β1, HGF, and IGF-1, whereas A2780 and SKOV-3 cells via exclusively Smad 2/3 and HGF. The conditioned medium from PMCs undergoing MMT promoted the progression of cancer cells and the effects exerted by the cells triggered to undergo MMT by the HGSOCs were significantly stronger than those related to the activity of their less aggressive counterparts. Our findings indicate that MMT in PMCs provoked by HGSOCs is stronger, proceeds via different mechanisms and has more procancerous characteristics than MMT provoked by less aggressive cancer histotypes, which may at least partly explain high aggressiveness of HGSOCs.

Layer-by-layer nanoparticles for novel delivery of cisplatin and PARP inhibitors for platinum-based drug resistance therapy in ovarian cancer.

Advanced staged high‐grade serous ovarian cancer (HGSOC) is the leading cause of gynecological cancer death in the developed world, with 5‐year survival rates of only 25–30% due to late‐stage diagnosis and the shortcomings of platinum‐based therapies. A Phase I clinical trial of a combination of free cisplatin and poly(ADP‐ribose) polymerase inhibitors (PARPis) showed therapeutic benefit for HGSOC. In this study, we address the challenge of resistance to platinum‐based therapy by developing a targeted delivery approach. Novel electrostatic layer‐by‐layer (LbL) liposomal nanoparticles (NPs) with a terminal hyaluronic acid layer that facilitates CD44 receptor targeting are designed for selective targeting of HGSOC cells; the liposomes can be formulated to contain both cisplatin and the PARPi drug within the liposomal core and bilayer. The therapeutic effectiveness of LbL NP‐encapsulated cisplatin and PARPi alone and in combination was compared with the corresponding free drugs in luciferase and CD44‐expressing OVCAR8 orthotopic xenografts in female nude mice. The NPs exhibited prolonged blood circulation half‐life, mechanistic staged drug release and targeted codelivery of the therapeutic agents to HGSOC cells. Moreover, compared to the free drugs, the NPs resulted in significantly reduced tumor metastasis, extended survival, and moderated systemic toxicity.

A transcriptome-wide association study of high-grade serous epithelial ovarian cancer identifies new susceptibility genes and splice variants.

We sought to identify susceptibility genes for high-grade serous ovarian cancer (HGSOC) by performing a transcriptome-wide association study (TWAS) of gene expression and splice junction usage in HGSOC-relevant tissue types (N = 2,169) and the largest GWAS available for HGSOC (N = 13,037 cases/40,941 controls). We identified 25 TWAS significant genes, 7 at the junction level only, including LRRC46 at 19q21.32, (P = 1 × 10−9), CHMP4C at 8q21 (P = 2 × 10−11), and a PRC1 junction at 15q26 (P = 7 × 10−9). In vitro assays for CHMP4C showed the associated variant induces allele specific exon inclusion (P = 0.0024). Functional screens in HGSOC cell lines found evidence of essentiality for three of the novel genes we identified: HAUS6, KANSL1 and PRC1, with the latter comparable to CMYC. Our study implicated at least one target gene for 6/13 distinct GWAS regions, identifying 23 novel candidate susceptibility genes for HGSOC.

ALPL regulates the aggressive potential of high grade serous ovarian cancer cells via a non-canonical WNT pathway

The ALPL gene is linked to hypophosphatasia, a rare genetic disease. Owing to the inverse relationships between ALPL expression and both the International Federation of Gynecology and Obstetrics (FIGO) stages and histological grades assigned to patients with serous ovarian cancer (SOC), this study was designed to explore the role and possible mechanisms of ALPL in cell motility of high grade SOC (HGSOC). The effects of ALPL overexpression on migration and invasion were detected in HGSOC cell lines SKOV3 and HEY. Gene ontology analysis for differential genes with ALPL overexpression identified several biological processes, including EMT, correlated with cell motility. Genes potentially implicated in EMT and associated with ALPL were screened using The Cancer Genome Atlas (TCGA) database. The WNT receptor Frizzled2 (FZD2) was identified and its role in HGSOC cell motility and survival was investigated. It was found that forced expression of ALPL could inhibit migration, invasion, and EMT in HGSOC cells. It also reduced the expression of FZD2 and its ligand WNT5A, accompanied by suppressed expression of their downstream target phosphorylated-STAT3 (pSTAT3). These effects were initiated via the FZD2 knockdown using siRNA and reversed by recombinant WNT5A protein. The relationship between FZD2 expression and poor HGSOC patient survival was also investigated. This data supports that ALPL might restrict the function of WNT5A-FZD2-STAT3 axis, a non-canonical WNT pathway for promoting EMT progression, which results in attenuated migration and invasion in HGSOC cells and improves survival in patients with SOC.

