Ovarian Tumor-initiating Cells Research Articles

Abstract 4264: The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment

Abstract High grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy but a majority relapse with chemoresistant disease. Research supports the notion that tumor-initiating cells (TICs), a subpopulation of drug resistant stem-like tumor cells, are responsible for facilitating relapse and therapies targeting these fecund cells may prolong remission. The tumor microenvironment (TME) plays a crucial role in therapy resistance, and tumor relapse. Cytotoxic chemotherapies have been shown to modify the stroma of the TME which may favor the survival of TICs displaying specific adhesion proteins. We and others have shown that NF-kB transcription factors RelA and RelB respond to signals from the ovarian TME to promote proliferation, chemoresistance, and survival. Our results further show that RelA and RelB regulate expression of specific integrins. Recently we found that TNF-like inducer of apoptosis (TWEAK), a cytokine involved in tissue repair, and its only known receptor Fn14, are enriched following chemotherapy, leading to enhanced chemoresistance and survival of TICs. TWEAK also induces expression of specific integrins in ovarian TICs. Therefore, we hypothesize that NF-kB mediated integrin expression enables adhesion and survival of TICs to a chemotherapy modified extracellular matrix (ECM) to facilitate tumor repopulation and relapse. Using magnetic activated cell sorting (MACS) for CD117+ TICs, we found that a significant majority are enriched for integrin AVB3. Supporting a role for TWEAK-Fn14-NF-kB in mediating integrin expression we further show that integrin AVB3, but not AVB5 or A5B1, was significantly increased in CD117+ TICs in the presence of TWEAK and was dependent on Fn14 as demonstrated through knockdown experiments. RNA-sequencing analysis of tumor cells grown in spheroid conditions with RelA or RelB shRNA knockdown relative to control, show ITGAV is dependent on RelA while ITGB3 expression is dependent on RelB. Taken together, our findings suggest CD117+ TICs have enhanced expression of AVB3 and is further induced by TWEAK mediated NF-kB activity for adaptation to a stressful post-chemotherapy environment. Ongoing in vitro and in vivo studies are investigating the therapeutic benefit of targeting AVB3 or TWEAK in CD117+ TICs for prevention of adhesion and tumor repopulation following chemotherapy. These studies will uncover interactions between TICs and ECM through their unique expression of integrins and clarifies the role of NF-kB transcription factors RelA and RelB in regulating TIC survival in the post-chemotherapy TME. Citation Format: Omar Lujano Olazaba, Mikella Robinson, Cecilia Gallo, Mena Shammas, Katelyn Shelby, Luisjesus Cruz, Samuel F. Gilbert, Carrie D. House. The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment [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 4264.

Immunization with Embryonic Stem Cells/Induced Pluripotent Stem Cells Induces Effective Immunity against Ovarian Tumor-Initiating Cells in Mice.

Cancer stem cells (CSCs) express pluripotent markers and share many features with normal pluripotent stem cells. It is possible that immunity induced by embryonic stem cells (ESCs) and induced pluripotent stem cells- (IPSCs-) based vaccines may selectively target CSCs. In our study, cells expressing the pluripotent marker CD133 in the murine ovarian cancer cell-line ID8 were isolated and identified as CSCs. We investigated the preventive efficacy of ESCs and IPSCs-based vaccines against the development of ovarian cancer in vivo and evaluated the humoral and cellular immunities targeting CSCs in vitro. Our study showed that preimmunization with both mouse-derived embryonic stem cells (mESCs) and mouse-induced pluripotent stem cells (mIPSCs) lysates, combined with an immunostimulatory adjuvant CpG, elicited strong humoral and cellular responses. These responses effectively suppressed the development of CSC-derived tumors. Immune sera collected from mESCs and mIPSCs-vaccinated mice contained antibodies that were capable of selectively targeting CSCs, resulting in the lysis of CSCs in the presence of complement. Cytotoxic T-lymphocytes generated from splenocytes of mESCs and mIPSCs-vaccinated hosts could secrete interferon- (IFN-) γ in response to CSCs and kill CSCs in vitro. These findings indicate that vaccines based on mESCs and mIPSCs can elicit effective antitumor immunities. These immunities are related to the conferring of humoral and cellular responses that directly target CSCs.

