Ovarian Cancer Stem Research Articles

STAU1-mediated CNBP mRNA degradation by LINC00665 alters stem cell characteristics in ovarian cancer

BackgroundTo investigate the role of lncRNA LINC00665 in modulating ovarian cancer stemness and its influence on treatment resistance and cancer development.MethodsWe isolated ovarian cancer stem cells (OCSCs) from the COC1 cell line using a combination of chemotherapeutic agents and growth factors, and verified their stemness through western blotting and immunofluorescence for stem cell markers. Employing bioinformatics, we identified lncRNAs associated with ovarian cancer, with a focus on LINC00665 and its interaction with the CNBP mRNA. In situ hybridization, immunohistochemistry, and qPCR were utilized to examine their expression and localization, alongside functional assays to determine the effects of LINC00665 on CNBP.ResultsLINC00665 employs its Alu elements to interact with the 3’-UTR of CNBP mRNA, targeting it for degradation. This molecular crosstalk enhances stemness by promoting the STAU1-mediated decay of CNBP mRNA, thereby modulating the Wnt and Notch signaling cascades that are pivotal for maintaining CSC characteristics and driving tumor progression. These mechanistic insights were corroborated by a series of in vitro assays and validated in vivo using tumor xenograft models. Furthermore, we established a positive correlation between elevated CNBP levels and increased disease-free survival in patients with ovarian cancer, underscoring the prognostic value of CNBP in this context.ConclusionslncRNA LINC00665 enhances stemness in ovarian cancer by mediating the degradation of CNBP mRNA, thereby identifying LINC00665 as a potential therapeutic target to counteract drug resistance and tumor recurrence associated with CSCs.

Unveiling stem-like traits and chemoresistance mechanisms in ovarian cancer cells through the TGFβ1-PITX2A/B signaling axis.

Ovarian cancer (OC) is the deadliest gynecological malignancy, having a high mortality rate due to its asymptomatic nature, chemoresistance, and recurrence. However, the proper mechanistic knowledge behind these phenomena is still inadequate. Cancer recurrence is commonly observed due to cancer stem cells which also show chemoresistance. We aimed to decipher the molecular mechanism behind chemoresistance and stemness in OC. Earlier studies suggested that PITX2, a homeobox transcription factor and, its different isoforms are associated with OC progression upon regulating different signaling pathways. Moreover, they regulate the expression of drug efflux transporters in kidney and colon cancer, rendering chemoresistance properties in the tumor cell. Considering these backgrounds, we decided to look for the role of PITX2 isoforms in promoting stemness and chemoresistance in OC cells. In this study, PITX2A/B has been shown to promote stemness and to enhance the transcription of ABCB1. PITX2 has been discovered to augment ABCB1 gene expression by directly binding to its promoter. To further investigate the regulatory mechanism of PITX2 gene expression, we found that TGFβ signaling could augment the PITX2A/B expression through both SMAD and non-SMAD signaling pathways. Collectively, we conclude that TGFβ1-activated PITX2A/B induces stem-like features and chemoresistance properties in the OC cells.

Abstract A098: Microenvironment-regulated matrix Gla protein drives ovarian cancer stemness

Abstract The clinical evolution of ovarian cancer (OC), with peritoneal dissemination, tumor relapse and chemoresistance, seems to be accounted for by a small population of ovarian cancer stem cells (OCSC), thus making them an attractive target for more efficacious treatments. However, this could only be achieved through the detailed characterization of the molecular and functional traits of this elusive cell subpopulation. Therefore, the identification of novel OCSC-associated targets and therapeutic approaches may offer alternative treatment options. We established a new platform of patient-derived organotypic models of OC and found that Matrix Gla Protein (MGP) was upregulated in cancer cells upon contact with the peritoneal tumor microenvironment. MGP is classically known as a vitamin K-dependent calcium chelator, mainly associated with the inhibition of tissue calcification and cardiovascular disease. In addition, MGP is also aberrantly expressed in different cancer types, but the biological mechanisms that regulate its functional contribution to cancer progression is still unknown. Our transcriptomic profiling of primary OCSC versus their non-stem counterpart pointed to MGP as a potential driver of cancer stemness. Indeed, we obtained evidence that MGP promotes, and is required for, stemness-associated biological features in vitro and in animal models, including sphere formation and tumor initiation. MGP was also implicated in the regulation of the expression of several key genes involved in stemness and/or EMT. Finally, MGP was upregulated in OC patients following chemotherapy, suggesting that the protein may be involved in OCSC-driven chemoresistance. The molecular mechanisms underlying MGP-regulated OCSC pathophysiology were investigated by RNA sequencing experiments on MGP-manipulated OC cells, which unveiled genetic networks and pathways that are orchestrated by MGP to promote OCSC function. The biological role of MGP in OC provides novel insight into the biology of OCSC, thus defining new pathways involved in OC stemness and paving the way to innovative therapeutic strategies for OC eradication. Citation Format: Valentina Nieddu, Valentina Melocchi, Chiara Battistini, Giulia Franciosa, Michela Lupia, Jesper Olsen, Nicoletta Colombo, Fabrizio Bianchi, Ugo Cavallaro. Microenvironment-regulated matrix Gla protein drives ovarian cancer stemness [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A098.

