Ovarian Cancer Stem Cell Markers Research Articles

Baicalin Attenuates YAP Activity to Suppress Ovarian Cancer Stemness.

PurposeThis study aims to reveal the mechanism underlying baicalin-suppressing ovarian cancer stemness.MethodsOVCAR-3 and the primary ovarian cancer cells were used for cell model. The ovarian cancer stem cells were isolated by suspension culture. Cell viability and clonogenicity were examined by CCK-8 assay and colony formation assay. The self-renewal of the cells was evaluated by the determination of sphere-forming capacity and the frequency of in vitro sphere-forming and in vivo tumor-initiating cells. The mRNA and protein levels were relatively quantified by qRT-PCR and Western blot. The transcription regulation of target genes was tested by luciferase reporter assay and a modified nuclear rn-on qRT-PCR assay.ResultsTreatment with a non-toxic dose of baicalin significantly inhibited the spherogenicity of ovarian cancer cells. Moreover, a non-toxic dose of baicalin treatment suppressed the frequency of sphere-forming and tumor-initiating ovarian cancer cells. Furthermore, the expression of ovarian cancer stem cell markers (CD133 and ALDH1A1) was inhibited by a non-toxic dose of baicalin treatment. Baicalin inhibits YAP activity and suppresses RASSF6, a positive regulator of YAP, at the transcriptional level. Overexpression of both YAP and RASSF6 abolished the inhibitory effect of baicalin on the proliferation and stemness of ovarian cancer cells.ConclusionThe results in this study demonstrated that baicalin suppresses the stemness of ovarian cancer cells by attenuating YAP activity via inhibiting RASSF6 at the transcriptional level. This finding revealed baicalin as a novel YAP inhibitor that could serve as an anti-cancer drug for eradicating ovarian cancer stem cells.

Metabolic Plasticity in Ovarian Cancer Stem Cells.

Ovarian Cancer is the fifth most common cancer in females and remains the most lethal gynecological malignancy as most patients are diagnosed at late stages of the disease. Despite initial responses to therapy, recurrence of chemo-resistant disease is common. The presence of residual cancer stem cells (CSCs) with the unique ability to adapt to several metabolic and signaling pathways represents a major challenge in developing novel targeted therapies. The objective of this study is to investigate the transcripts of putative ovarian cancer stem cell (OCSC) markers in correlation with transcripts of receptors, transporters, and enzymes of the energy generating metabolic pathways involved in high grade serous ovarian cancer (HGSOC). We conducted correlative analysis in data downloaded from The Cancer Genome Atlas (TCGA), studies of experimental OCSCs and their parental lines from Gene Expression Omnibus (GEO), and Cancer Cell Line Encyclopedia (CCLE). We found positive correlations between the transcripts of OCSC markers, specifically CD44, and glycolytic markers. TCGA datasets revealed that NOTCH1, CD133, CD44, CD24, and ALDH1A1, positively and significantly correlated with tricarboxylic acid cycle (TCA) enzymes. OVCAR3-OCSCs (cancer stem cells derived from a well-established epithelial ovarian cancer cell line) exhibited enrichment of the electron transport chain (ETC) mainly in complexes I, III, IV, and V, further supporting reliance on the oxidative phosphorylation (OXPHOS) phenotype. OVCAR3-OCSCs also exhibited significant increase in CD36, ACACA, SCD, and CPT1A, with CD44, CD133, and ALDH1A1 exhibiting positive correlations with lipid metabolic enzymes. TCGA data show positive correlations between OCSC markers and glutamine metabolism enzymes, whereas in OCSC experimental models of GSE64999, GSE28799, and CCLE, the number of positive and negative correlations observed was significantly lower and was different between model systems. Appropriate integration and validation of data model systems with those in patients’ specimens is needed not only to bridge our knowledge gap of metabolic programing of OCSCs, but also in designing novel strategies to target the metabolic plasticity of dormant, resistant, and CSCs.