Abstract 4960: Sox2, Oct4, and Nanog expression for identification of ovarian cancer tumor initiating cells

Abstract High Grade Serous Ovarian Cancer (HGSOC) is the most lethal gynecological disease in women, with over 70% of advanced stage disease relapsing within 18 months. Recent studies suggest that tumor-initiating cells (TICs) support tumor heterogeneity, chemoresistance, and relapse in HGSOC. Identification of TICs has traditionally relied on expression of surface markers such as CD133, CD44, and CD117 and/or enzymes such as aldehyde dehydrogenase (ALDH). Unfortunately, these markers are often cell type specific and not reproducible across patient samples. More reliable markers of TICs may include stem cell transcription factors such as Sox2, Oct4, and Nanog that function to support long-term self-renewal, multipotency, and quiescence, although these factors have not been comprehensively evaluated in ovarian cancer. We have previously shown that ovarian cancer cells cultured in 3D spheroid-supporting conditions have enhanced expression of Sox2, Oct4, and Nanog and are more tumorigenic than cells cultured in 2D as a monolayer. We hypothesize that Sox2, Oct4, and Nanog support spheroid formation, chemotherapy resistance, and tumorigenicity more reliably than traditional TIC markers. To investigate this hypothesis we evaluated five HGSOC cell lines (CAOV3/CAOV4/OV90/OVCAR4/OVCAR8), two undefined serous lines (ACI23, OVCAR5) and one non-serous line (SKOV3) in 2D vs 3D conditions and quantitated Sox2, Oct4, and Nanog expression, spheroid formation, chemotherapy resistance and tumorigenicity. Our data show that OVCAR4, OVCAR5, and OVCAR8 had the highest expression of Sox2, Oct4, and Nanog in 3D conditions and at least half of the cell lines had enriched expression of all three factors after chemotherapy treatment. A correlation analysis of the traditional TIC markers shows that the surface marker CD133 positively correlated with expression of Sox2, Oct4, and Nanog. Analysis of TCGA data shows that Sox2, Oct4, and Nanog are prone to amplification or gain of function events in 21%, 4%, and 7% of patients, respectively, and these patients are significantly more likely to have recurrent disease. Overall our findings suggest that Sox2, Oct4, and Nanog correlate with TIC features and may be more reliable markers for the identification of cells with enhanced tumor-initiation capacity and drug resistance. Our data further clarify specific ovarian cancer cell lines that have greater dependence on these factors. More reliable and consistent identification of ovarian TICs will improve our understanding of TIC biology and enable the design of therapies to overcome chemotherapy resistance and disease relapse. Citation Format: Mikella Robinson, Samuel F. Gilbert, Logan J. Alexander, Samuel E. Green, Omar Lujano-Olazaba, Omid Patrus, Jennifer A. Waters, Jacqueline M. Lara, Carrie D. House. Sox2, Oct4, and Nanog expression for identification of ovarian cancer tumor initiating cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4960.

Abstract A53: Energetic regulation of the Cyclin E oncoprotein in mitochondria-dependent ovarian tumor initiating cells