Cancer stemness kinase inhibitor amcasertib: a promising therapeutic agent in ovarian cancer stem and cancer cell models with different genetic profiles.

Ovarian cancer, often referred to as the 'silent killer,' is a significant contributor to mortality rates. Emerging evidence implicates Nanog as a potential therapeutic target in ovarian cancer. Amcasertib (BBI-503) is an orally administered primary class stemness kinase inhibitor that effectively targets NANOG and various cancer stem cell pathways by specifically inhibiting serine-threonine stemness kinases. This study aimed to evaluate the antineoplastic effects of Nanog inhibition, a critical transcription factor associated with pluripotency and its role in ovarian cancer tumorigenesis, using the novel therapeutic agent Amcasertib in ovarian cancer cells characterized by distinct genetic profiles. The cytotoxicity of Amcasertib was assessed in both ovarian cancer and cancer stem cell models utilizing the Xelligence-RTCA system. The impact of the determined IC50 dose on apoptosis, invasion, migration, epithelial-mesenchymal transition (EMT), cell cycle progression, colony formation, and spheroid growth was evaluated using appropriate analytical techniques. Our findings revealed that Amcasertib exhibited significant antiproliferative effects and induced apoptosis in ovarian cancer and cancer stem cells. Moreover, Amcasertib caused G1 phase arrest and impeded colony formation in MDAH-2774 cells. Additionally, Amcasertib effectively inhibited spheroid growth in OVCAR-3 and OCSC cells. Notably, it demonstrated the ability to suppress invasion and migration in MDAH-2774 and OCSC cells. Furthermore, the suppression of Nanog-mediated stem cell-like features by Amcasertib was particularly pronounced in ER-negative ovarian cancer and cancer stem cells, highlighting its high anticancer efficacy in this subgroup. These results suggest that Amcasertib holds promise as a potential standalone or combination therapy agent for the treatment of ER-negative ovarian cancer.

Scoulerine promotes cytotoxicity and attenuates stemness in ovarian cancer by targeting PI3K/AKT/mTOR axis.

In women, ovarian cancer is a common gynecological cancer associated with poor prognosis, reoccurrence and chemoresistance. Scoulerine, a benzylisoquinoline alkaloid, has been reported effective against several carcinomas. Thus, we investigated the impact of scoulerine on ovarian cancer cells (OVCAR3). Cell viability was assessed by MTT assay, migration was determined by Boyden Chamber assay, while the invasion was monitored by Boyden Chamber assay using the matrigel. The stemness properties of OVCAR3 cells were observed by tumorsphere assay. Epithelial to mesenchymal transition (EMT) and stemness-related protein markers were monitored by real-time PCR analysis and immunoblotting. Scoulerine inhibits the viability of OVCAR3 cells with the IC 50 observed at 10 µmol L-1 after 48 h treatment. Scoulerine inhibited the colony-forming ability, migration and invasiveness of OVCAR3 cells in a dose-dependent fashion. Scoulerine treatment also drastically reduced the spheroid-forming ability of OVCAR3 cells. The mesenchymal and stemness--related markers like N-cadherin, vimentin, CD-44, Oct-4, Sox-2 and Aldh1A1 were downregulated, whereas the epithelial markers like E-cadherin and CD-24 were upregulated in scoulerine-treated cells. The upstream PI3K/Akt/mTOR-axis was downregulated in scoulerine-treated cells. We concluded that scoulerine successfully perturbs the cancerous properties of OVCAR3 cells by targeting the PI3K/Akt/mTOR axis. In vivo studies revealed a substantial decrease in tumor mass and volume after scoulerine treatment. Furthermore, scoulerine treatment was found to decrease oxidative stress factors in ovarian cancer mice model. Scoulerine is a potential anticancer agent against ovarian cancer and can be considered as a lead molecule for this malignancy, provided further investigations are performed.