Mobilization of Intracellular Calcium Stores and ER‐Mitochondrial Coupling in High‐grade Serous Ovarian Cancer (HGSOC) Cells

Ca2+ signaling plays crucial roles in cancer metastasis by participating in proliferation, migration, invasion and transformation. However, specific involvements of various intracellular Ca2+ stores in these processes are not clearly defined, and the characteristics of the Ca2+ release mechanisms have not been determined in high‐grade ovarian cancer (HGSOC) cells. In the present study, we sought to determine the functional properties of the IP3‐receptor (IP3R), ryanodine receptor (RyR), and nicotinic acid adenine dinucleotide phosphate (NAADP) gated Ca2+ release pathways in the HGSOC cell line OVCA433. Subcellular Ca2+ signals in OVCA433 cells were monitored using confocal fluorescence microscopy with the cytoplasmic Ca2+ fluorescent dye Fluo‐4 AM. Activation of IP3R by photorelease of cell‐permeant caged‐IP3 using a 405 laser elicited a dramatic increase in cytosolic [Ca2+] indicating robust IP3R‐gated Ca2+ stores. Activation of RyRs using the common agonist caffeine or 4‐chloro‐m‐cresol (4CmC) activated rapid Ca2+ release, indicating the presence of functional RyR‐gated Ca2+ stores in these non‐excitable cells. Moreover, application of the cell permeant NAADP‐AM also caused intracellular Ca2+ release. These results demonstrated the presence of functional IP3R‐, RyR‐, and NAADP‐gated Ca2+ stores in OVCA433 cells. Super‐resolution confocal live cell imaging were preformed in OVCA433 cells loaded with Bodipy‐FI‐X ryanodine, CellLight ER‐RFP, Lysotracker Deep Red, and/or Mitotracker Orange, to further examined the subcellullar organization of the intracellular Ca2+ stores. We found that RyRs are expressed in a subpopulation of ER occupying special locations in the central and peripheral regions, and the ERs in the central region are closely associated with mitochondria. The lysosomal acidic stores are distributed in a random manner. The close association of central ERs and mitochondria suggests that ER‐mitochondrial coupling may occur in OVCA433 cells. This possibility was confirmed by photorelease of caged‐IP3 in a small region inside the cells by an UV laser which elicited significant increase in [Ca2+]mito measured by Rhod‐2. Application of caffeine to OVCA433 cells also elicited a rapid increase in cytosolic [Ca2+], which was followed by sustained increase in [Ca2+]mito. Moreover, caffeine‐induced Ca2+ release activated superoxide bursts in mitochondria of OVCA433 cells loaded with the mitochondrial ROS indicator dye MitoSOX Red. Our results, hence, for the first time, characterized the IP3R‐, RyR‐, and NAADP‐mediated Ca2+ release, and demonstrated IP3R and RyR‐dependent ER‐mitochondrial Ca2+ transfer and coupling in OVCA433 cells. The results provide the fundamental information on intracellular Ca2+ signaling in HGSOC cells and could be important for the study of Ca2+ signaling in ovarian cancer metastasis.Support or Funding InformationSupported by DOD W81XWH‐17‐1‐0146This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clonealign: statistical integration of independent single-cell RNA and DNA sequencing data from human cancers

Measuring gene expression of tumor clones at single-cell resolution links functional consequences to somatic alterations. Without scalable methods to simultaneously assay DNA and RNA from the same single cell, parallel single-cell DNA and RNA measurements from independent cell populations must be mapped for genome-transcriptome association. We present clonealign, which assigns gene expression states to cancer clones using single-cell RNA and DNA sequencing independently sampled from a heterogeneous population. We apply clonealign to triple-negative breast cancer patient-derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either sequencing method alone.

Exposure of the extracellular matrix and colonization of the ovary in metastasis of fallopian-tube-derived cancer.

