Ovarian Cancer-initiating Cells Research Articles

CDC50A might be a novel biomarker of epithelial ovarian cancer-initiating cells

BackgroundThe aim of this work was to screen and validate biomarkers of ovarian cancer-initiating cells to detect the mechanisms of recurrence of epithelial ovarian cancer (EOC).MethodsStably labelled the amino acid in side population (SP) cells of epithelial ovarian cancer which were rich in cancer-initiating cells and non-SP cells with isotope in culture and differentially expressed cellular membrane proteins in SP cells were identified through proteomics technology. The new candidate biomarker was screened and validated through RT-PCR and western blot. Both in cell lines and primary EOC, cancer-initiating biofunctions of CDC50A positive cells were validated. Moreover, the characteristics of mesenchymal transition (EMT) was also detected and the correlation between the biomarker and clinical prognosis was observed.ResultsThrough proteomics technology, candidate protein CDC50A was screened, and its significantly differential expression in SP cells was validated. CDC50A-positive cells from cell lines and primary ovarian cancer tissues were validated to show characteristics of cancer-initiating cells both in vitro and in vivo, including sphere-forming, self-renewal, differentiation, tumor metastasis and tumorigenicity in mice. The relationship between CDC50A-positive cells from primary tissues and tumour metastasis was confirmed based on their mesenchymal transition characteristics. Among 16 high-grade ovarian serous cancer patients, a high ratio of CDC50A-positive cells in primary tumours was correlated with a shorter platinum-free interval (p = 0.031, HR 0.260, 95% CI 0.77 ~ 0.885).ConclusionCDC50A could be used to screen ovarian cancer-initiating cells and might be a new target to resolve tumour development in EOC patients.