Abstract 5023: PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling

Abstract Background: Ovarian cancer is one of the most aggressive malignancies threatening women’s health worldwide. Its high mortality rate is largely due to symptoms not appearing until late in the disease, usually after the cancer has metastasized. Despite current state-of-the-art treatments, the prognosis for patients remains poor. New effective treatments are urgently needed to improve patient survival. High lactate production and low glucose oxidation, independent of oxygen availability, is known as the Warburg effect and is common in cancer. Phosphofructokinase 1 (PFK-1) irreversibly converts fructose 6-phosphate to fructose 1,6-bisphosphate, the first step in the Warburg effect. The platelet isoform of PFK (PFKP) regulates glycolysis and plays roles in tumorigenesis in other human cancers, the precise mechanisms has yet to be defined. Moreover, PFKP in ovarian cancer remain unknown. We hypothesize that PFKP modulates metastasis and stemness in ovarian cancer. Methods: Expression of PFKP in clinical samples, ascites-derived tumor cells, normal ovarian epithelial cells (HOSEs) and ovarian cancer cell lines was determined by immunohistochemistry/immunofluorescence and qPCR/immunoblotting, respectively. The correlation between PFKP and clinicopathological parameters was analyzed. Ectopic expression or knockdown of PFKP followed by migration, invasion, sphere formation and soft agar assays were performed in ascites-derived tumor cells and ovarian cancer cell lines to investigate the effects on metastasis, stemness and malignant transformation respectively. Additionally, the related signaling pathway was explored by qPCR and immuoblotting. Results: Up-regulation of PFKP in clinical samples, ascites-derived tumor cells and ovarian cancer cell lines was found when compared to HOSEs. Higher PFKP was correlated to shorter overall and disease-free survival. Functionally, knockdown of PFKP by siRNA/shRNA approaches in ascites-derived tumor cells, ES-2 and HeyA8 cells decreased migration, invasion, sphere formation and colony formation in soft agar, which was accompanied by decreased expression α5 integrin, pERK and MMP9. Ectopic expression of PFKP in SKOV-3 cells showed opposite effects. Intriguingly, we found that MEK-1 inhibitor (U0126) or an anti-MMP9 neutralizing antibody could block PFKP-mediated migration/sphere formation abilities. Conclusion: PFKP was overexpressed in ovarian cancer and regulates metastasis, stemness and anchorage-independent growth. α5 integrin and ERK/MMP9 signaling were involved in such functional effects. PFKP could be a potential prognostic marker and therapeutic target for ovarian cancer. Citation Format: Ruiqian Zhang, Michelle K.Y. Siu, Xuetang Mo, Mingo M.H. Yung, Jingjing Wang, Yuxin Jiang, Annie N.Y. Cheung, Hextan Y.S. Ngan, Karen K.L. Chan. PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5023.

A Novel ALDH1A1 Inhibitor Blocks Platinum-Induced Senescence and Stemness in Ovarian Cancer.

Simple SummaryOvarian cancer is the deadliest amongst the gynecologic malignancies. Most ovarian cancer patients initially respond to chemotherapy but will eventually relapse and become chemoresistant. A specialized subpopulation of cells within the tumor known as cancer stem cells are known to contribute to recurrence and chemoresistance. Ovarian cancer stem cells have a high expression of ALDH1A1, and patients with a high level of ALDH1A1 in their tumor have worse survival. Thus, specifically targeting ALDH1A1 could be an effective strategy to inhibit cancer stemness and disease relapse. We describe the discovery of a novel ALDH1A1 inhibitor called 974 and show that targeting ALDH1A1 with 974 decreases the population of ovarian cancer stem cells. Furthermore, inhibiting ALDH1A1 suppresses chemotherapy-induced senescence and stemness. Collectively, our data demonstrate that targeting ALDH1A1 in cancer stem cells could be an effective strategy to overcome chemotherapy resistance in ovarian cancer.Ovarian cancer is a deadly disease attributed to late-stage detection as well as recurrence and the development of chemoresistance. Ovarian cancer stem cells (OCSCs) are hypothesized to be largely responsible for the emergence of chemoresistant tumors. Although chemotherapy may initially succeed at decreasing the size and number of tumors, it leaves behind residual malignant OCSCs. In this study, we demonstrate that aldehyde dehydrogenase 1A1 (ALDH1A1) is essential for the survival of OCSCs. We identified a first-in-class ALDH1A1 inhibitor, compound 974, and used 974 as a tool to decipher the mechanism of stemness regulation by ALDH1A1. The treatment of OCSCs with 974 significantly inhibited ALDH activity, the expression of stemness genes, and spheroid and colony formation. An in vivo limiting dilution assay demonstrated that 974 significantly inhibited CSC frequency. A transcriptomic sequencing of cells treated with 974 revealed a significant downregulation of genes related to stemness and chemoresistance as well as senescence and the senescence-associated secretory phenotype (SASP). We confirmed that 974 inhibited the senescence and stemness induced by platinum-based chemotherapy in functional assays. Overall, these data establish that ALDH1A1 is essential for OCSC survival and that ALDH1A1 inhibition suppresses chemotherapy-induced senescence and stemness. Targeting ALDH1A1 using small-molecule inhibitors in combination with chemotherapy therefore presents a promising strategy to prevent ovarian cancer recurrence and has the potential for clinical translation.