Thy-1 predicts poor prognosis and is associated with self-renewal in ovarian cancer

BackgroundOvarian cancer is the leading cause of gynecologic cancer death in the United States despite effective first-line systemic chemotherapy. Cancer stem cells (CSCs) retain the ability to self-renew and proliferate and may be a means of harboring disease that evades standard treatment strategies. We previously performed a high-throughput screen to assess differential protein expression in ovarian CSCs compared to non-CSCs and observed that Thy-1 was more highly expressed in CSCs. Our primary aim was to validate Thy-1 (CD90) as a cancer stem cell (CSC) marker in epithelial ovarian cancer (EOC), correlate with clinical outcomes, and assess as a potential therapeutic target.ResultsKaplan Meier (KM) Plotter data were correlated with survival outcomes. Quantitative real-time PCR, flow cytometry, and immunoblots assessed RNA and protein expression. Limiting dilution assays assessed self-renewal capacity and proliferation assays assessed proliferative capacity. RNA in-situ hybridization was performed on patient specimens to assess feasibility. Thy-1 (CD90) is more highly expressed in ovarian CSCs than non-CSCs, in EOC compared to benign ovarian epithelium (P < 0.001), and is highest in serous EOC (P < 0.05). Serous ovarian cancers with high Thy-1 expression have poorer outcomes (median PFS 15.8 vs. 18.3 months, P = 0 < 0.001; median OS 40.1 v. 45.8 months, P = 0.036). Endometrioid ovarian cancers with high Thy-1 have poorer PFS, but no difference in OS (upper quartile PFS 34 v. 11 months, P = 0.013; quartile OS not reached, P = 0.69). In vitro, Thy-1 expression is higher in CSCs versus non-CSCs. EOC cells with high Thy-1 expression demonstrate increased proliferation and self-renewal. Thy-1 knockdown in EOC cells decreases proliferative capacity and self-renewal capacity, and knockdown is associated with decreased expression of stem cell transcription factors NANOG and SOX2. RNA in situ hybridization is feasible in ovarian cancer tissue specimens.ConclusionsThy-1 is a marker of ovarian CSCs. Increased expression of Thy-1 in EOC predicts poor prognosis and is associated with increased proliferative and self-renewal capacity. Thy-1 knockdown decreases proliferative and self-renewal capacity, and represents a potential therapeutic target.

Abstract NT-107: CHEMORESISTANT OVARIAN CANCER STEM CELLS REVEAL NOVEL THERAPEUTIC TARGETS

Abstract More than 60% of patients with ovarian cancer respond to first-line therapy, often achieving complete remission. However, these encouraging clinical outcomes are quickly overshadowed by high rates of treatment failure. More than half of advanced-stage patients relapse within five years, and these patients face poor prognoses. We sought to better understand the cause of ovarian cancer treatment failure with the hope of improving patient outcomes. We hypothesized that ovarian cancer stem cells may resist chemotherapy allowing them to regrow tumors causing relapse, metastasis and ultimately death. We further postulated that characterization of these cells would reveal therapeutic targets that may be exploited to achieve more durable remissions. To begin to test this hypothesis, we proposed that high aldehyde dehydrogenase I activity (ALDH1high) would identify stem-like ovarian cancer cells that would resist chemotherapy treatment and be capable of regenerating tumors. We compared ALDH1high cells to ALDH1low cells in three ovarian cancer cell lines: the human SKOV3 cell line, the rat FNAR-C1 cell line, and the murine ID8 cell line. Consistent with our predictions, we found that ALDH1high cells were resistant to several drugs commonly used to treat ovarian cancer. The ALDH1high population was also the only fraction of cells capable of forming tumors in vivo. In keeping with their proposed cancer stem cell phenotype, ALDH1high cells were smaller in size, relatively quiescent, and able to regenerate the phenotypic diversity of the cell line. Furthermore, they lacked contact inhibition and were capable of nonadherent growth. Significant controversy exists concerning how best to identify ovarian cancer stem cells. Many markers have been proposed with data to both support and refute their use in the literature. Resolving these uncertainties is hampered by the lack of systematic evaluation of each marker in proposed cancer stem cell pools. We used microarray analysis and quantitative real-time RT-PCR to measure the expression of ABCG2, CD24, CD44, CD133 and KIT in our ALDH1high cells compared to their ALDH1low counterparts and found no consistent differential regulation in the ALDH1high cells across all three cell lines. Further work remains needed to more clearly define the relationships between each of these proposed ovarian cancer stem cell markers. Gene expression profiles identified several possible vulnerabilities that could be exploited therapeutically. ALDH1high cells have upregulation of the mTOR pathway, FGF18 and CD47. They also express Her-2/neu at similarly high levels as ALDH1low cells. Drugs are already clinically available to target mTOR and Her-2/neu, which would speed their implementation. FGF18 signals through the FGFR3 receptor. Although FGF18 is upregulated in the cancer stem cell pool, both cancer stem and non-stem cells express its receptor, FGFR3, at similar levels. Several approaches to target FGFR3 are in clinical trials, as are antibodies targeting CD47. mTOR and CD47 inhibition would potentially be selective for ovarian cancer stem cells, but Her-2/neu and FGFR3 inhibition could also target the more differentiated, non-stem cancer cells. Further work is necessary to validate these treatment approaches. Citation Format: Allison C. Sharrow, Richard J Jones, Lily Wu. CHEMORESISTANT OVARIAN CANCER STEM CELLS REVEAL NOVEL THERAPEUTIC TARGETS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-107.