High-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube epithelium (FTE), but the role of the ovary in these tumors is unclear. Tumorigenic murine oviductal epithelial (MOE) cells allografted in the ovarian bursa resulted in aggressive tumors that spread throughout the peritoneum whereas intraperitoneal xenografting the same number of cells did not form tumors, indicating that colonization of the ovary may play a role in metastasis. Physical tearing of the ovarian surface to mimic rupture of the ovary during ovulation (independent of hormonal changes) resulted in more MOE and HGSOC cells adhering to the ovary compared with intact ovaries. More MOE cells also adhered to three-dimensional (3D) collagen and primary ovarian stromal cells than to ovarian surface epithelia, indicating that FTE cells adhered to the extracellular matrix exposed during ovulation. However, plating cells on 3D collagen reduced the viability of normal FTE but not cancer cells. Mutation of p53 (R273H or R248W) and activation of Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) (G12V) did not increase the viability of MOE cells on 3D collagen. In contrast, loss of phosphatase and tensin homolog (PTEN) allowed MOE cells to retain normal viability on 3D collagen. Loss of PTEN activated AKT and RAC1/c-jun N-terminal kinase signaling that each contributed to the increased viability, invasion and attachment in the collagen rich ovarian microenvironment. These results show that loss of PTEN activates multiple pathways that together enhance colonization of the ovary due to access to 3D collagen, which is a critical organ in the colonization of FTE-derived HGSOC.

Sprague Dawley Rag2-Null Rats Created from Engineered Spermatogonial Stem Cells Are Immunodeficient and Permissive to Human Xenografts.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2 tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non-small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481-9. ©2018 AACR.

158 - Mn porphyrin/ascorbate sensitizes serous epithelial ovarian cancer to chemotherapy

Ovarian cancer is the most lethal gynecologic malignancy. Even with aggressive frontline treatment, the majority of patients with advanced disease will develop a recurrence within the first 3 years and ultimately die of disease. Serous epithelial ovarian cancers [high-grade serous ovarian cancers(HGSC) and low-grade serous ovarian cancers(LGSC)] are especially challenging to treat due to primary and/or secondary chemoresistance that ultimately result in death. Oxidative stress plays important role in the carcinogenesis, cancer progression and metastases. Mn porphyrin(MnP)-based SOD mimics, when combined with ascorbate (Asc) and/or radiation (RT), largely suppress 4T1 mouse breast tumor growth through MnP-catalyzed H2O2/GSH-mediated oxidative modification of signaling proteins, with NF-kB/TAB3/p38MAPK/p38α(MAPK14)/HSP60 as a key S-glutathionylated network. Here we extended our studies of MnP/Asc tumor therapy to ovarian tumor. Genomic RNA microarray data from HGSC patients point to the key role of Bcl-2, which is transcriptionally regulated by NF-kB. Bcl-2 was downregulated by MnP/RT in our mouse glioma study. Bcl2 was overexpressed in HGSC specimens from women with shorter survival. We tested here MnTnBuOE-2-PyP5+(BMX-001) which is presently in 3 clinical trials as a normal tissue radioprotector. We used LGSC cell line HOC7 and HGSC cell line Caov2. Caov2 harbors a p53 mutation, characteristic of the genetic profile of HGSCs. Caov2 also has a high taxane- (paclitaxel and docetaxel) and moderate platinum resistance. Combination of BMX-001/Asc, with either chemotherapy (carboplatin or paclitaxel) resulted in a complete loss of HOC7 cell viability. 25% and 40% reduction in Coav2 cell viability was seen when BMX-001/Asc was combined with carboplatin or paclitaxel, respectively. BMX-001 was 1 µM and Asc 0.5 mM. Carboplatin and paclitaxel concentrations were 5.4 µM, and 1.8 nM. Our data point to the clinical potential of MnP/Asc system on suppression of ovarian tumor growth. Work was supported by Charles B. Hammond Grant, NC Biotechnology grant 2016-BIG-6518 and philanthropic funds.

Imaging Mass Spectrometry Reveals Crosstalk between the Fallopian Tube and the Ovary that Drives Primary Metastasis of Ovarian Cancer

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer deaths among women. New evidence suggests that HGSOC arises in the fallopian tube and then colonizes the ovary before spreading into the peritoneal space. Therefore, due to the proximity of this metastasis, an experimental design was optimized using imaging mass spectrometry to capture the spatial composition of small molecules uniquely expressed when fallopian-tube-derived tumor cells were grown in the microenvironment of the ovary as a model of primary metastasis. The observed mass-to-charge ratios (m/z’s) that were induced specifically in coculture represent small molecules that may contribute to the metastasis of HGSOC selectively to the ovary. Human fallopian tube epithelial HGSOC and tumorigenic murine oviductal epithelial cells, but not normal cell types, repeatedly induced a signal from the ovary at m/z 170. This signal was identified as norepinephrine, which was confirmed to stimulate invasion of ovarian cancer cells lacking wild-type p53. These molecules may reveal pathways that contribute to metastasis and biological targets for therapeutic intervention to block ovarian metastasis of fallopian-tube-derived HGSOC. The developed mass spectrometry method can be adapted to other mammalian-based model systems for investigation of untargeted metabolomics that facilitate metastasis.