Abstract 6063: FOXK2 promotes stemness in ovarian cancer by regulating unfolded protein response signaling

Abstract Introduction: Cancer stem cells (CSCs), a rare cell population within tumors that possesses self-renewal and tumor initiation capacity, have been implicated in cancer relapse and resistance to chemotherapy. In ovarian cancer (OC), CSCs are characterized by a high expression of aldehyde dehydrogenase (ALDH). Understanding the regulatory programs promoting stemness could identify new treatments. We identified FOXK2 as a key transcription factor (TF) that promotes stemness in OC. Methods: We generated FOXK2 knockdown OC models by stable transduction of shRNA targeting this TF and measured the population of ALDH+ CSCs, expression of stemness-associated genes, and in vivo tumor initiation capacities. Transcriptomic and ChIP-seq analyses were used to identify key FOXK2 targets. CRISPR-dCas9 genome editing was used to verify the regulatory function of FOXK2. Rescue experiments validated the roles of a newly discovered target gene of FOXK2. Results: Through gene expression analyses in CSCs vs. non-CSCs, we identified FOXK2 as a highly expressed stemness-specific TF in OC. Genetic depletion of FOXK2 diminished stemness features and reduced in vivo tumor initiation capacity. Through ChIP-sequencing we discovered that FOXK2 directly regulates IRE1α (ERN1 gene) expression, a key sensor for the unfolded protein response (UPR). ChIP-sequencing analysis further revealed that FOXK2 binds to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a CRISPR/dCas9 diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 mRNA splicing and activation of stemness pathways, while genetic or pharmacological blockade of IRE1α inhibited CSCs in OC models. Conclusion: Our data establish a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in ovarian CSCs. Citation Format: Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela E. Matei. FOXK2 promotes stemness in ovarian cancer by regulating unfolded protein response signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6063.

MiR-600 promotes ovarian cancer cells stemness, proliferation and metastasis via targeting KLF9

Previous studies have revealed that miRNAs participate in the pathogenesis of ovarian cancer; however, whether miR-600 is also involved remains unclear. In this study, we aimed to investigated the role of miR-600 in ovarian cancer progression. Here, miR-600 expression was significantly upregulated in ovarian cancer tissues and stem cells. Functional studies showed that miR-600 promoted ovarian cancer cell stemness, proliferation and metastasis. Mechanistic studies revealed that Kruppel like factor 9 (KLF9) was indicated as the target of miR-600. The luciferase reporter assay suggested that miR-600 directly bound to the 3′-untranslated region of KLF9. Additionally, miR-600 expression was negatively associated with KLF9 expression in human ovarian cancer tissues. Si-KLF9 partially abolished the discrepancy of self-renewal, growth and metastasis capacity between miR-600 knockdown ovarian cancer cells and control cells. In conclusion, our results suggest that miR-600 promotes ovarian cancer cell stemness, proliferation and metastasis via directly downregulating KLF9, and impairing miR-600 levels may be a new treatment strategy for ovarian cancer in the future.

PFKFB3 Regulates Chemoresistance, Metastasis and Stemness via IAP Proteins and the NF-κB Signaling Pathway in Ovarian Cancer.