A Distinct Oncogenerative Multinucleated Cancer Cell Serves as a Source of Stemness and Tumor Heterogeneity.

The effects of anticancer treatments on cell heterogeneity and their proliferative potential play an important role in tumor persistence and metastasis. However, little is known about de-polyploidization, cell fate, and physiologic stemness of the resulting cell populations. Here, we describe a distinctive cell type termed "pregnant" P1 cells found within chemotherapy-refractory ovarian tumors, which generate and gestate daughter generation Gn cells intracytoplasmically. Release of Gn cells occurred by ejection through crevices in the P1 cell membrane by body contractions or using a funiculus-like structure. These events characterized a not yet described mechanism of cell segregation. Maternal P1 cells were principally capable of surviving parturition events and continued to breed and nurture Gn progenies. In addition, P1 cells were competent to horizontally transmit offspring Gn cells into other specific proximal cells, injecting them to receptor R1 cells via cell-cell tunneling. This process represents a new mechanism used by tumor cells to invade surrounding tissues and ensure life cycles. In contrast to the pregnant P1 cells with low expression of stem cell markers despite their physiologic stemness, the first offspring generations of daughter G1 cells expressed high levels of ovarian cancer stem cell markers. Furthermore, both P1 and Gn cells overexpressed multiple human endogenous retroviral envelope proteins. Moreover, programmed death-ligand 1 and the immunosuppressive domain of the retroviral envelope proteins were also overexpressed in P1 cells, suggesting effective protection against the host immune system. Together, our data suggest that P1 oncogenerative cancer cells exhibit a not yet described cell biological mechanism of persistence and transmission of malignant cells in patients with advanced cancers.Significance: P1 oncogenerative cell entities express low levels of CSC markers, which are characteristic of their histological origin. Cancer Res; 78(9); 2318-31. ©2018 AACR.

The enhanced delivery of salinomycin to CD133+ ovarian cancer stem cells through CD133 antibody conjugation with poly(lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles.