750 AK119, a CD73 targeting antibody with dual mechanism of action

BackgroundAK119 is an Fc-engineered humanized IgG1 monoclonal antibody targeting human CD73. CD73-extracellular adenosine pathway regulates conversion of pro-inflammatory and immuno-stimulatory extracellular adenosine ATP into immunosuppressive adenosine. CD73 expresses on cancer cells, endothelial cells, fibroblasts, lymphocytes and myeloid cells. CD73 upregulated can be a result of tissue hypoxia,1 epithelial-to-mesenchymal transition,2 inflammation3 and/or cytotoxic stress.4 Also, increasing immune response may lead to faster viral clearance, shorter recovery time, less complications, longer immunity and protection from re-infection. Inhibiting CD73 was reported to evoke B cells activation and shows anti-fibrotic effects. The ability of enhancing immune response provides a potential opportunity to treat COVID-19. Thus, we investigated pharmacological activity of AK119 as an agent treating cancers, COVID-19 and fibrosis.MethodsAK119 inhibition of CD73 enzymatic activity was tested in human PBMCs based assay. The ability of AK119 to enhance B cells immune response was detected by cell-based assay. PBMCs were incubated overnight with APCP (inhibitor of CD73 enzyme acitvity) or AK119, CPI006 or MEDI9447. Flow cytometry analysis was performed with gating on B cells (CD19+CD3-) and MFI and positive percent were reported for antibody staining of CD69 or CD83, as well as HLA-DR and IgM. Enhancement of anti-SARS-CoV-2 antibody production was studied using human CD73 transgenic mouse immunized with SARS-CoV-2 spike protein. The in-vivo activity of AK119 was further studied in bleomycin-induced pulmonary fibrosis model in human CD73 transgenic mouse.ResultsAK119 shows a more potent antigen binding (figure 1) and completely CD73 enzyme inhibition activity (figure 2). AK119 promotes B cell proliferation, and upregulating CD69, CD83, HLA-DR and IgM that are markers of B cell activation (figure 3). B cell activation induced by AK119 is independent of adenosine. AK119 show significantly higher bioactivity to induce B cells activation in comparison with MEDI9447 or CPI006 (figure 4). In human CD73 transgenic mice, AK119 increased secretion of anti-S protein IgG (figure 5). In pulmonary fibrosis mouse model, number of inflammatory cell in broncholveolr lavage fluid of AK119 was significantly decreased, and decreased HYP representing collagen content in lung tissue homogenate of mice was found in both AK119 50 mg/kg and 10 mg/kg group (figure 6).ConclusionsAK119 selectively binds to and inhibits the ectonucleotidase activity of CD73 thus reducing adenosine accumulation. Results from non-clinical pharmacology studies reveal potent bioactivities as well as favorable safety properties of AK119. AK119 is intended for advanced solid tumors, pulmonary fibrosis and therapy of COVID-19.ReferencesBullen JW, Tchernyshyov I, Holewinski RJ, DeVine L, Wu F, Venkatraman V, Kass DL, Cole RN, Van Eyk J, Semenza GL, Protein kinase A-dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1. Sci Signal 2016;9(430):ra56.Lupia M, Angiolini F, Bertalot G, Freddi S, Sachsenmeier KF, Chisci E, Kutryb-Zajac B, Confalonieri S, Smolenski RT, Giovannoni R, Colombo N, Bianchi F, Cavallaro U. CD73 regulates stemness and epithelial-Mesenchymal transition in ovarian cancer-initiating cells, Stem Cell Rep 2018;10(4):1412–1425.Reinhardt J, Landsberg J, Schmid-Burgk JL, Ramis BB, Bald T, Glodde N, Lopez-Ramos D, Young A, Ngiow SF, Nettersheim D, Schorle H, Quast T, Kolanus W, Schadendorf D, Long GV, Madore J, Scolyer RA, Ribas A, Smyth MJ, Tumeh PC, Tuting T, Holzel M. MAPK signaling and inflammation link melanoma phenotype switching to induction of CD73 during immunotherapy. Cancer Res 2017;77(17):4697–4709.Samanta D, Park Y, Ni X, Li H, Zahnow CA, Gabrielson E, Pan F, Semenza GL. Chemotherapy induces enrichment of CD47(+)/CD73(+)/ PDL1(+) immune evasive triple-negative breast cancer cells, Proc Natl Acad Sci USA. 2018;115(6):E1239–E1248.Abstract 750 Figure 1Binding activity of AK119 to human PBMCs. Binding Curve of AK119 to CD73 expressed on (A) CD8+ T cells and (B) CD19+ B cells in human PBMCsAbstract 750 Figure 2Inhibition activity of CD73 on human PBMCs. AK119 Inhibits Enzymatic Activity of CD73 Expressed on human PBMCsAbstract 750 Figure 3Effect of upregulating B cell markers by AK119. AK119 Upregulates (A) CD69, (B) CD83, (C) HLA-DR and (D) IgM Expression on B cellsAbstract 750 Figure 4Stimulation of B cell Proliferation by AK119Abstract 750 Figure 5Therapeutic activity in the COVID-19 mouse model. Serum Concentration of S protein-specific IgG in Mouse Model of COVID-19Abstract 750 Figure 6Therapeutic activity in the asthma mouse model. (A) AK119 relieves the increased airway resistance and restore the lung function. (B) Reduction of the inflammatory cells in BALF by AK119

Abstract A08: A new driver pathway in ovarian cancer stem cells

Abstract Ovarian cancer stem cells (OCSC) are believed to fuel tumor metastasis, recurrence, and chemoresistance. Therefore, understanding the molecular players involved in OCSC function represents an urgent need towards the design of innovative and more efficacious treatments of this neoplasm. Our molecular profiling of primary OCSC isolated from high-grade serous ovarian cancer patients revealed a series of novel genes and pathways specifically associated with this cell subpopulation. Among these, we initially focused on CD73, a cell-surface 5’-ectonucleotidase that catalyzes the production of extracellular adenosine. Our data revealed the new role of CD73 as a driver of OC stemness and a potential target for OCSC-eradicating treatments. More recently, we investigated the functional role in OCSC of the immunoglobulin-like cell adhesion molecule L1 (L1CAM), a transmembrane protein that we have previously implicated in OC malignancy (Zecchini et al., 2008). Loss- and gain-of-function studies were performed by manipulating the expression of L1CAM in OC cells. These experiments revealed the causal role of L1CAM not only in stemness-related properties such as anoikis resistance, clonogenic proliferation under low-attachment conditions and self-renewal, but also in OCSC-driven tumor initiation. Mechanistically, L1CAM appeared to act through the induction, activation, and nuclear translocation of STAT3, all events that occurred predominantly in the OCSC compartment rather than in the bulk tumor cell population. Indeed, the inhibition of STAT3 activity abolished L1-induced OC stemness. Of note, L1-dependent activation of STAT3 was not mediated by the canonical JAK pathway, but rather by the nonreceptor tyrosine kinase SRC. Accordingly, SRC inhibition prevented the induction of stemness-related features by L1CAM. Finally, to identify the molecular mechanisms that link L1CAM with SRC, we built on our previous findings that the L1CAM-related molecule NCAM activates FGFR1 signaling. This enabled us to find a crosstalk between L1CAM and FGFR1 which, in turn, accounts for the stimulation of SRC-mediated activation of STAT3 and, ultimately, OCSC function. Finally, we provide evidence that the inactivation of L1CAM enhances the sensitivity of OCSC to chemotherapeutics, implicating L1CAM as a potential target to overcome chemoresistance. In conclusion, our results uncovered a novel L1/FGFR1/SRC/STAT3 pathway that acts as a driver in OC stemness, shedding light on the pathophysiology of OCSC and opening new therapeutic perspectives for the eradication of such a devastating disease.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells

SummaryCancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5′-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication.

Abstract 950: Bistable switching of c-KIT by estrogen-mediated ceRNA and epigenetic silencing of miR-193a predicts survival in ovarian cancer

Abstract Ovarian cancer is one of the most lethal cancers in the female reproductive system. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by the surface antigen CD44 and c-KIT (CD117). Previous study also suggested that long term treatment of estrogen such as hormonal replacement therapy (HRT) may increase the risk of ovarian cancer, however the role of estrogen in ovarian carcinogenesis is still controversial. To unravel this complexity, we propose a mathematical model to explore how the ER signaling pathway contribute to c-KIT expression during ovarian carcinogenesis: one through a ceRNA competition of an ER target,E2F6 and c-KIT for their targeted miRNA, miR-193a; second by binding of E2F6 protein, in association with the polycomb complex, to the promoter of miR-193a to downregulate miR-193a transcription by epigenetic modifications. Our model found that epigenetic silencing of miR-193a generates a bistable switching of c-KIT during ovarian carcinogenesis based on the level of EZH2. To confirm our results, we performed ectopic expression of miR-193a and 3’UTR luciferase in ovarian cancer cell lines and confirmed that E2F6 and c-KIT are the targets of miR-193a. Importantly, treatment of E2 or bisphenol A (BPA) resulted in the up-regulation of E2F6 and c-kit mRNA in IOSE cells in which no or low methylation at the promoter CpG island of miR-193a was found. On the contrary, promoter hypermethylation of miR193a could be observed in miR-193a-underexpressed CP70 ovarian cancer cells but not in HeyC2 cells which showed similar expression level of miR-193a as in IOSE cells. Treatment of demethylating agent (5azaDC) or EZH2 inhibitor (GSK343) resulted in a reexpression of miR-193a in CP70 ovarian cancer cells. Overexpression of miR-193a inhibited tumor growth in vitro and in an animal model. Further ChIP-PCR assay also found that open chromatin mark H3K4me3 was enriched in the promoter region of miR-193a in HeyC2 but not in CP70 cells. On the contrary, repressive chromatin marks H3K9me3 and H3K27me3 were only enriched in CP70 cells. Clinically, ovarian cancer patients (n = 109) with higher promoter methylation of miR-193a were associated with poor survival (p>0.05). Additional analysis of the TCGA ovarian cancer dataset demonstrated that ovarian cancer patients with low expression of EZH2, a polycomb-group protein, showed positive correlation (p<0.05) between E2F6 and c-KIT which resembles the ceRNA phenomenon between these two mRNAs. Importantly, ovarian cancer patients with low expression of EZH2 tended to have lower expression of c-KIT. In conclusion, our mathematical model and experimental data suggests that miR-193a can be epigenetically regulated by both ceRNA and promoter methylation. Simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer. Citation Format: Frank Hsueh-Che Cheng, Baltazar D. Aguda, Hon-Yi Lin, Je-Chiang Tsai, Marek Kochanczyk, Ru-Inn Lin, Jora M. J. Lin, Gary C. W. Chen, Cheng-Chang Chang, Hung-Cheng Lai, Kenneth P. Nephew, Tzy-Wei Hwang, Michael W. Y. Chan. Bistable switching of c-KIT by estrogen-mediated ceRNA and epigenetic silencing of miR-193a predicts survival in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 950.