Abstract Study Purpose: Ovarian tumor-initiating (stem) cells (ovTICs) contribute to EOC chemoresistance. Thus, understanding their vulnerabilities may reveal novel therapeutic strategies. Overactivation of the cell cycle regulator Cyclin E is linked to chemoresistance of a subpopulation of epithelial ovarian carcinoma (EOC). However, role of Cyclin E in ovTIC maintenance is not clear. Energetics of TICs are rewired and Cyclin E function is uniquely linked to energetics and metabolism, which has not been tested in EOC. In nontumorigenic models, we reported that protein levels of Cyclin E (and certain stem cell markers) are elevated by boost in mitochondrial energetics caused by repression of the mitochondrial fission protein, Drp1. In EOC cells, Drp1 repression elevates the functional ovTIC marker Aldh, while active ovTICsAldh+ is dependent on mitochondrial energetics unlike the bulk tumor cells (BTCs Aldh-). Moreover, ovTICAldh+ potency is higher in cells with higher mitochondrial transmembrane potential. Therefore, we are investigating how mitochondrial energetics modulated by Drp1 regulates Cyclin E for ovTIC maintenance, which may reveal energetic vulnerabilities of ovTICs. Experimental Procedures: We sort 4 cell populations based on Aldh activity and mitochondrial membrane potential and then study mitochondrial and Cyclin E properties. Here, we use biochemical and cell biologic techniques, including our unique high-resolution microscopy-based techniques. We isolate mitochondria by biochemical cell fractionation. We manipulate genes by shRNA mediated knockdown. Unpublished Data: First, we confirmed the existence of the Aldh+ cell population with higher or lower mitochondrial membrane potential in mouse xenografts and patient EOC cells, and also confirmed a noncanonical mitochondrial pool of Cyclin E in EOC cells and xenografts as we reported for other cells. Interestingly, chemoresistance related ovTICAldh+ enrichment happens with increase in Cyclin E protein levels only when Drp1 is repressed to allow boost in mitochondrial energetics. Indeed, Cyclin E levels are higher in isolated quiescent ovTICsAldh+ than the ovBTCs Aldh-. More importantly, activation of TIC properties maintains the elevated Cyclin E levels in the more potent ovTICsAldh+ with higher mitochondrial potential, but not in the less potent ovTICsAldh+ with lower mitochondrial potential. This Cyclin E dependence on mitochondrial energetics in ovTICsAldh+ may be the underlying mechanism of the attenuation of chemoresistance with mitochondrial inhibitors, as we observed. Conclusions: We propose the novel concept that ovTICsAldh+ “primed” by Drp1 regulated mitochondrial energetics maintain elevated Cyclin E levels, which may involve mitochondrial translocation and release of Cyclin E. We speculate that inclusion of specific mitochondrial inhibitors in the chemotherapeutic regime may eliminate mitochondria dependent ovTICAldh+ that are maintained by mitochondria regulated Cyclin E. Citation Format: Kasturi Mitra, Brian Spurlock. Energetic regulation of the Cyclin E oncoprotein in mitochondria-dependent ovarian tumor initiating cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A53.

L1 Cell Adhesion Molecule Confers Radioresistance to Ovarian Cancer and Defines a New Cancer Stem Cell Population

Many solid tumors, including ovarian cancer, contain small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and play a role in disease recurrence. We demonstrated that the L1 cell adhesion molecule (L1CAM) is a new CSC target in ovarian cancer, triggering radioresistance. Using fluorescence-activated cell sorting, specific cell populations expressing L1CAM alone or in combination with the established CSC marker CD133 were isolated from three ovarian cancer cell lines. Double-positive L1CAM+/CD133+ cells displayed higher spherogenic and clonogenic properties in comparison to L1CAM−/CD133− cells. Furthermore, L1CAM+/CD133+ cells retained highest clonogenic capacity after irradiation and exhibited up-regulation of some CSC-specific genes, enhanced tumor-initiating capacity, self-renewal and higher tumor take rate in nude mice when compared with other cell populations. Superior radioresistance by L1CAM expression was confirmed by deletion of L1CAM using CRISPR-Cas9 technology. Moreover, we found expression signatures associated with epithelial-to-mesenchymal transition phenotype in L1CAM deleted cells. These results indicate that L1CAM in combination with CD133 defines a new cancer cell population of ovarian tumor-initiating cells with the implication of targeting L1CAM as a novel therapeutic approach for ovarian CSCs.