Ovarian cancer is the most lethal gynecologic malignancy, and ovarian cancer stem cells (CSCs) serve a pivotal function in the metastasis and recurrence of ovarian cancer. Multiple previous studies have validated CD133 as a marker of ovarian CSCs. Although salinomycin is a promising therapeutic agent that has been demonstrated to kill CSCs in various types of cancer, poor aqueous solubility hampers its clinical application. The present study used salinomycin-loaded poly(lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles conjugated with CD133 antibodies (CD133-SAL-NP) to eliminate CD133+ ovarian CSCs. The results revealed that CD133-SAL-NPs were of an appropriate size (149.2 nm) and exhibited sustained drug release. CD133-SAL-NPs efficiently bound to CD133+ ovarian cancer cells, resulting in an increased cytotoxic effect in CD133+ ovarian cancer cells, compared with the untargeted SAL-NPs and salinomycin. CD133-SAL-NPs reduced the percentage of CD133+ ovarian CSCs in ovarian cells more effectively than treatment with salinomycin or SAL-NPs, suggesting that CD133-SAL-NP targeted CD133+ ovarian CSCs. In nude mice bearing ovarian cancer xenografts, CD133-SAL-NPs exerted improved therapeutic effects compared with SAL-NPs and salinomycin. Thus, CD133 was demonstrated to be a promising target for drug delivery to ovarian CSCs, and may be useful as an agent to inhibit the growth of ovarian cancer by targeting CD133+ ovarian CSCs. CD133-SAL-NPs may therefore represent a promising approach for the treatment of ovarian cancer.

Abstract MIP-052: THE ROLES OF CD24 IN OVARIAN CANCER DEVELOPMENT

Abstract OBJECTIVES: CD24 is recently reported as ovarian cancer stem cell markers, and the expression of CD24 is increased from borderline tumors to invasive ovarian carcinomas. However, the underlying pathways controlled by CD24 in ovarian pathogenesis were poorly understood. We investigated the potential roles of CD24 in normal fallopian tube epithelial (FTE) cells and ovarian cancer development. METHODS: Lentiviral particles harboring CD24 shRNAs targeting CD24 expression were used to establish FTE cell lines and ovarian cancer cell lines with CD24 knockdown. The resulting cell lines were compared with the correspondent cells with non target shRNA control in colony formation assay, BrdU incorporation, cell growth and drug resistance. Western blot was used for analyzing downstream pathways affected by the knockdown of CD24. Expression of CD24 and its downstream effectors identified from western blot, including Pax 2 and Gli1 were studied in normal FTE, non–serous and serous tumor tissues by immunohistochemistry (IHC) to confirm whether the in vitro study is clinically relevant. RESULTS: Western blot analysis showed that CD24 expression was absent in normal ovarian surface epithelial (OSE) cells but was relatively high in normal FTE cells. In addition, serous cancer cell lines have a lower CD24 expression when compared to non–serous cancer cell lines. Knockdown of CD24 in FTE cells caused slower growth, lower ability in colony formation, reduced percentage of cells in S phase and decrease in expression of p–stat3 in JAK pathway and the JAK downstream targets Nanog and OCT3/4; and Pax2, which is normally expressed in FTE but lost in FTE secretory cell outgrowth (SCOUT). The loss of Pax2 in SCOUT is associated with serous carcinoma. IHC showed cytoplasmic co–localized expression of CD24 and Pax2 in the normal FTE tissue, suggesting the possibility of interaction between these two proteins in vivo. In contrast, knockdown of CD24 in ovarian cancer cells caused faster growth, higher ability in colony formation, increased percentage of cells in S phase, more resistant to carboplatin and higher expression of Shh and Gli1 in Sonic hedgehog (SHH) pathway. The above effects were more obvious in the non–serous cancer cell line. Activation of SHH pathway requires translocation of Gli1 into the nucleus. Concurrent with the in vitro results, IHC showed that the expression of Gli1 was found inside the nucleus in the non–serous tumor tissues but in the cytoplasm in serous–tissue, suggesting that the effect brought by SHH pathway is more prominent in the non–serous cancer cells. CONCLUSION: Our study is the first study revealing the linkage between CD24 and PAX2, and suggesting normal FTE cells depends on CD24/JAK pathway for growth and the loss of CD24 expression and hence, the loss of PAX2 may be associated with serous type tumor development, basing on other studies that SCOUTs were significantly associated with serous carcinoma. Moreover, the study also suggests CD24 pathway affects Type I and Type II (HGSOC) tumors differently. The CD24/SHH pathways may be responsible in controlling cell growth and drug resistance in ovarian cancer cells, hence, warrant further studies. Citation Format: Pui–Wah Choi, Brooke E. Howitt, Christopher P. Crum, Ross S. Berkowitz, and Shu–Wing Ng. THE ROLES OF CD24 IN OVARIAN CANCER DEVELOPMENT [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 MIP-052.