A mathematical model of bimodal epigenetic control of miR-193a in ovarian cancer stem cells.

Accumulating data indicate that cancer stem cells contribute to tumor chemoresistance and their persistence alters clinical outcome. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by surface antigen CD44 and c-KIT (CD117). It has been experimentally demonstrated that a microRNA, namely miR-193a, targets c-KIT mRNA for degradation and could play a crucial role in ovarian cancer development. How miR-193a is regulated is poorly understood and the emerging picture is complex. To unravel this complexity, we propose a mathematical model to explore how estrogen-mediated up-regulation of another target of miR-193a, namely E2F6, can attenuate the function of miR-193a in two ways, one through a competition of E2F6 and c-KIT transcripts for miR-193a, and second by binding of E2F6 protein, in association with a polycomb complex, to the promoter of miR-193a to down-regulate its transcription. Our model predicts that this bimodal control increases the expression of c-KIT and that the second mode of epigenetic regulation is required to generate a switching behavior in c-KIT and E2F6 expressions. Additional analysis of the TCGA ovarian cancer dataset demonstrates that ovarian cancer patients with low expression of EZH2, a polycomb-group family protein, show positive correlation between E2F6 and c-KIT. We conjecture that a simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.

YAP/TEAD co-activator regulated pluripotency and chemoresistance in ovarian cancer initiated cells.

Recent evidence suggests that some solid tumors, including ovarian cancer, contain distinct populations of stem cells that are responsible for tumor initiation, growth, chemo-resistance, and recurrence. The Hippo pathway has attracted considerable attention and some investigators have focused on YAP functions for maintaining stemness and cell differentiation. In this study, we successfully isolated the ovarian cancer initiating cells (OCICs) and demonstrated YAP promoted self-renewal of ovarian cancer initiated cell (OCIC) through its downstream co-activator TEAD. YAP and TEAD families were required for maintaining the expression of specific genes that may be involved in OCICs' stemness and chemoresistance. Taken together, our data first indicate that YAP/TEAD co-activator regulated ovarian cancer initiated cell pluripotency and chemo-resistance. It proposed a new mechanism on the drug resistance in cancer stem cell that Hippo-YAP signal pathway might serve as therapeutic targets for ovarian cancer treatment in clinical.

858: Simvastatin modifies the expression of stemness and epithelial–mesenchymal transition (EMT) markers in ovarian cancer-initiating cells resulting in decreased cell migration

858: Simvastatin modifies the expression of stemness and epithelial–mesenchymal transition (EMT) markers in ovarian cancer-initiating cells resulting in decreased cell migration

Cisplatin targets the stromal cell-derived factor-1-CXC chemokine receptor type 4 axis to suppress metastasis and invasion of ovarian cancer-initiating cells

In ovarian cancer, CD44+/CD117+ stem cells, also known as cancer-initiating cells (CICs), are highly proliferative and invasive. Therefore, the CD44+/CD117+ subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the effects of cisplatin (CDDP) on metastasis and invasion suppression of ovarian CICs by targeting the CXC chemokine receptor-4 (CXCR4) signaling pathway in vitro and in vivo. CD44+/CD117+ ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometry sorting. A 3-(4,5-dimethylthiazol-2-yl)-2.5-dipheny-tetrazolium bromide (MTT) assay revealed significant inhibition of proliferation of ovarian CICs with increasing CDDP drug concentrations. Moreover, colony formation and transwell migration assays indicated that CDDP significantly suppressed the invasive capacity of ovarian CICs in vitro. The expression levels of stromal cell-derived factor (SDF)-1, CXCR4, matrix metalloproteinase (MMP) 2, and MMP9 mRNA and protein levels were significantly reduced in CDDP-treated cells compared to untreated ovarian CICs. Furthermore, xenograft experiments confirmed that CDDP suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. In addition, CXCR4 agonist (diprotin A) treatment of ovarian CICs weakened the effects of CDDP and enhanced SDF-1-CXCR4 axis expression in ovarian CICs. Thus, the SDF-1-CXCR4 axis is an important mediator of proliferation and invasion in CXCR4-overexpressing ovarian cancer-initiating cells (OCICs). Furthermore, CDDP inhibits invasion and metastasis of OCICs by targeting SDF-1-CXCR4 axis expression.