Abstract B66: miRNA 3’UTR activity driven enrichment of ovarian tumor-initiating cells (TICs) to overcome the barriers of heterogeneity and TIC plasticity

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Tumor recurrence is a major barrier towards improving patient outcome in EOC. Tumor-initiating cells (TICs) survive platinum-based therapies and contribute to tumor recurrence, at which stage the tumor is often difficult to eradicate, and thus TICs form the root cause of poor patient survival in EOC. Unfortunately, no TIC-targeting drugs to date are being evaluated in EOC patient trials. Isolating majority of TIC clones present in EOC tumors for functional analysis remains elusive to date and hence, understanding the complexity of TIC functioning remains a hurdle to be overcome in TIC therapeutics. TIC regulators can differ between tumors across patients and also in tumors within a patient. Tumor heterogeneity and TIC plasticity are the major challenges towards deciphering TIC functioning in EOC. Studying EOC cell populations with TIC properties is often limited to approaches driven by enrichment based on expression of stem-cell markers, which do not faithfully reflect TIC functioning in the context of heterogeneity and plasticity. Hence, pathway-based enrichment of TICs provide a reliable approach to functionally enrich for TIC subpopulations in EOC. miRNAs are small RNA molecules that regulate post-transcriptional gene silencing by binding mostly to the 3’UTR of their potential targets and targeting them for degradation through miRNA-induced silencing complexes (miRISCs). Isolation and characterization of TIC subpopulations in EOC based on miRNA activity can offer a reliable approach to functionally enrich for TICs in EOC. We have developed a novel miRNA-sensor based functional platform driven by miR-181a 3’UTR activity with mCHERRY fluorescence as the readout. miR-181a expression was upregulated in primary recurrent EOC tumors that were clinically resistant to platinum chemotherapy, suggesting a potential role for this miRNA in regulating TIC properties in EOC. Accordingly, miR-181a high subpopulation isolated from both cisplatin-sensitive and cisplatin-resistant/recurrent primary high-grade serous (HGSOC) ovarian tumor cells using miR-181a sensor were enriched in TIC properties in vitro and in vivo. Cisplatin treatment of primary EOC cells enriched miR-181a high subpopulation and reprogrammed miR-181a low non-TICs into miR-181a high TICs. Mechanistically, Wnt signaling was upregulated in miR-181a high ovarian tumor cells, showing that miRNA sensor platform can enrich TICs based on stem-cell pathway activity. Taken together, we provide the first evidence of TIC enrichment and analysis in EOC based on miRNA 3’UTR activity that provides with a valuable tool to broaden the understanding of TIC regulatory mechanisms in EOC in “real time,” with potential extension to other cancers by overcoming the barriers of tumor heterogeneity and TIC plasticity. miRNA sensor platform will provide a TIC pharmacologic screening tool to enable the discovery of novel miRNA-inhibiting TIC-targeting drugs in EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Matthew Knarr, Olga Kovalenko, Arshia Surti, Analisa DiFeo. miRNA 3’UTR activity driven enrichment of ovarian tumor-initiating cells (TICs) to overcome the barriers of heterogeneity and TIC plasticity. [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 B66.

Abstract 1910: NF-kappaB classical versus alternative pathways support distinct populations of ovarian cancer tumor-initiating cells

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions defined by low attachment flasks and serum free media. Preliminary data confirm that these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. These TIC-enriched culture conditions enhance NF-kappaB expression and activity. More specifically, alternative NF-kappaB signaling through the RelB transcription factor supports TIC populations by regulating aldehyde dehydrogenase (ALDH), an enzyme with high activity in TICs. Using an inducible shRNA targeting RelB we show that spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in both subcutaneous and intrabursal xenograft models, significantly decreased in the absence of RelB. Interestingly, loss of classical NF-kappaB signaling, through an shRNA targeting the RelA transcription factor, was less effective in targeting TICs, but more effective at targeting a proliferative subpopulation with high Ki67 staining. We conclude that classical and alternative NF-kappaB signaling support ovarian tumor cells with distinct phenotypes and the collaboration of these pathways may be critical for supporting tumor repopulation following chemotherapy. Current studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to each NF-kappaB pathway in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and further focus therapeutic strategies to prevent relapse. Citation Format: Carrie D. House, Christina M. Annunziata. NF-kappaB classical versus alternative pathways support distinct populations of ovarian cancer tumor-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2017-1910

