Lipid Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells

OBJECTIVES: Lack of sensitive single-cell analysis tools has limited the characterization of the metabolic activity of cancer stem cells (CSCs). The objectives of this study were to identify and target metabolic pathways specifically deregulated in ovarian CSCs. METHODS: We developed a new technique based on hyperspectral stimulated Raman scattering imaging of single living cells coupled with electrospray ionization mass spectrometry (ESI-MS) analysis of extracted lipids to identify unique metabolic spectra of ovarian CSCs. SiRNA knockdown and pharmacological inhibitors were used to block fatty acid desaturases and to measure effects on the ovarian CSCs charcateristics. RESULTS: We report significantly increased levels of unsaturated lipids in flow-sorted ovarian CSCs (ALDH+CD133+) compared to non-CSCs (ALDH-CD133-). Higher lipid unsaturation levels were also detected in CSC-enriched spheroids compared to monolayer cultures of ovarian cancer cell lines or primary cells derived from human ovarian tumors. ESI-MS identified that the elevated unsaturation in lipid droplets involved an enhanced conversion from saturated fatty acids (18:0) to unsaturated fatty acids (18:1). Expression of fatty acid desaturases was increased in ovarian CSCs compared to non-CSCs. Pharmacological inhibition or siRNA knockdown of lipid desaturases Δ9 (SCD1) or Δ6 effectively eliminated CSCs, suppressed sphere formation in vitro, and blocked tumor initiation capacity in vivo, as measured by a tumor initiation dilution assay. Mechanistically, the lipid desaturase inhibitors suppressed ovarian cancer cell stemness by blocking the NF-κB survival pathway. Chromatin immunoprecipitation and overexpression of RelA demonstrated that NF-κB transcriptionally regulates SCD1 expression. Collectively, our data suggest that a positive feedback loop involving lipid desaturases, NF-κB, and the stem cell marker ALDH1 can be effectively blocked by SCD1 inhibitors, suppressing ovarian CSCs. CONCLUSIONS: These results demonstrate that increased lipid unsaturation is a novel metabolic marker for ovarian CSCs and a target for CSC-specific therapy. Citation Format: Junjie Li, Salvatore Condello, Jessica Thomes-Pepin, Xiaoxiao Ma, Yu Xia, Thomas D. Hurley, Ji-Xin Cheng, Daniela Matei. LIPID DESATURATION IS A METABOLIC MARKER AND THERAPEUTIC TARGET OF OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP21.

Ovarian cancer (OC) is the most lethal gynecologic cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The metastatic progression and post-chemotherapy recurrence of OC is linked to a small population of cancer stem cells (CSCs) detectable as ALDH+/CD133+ phenotype. In OC, the tumor microenvironment (TME) provides a favorable milieu that protects quiescent CSCs during chemotherapy and supports their tumorigenic functions. The multifunctional protein tissue transglutaminase (TG2), with enzymatic and scaffold functions, is an important molecule secreted in the TME where it modulates oncogenic signaling by interacting with extracellular matrix (ECM) components, such as fibronectin (FN) and integrins. Its aberrant expression is correlated with OC progression and with the tumorigenic OCSC phenotype. Here, we hypothesized that the formation of the TG2/FN/integrins ternary complex at the cell membrane promotes survival of CSCs and supports OC spheroid formation by regulating the Wnt/beta-catenin pathway. Our data demonstrate that TG2, FN, and integrin β1 mRNA expression is markedly increased in OCSCs (ALDH+/CD133+) compared with non-CSC (ALDH-/CD133-) and highly enriched in OC cells grown as spheroids compared with monolayers (p<0.01). Immunofluorescence (IF) staining showed abundant complexes of TG2 with integrin β1 and FN in OC cells grown as spheroids, and the protein-protein interactions were detected in human tumors by proximity ligation assay. TG2 expression was strongly correlated with ITGB1 (R=0.23, P<0.0001) and with FN1 (R=0.39, P<0.0001) in the TCGA ovarian cancer database. Use of function-inhibiting antibody against the FN-binding domain of TG2 (4G3) dramatically suppressed spheroid proliferation, TG2/FN/integrin complex formation, and