Topoisomerase I inhibitor suppress tumor growth in chemoresistant ovarian cancer-initiating cells

Background: To investigate the role of a topoisomerase I inhibitor (topotecan) in chemoresistant ovarian cancer-initiating cells. Materials and Methods: We isolated ovarian cancer-initiating cells (CP70 side-population cells) from the CP70 cell line using FACS Aria-based sorting and cultured them in suspension to form spheroids (CP70 side-population sphere [SPS]). Gene expression was assessed by microarray, to identify potentially effective chemotherapeutic drugs. An MTS assay was used to evaluate cell growth. Results: CP70 SPS cells showed significant resistance to the chemotherapeutic drugs cisplatin and paclitaxel. Microarray analysis demonstrated a high expression of topoisomerase-related genes in CP70 SPS cells. Topotecan inhibited ovarian cancer-initiating cells (CP70 SPS) in vitro more than it did their parental CP70 cells. This result was confirmed in tissues from human patients. Conclusions: Chemoresistant ovarian cancer-initiating cells exhibited high expression levels of topoisomerase, which could be an alternative target of adjuvant therapy for patients with chemoresistant ovarian cancer.

Investigating molecular profiles of ovarian cancer: an update on cancer stem cells.

Currently we are more and more improving our knowledge about the characteristics and the role of cancer stem cells in human cancer. Particularly we have realized that self-renewing ovarian cancer stem cells (CSCs) or ovarian cancer-initiating cells, and mesenchymal stem cells (SCs) too, are probably implicated in the etiopathogenesis of epithelial ovarian cancer (EOC). There is clear evidence that these cells are also involved in its intra- and extra-peritoneal diffusion and in the occurrence of chemo-resistance. In assessing the molecular characteristics of ovarian CSCs, we have to take note that these cellular populations are rare and the absence of specific cell surface markers represents a challenge to isolate and identify pure SC populations. In our review, we focused our attention on the molecular characteristics of epithelial ovarian CSCs and on the methods to detect them starting from their biological features. The study of ovarian CSCs is taking on an increasingly important strategic role, mostly for the potential therapeutic application in the next future.

Abstract B43: Xenograft mouse models for ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy in developed countries. The incidence is 1 in 70 women, and 5-year survival rate below 50 %. Although major achievements have been reached in understanding the molecular alterations underlying the disease, the discoveries need yet to be integrated into clinical practice. A major obstacle in this process may be the translation of in vitro findings into diagnostic tests and novel therapeutic approaches. Well-characterized and representative preclinical mouse models that recapitulate the heterogeneity of tumors in patients are crucial in narrowing the gap between experimental and clinical studies. The aim of this work was to test and validate various mouse xenograft models for their applicability in ovarian cancer research. We tested several xenograft mouse models using (1) established commercially available ovarian cancer cell lines, (2) primary cell lines originating from high grade serous ovarian cancers or ascitic fluid, and (3) primary or metastatic tumor tissue directly from operation. Cell growth, tumor take rate and histopathological correlation between mouse and human tumors were applied for selection of commercial cell lines in xenograft experiments into athymic nude mice. Intrabursal orthotopic inoculations were used to model early, and intraperitoneal inoculations to model late stage ovarian cancer. Translational aspects were studied by comparing the histopathology between mouse tumors and human ovarian cancer specimens and by testing the utility of serum biomarkers CA-125 and HE-4 in mouse models. The mouse xenograft models faithfully represented the morphology of human serous ovarian cancer and many of the tissue markers were similarly expressed in human and mouse tumors. Importantly, the serum biomarker analysis developed in this study allowed follow-up of the tumor burden ex vivo. Such biomarker will be useful in studying drug responses and other tumor modifying therapies. The primary cell lines were inoculated subcutaneously to immunodeficient NOD-SCID and NOG mice in order to enrich and characterize ovarian cancer initiating cells (OCICs). Due to the low rate of tumor formation of in vitro cultured OCICs, we have started to use patient derived tumor xenograft (PDTX) transplantations. In this model fresh surgical tissue, sectioned in app. 3 mm3 pieces are implanted into the flanks of immunodeficient NOG mice. The preliminary results show a take rate of 75 % with PDTXs. Histopathological comparisons of original human specimen to mouse tumors reveals that this model also recapitulates the human microenvironment in PDTX modelling. Also, the PDTX model appears to increase the probability for successfully establishing novel primary cell lines. In conclusion, mouse xenograft models offer translational and important validation methods for pre-clinical cancer drug development and diagnostics. Citation Format: Tarja Lamminen, Katja Kaipio, Piia Mikkonen, Pia Roering, Olli Carpén. Xenograft mouse models for ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B43.