Abstract AP13: NF-KAPPAB CLASSICAL VERSUS ALTERNATIVE PATHWAYS SUPPORT DISTINCT POPULATIONS OF OVARIAN CANCER TUMOR-INITIATING CELLS

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions defined by low attachment flasks and serum free media. Preliminary data confirm that these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. These TIC-enriched culture conditions enhance NF-kappaB expression and activity. More specifically, alternative NF-kappaB signaling through the RelB transcription factor supports TIC populations by regulating aldehyde dehydrogenase (ALDH), an enzyme with high activity in TICs. Using an inducible shRNA targeting RelB we show that spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in both subcutaneous and intrabursal xenograft models, significantly decreased in the absence of RelB. Interestingly, loss of classical NF-kappaB signaling, through an shRNA targeting the RelA transcription factor, was less effective in targeting TICs, but more effective at targeting a proliferative subpopulation with high Ki67 staining. We conclude that classical and alternative NF-kappaB signaling support ovarian tumor cells with distinct phenotypes and the collaboration of these pathways may be critical for supporting tumor repopulation following chemotherapy. Current studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to each NF-kappaB pathway in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and further focus therapeutic strategies to prevent relapse. Citation Format: Carrie D. House, Elizabeth A. Jordan, Christina M. Annunziata. NF-KAPPAB CLASSICAL VERSUS ALTERNATIVE PATHWAYS SUPPORT DISTINCT POPULATIONS OF OVARIAN CANCER TUMOR-INITIATING CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP13.

Abstract A72: Dissecting the role of NF-kappaB signaling in ovarian cancer tumor-initiating cells.

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions. Preliminary data show these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. Preliminary data show TIC-enriched culture conditions increased NF-kappaB expression and activity in TICs and stimulation of NF-kappaB signaling enhanced chemoresistance and stem cell marker expression. Current studies focus on dissecting the role of individual NF-kappaB proteins in TICs using inducible shRNAs to measure changes in spheroid formation, chemoresistance, tumorigenesis, and disease relapse following chemotherapy in a xenograft model. Future studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to NF-kappaB in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and may lead to improved therapeutic strategies. Citation Format: Carrie D. House, Christina M. Annunziata. Dissecting the role of NF-kappaB signaling in ovarian cancer tumor-initiating cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A72.

Abstract A64: β-catenin as a TIC therapeutic target in epithelial ovarian cancer.