Replication Protein A Availability during DNA Replication Stress Is a Major Determinant of Cisplatin Resistance in Ovarian Cancer Cells.

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561-73. ©2018 AACR.

SQ1274, a novel microtubule inhibitor, inhibits ovarian and uterine cancer cell growth

ObjectivePaclitaxel, a microtubule inhibitor, is subject to tumor resistance while treating high-grade serous ovarian and uterine cancer. This study aims to directly compare the effects of SQ1274, a novel microtubule inhibitor that binds to the colchicine-binding site on tubulin, and paclitaxel in high-grade serous ovarian and uterine cancer cell lines both in vitro and in vivo. MethodsWe assessed the sensitivity of ovarian (OVCAR8) and uterine (ARK1) cancer cell lines to SQ1274 and paclitaxel using XTT assays. We used western blot and quantitative real-time PCR to analyze changes in AXL RNA and protein expression by SQ1274 and paclitaxel. Differences in cell-cycle arrest and apoptosis were investigated using flow cytometry. Finally, we treated ovarian and uterine xenograft models with vehicle, paclitaxel, or SQ1274. ResultsFirst, we demonstrate that SQ1274 has a much lower IC50 than paclitaxel in both ARK1 (1.26 nM vs. 15.34 nM, respectively) and OVCAR8 (1.34 nM vs. 10.29 nM, respectively) cancer cell lines. Second, we show SQ1274 decreases both RNA and protein expression of AXL. Third, we show that SQ1274 causes increased cell-cycle arrest and apoptosis compared to paclitaxel. Finally, we report that SQ1274 more effectively inhibits tumor growth in vivo compared to paclitaxel. ConclusionsSQ1274 presents as a viable alternative to paclitaxel for treating ovarian and uterine cancer. This study supports the development of SQ1274 as a chemotherapeutic to treat ovarian and uterine cancer.

FTY720 enhances the anti-tumor activity of carboplatin and tamoxifen in a patient-derived xenograft model of ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related deaths among women in the United States. Although most patients respond to frontline therapy, virtually all patients relapse with chemoresistant disease. This study addresses the hypothesis that carboplatin or tamoxifen + FTY720, a sphingosine analogue, will minimize or circumvent drug-resistance in ovarian cancer cells and tumor models.In vitro data demonstrate that FTY720 sensitized two drug-resistant (A2780. cp20, HeyA8. MDR) and two high-grade serous ovarian cancer cell lines (COV362, CAOV3) to carboplatin, a standard of care for patients with ovarian cancer, and to the selective estrogen receptor modulator tamoxifen. FTY720 + tamoxifen was synergistic in vitro, and combinations of FTY720 + carboplatin or + tamoxifen were more effective than each single agent in a patient-derived xenograft model of ovarian carcinoma. FTY720 + tamoxifen arrested tumor growth. FTY720 + carboplatin induced tumor regressions, with tumor volumes reduced by ∼86% compared to initial tumor volumes. Anti-tumor efficacy was concomitant with increases in intracellular proapoptotic lipid ceramide. The data suggest that FTY720 + tamoxifen or carboplatin may be effective in treating ovarian tumors.

LY2157299 Monohydrate, a TGF-βR1 Inhibitor, Suppresses Tumor Growth and Ascites Development in Ovarian Cancer.

Transforming growth factor beta (TGF-β) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-β receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-β1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-β1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-β signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-β signaling-directed therapy in ovarian cancer.