Methylomic profiling of ovarian cancer-initiating cells identifies DNA methylation as a prognostic biomarker

Objective: Epigenetic changes of ovarian cancer-initiating cells remain unexplored. In this study, we aimed to discover novel DNA methylation in ovarian cancer initiation cells and tested its potential as a prognostic biomarker.

Abstract 3730: 2-Methoxyestradiol induces differentiation of epithelial ovarian cancer-initiating cells sensitizing them to chemotherapy.

Abstract Ovarian cancer is the most deadly gynecological malignancy. Its lethality is mainly due to late diagnosis and acquisition of resistance to available therapies. Current evidence considers “tumor cell heterogeneity” as a cancer hallmark. A subset of cancer cells, named "cancer-initiating cells" (CICs), possesses stem cell-like capabilities allowing self-renewal, differentiation and dedifferentiation into other cell types, some of them evolved to a more invasive and resistant phenotype. Thus, the existence of CICs allows tumor to overcome therapy and regrowth as resistant kind over time. Finding therapeutic strategies effective against this subset of cells remain a major goal in cancer therapeutics. Previously we have shown that 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, induces cell death in ovarian cancer cells. Objective: In the present work we investigated the potential effects of 2-ME on CICs isolated from either ovarian cancer cell lines or primary tissue cultures obtained from ovarian cancer patients. Methods: CICs were isolated from both ovarian cancer cell lines (SKOV3, UCI 101, HEY) as well as primary tissue cultures established from ascites samples. Stemness, epithelial-mesenchymal transition (EMT), and anti-apoptotic markers were studied by immunoblotting (W-B), RT-PCR and immunohistochemistry in CICs cultured under stem-selection medium/non-attachment conditions and after treatment with different concentrations of 2-ME (5 to 10 uM, up to 24h). CICs treated under similar conditions were then exposed to different concentrations of chemotherapeutic reagents (i.e. paclitaxel, 0,5-5 uM, for up to 48 h). Cell death was measured by Tunel assay and PARP cleavage (measured by W-B). Results: Isolated CICs grow forming ovospheres that express higher levels of Oct-4, Nanog, Snail, N-Cadherin, CD44, CD133, survivin, and lower levels of E-cadherin compared with non-selected cell population (whole cell population, WP). In contrast to WP, CICs did not undergo apoptosis upon 2-ME exposure (5 uM for 24 h) measured by PARP cleavage. However, 2-ME treatment results in a decrease of Nanog, N-cadherin, Snail, Survivin and an increase in E-cadherin protein expression levels. In addition, a decrease in mRNA levels of Twist, Zeb2 (but not Zeb1) is observed under similar conditions (5 uM for 6 h). 2-ME pretreatment increases paclitaxel-induced cell death in CICs compared to those treated with paclitaxel alone, measured by Tunel assay. Conclusions: 2-ME induces the loss of stemness and EMT markers in epithelial ovarian CICs. This differentiating effect induced by 2-ME results in sensitization of this subset of resistant cells to chemotherapeutic agents. Further studies might be conducted to assess the clinical usefulness of this combination. (FONDECYT 1120292, and BMRC CTU06) Citation Format: Mauricio A. Cuello, Sumie Kato, Katherine Hormazábal, Lorena Abarzua, Loreto Bravo, Isabel Barriga, Nicanor Barrena, Jorge Brañes, Erasmo Bravo, Roger Gejman, Enrique Castellón, Gareth Owen. 2-Methoxyestradiol induces differentiation of epithelial ovarian cancer-initiating cells sensitizing them to chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3730. doi:10.1158/1538-7445.AM2013-3730