Glycolysis has been reported to be critical for cancer stem cells (CSCs), which are associated with tumor chemoresistance, metastasis and recurrence. Thus, selectively targeting glycolytic enzymes may be a potential therapy for ovarian cancer. 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 3 (PFKFB3), the main source of fructose-2,6-bisphosphate, controls the first committed step in glycolysis. We investigate the clinical significance and roles of PFKFB3 in ovarian cancer using in vitro and in vivo experiments. We demonstrate that PFKFB3 is widely overexpressed in ovarian cancer and correlates with advanced stage/grade and poor outcomes. Significant up-regulation of PFKFB3 was found in ascites and metastatic foci, as well as CSC-enriched tumorspheres and ALDH+CD44+ cells. 3PO, a PFKFB3 inhibitor, reduced lactate level and sensitized A2780CP cells to cisplatin treatment, along with the modulation of inhibitors of apoptosis proteins (c-IAP1, c-IAP2 and survivin) and an immune modulator CD70. Blockade of PFKFB3 by siRNA approach in the CSC-enriched subset led to decreases in glycolysis and CSC properties, and activation of the NF-κB cascade. PFK158, another potent inhibitor of PFKFB3, impaired the stemness of ALDH+CD44+ cells in vitro and in vivo, whereas ectopic expression of PFKFB3 had the opposite results. Overall, PFKFB3 was found to mediate metabolic reprogramming, chemoresistance, metastasis and stemness in ovarian cancer, possibly via the modulation of inhibitors of apoptosis proteins and the NF-κB signaling pathway; thus, suggesting that PFKFB3 may be a potential therapeutic target for ovarian cancer.

Transcriptomic Analyses Reveal B-Cell Translocation Gene 2 as a Potential Therapeutic Target in Ovarian Cancer.

Ovarian cancer is the most common and aggressive type of tumor of the female reproductive system. Two factors account for this detrimental clinical presentation: (i) the lack of early detection methods and (ii) the inherently aggressive nature of this malignancy. Currently, transcriptomic analyses have become important tools to identify new targets in different cancer types. In this study, by measuring expression levels in ovarian cancer samples and stem cell samples, we identified 24 tumor suppressor genes consistently associated with poor prognosis. Combined results further revealed a potential therapeutic candidate, BTG2, which belongs to the antiproliferative gene family. Our results showed that BTG2 expression regulated ovarian cancer cell proliferation via G1/S phase cell cycle arrest by regulating Cyclin D1, CDK4, p-AKT, and p-ERK expression. BTG2 also inhibited cell migration by modulating MMP-2 and MMP-9 expression. Furthermore, xenograft models confirmed a growth inhibitory effect of BTG2 in ovarian cancer in vivo. BTG2 was significantly associated with ovarian cancer FIGO stage and grade in the clinic. Our findings indicated that BTG2 exerts a suppressive impact on ovarian cancer and could be a potential biomarker.

Overexpressed COL5A1 is correlated with tumor progression, paclitaxel resistance, and tumor-infiltrating immune cells in ovarian cancer.

Ovarian cancer (OC) remains the leading cause of cancer-related death among gynecological cancers. The present study examined the role of collagen type V alpha 1 (COL5A1) and the characteristics of COL5A1 as an oncogenic protein in OC. The association of COL5A1 with paclitaxel (PTX)-resistance and stemness in OC was also studied and the multidatabase and big data analyses of the prognostic value, coexpression network, genetic alterations, and tumor-infiltrating immune cells of COL5A1 were elucidated. We found that COL5A1 expression was high in OC cells and tissues. Knockdown of COL5A1 inhibited the proliferation and migration of OC cells. Further study also showed that COL5A1 was overexpressed in PTX-resistant OC cells compared to respective PTX-sensitive cells. Additionally, COL5A1 was more enriched in OC stem cell-like cells. Silencing COL5A1 expression decreased the OC cell resistance to PTX and inhibited the ability of OC-spheroid formation. Survival analysis predicted that the elevated COL5A1 expression was associated with a worse survival outcome and correlated to the tumor stage of OC patients. The estimating relative subsets of RNA transcripts (CIBERSORT) algorithm analysis also unveiled the correlation of several tumor-infiltrating immune cells with the expression of COL5A1. Taken together, our data demonstrate that COL5A1 is a biomarker to predict OC progression and PTX-resistance and represents a promising target for OC treatment.

Abstract A55: DCLK1 mediates tumor stemness and platinum resistance in high-grade serous epithelial ovarian cancer