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. The most common and lethal form of EOC which makes up greater than 75% of cases is high-grade serous cancer (HGSOC). Platinum-based drugs are used as first-line chemotherapeutics for all women diagnosed with HGSOC. Unfortunately, ~20% do not respond to this treatment and up to 80% of those patients which do respond recur within 5 years at which point the cancer is particularly difficult to eradicate. Emerging evidence supports the concept that platinum based therapies are able to eliminate the bulk of differentiated cancer cells but are unable to eliminate tumor-initiating cells (TICs) which are rare populations of tumor cells characterized by their ability to self-renew indefinitely and thus drive the expansion of drug-resistant tumors. Hence, there is immediate need of novel TIC targeted therapeutic approaches that can overcome chemo-resistance in EOC. Thus, identifying critical drivers of the stem-like phenotype that can be pharmacologically modulated in ovarian TICs is required. To date, no TIC targeting drugs have successfully completed clinical trials in EOC. Using primary HGSOC cells derived from HGSOC patient derived xenograft (PDX) models, we have recently identified β-catenin as a critical driver of HGSOC TIC phenotype in-vitro. β-catenin knockdown decreased stem-cell frequency and induced chemo-sensitivity in-vitro in platinum resistant HGSOC PDX derived primary cells. ALDH positive Ovarian TIC subpopulation with high Wnt reporter activity was platinum resistant and exhibited increased expression of β-catenin regulated stem-cell markers clearly identifying β-catenin regulated stem-cell transcriptional axis as a driver of chemo-resistance in Ovarian TICs. Accordingly, the β-catenin stem-cell transcriptional axis specific inhibitor iCG-001 (PRI-724) decreased stem-cell frequency and induced chemo-sensitivity in platinum resistant ovarian TICs, suggesting that pharmacologic inhibition of β-catenin could be a potential novel approach aimed at eradicating TICs in EOC. Furthermore, in-vivo tumor-initiation assays showed a robust decrease in tumor-initiation capacity upon β-catenin knockdown in platinum resistant HGSOC cells, thus establishing β-catenin as a driver of TIC phenotype in HGSOC. In sum, we have identified that pharmacologic inhibition of β-catenin can be a novel TIC targeted therapy approach to overcome chemo-resistance and improve patient outcome in in EOC. Our results suggest that the β-catenin specific inhibitor PRI-724, that has shown promising results in phase I clinical trials in solid tumors, can be a potential TIC targeting drug in EOC. These results will form a solid foundation for clinical trials with PRI-724 in patients with chemo-recurrent EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Olga Kovalenko, Sareena Singh, Amy Armstrong, Analisa DiFeo. β-catenin as a TIC therapeutic target in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A64.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions.

Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.

Abstract 5425: An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression

Abstract Triple-negative breast cancer (TNBC) and ovarian cancer comprise heterogeneous tumors, and neither targeted therapies nor traditional chemotherapies have provided consistent clinical benefit. Novel therapies that target and actively eradicate the subpopulation of tumor cells that mediate drug resistance and tumor relapse could significantly improve patient survival. Tumor-initiating cells (TIC) are functionally defined as the subpopulation of cells that drive long-term tumor growth, resistance to therapy and disease relapse. We herein identified CD324 as a surface antigen able to reproducibly enrich for TIC in well annotated, low passage TNBC and ovarian cancer patient-derived xenografts (PDXs). Gene expression analysis of TIC led to the identification of Ephrin-A4 as a prospective therapeutic TIC target. Humanized Ephrin-A4-specific monoclonal antibodies (mAbs) were generated and demonstrated to internalize to mediate the delivery of potent cytotoxins. An antibody-drug conjugate (ADC) comprising a humanized anti-Ephrin-A4 mAb conjugated to the DNA damaging agent calicheamicin achieved sustained tumor regressions in vivo in both TNBC and ovarian cancer PDX. Anti-Ephrin-A4-ADC (PF-06647263) actively reduced TIC frequency as evidenced by limiting dilution analysis in serial transplantation assays. Unexpectedly, TNBC tumors of the non-Claudin low molecular subtype exhibited higher Ephrin-A4 expression and more robust responses to the ADC than other breast cancer subtypes, which suggests a specific translational application for breast tumor subtype classification. Together these findings demonstrate the potential of the Ephrin-A4-targeted calicheamicin conjugate as a first-in-class compound designed to eradicate TIC and improve long-term survival of cancer patients. PF-06647263 is currently being evaluated in a Phase I clinical trial. Citation Format: Marc Damelin, Alex Bankovich, Albert Park, Jorge Aguilar, Wade Anderson, Marianne Santaguida, Sarah Fong, Monette Aujay, Kiran Khandke, Virginia Pulito, Elana Ernstoff, Paul Escarpe, Jeff Bernstein, Marybeth A. Pysz, Wenyan Zhong, Erik Upeslacis, Judy Lucas, Justin Lucas, Timothy Nichols, Kathryn Loving, Orit Foord, Johannes Hampl, Robert Stull, Frank Barletta, Hadi Falahatpisheh, Puja Sapra, Hans Peter Gerber, Scott J. Dylla. An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2015-5425