tumor growth in a xenograft model. Disruption of TG2/FN complex altered key oncogenic signaling pathways, and in particular blocked the canonical Wnt/β-catenin signaling pathway essential to sustaining cancer cell stemness. The Wnt receptor Frizzled genes (Fzd1, Fzd7 and Fzd8) showed the greatest difference in gene expression, indicating a direct correlation between TG2/FN complex and Wnt pathway activation. Co-immunoprecipitation and IF staining demonstrated that TG2 co-localizes and directly binds to Fzd7. Furthermore, protein docking and peptide inhibition demonstrated that the interaction between TG2 and Fzd7 overlapped with the FN-binding domain of TG2. Finally, blockade of TG2/FN complex and shRNA-mediated Fzd7 knockdown attenuated spheroids proliferation and targeted gene expression induced by Wnt3a/7a. This study provides strong evidence supporting a key function of TG2/FN complexes in OCSCs. By demonstrating that TG2 directly binds Fzd7, we identified a novel function of TG2 and a new mechanism promoting CSCs’ proliferation and tumorigenicity. These results point to TG2/FN clusters or the newly discovered TG2/Fzd7 complex as potential new therapeutic CSC targets. Citation Format: Salvatore Condello, Livia Sima, Cristina Ivan, Horacio Cardenas, Gary Schiltz, Rama Mishra, Daniela Matei. Tissue transglutaminase/Frizzled receptor clusters regulate WNT transcriptional activity in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B56.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors and plays important roles in tumorigenesis. Amplification and overexpression of FAK have been observed in aggressive human cancers including ovarian and breast cancers. Defactinib is a potent, selective, and orally active FAK inhibitor with demonstrated tolerability and preliminary clinical activity as a single agent in a Phase 1 clinical trial. In an ongoing Phase 1/1b clinical trial (NCT01778803), we have demonstrated that defactinib can be combined with paclitaxel and have observed preliminary signs of clinical activity. We report here preclinical data supporting the clinical testing of defactinib in combination with paclitaxel in patients with ovarian cancer. In human ovarian cancer cell lines TOV-21G and OV-7, defactinib was found to enhance the efficacy of paclitaxel. Combination Index analyses demonstrated synergistic inhibition of tumor cell proliferation/survival with combination of defactinib and paclitaxel. We find that FAK inhibitors preferentially target breast and ovarian cancer stem cells (CSCs) relative to bulk tumor cells as evidenced by a diminished proportion of CSCs in multiple orthogonal CSC assays. In direct contrast, the standard-of-care cytotoxic agents, paclitaxel and carboplatin, increased the percentage of CSCs, indicating that these agents do not effectively target CSCs. Importantly, combination of FAK inhibitors with cytotoxic agents attenuated the enrichment of CSCs induced by the cytotoxic agents. We subsequently determined if defactinib reduces CSCs in tumor specimens from ovarian cancer patients who were previously treated with taxane and platinum. Ex vivo treatment of ovarian tumor tissue fragments with defactinib reduced the percentage of CSCs as assessed by CD24hi/CD117hi CSC markers and the Aldefluor assay. To assess drug effects on tumor-initiating capability, the ‘gold standard’ for CSC activity, TOV-21G cells were pretreated in vitro with the FAK inhibitor, paclitaxel or the combination of both agents and 1,000 of the resulting cells were injected into immunodeficient mice. The FAK inhibitor alone or in combination with paclitaxel prevented tumor initiation, while paclitaxel alone did not reduce tumor initiation, suggesting that treatment with a FAK inhibitor could effectively eliminate tumor initiating CSCs. In summary, our data indicate that defactinib preferentially targets cancer stem cells and enhances the activity of paclitaxel in preclinical models of ovarian cancer. These results provide additional support for the current clinical development of defactinib in combination with paclitaxel for the treatment of ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C271. Citation Format: Christian M. Vidal, Vihren N. Kolev, Quentin G. Wright, Winnie F. Tam, David T. Weaver, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. FAK inhibitor defactinib (VS-6063) enhances the efficacy of paclitaxel and preferentially targets ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C271.