Abstract B36: FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer

Abstract Purpose: FOXM1 is located at 12p13.33, which shows copy number gains in the majority of high-grade serous ovarian cancer (HGSC) cases. The 12p13.33 amplicon contains 33 genes, some of which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 overexpression is associated with genomic instability in cancer, whether FOXM1 contributes directly to this phenotype is unclear. We hypothesized that FOXM1 induces DNA replication stress (DRS), resulting in ATR-CHK1 activation. Intriguingly, we noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 activation, is contained within the 12p13.33 amplicon and is arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1/RHNO1 bidirectional promoter, FOXM1/RHNO1 expression, and determined the role of FOXM1/RHNO1 in DRS, the DRS response, and clonogenic growth of HGSC cells. Methods: We used reporter assays to characterize the FOXM1/RHNO1 bidirectional promoter and FOXM1 transcriptional activity. We analyzed copy number, mRNA, and protein expression datasets in TCGA, CCLE, and GTEX. We measured FOXM1/RHNO1 expression in HGSC cell lines and tumors. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to DRS and DNA damage. We knocked down RHNO1 in HGSC cells to determine its contribution to the DRS response and clonogenic growth. We performed Western blot analyses to measure markers of DRS and DNA damage. We characterized FOXM1-induced DNA damage at single-cell resolution using Comet assays and flow cytometry measurement of γ-H2AX. Results: FOXM1/RHNO1 showed copy number gains or amplifications in 58% of TCGA HGSC tumors, and FOXM1/RHNO1 mRNA expression significantly correlated in TCGA HGSC tumors, CCLE HGSC lines, and GTEX normal tissues. The FOXM1/RHNO1 bidirectional promoter showed balanced activity in each direction in FTE and HGSC cells, and bidirectional promoter activity correlated with endogenous mRNA expression. TCGA HGSC data indicated that FOXM1 expression associates with markers of DNA replication, global copy number alterations, and CHK1-Ser345-P, a canonical marker of DRS. In agreement, FOXM1 overexpression in FTE cells induced phosphorylation of ATR-Thr1989, CHK1-Ser317/345, H2AX-Ser139, and the proportion of DNA in Comet tails. In contrast, a DNA-binding domain mutant of FOXM1 did not induce DRS or DNA damage. RHNO1 knockdown attenuated CHK1-Ser345-P in response to DRS and RHNO1 shRNA knockdown or CRISPR-Cas9 knockout severely reduced HGSC cell clonogenic growth. Importantly, the effects of RHNO1 disruption were rescued by WT RHNO1 expression, but not by a RHNO1 mutant defective for interaction with the 9-1-1 complex. Conclusion: Our data reveal that FOXM1/RHNO1 share a bidirectional promoter and that this may account for their frequent coexpression in HGSC. FOXM1 induces DRS, and this is dependent on its ability to bind DNA. Conversely, RHNO1 contributes to ATR-CHK1 signaling and promotes the clonogenic growth of HGSC cells. We hypothesize that balanced FOXM1/RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1/RHNO1 on DRS and genomic instability in model systems. Finally, our data suggest that the ATR-CHK1 pathway is a synthetic lethal vulnerability in HGSC tumors that overexpress FOXM1. Citation Format: Carter J. Barger, Linda Chee, Connor Branick, Kunle Odunsi, Ronny Drapkin, Adam R. Karpf. FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B36.

Abstract B53: Estrogen receptor beta 2 (ERβ2)- p53- FOXM1 signaling axis in high-grade serous ovarian cancer (HGSOC): Underlying mechanisms and implications for resistance to therapy