Abstract 1923: NF-κB signaling supports a sub-population of ovarian cancer tumor-initiating cells

Abstract Although most patients respond favorably to first-line chemotherapy, ovarian cancer remains the most lethal gynecological cancer in the United States primarily because of a high incidence of disease recurrence. The inability of traditional chemotherapy to eradicate tumor-initiating cells (TICs) is a plausible explanation for tumor relapse and we have begun to characterize TIC populations in ovarian cancer cell lines. Importantly, we are investigating the role of the NF-κB pathway in regulating this system. We have identified conditions that sustain a highly tumorigenic TIC population. Preliminary data suggest that NF-κB promotes and sustains TICs in ovarian cancer and current efforts are underway to characterize the molecular mechanisms underlying this preference. We will correlate activity of this pathway with expression of ovarian TIC protein markers such as CD133, Sox2, Nanog, and Oct-4 in TIC cell cultures and xenografts. Moreover we will use TICs to investigate effects of inhibiting this pathway on marker expression, proliferation, resistance to chemotherapy, and the ability to form tumors in mice. Clarifying the specific role of the NF-κB network in TIC populations will further our understanding of ovarian cancer initiation, progression, and relapse and may lead to improved therapeutic strategies for women in first remission, to prevent subsequent relapse to incurable disease. Citation Format: Carrie D. House, Christina M. Annunziata. NF-κB signaling supports a sub-population of ovarian cancer tumor-initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1923. doi:10.1158/1538-7445.AM2014-1923

Ovarian tumor-initiating cells display a flexible metabolism

An altered metabolism during ovarian cancer progression allows for increased macromolecular synthesis and unrestrained growth. However, the metabolic phenotype of cancer stem or tumor-initiating cells, small tumor cell populations that are able to recapitulate the original tumor, has not been well characterized. In the present study, we compared the metabolic phenotype of the stem cell enriched cell variant, MOSE-LFFLv (TIC), derived from mouse ovarian surface epithelial (MOSE) cells, to their parental (MOSE-L) and benign precursor (MOSE-E) cells. TICs exhibit a decrease in glucose and fatty acid oxidation with a concomitant increase in lactate secretion. In contrast to MOSE-L cells, TICs can increase their rate of glycolysis to overcome the inhibition of ATP synthase by oligomycin and can increase their oxygen consumption rate to maintain proton motive force when uncoupled, similar to the benign MOSE-E cells. TICs have an increased survival rate under limiting conditions as well as an increased survival rate when treated with AICAR, but exhibit a higher sensitivity to metformin than MOSE-E and MOSE-L cells. Together, our data show that TICs have a distinct metabolic profile that may render them flexible to adapt to the specific conditions of their microenvironment. By better understanding their metabolic phenotype and external environmental conditions that support their survival, treatment interventions can be designed to extend current therapy regimens to eradicate TICs.

Hypomethylation signature of tumor-initiating cells predicts poor prognosis of ovarian cancer patients

DNA methylation contributes to tumor formation, development and metastasis. Epigenetic dysregulation of stem cells is thought to predispose to malignant development. The clinical significance of DNA methylation in ovarian tumor-initiating cells (OTICs) remains unexplored. We analyzed the methylomic profiles of OTICs (CP70sps) and their derived progeny using a human methylation array. qRT-PCR, quantitative methylation-specific PCR (qMSP) and pyrosequencing were used to verify gene expression and DNA methylation in cancer cell lines. The methylation status of genes was validated quantitatively in cancer tissues and correlated with clinicopathological factors. ATG4A and HIST1H2BN were hypomethylated in OTICs. Methylation analysis of ATG4A and HIST1H2BN by qMSP in 168 tissue samples from patients with ovarian cancer showed that HIST1H2BN methylation was a significant and independent predictor of progression-free survival (PFS) and overall survival (OS). Multivariate Cox regression analysis showed that patients with a low level of HIST1H2BN methylation had poor PFS (hazard ratio (HR), 4.5; 95% confidence interval (CI), 1.4-14.8) and OS (HR, 4.3; 95% CI, 1.3-14.0). Hypomethylation of both ATG4A and HIST1H2BN predicted a poor PFS (HR, 1.8; 95% CI, 1.0-3.6; median, 21 months) and OS (HR, 1.7; 95% CI, 1.0-3.0; median, 40 months). In an independent cohort of ovarian tumors, hypomethylation predicted early disease recurrence (HR, 1.7; 95% CI, 1.1-2.5) and death (HR, 1.4; 95% CI, 1.0-1.9). The demonstration that expression of ATG4A in cells increased their stem properties provided an indication of its biological function. Hypomethylation of ATG4A and HIST1H2BN in OTICs predicts a poor prognosis for ovarian cancer patients.