The inhibitory effect of hypoxic cytotoxin on the expansion of cancer stem cells in ovarian cancer

While an increase in progression free survival time is seen when an angiogenesis inhibitor is used in the treatment of high-relapse rate ovarian cancer, it has little effect on overall survival. A possible cause of treatment-resistance to angiogenesis inhibitors is the growth of stem cells in a hypoxic microenvironment built inside the tumor tissue by angiogenesis inhibition. In this study, we examined the possible suppression of stem cell and cancer stem cell (CSC) expansion by hypoxic cytotoxin, TX-402.TX-402, an analogue of tirapazamine, has been developed as a hypoxia selective prodrug with inhibitory effects of HIF-1 and angiogenesis. We considered TX-402 as a possible molecular-target drug candidate for ovarian cancer due to its inhibition of CSC expansion. In this study, we found that the expressions of HIF-1α and HIF-2α were increased under hypoxia in serous ovarian cancer cell lines. The expressions of HIF-1α and HIF-2α induced under hypoxia were repressed by TX-402 in a dose-dependent manner. Next, we investigated the effects of hypoxia on the expression levels of stem cell factors, Oct4, Nanog, Sox2 and Lin28, and showed that their expressions were induced by hypoxia. It was also observed that the expressions of putative ovarian cancer stem cell markers, CD133 and CD44 were induced under hypoxia. Furthermore, TX-402 was found to dose-dependently inhibit the expressions of CSC markers and stem cell factors.Oct4 expression was repressed by HIF-2α silencing, but not by HIF-1α silencing, indicating that TX-402 may repress the expression of Oct4 by inhibiting HIF-2α.We constructed CaOV3 spheroids as a 3-dimensional hypoxia model, in which the internal hypoxic region contained CSC-like cells expressing Oct4. The internal hypoxic region, which contained Oct4 expressing cells, disappeared following TX-402 treatment.In conclusion, hypoxia promoted the expansion of CSCs expressing CD133 and CD44 accompanied by an increase of stem cell factors. Its inhibition of hypoxia-induced CSC expansion makes TX-402 promising agent usable in combination for ovarian cancer therapy.

Targeting platinum resistant disease in ovarian cancer.

Ovarian cancer is an extremely aggressive disease in which the vast majority of patients face a very poor prognosis. Although most patients initially respond to current chemotherapeutic regimens that include a combination of platinum- based therapy (cisplatin/carboplatin) and paclitaxel, the vast majority of them quickly relapse and develop increased resistance to available treatments. Thus, intrinsic and acquired chemotherapy resistance is a major obstacle in the treatment of ovarian cancer patients. Consequently, the priorities for basic and translational ovarian cancer research need to include the identification of novel therapies directed against key molecular targets and signaling pathways in platinum resistant disease. At the same time, we need to develop novel systems for drug delivery aimed at increasing the efficacy and reducing the toxicity of platinum-based treatments. Improving the current responses to platinum chemotherapy is critical not only for achieving a better outcome clinically, including a longer survival, but also for allowing patients to have a better quality of life while in treatment.

Ovarian cancer stem cell markers: Prognostic and therapeutic implications

Cancer stem cells are rare chemotherapy resistant cells within a tumor which can serve to populate the bulk of a tumor with more differentiated daughter cells and potentially contribute to recurrent disease. Ovarian cancer is a disease for which at the time of initial treatment we can obtain complete clinical remission in the majority of patients. Unfortunately, most will relapse and succumb to their disease. This clinical course is in line with the cancer stem cell model. In the past 5years a significant amount of work has been done to identify cells with characteristics of ovarian cancer stem cells. This review will focus specifically on the markers used to define human ovarian cancer stem cells, the prognostic implications of the expression of these cancer stem cell markers in patient’s primary tumors, and the potential of these cancer stem cell markers to serve as therapeutic targets.