Abstract Background and Goals: HGSOC is the most aggressive ovarian carcinoma and accounts for two-thirds of deaths from ovarian cancer. The Cancer Genome Atlas (TCGA) analysis showed that p53 is mutated in about 96% of HGSOC and forkhead box M1 (FOXM1) protein signaling network is altered in 87% of cases. These abnormalities significantly contribute to resistance of tumors to chemotherapy. Recent studies have also shown that estrogen receptor beta (ERβ) is expressed at high levels in HGSOC; however, there is no consensus weather ERβ is pro- or anti-tumorigenic. Due to the established roles of FOXM1 in chemotherapy resistance and our observation that ERβ2 regulates FOXM1 expression, we hypothesized that ERβ2 status may be of clinical relevance for the treatment of HGSOC. The objective of the current study was to delineate the mechanisms by which ERβ2-mutant p53-FOXM1 signaling axis impacts resistance to carboplatin therapy. Experimental Procedures: Proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were used to analyze protein-protein interactions. Transcription factor binding to endogenous gene promoters was determined by chromatin immunoprecipitation (Ch-IP) assay. RNAi technology was used to downregulate expression of specific proteins. Endogenous gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Apoptosis was analyzed by Annexin V staining and PARP-cleavage assays. Cell proliferation was measured by colony formation assay. Expression levels of ERβ, p53, and FOXM1 in HGSOC patient tumor tissue microarrays (TMAs) were determined by immunohistochemistry (IHC). Unpublished Results: Co-IP showed that ERβ2 binds to mutant p53. PLA demonstrated that ERβ2 is in a protein complex with mutant p53 in situ and qRT-PCR showed that ERβ2 in concert with mutant p53 upregulates FOXM1 gene transcription. Effect of ERβ2 on FOXM1 transcription was abrogated in cells that are p53-null. ChIP assay showed ERβ2 binding to FOXM1 gene promotor and regulating its gene expression. Concomitantly, expression of downstream targets of FOXM1 that are responsible for cell proliferation and DNA damage repair was upregulated. Furthermore, ERβ and p53 regulate expression of each other in a feedback loop. Knocking down ERβ2 in combination with carboplatin resulted in increased apoptosis and deceased proliferation in HGSOC cells. Importantly, APR-246 (PRIMA-1MET) (a small molecule that binds and converts mutant p53 to wild-type p53 conformation) in combination with ERβ2 knockdown led to decreased FOXM1 protein levels and increased apoptosis. Consistent with our data from cell model studies, IHC data on patient tumor TMAs showed that ERβ2 expression has linear correlation with FOXM1 expression in the primary and metastatic tumors. Analysis of correlation of these data with clinical characteristics such as therapeutic response and patient survival is under way. Conclusions: Our experiments have uncovered a novel (ERβ2)-p53-FOXM1 signaling axis in driving HGSOC and contributing to resistance to therapy. The data suggest that this signaling axis could be a potential therapeutic target. Citation Format: Chetan Oturkar, Austin Miller, Emese Zsiros, Gokul M. Das. Estrogen receptor beta 2 (ERβ2)- p53- FOXM1 signaling axis in high-grade serous ovarian cancer (HGSOC): Underlying mechanisms and implications for resistance to therapy. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B53.

Abstract A26: PTEN and colonization of the ovary in metastasis of fallopian tube-derived ovarian cancer

Abstract It is now clear that high-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube epithelium (FTE), making the role of the ovary in these tumors unclear. Therefore, the objectives of this research were to 1) determine if colonization of the ovary was an important step in the spread of FTE-derived tumors to the peritoneum and 2) explore the role of ovarian rupture, independent of hormonal changes, in tumor cell colonization of the ovary. First, 50,000 murine oviductal epithelial (MOE) cells stably expressing PTENshRNA+KRASG12V were allografted into the left ovarian bursa (IB) or peritoneal space (PS). IB-grafted mice developed tumors, with all mice requiring sacrifice within 90 days. Tumors were detected throughout the peritoneum and in the contralateral ovary. In contrast, PS-grafted mice failed to develop tumors for the duration of the experiment (150 days). To determine if rupture of the ovary was important in colonization of the ovary, an ex vivo attachment assay was developed in which ovaries was left intact or cut with a scalpel to rupture the ovarian surface (ovulation mimetic) without associated hormonal changes. Ovulation mimetic ovaries had significantly more MOE GFP and OVCAR8 RFP cells attached, particularly in the wounded areas. To confirm that increased attachment was due to exposure of the ovarian stroma, MOE GFP cells were plated on monolayers of murine ovarian surface epithelium (MOSE), monolayers of murine ovarian stroma (MOST), or three-dimensional (3D) type I collagen gels (the primary extracellular matrix protein in the ovarian stroma). MOE GFP cells attached to MOST more than MOSE and to 3D collagen than MOST, supporting the contention that FTE cells adhere to the extracellular matrix (ECM), such as collagen, exposed during ovulation. We next sought to test the ability of cells to survive in an environment rich in 3D collagen. 3D collagen reduced the viability of normal epithelial cells (MOSE, MOE, and IOSE80) but had no effect on HGSOC cells (OVCAR3, OVCAR4, OVCAR5, OVAR8, and OVSAHO), indicating that transformation of FTE is important for survival in the collagen-rich ovarian stroma. RNA-seq showed that 3D collagen decreases genes associated with focal adhesion, regulation of actin cytoskeleton, and ECM-receptor interaction, supporting the idea that normal epithelial cells respond poorly to a microenvironment rich in 3D collagen. In contrast, genes associated with p53 signaling and suppression of MAPK/AKT were increased when cells were grown on 3D collagen. However, plating cells on 3D collagen did not lead to stabilization of p53 and expression of mutant p53 had no effect on viability of MOE cells on 3D collagen. KRASG12V tended to increase viability, but only PTENshRNA completely rescued viability on 3D collagen as compared to 2D. PTENshRNA had no effect on adhesion to 3D collagen, but it did increase invasion through collagen. Cells expressing PTENshRNA showed increased attachment to 3D ovaries that were wounded to mimic ovulation; however, the AKT inhibitor MK2206 did not reduce this adhesion. Interestingly, after 7 days of culture on 3D collagen, MOE PTENshRNA cells formed spheroids while controls did not, leading to the hypothesis that spheroids may mediate metastasis from the fallopian tube to the ovary. In support of this contention, spheroids were observed in the fallopian tube of PAX8+/cre PTENflox/flox mice, which develop fallopian tube-derived ovarian cancer. Furthermore, OVCAR8 RFP spheroids preferentially attached to wounded sites mimicking ovulation relative to intact ovaries. In conclusion, FTE cells attached to exposed ECM of the ovary and colonization of the ovary enhances further spread of FTE-derived tumors beyond the reproductive tract. Inhibition of ovarian colonization may be a new approach to inhibit peritoneal spread of cancer in women at high risk of developing HGSOC. Citation Format: Matthew Dean, Vivian Jin, Angela Russo, Joanna E. Burdette. PTEN and colonization of the ovary in metastasis of fallopian tube-derived ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A26.