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Purpose: While chemotherapy may succeed initially at decreasing the number of ovarian cancer (OC) cells, it leaves behind tumors enriched in ovarian cancer stem cells (OCSCs), which most likely drive chemoresistance and tumor relapse. An emerging model indicates that non-OCSCs may dedifferentiate and acquire stem cell properties under certain circumstances. Recent studies in OC implicate a critical role for aberrant cytokine interleukin-6 (IL-6) secretion and the IL-6 signaling pathway may play a critical role in converting non-CSCs to CSC. In the current study, we investigated the mechanism contributing to OCSC enrichment after platinum treatment in vivo. We hypothesize that platinum-induced IL-6 secretion activates STAT3 signaling-mediated expression of the cancer stem cell marker ALDH1A1, resulting in CSC enrichment. In addition, based on our previous study in OCSCs (Wang et al., 2014, Cancer Res.), we sought to examine the mechanism by which guadecitabine (SGI-110), a second generation hypomethylating agent HMA, inhibits OCSCs in OC xenograft residuals after platinum treatment. Methods: OC cells (OVCAR4 and A2780) were cultured alone or co-cultured with normal omental fibroblasts (NOFs) or guadecitabine (100nM, 3days)-treated NOFs in the starving condition for 24h and then treated with 3h cisplatin (CDDP; respective IC50 doses of 27.4 and 14.7μM), guadecitabine, IL-6 (100ng/ml), neutralizing antibody (Nab) against IL-6 (IL-6-Nab, 200ng/mL) or IL-6+IL-6-Nab. IL-6 secretion levels were measured by ELISA. FACS was used to analyze OCSC population marked as aldehyde dehydrogenase (ALDH)+ cells, and western blot was used to examine ALDH1A1 and pSTAT3 levels in cells treated as described above. An ALDH1A1 promoter-luciferase reporter (pGL3-ALDH1A1-Luc; -1 to -1031) assay was used to determine whether IL-6 transactivates ALDH1A1 expression in OC cells. OC cell migration under the described conditions was determined by transwell migration assays. Results: We found that CDDP induced (P<0.05) IL-6 secretion by OCs up to 48h post-treatment and up to 96h by NOFs or by co-cultured cells, suggesting a role of the tumor microenvironment in platinum-induced IL-6 secretion. Guadecitabine inhibited (P<0.05) CDDP-induced IL-6 secretion by NOFs alone or in co-culture with OVCAR4, suggesting an inhibitory role of the HMA on IL-6 signaling. By assaying FACS sorted ALDH+/- cells, we determined that ALDH+ cells express increased (P<0.05) levels of the IL-6 receptor and secrete higher (P<0.05) levels of IL-6 in the conditioned media compared to ALDH- cells. Treatment of OC cells with IL-6 enriched the percentage of ALDH+, which was inhibited by IL-6-Nab. Luciferase assay results revealed that IL-6 transactivated (p<0.05) ALDH1A1 reporter gene expression in ALDH- cells, which was blocked by IL-6-Nab. Further, we observed that IL-6- or CDDP-treated OC cells increased (P<0.05) ALDH1A1 and pSTAT3 protein expression, and guadecitabine treatment inhibited (P<0.05) expression of ALDH1A1 and pSTAT3 in OCs. ALDH+ cells showed greater migration potential than ALDH- cells, and guadecitabine inhibited (P<0.05) migration of both ALDH+ and ALDH- cells. Guadecitabine, alone or combination with IL-6-Nab, inhibited (P<0.05) CDDP-induced OVCAR4 migration in co-culture with NOFs. Conclusion: Our data indicate that IL-6 is a potent regulator of ALDH1A1 expression and the OCSC phenotype and further suggest an inhibitory effect of guadecitabine on the conversion of non-OCSCs to OCSCs. The data support a role for IL-6 in OCSC enrichment after platinum treatment and suggest that a combination approach of IL-6 neutralizing antibody with guadecitabine could represent a novel maintenance strategy after chemotherapy for eradicating OCSCs and preventing tumor recurrence. Citation Format: Yinu Wang, Anirban Kumar Mitra, Kenneth P. Nephew. Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A57.