Description of the CD133+ subpopulation of the human ovarian cancer cell line OVCAR3

Cancer stem cells (CSCs) form a very rare population within tumors and possess the ability to proliferate and self-renew indefinitely. The cluster of differentiation (CD) 133+ ovarian CSCs (OCSCs) have been identified recently and their clinical implications are about to be clarified. In this context, we use the CD133 antigen as a marker of OCSCs in OVCAR3 cells and show that microRNAs (miRNAs) are aberrantly expressed in this subpopulation. The OCSCs in the OVCAR3 cell line were identified by the monoclonal mouse anti-CD133-1 antibody (clone CD133). Microarray and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were used to identify miRNAs with altered expression in CD133+ cells. The expression levels of dysregulated miRNAs, namely, miR-204, miR-206, miR-223, miR-9, miR-100, and miR-200c, were examined using TaqMan PCR. The RNA and protein levels of stem cell-specific genes were examined by real-time RT-PCR and western blot analyses. Our results of microarray and real-time RT-PCR analyses revealed distinct miRNA expression profiles between CD133+ and CD133- OVCAR3 cells. The expression of stem cell-specific genes, namely, Oct3/4, Sox2, and Nanog, was higher in CD133+ cells than in CD133- cells while that of FoxD3, was lower in CD133+ cells than in CD133- cells. In conclusion, our data indicate that CD133 expression defines a tumor-initiating subpopulation of cells in the OVCAR3 cell line. The overall miRNA expression profile of CD133+ OVCAR3 cells was clearly distinct from that of CD133- OVCAR3 cells, indicating that miRNAs are involved in the development of this neoplasia and may serve as pertinent chemotherapeutic targets.

Abstract 10: Characterization of ovarian CSC using ALDH and CD133 identifies a cancer stem cell hierarchy

Abstract Recent studies in ovarian cancer suggest that the protein CD133 may be a marker of cancer stem cells (CSC). CSC studies in several solid tumors have identified Aldehyde dehyrogenase (ALDH) enzymatic activity as a CSC marker, however ALDH has not been studied in ovarian cancer. We sought to determine if ALDH alone or in combination with CD133 could better define CSC in ovarian cancer. We analyzed the expression of these markers in 13 consecutive primary human ovarian tumor specimens and 8 cell lines. 70% of primary ovarian tumors analyzed had CD133+ cells detectable in low number in, and many tumor cell lines lacked CD133 expression. In contrast, 100% of the primary human ovarian tumor specimens and ovarian cancer cell lines demonstrated ALDH activity. ALDH+ cells isolated from 6 ovarian cancer cell lines preferentially grew larger tumors at a faster rate compared to ALDH− cells. In some cases ALDH− cells were incapable of generating tumors, suggesting ALDH is a good marker of ovarian CSC in cell lines. Importantly, as few as 1000 ALDH+ cells directly isolated from human ovarian tumors were capable of generating tumors in immune deficient mice. ALDH− cells did not form tumors in mice. Interestingly, when ALDH was used in combination with CD133 to analyze ovarian cancer cell lines we observed greater growth in the ALDH+CD133+ cells compared to ALDH+CD133− cells suggesting a potential hierarchy of the stem cells. Consistent with this, as few as 11 ALDH+CD133+ cells freshly isolated from human tumors were capable of forming tumors in mice. Consistent with a stem cell hierarchy, tumors formed from ALDH+CD133+ cells demonstrated a poorly differentiated histology, whereas tumors formed from ALDH+ cells alone demonstrated a more differentiated histology. Finally analysis of tumors generated from ALDH+ and ALDH+CD133+ tumors demonstrated a significant increase in microvascular density compared to ALDH− tumors. qRT-PCR analysis demonstrated that ALDH+ cells compared to ALDH− cells preferentially express numerous angiogenic factors. Taken together our studies: (1) indicate ALDH as a marker of ovarian CSC, (2) suggest a hierarchy of ovarian cancer stem cell differentiation state, and (3) demonstrates that ovarian CSC are highly angiogenic to recruit vasculature to create a stem cell niche. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 10.