Abstract B07: Targeting DAB2IP in ovarian cancer stem cells

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancies. Failure to eliminate ovarian cancer stem cells (OCSCs) by the end of conventional chemotherapy contributes to tumor relapse and metastasis. Understanding the molecular features of OCSCs may allow for effective targeting and eradicating these cells and tumor remission. We reported tumor-initiation and expansion capacity by ALDH+ OCSCs after platinum-based treatment of ovarian cancer xenografts. In addition, loss of DAB2IP in prostate and colon cancer promoted CSC-like features, suggesting a critical role in modulating CSC maintenance. DAB2IP, a novel member of Ras GTPase-activating (GAP) protein family, was identified as a potent tumor suppressor that interfered with many aspects of cancer progression and was frequently downregulated in tumors. In the current study, we aimed to elucidate the tumor-suppressor role of DAB2IP in ovarian cancer in the context of OCSCs and explore epigenetic approaches to upregulate its expression. OCSCs and non-CSCs subpopulations were isolated from multiple high-grade serous ovarian cancer cell lines (Kuramochi, OVCAR3, and COV362). Reduced (p<0.05) DAB2IP expression in OCSCs compared to non-CSC counterparts was observed, suggesting that DAB2IP suppresses OCSC maintenance. Overexpression of DAB2IP in OCSCs derived from Kuramochi decreased (p<0.05) OCSC population, and DAB2IP overexpression inhibited stemness-related phenotypes, including reduced (P<0.05) spheroid formation ability after 14-day incubation under stem cell conditions and decreased (P<0.05) colony formation. Decreased expression of stem cell markers (Oct4, Nanog, and Twist) in DAB2IP-overexpressing cells was also observed, indicating potential key effectors downstream of DAB2IP. Furthermore, elevated DAB2IP expression in OCSCs decreased (p<0.05) cisplatin IC50 and cell migration capacity in transwell assay, suggesting DAB2IP plays a role in OCSC function. Aberrant DNA promoter methylation and histone modifications were previously shown to be two major mechanisms of DAB2IP inactivation in cancer. We treated OCSCs with next-generation DNMT inhibitor guadecitabine and EZH2 inhibitor GSK-126, either alone or in combination. Single-agent guadecitabine or GSK-126 decreased ALDH expression with greater (p<0.05) effect in combination. Single GSK-126 was sufficient to increase DAB2IP expression (p<0.05) with additional increase when combined with guadecitabine, correlating with decreased ALDH expression. Collectively, our data suggested a possible combination epigenetic therapy to eliminate OCSCs, potentially through targeting DAB2IP, an important OCSC regulator. Citation Format: Xingyue Zong, Ali R. Ozes, Kenneth P. Nephew. Targeting DAB2IP in ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B07.