Abstract Background: Ovarian cancer is the fifth leading cause of cancer death in the United States, with an anticipated 13,000 deaths in the United States in 2017. High-grade serous (HGSOC) sub-type is both the most common and the most lethal histopathology. As more than 85% of patients present with metastatic disease, the 5-year overall survival remains dismal at only 30%. It is known that human cancers are initiated and maintained by self-renewing stem cells. Cancer stem cell related protein Doublecortin-like kinase 1 (DCLK1) is a major regulator of stemness and EMT, promoting drug resistance, tumor dormancy, progression, and metastasis in solid tumors. The molecular mechanisms of DCLK1 in tumorigenesis and drug resistance remain limited but DCLK1 expression is increased in cancer, especially in the drug-resistant setting. Results: Our lab has made the discovery that DCLK1 is overexpressed in HGSOC where it regulates tumor progression, tumor stemness, and EMT via noncoding RNA regulation of downstream oncogenic pathways. Our study involved assessment of DCLK1 expression in tissue culture and xenografts created from primary patient-derived cell lines (PDACs). Expression of the DCLK1 protein was assessed by immunoblot analysis in lysates of cell lines representing HGSOC and PDACs obtained from the ascites of patients at the University of Oklahoma at the time of diagnosis, prior to initiation of chemotherapy. FTE-187, FTE-188, and OSE cells were used as normal controls. Elevated levels of DCLK1 expression were observed in the panel of HGSOC cell lines and in PDAC lines. Our in vitro studies confirmed DCLK1 upregulation in the tissue and stroma of PDAC lines and our in vivo studies with PDAC xenografts revealed that siRNA knockdown of DCLK1 negatively regulated tumor suppressor miRNAs including mi-143/145, let-7a, and miR-200a-c resulting in downregulation of pluripotency, EMT, and proangiogenic markers including OCT4, SOX2, KLF4, NANOG, LIN28B, NOTCH, KRAS, and c-MYC. This was most pronounced in the setting of platinum resistance. For this, lysates from paired cells lines with known platinum-sensitive and platinum-resistant variants were assessed (Tyk-nu/Tyk-nu CPR and A2780/A2780.cp). An increase in DCLK1 expression was observed in both of the platinum-resistant lines compared to the platinum-sensitive lines. This was confirmed in PDACs where clinical platinum status correlated with DCLK1 expression, suggesting the role of DCLK1 in inherent platinum-resistant tumor clones. Conclusions: Our data implicate DCLK1 in ovarian cancer pathogenesis and platinum resistance and indicate that DCLK1 knockdown via may suppress malignant progression and resensitize tumors to platinum chemotherapy. These data could have a significant impact on future therapeutic strategies involving DCLK1 inhibitors in treating platinum-resistant patients. Given the high recurrence rate and inevitable development of platinum-resistant disease, such compounds have high potential to impact clinical outcomes in this lethal malignancy. Citation Format: Katherine M. Moxley, JiHee Ha, Daniel Andrade, Courtney Houchen, Danny Dhanasekaran. DCLK1 mediates tumor stemness and platinum resistance in high-grade serous epithelial ovarian cancer [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 A55.

Abstract A76: Role of frizzled-7 in platinum tolerance ovarian cancer

Abstract While ovarian cancer (OC) is considered as a highly chemoresponsive tumor, 75% women experience tumor relapse associated with chemoresistance. It has been hypothesized that ovarian cancer stem cells (OCSCs) are responsible for developing chemoresistant recurrent tumors and sharing characteristics with normal SCs. Measuring aldehyde dehydrogenase 1 (ALDH1) enzymatic activity has been demonstrated as an established method for OCSC identification and isolation. Our previous studies indicated that while chemotherapy initially “debulks” the mass of OC, residual tumors are enriched in OCSCs. Here, we aimed to identify new markers for CSC enriched platinum-resistant OCs using in vitro and in vivo models. We found that the surviving cells after repeated treatment with platinum were enriched in ALDH(+) cells, formed more spheroids and contained cells expressing CSC-related transcription factors (Sox 2) compared to parental cells, supporting that repeated platinum treatment selects cells with stem-like properties. Next, CSC RT-PCR based platform was used to compare wild-type and chemoresistant OCs and to rule out candidate genes that may drive stemness and chemoresistance. CSC array showed that Frizzled 7 receptor (FZD7), a transmembrane receptor involved in canonical Wnt/β-catenin, emerged as a top highly expressed marker in platinum-resistant compared to parental cells and was next studied as a putative new marker for CSC-enriched chemoresistant OCs. FZD7 expression was increased in platinum-resistant cells compared to parental cells at mRNA and protein levels. FZD7(+) cells were detectable and represent ~2-15% on OC cell lines and cells dissociated from human tumors. FZD7(+) population enriched in the platinum-resistant OCs and xenografts. In addition, KD FZD7 in chemoresistant OC cells restored chemosensitivity of OCs, decreased spheroid formation, and delayed tumor initiation, suggesting the role of FZD7 in regulating chemoresistance and stemness of OCs. RNA sequencing results of FZD7(+)/FZD7(-) OCs revealed that FZD7(+) cells downregulated genes associated to DNA damage response, and upregulated “persister cells” related genes, including EMT and stemness-associated genes. Importantly, mitochondrial dysfunction and oxidative phosphorylation were enriched in FZD7(-) OCs compared with FZD7(+) cells. As “persister cells” have been demonstrated to be dependent on antioxidant protein, glutathione peroxidase 4 (GPX4), for cell survival and maintaining mitochondrial oxidative phosphorylation in other cancer models, we checked GPX4 expression in FZD7(+) OCs and platinum-resistant OCs; the expression of GPX4 is correlated with FZD7 expression associated with chemoresistance. Platinum-resistant cells are more sensitive to GPX4 inhibitor treatment and KD FZD7 decreased GPX4 expression and blunt sensitivity of OCs to GPX4 inhibitor treatment, further supporting that platinum-resistant OCs are “persister like cells,” which are protected by GPX4 from lipid peroxidation stress and ferroptosis. Citation Format: Yinu Wang, Guanyuan Zhao, Hao Huang, Salvatore Condello, Jianjun Wei, Daniela Matei. Role of frizzled-7 in platinum tolerance ovarian cancer [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 A76.

Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410–420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.

Wnt Signaling in Ovarian Cancer Stemness, EMT, and Therapy Resistance.

Ovarian cancers represent the deadliest among gynecologic malignancies and are characterized by a hierarchical structure with cancer stem cells (CSCs) endowed with self-renewal and the capacity to differentiate. The Wnt/β-catenin signaling pathway, known to regulate stemness in a broad spectrum of stem cell niches including the ovary, is thought to play an important role in ovarian cancer. Importantly, Wnt activity was shown to correlate with grade, epithelial to mesenchymal transition, chemotherapy resistance, and poor prognosis in ovarian cancer. This review will discuss the current knowledge of the role of Wnt signaling in ovarian cancer stemness, epithelial to mesenchymal transition (EMT), and therapy resistance. In addition, the alleged role of exosomes in the paracrine activation of Wnt signaling and pre-metastatic niche formation will be reviewed. Finally, novel potential treatment options based on Wnt inhibition will be highlighted.

Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance

Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Method: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. Conclusion: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Abstract 3678: Ovarian cancer stemness is driven by a novel L1/Stat3/Src axis

Abstract Introduction Ovarian cancer (OC) is the most lethal gynaecological malignancy due to the lack of peculiar symptoms in its early phase (Bowtell, 2010). OC often relapses as a chemoresistant disease within 3 years after surgical debulking (Lengyel, 2010; Sun et al, 2007). These pathological hallmarks raised the hypothesis that OC is a cancer-stem cell (CSC)-driven disease. Indeed, a “stem-like” chemoresistant subset of OC cells is more tumorigenic than the bulk tumor cell population and is able to form spheroids in vitro (Bapat et al, 2005; Pan et al 2012). This pointed to ovarian CSC (OCSC) as an attractive target for OC-eradicating therapies. The cell adhesion molecule L1 has been implicated in OC progression (Zecchini et al, 2008). While a few reports implicated L1 in stemness, its role in OCSC has not been investigated. Based on these observations, we aimed to investigate the functional role of L1 in the physiopathology of OCSC. Experimental procedures OVCAR3 and Ov90, two OC cell lines with high and low endogenous L1 levels, respectively, were employed for loss- and gain-of-function studies. Sphere-forming efficiency (SFE) was determined as the fraction cells within the bulk population able to overcome anoikis and proliferate in suspension cultures, and used as an in vitroproxy for CSC frequency. Tumorigenesis in NSG mice was employed as in vivo assays for cancer stemness. BBI608 and SU6656 were used as STAT3 and SRC activity inhibitor, respectively. Results L1 was both required and sufficient for self-renewal in OC. Indeed, L1 silencing prevented tumor formation while its forced expression in OCSC-enriched sphere cultures promoted tumor initiation in vivo. Mechanistically, L1 increased dramatically the expression and the activation of STAT3 in OCSC and promoted its nuclear localization. L1 per se was sufficient for OC sphere formation even under very stringent culture conditions in which, once again, STAT3 was activated in L1-expressing OCSC only. Moreover, STAT3 activity was required for L1-induced clonogenic advantage, which occurred in a JAK-independent manner. Alternatively, L1-induced SFE was mediated by SRC-dependent STAT3 activation. SRC inhibition, in turn, reduced FAK activation in L1-expressing OCSC. Thus, a novel L1/STAT3/SRC axis appears to sustain OCSC pathophysiology. L1 is also an optimal target for OC eradication since it confers chemoresistance to OCSC while its targeting reduces OCSC frequency. On these premises, L1 targeting should be explored in combined treatments with chemotherapeutic agents. Conclusion L1 can be considered as a new player and potential target in the context of OCSC, and the L1/STAT3/SRC interplay emerges as a novel mechanism in OC initiation and progression. Therefore, this work might pave the way to novel therapeutic strategies for the eradication of such a devastating disease. Citation Format: Marco Giordano, Alessandra Villa, Giovanni Bertalot, Fabrizio Bianchi, Stefano Freddi, Ugo Cavallaro. Ovarian cancer stemness is driven by a novel L1/Stat3/Src axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3678.