Drug screening identifies growth inhibition of ovarian tumor-initiating cells by niclosamide

Objective: Recent studies underscore the need to target tumor progenitors that have been shown to be resistant to conventional chemotherapeutic agents. The present study was designed to discover novel compounds with activity against drug-resistant ovarian tumor-initiating cells (OTICs).

Growth Inhibition of Ovarian Tumor–Initiating Cells by Niclosamide

A recent hypothesis for cancer chemoresistance posits that cytotoxic survival of a subpopulation of tumor progenitors drives the propagation of recurrent disease, underscoring the need for new therapeutics that target such primitive cells. To discover such novel compounds active against drug-resistant ovarian cancer, we identified a subset of chemoresistant ovarian tumor cells fulfilling current definitions of cancer-initiating cells from cell lines and patient tumors using multiple stemness phenotypes, including the expression of stem cell markers, membrane dye efflux, sphere formation, potent tumorigenicity, and serial tumor propagation. We then subjected such stem-like ovarian tumor-initiating cells (OTIC) to high-throughput drug screening using more than 1,200 clinically approved drugs. Of 61 potential compounds preliminarily identified, more stringent assessments showed that the antihelmintic niclosamide selectively targets OTICs in vitro and in vivo. Gene expression arrays following OTIC treatment revealed niclosamide to disrupt multiple metabolic pathways affecting biogenetics, biogenesis, and redox regulation. These studies support niclosamide as a promising therapy for ovarian cancer and warrant further preclinical and clinical evaluation of this safe, clinically proven drug for the management of this devastating gynecologic malignancy.

C-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/β-catenin–ATP-binding cassette G2 signaling

Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.

Phenotypic heterogeneity and instability of human ovarian tumor-initiating cells

The cancer stem cell (CSC) model proposes that tumors have a hierarchical organization in which only some cells indefinitely self-renew and thereby sustain tumor growth. In addition, the CSC model requires that tumor-initiating cells (TICs) be prospectively isolatable on the basis of their phenotype. Previous studies have suggested that serous ovarian cancer (SOC) conforms to the CSC model, but these used arguably nonfidelitous immortalized cell lines, cultured primary cells, or passaged xenografts as the source of tumor cells. We developed a robust assay for quantifying TICs from primary SOC. Using this assay, we find that TICs are rare when assayed in either NOD/SCID or NOD/SCID/IL2Rγ(-/-) (NSG) mice. TIC frequency (TICf) varies substantially between patients, although it is similar in primary ovarian masses and omental metastases, suggesting that TICf is an intrinsic property of ovarian tumors. CD133 marks all TICs from several primary SOC cases. However, in other cases, substantial TIC activity is found in both the CD133(+) and CD133(-) fractions, whereas still other cases have exclusively CD133(-) TICs. Furthermore, the TIC phenotype can change in xenografts: primary tumors in which all TICs are CD133(+) can give rise to xenografts that contain substantial numbers of CD133(-) TICs. Our results highlight the need for quantitative rigor in the evaluation of TICs and for caution when using passaged xenografts for such studies. Furthermore, although our data suggest that SOC conforms to the CSC hypothesis, the heterogeneity of the TIC phenotype may complicate its clinical application.