Abstract B64: The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer

Abstract The role and regulation of ZIP4, a zinc (Zn) transporter, have been studied in the context of extracellular Zn transporting. Factors other than Zn with ZIP4 regulatory effects are essentially unknown. In addition, its role in cancer stem cell (CSC) has not been reported in any type of cancer. RNA-seq analyses were conducted to compare genes differentially expressed in less and more aggressive epithelial ovarian cancer (EOC) cells. ZIP4 expression and its regulation was assessed by immunoblotting, quantitative PCR, or immunofluorescence staining in human ovarian tissues. CSC-related activities were examined to evaluate the role of ZIP4 in human high-grade serous ovarian cancer (HGSOC) cells. RNAi and CRISPR techniques were used to knock down or knock out ZIP4 and related genes. EOC tumors overexpressed ZIP4 when compared with normal and benign tissues. ZIP4 knockout significantly reduced several CSC-related activities in EOC cells, including proliferation, anoikis resistance, colony formation, spheroid formation, drug resistance, and side-population (SP). Only ZIP4-expressing SP highly expressed known CSC markers ALDH1 and OCT4. ZIP4 knockout dramatically reduced tumorigenesis and ZIP4 overexpression increased tumorigenesis in vivo. In addition, the ZIP4-expressing SP population had the tumor initiating activity. Moreover, the oncolipid lysophosphatic acid (LPA) effectively upregulated ZIP4 expression via the nuclear receptor peroxisome proliferator-activated receptor gamma, and LPA’s promoting effects in CSC-related activities in HGSOC cells was at least partially mediated by ZIP4 in an extracellular Zn-independent manner. Our critical data imply that ZIP4 is a new and important cancer stem cell regulator in EOC. Our data also provide an innovative interpretation for the apparent disconnection between low levels of zinc and upregulation of ZIP4 in EOC tissues. Citation Format: Yan Xu, Qipeng Fan, Qingchun Cai, Kenneth P. Nephew. The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B64.

Abstract A57: Drug sensitivity and resistance testing (DSRT) of clinically important compounds on primary ovarian cancer cell lines

Abstract The heterogeneity of chemoresponses in high-grade serous ovarian cancer (HGS-OvCa) presents a major clinical challenge. The inherited or acquired nonresponsiveness to current therapies is likely to be one reason for relapse and treatment failure. Better models for predicting effective treatment options and drug combinations are needed. Although most tumors initially respond to standard platinum-taxane combination therapy, treatment resistance eventually evolves in 80-90% of the patients. Drug sensitivity and resistance testing (DSRT) was performed by using high-throughput screening (HTS) of 306 small-drug molecules on six primary cell lines from patients with disseminated HGS-OvCa. Commonly available ovarian cancer cell line OVCAR8 was used as a control. The drug effect was investigated with help of a quantitative scoring approach, drug sensitivity score (DSS). Sixteen drugs of clinical interest were investigated in more detail. Drug sensitivity varied greatly within primary cell lines. Out of 306 compounds, 29 were effective on the most resistant cell line, whereas 102 compounds showed effect on the most sensitive line. We found that 9 out of the 16 clinically important compounds gave no response on the tested cell lines. Seven compounds provided response on the screened cell lines: four HSP90 inhibitors, an HSP70 inhibitor, a Wee1 inhibitor, and a proteasome inhibitor. Of these seven compounds, the most effective drug compounds (the Wee1 inhibitor and two HSP90 inhibitors) were chosen for validation on primary HGS-OvCa cell lines to verify the HTS result. The effectiveness of currently used clinical treatment-line drugs, cisplatin and paclitaxel, was compared to the putative novel drug compounds on the primary cell lines and the control cell line. One primary cell line was completely resistant to the validated drug compounds as well as to cisplatin and paclitaxel combination treatment. Five cell lines were sensitive to cisplatin-paclitaxel treatment, but the sensitivity to the three most effective compounds varied greatly. The commonly available cell line OVCAR8 was the most sensitive of all tested cell lines. In conclusion, we found three potential drug compounds of clinical interest effective for disseminated HGS-OvCa. The analyses demonstrate that patient-derived primary HGS-OvCa cell lines have unique heterogeneous characteristics that are likely to have an effect on the choice of best drug candidates for each individual cell line, and furthermore for each patient. Consequently, more combinatorial drug treatment strategies should be validated to find new specific personalized drug treatment strategies. Citation Format: Pia Roering, Piia Mikkonen, Swapnil Potdar, Krister Wennerberg, Johanna Hynninen, Seija Grénman, Annika Auranen, Olli Carpén, Katja Kaipio. Drug sensitivity and resistance testing (DSRT) of clinically important compounds on primary ovarian cancer cell lines. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A57.