1α,25‑Dihydroxyvitamin�D3 restrains stem cell‑like properties of ovarian cancer cells by enhancing vitamin�D receptor and suppressing CD44

Scientific evidence linking vitamin D with various cancer types is growing, but the effects of vitamin D on ovarian cancer stem cell‑like cells (CSCs) are largely unknown. The present study aimed to examine whether vitamin D was able to restrain the stemness of ovarian cancer. A side population (SP) from malignant ovarian surface epithelial cells was identified as CSCs, in vitro and in vivo. Furthermore, 1α,25‑dihydroxyvitamin D3 [1α,25(OH)2D3] treatment inhibited the self‑renewal capacity of SP cells by decreasing the sphere formation rate and by suppressing the mRNA expression levels of cluster of differentiation CD44, NANOG, OCT4, SOX2, Krüppel‑like factor 4 and adenosine triphosphate binding cassette subfamily G member 2. Additionally, 1α,25(OH)2D3 treatment decreased the expression of Cyclin D1, whereas it increased the expression of β‑catenin and vitamin D receptor (VDR). Notably, immunofluorescence staining verified that 1α,25(OH)2D3 promoted the expression of β‑catenin in the cytoplasm. Furthermore, vitamin D3 delayed the onset of tumor formation derived from injection of ovarian CSCs to nude mice, by reducing CD44 and enhancing β‑catenin expressions in vivo. In conclusion, 1α,25(OH)2D3 restrains the stem cell‑like properties of ovarian cancer cells by enhancing the expression of VDR, by promoting the expression of β‑catenin in the cytoplasm, and by suppressing the expression of CD44. These findings provide a novel insight into the functions of vitamin D in diminishing the stemness of cancer CSCs.

Abstract LB-048: c-Kit enhances ovarian cancer stemness via upregulating phospho-PHB in the lipid raft domain of the plasma membrane

Abstract Phosphorylation of prohibitin (PHB) at threonine 258 (phospho-PHBT258) and tyrosine 259 (phospho-PHBY259) in human cancer-cell membranes is associated with an increase in the invasiveness and metastasis of cancer cells. Phospho-PHBY259 can regulate phospho-PHBT258 but not vice versa. Here, we found that stably overexpressing c-Kit in SKOV3 ovarian cancer cells (SKOV3_c-Kit) increased phospho-PHBY259, phospho-PHBT258 and PHB levels in the lipid raft domain of the plasma membrane. On the other hand, inhibition of c-Kit expression decreased PHB expression in SKOV3_c-Kit and Kuramochi ovarian cancer cells. Immunoprecipitation analysis showed that c-Kit interacts with PHB. SKOV3_c-Kit cells or SKOV3 cells overexpressing PHB in the lipid raft had higher levels of Notch3, NICD3 (Notch3 intracellular domain) and ABCG2 protein expression as compared to wild-type SKOV3 cells. Moreover, generation of SKOV3-G4 cells through serial passage of SKOV3 cells into nude mice ovary 4 times, revealed that the levels of c-Kit, Notch3, NICD3, phospho-PHBY259, phospho-PHBT258 and PHB in the lipid raft domain of SKOV3-G4 were significantly higher than wild-type SKOV3 cells. The SKOV3-G4 cells formed spheroids with stem cell-like properties such as higher expression of stem cell markers (Oct4, Nanog, SOX2). In addition, SKOV3-G4 cells exhibited more clonogenic potential and migration/invasion ability than wild-type SKOV3. These results suggest that c-Kit interacts with PHB in the lipid raft domain to phosphorylate PHB to increase invasiveness and stemness in ovarian cancer. Citation Format: Shu-Mei Y. Liang, Chia-Hsun Fang, Pu-Yuan Chang, Chi-Ming Liang. c-Kit enhances ovarian cancer stemness via upregulating phospho-PHB in the lipid raft domain of the plasma membrane [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-048.