MicroRNA-155 is a novel suppressor of ovarian cancer-initiating cells that targets CLDN1

Previous cDNA microarrays indicated that CLDN1 (claudin-1) is an important gene for ovarian cancer-initiating cell (OCIC) invasion and adhesion. Here, we show that the downregulation of miR-155 in OCICs correlates with CLDN1 overexpression and the suppression of OCIC invasion. Luciferase assays indicate that miR-155 targets CLDN1 mRNA on the 3′ UTR. CLDN1 mRNA and claudin-1 protein expression were significantly decreased in miR-155-OCICs. Proliferation assays and Transwell migration assays show that miR-155 significantly suppresses the proliferative and invasive capacity of OCICs. Furthermore, miR-155 suppresses the growth of OCIC xenograft tumors. Thus, overexpression of miR-155 may prevent tumorigenesis in human ovarian cancer through downregulation of CLDN1.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.

MicroRNA‐199a targets CD44 to suppress the tumorigenicity and multidrug resistance of ovarian cancer‐initiating cells

In ovarian cancer, CD44(+) /CD117(+) stem cells, also known as cancer-initiating cells (CICs), are highly proliferative, have a low degree of differentiation, and are resistant to chemotherapeutics. Therefore, the CD44(+) /CD117(+) subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the role of microRNA-199a (miR-199a) in ovarian cancer stem cells. Luciferase reporter gene assays confirmed that miR-199a targets CD44 via an miR-199a-binding site in the 3'-UTR. CD44(+) /CD117(+) ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometric sorting. miR-199a was cloned and transfected into ovarian CICs. CD44 mRNA and protein expression was significantly decreased in miR-199a-transfected ovarian CICs as compared with miR-199a mutant-transfected and untransfected cells. Cell cycle analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide proliferation assays, the colony formation assay and the transwell migration assay indicated that miR-199a significantly affected cell cycle regulation and suppressed the proliferation and invasive capacity of ovarian CICs in vitro. miR-199a significantly increased the chemosensitivity of ovarian CICs to cisplatin, pacitaxel, and adriamycin, and reduced mRNA expression of the multidrug resistance gene ABCG2 as compared with miR-199a mutant-transfected and untransfected cells. The expression of stemness markers was also significantly reduced in miR-199a-transfected CICs as compared with miR-199a mutant-transfected and untransfected ovarian cells. Furthermore, xenograft experiments confirmed that miR-199a suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. Thus, expression of endogenous mature miR-199a may prevent tumorigenesis in human ovarian cancer by regulating expression of its target gene CD44.

Abstract 5206: Systems biology identification of processes/pathways facilitating the development of high-grade serous ovarian cancer

Abstract Previously, based on well-known stem cell phenotypes (sphere formation, potent tumorigenicity, embryonic gene expression, and chemoresistance), we used patient tumors to isolate high-grade serous (HGS) ovarian cancer-initiating cells (OCICs) that express the cell surface markers CD44 (a hyaluronate receptor) and CD117 (c-kit) (Zhang et al., 2008, Cancer Res 68:4311). Here, to identify biological pathways that underlie HGS ovarian tumorigenesis, including the specific role of OCICs in that process, we performed integrated analyses of global gene expression, DNA copy number, and DNA methylation patterns. To that end, OCICs and “bulk” (i.e., disaggregated but unsorted) tumor cells were compared to primary human normal ovarian surface epithelial (nOSE) cells that, along with fallopian tube epithelial (FTE) cells, represent hypothesized cells-of-origin for HGS pelvic cancers. Principle components analysis and unsupervised hierarchical clustering revealed distinct DNA methylation and gene expression profiles for replicate (n=3) samples of each cell type (i.e., OCIC, bulk tumor, and nOSE). Gene ontology and additional unsupervised analyses of “gene expression signatures” corresponding to the epithelial-to-mesenchymal transition (82 of 159 signature genes), TGF-beta (47 of 86 genes), and Wnt (75 of 151 genes) pathways demonstrated well-defined segregation of the three cell types, with mesenchymal and Wnt signaling hierarchies being nOSE > OCIC > bulk tumor cells. To examine early events in HGS ovarian tumorigenesis, previously published (Tone et al., 2008, Clin Cancer Res 14:4067) gene expression datasets were employed to show that nOSE, OCIC, and bulk ovarian tumor cell transcriptomes distinctly segregate from normal (FTE) and malignant fallopian epithelial transcriptomes. Interestingly, the largest divergence was between the nOSE and FTE gene expression profiles. Finally, DNA methylation and gene expression patterns were integrated, implicating specific networks/circuits in ovarian carcinogenesis. Taken together, these results support a mesenchymal-to-epithelial transition during ovarian tumorigenesis, reveal legitimate therapeutic targets (at the pathway level), and raise uncertainty that surface epithelial cells of the fallopian tube and ovary are reciprocal cells-of-origin for HGS pelvic carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5206. doi:1538-7445.AM2012-5206

Aberrant TGFβ/SMAD4 signaling contributes to epigenetic silencing of a putative tumor suppressor, RunX1T1 in ovarian cancer

Aberrant TGFβ signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGF-beta signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (P=0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (P<0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGFβ/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis.

Abstract 83: Hypomethylation of TGF-beta target gene, ABCA1 in ovarian cancer and cancer initialing cell and is associated with poor prognosis in cancer patients

Abstract Dysregulation of TGF-β signaling plays a crucial role in ovarian carcinogenesis and maintaining cancer stem cell properties. We have previously identified the TGF-β responsive targets in immortalized ovarian surface epithelial cells (IOSE) by expression microarray and found that ABCA1, a family members of ATP-binding cassette is up-regulated following TGF-β treatment (Qin et al, BMC Syst Biol 2009). Thus, we hypothesize that ABCA1 may be involved in ovarian cancer and its initiation. First, we compared the expression level of ABCA1 in IOSE and a panel of ovarian cancer cell lines and found that ABCA1 was up-regulated in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but down-regulated in A2780 and CP70 ovarian cancer cells. The down-regulation in A2780 and CP70 cells was associated with promoter hypermethylation as demonstrated by bisulphite sequencing and demethylation treatment. To investigate if ABCA1 is involved in ovarian cancer initiating cells (OCIC), we compared the methylation level of ABCA1 in ovarian tumor samples and their corresponding spheroids which is previously found to be enriched with OCIC. Lower methylation of ABCA1 could be detected in 2/6 OCIC samples as compared to their original tumors. We further analyzed the expression and methylation level of ABCA1 in CP70 spheroid (CP70sp) derived from suspension culture of CP70 ovarian cancer cells. Comparing to CP70, up-regulation of stem cell markers Nanog (3.8 fold) and Oct4 (12 fold) was detected in CP70sp cells suggesting that CP70sp may contain OCIC. Interestingly, up-regulation of ABCA1 (5.2 fold) concomitant with promoter hypomethylation was detected in CP70sp cells. Additionally, active demethylation may be involved in the hypomethylation of ABCA1 as expression of the methylcytosine dioxygenase, TET1 was up-regulated in the CP70sp cells (5.3 fold). Furthermore, ABCA1 was involved in drug resistance of ovarian cancer which is a feature of cancer initiating cells, as lenti-viral knockdown of ABCA1 in a platinum resistant MCP2 ovarian cancer cells resensitized the cells to cisplatin (IC50: shABCA1 vs shGFP: 0.267 ug/ml vs 0.402 ug/ml). We further analyzed the methylation level of ABCA1 in 97 ovarian cancer, 46 benign, and 4 normal samples using real-time quantitative MSP assay and found that higher methylation level of ABCA1 was detected in cancer than benign (P=0.051) and normal tissues (P&amp;lt;0.05). Importantly, cancer patients showing lower or no methylation of ABCA1 has significantly shorter progression free survival than patients with higher methylation (log rank, P=0.0463). In conclusion, ABCA1 is hypomethylated in a sub-set of ovarian cancer and its cancer initiating cells and is associated with poor prognosis in cancer patients. The role of ABCA1 in OCIC and the involvement of 5-hydroxymethylcytosine (5hmC) in the active demethylation of ABCA1 deserves further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 83. doi:10.1158/1538-7445.AM2011-83