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.

Anaplastic thyroid cancer (ATC) is one of the most aggressive solid cancers in humans, with limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity contributes to therapeutic resistance and recurrence of ATC. We show that the expression of the endogenous thyroid hormone receptor β gene (THRB) is silenced in ATC and demonstrate that the exogenously expressed TRβ suppresses CSC activity. Decitabine is one of the demethylation agents to treat myelodysplastic syndrome and acute myeloid leukemia patients and is currently in clinical trials for hematopoietic malignancies and solid tumors. We aim to show that the re-expression of the endogenous THRB gene by decitabine can attenuate CSC activity to block ATC tumor growth. We treated ATC cell lines derived from human ATC tumors (11T and 16T cells) with decitabine and evaluated the effects of the reactivated endogenous TRβ on CSC activity in vitro and in vivo xenograft models. We found that treatment of 11T and 16T cells with decitabine reactivated the expression of endogenous TRβ, as evidenced by western blot and immunohistochemical analyses. The expressed TRβ inhibited cell proliferation by arresting cells at the S phase, increased apoptotic cell death by upregulation of cleaved caspase-3, and markedly suppressed the expression of CSC regulators, including cMYC, ALDH, SOX2, CD44, and β-catenin. Decitabine also inhibited xenograft tumor growth by suppressing CSC activity, inhibiting cancer cell proliferation, and increasing apoptosis. Our findings suggest that re-expression of the endogenous TRβ is a novel therapeutic approach for ATC via suppression of CSC activity.

OBJECTIVE: Cancer stem cells (CSCs) represent a subpopulation of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance, drug resistance and metastasis of tumors. CSCs involved in drug resistance and relapse of cancers can significantly affect ovarian cancer therapy. The antibiotics salinomycin has recently been shown to be a potent compound to deplete chemoresistant cells in adenocarcinoma. The objective of the present study was to evaluate the effect of salinomycin on ovarian CSC whereby salinomycin mono-and combination treatment with paclitaxol regimend were analyzed. METHODS: The CD44+CD117+CSCs were isolated from the primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer by using immune magnetic-activated cell sorting system. To evaluate the effect of salinomycin on ovarian CSC, cells were treated in single and combined treatment. The expression was Nanog, Oct3/4, Sox2 and ABCG2 mRNA was determined by RT-PCR and protein expression was detected by Western blot analysis. The cell viability assay, gelatin zymography and apoptosis assay were applied to evaluate the effects of salinomycin compared with parental tumor cells and CSCs. RESULTS: The combination of salinomycin with paclitaxel (PTX) was enhanced change cell viability or activating apoptosis in CD44+CD117+ cells, whereas the salinomycin monotreatment did not cause significant changes. Salinomycin treatment reduced stemness gene expression and suppressed invasion of CSCs. CONCLUSIONS: Based on our findings, we concluded that anti-cancer effect of salinomycin with PTX reduced stemness and induced apoptosis in ovarian cancer and cancer stem cells. Citation Format: So-Jin Shin, Jin-Young Kim, Hyun-Gyo Lee, Eun-Ji Nam, Chi-Heum Cho. Salinomycin have antiproliferative and apoptotic effects on ovarian cancer stem-like cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2014-217

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors and plays important roles in tumorigenesis. Amplification and overexpression of FAK have been observed in aggressive human cancers including ovarian and breast cancers. VS-6063 and VS-4718 are potent, selective, and orally active FAK inhibitors. In a Phase 1 clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. In addition, we have demonstrated that VS-6063 can be combined with paclitaxel and preliminary signs of clinical activity, including CA-125 reduction to normal levels, were observed with this combination in patients with ovarian cancer. VS-4718 was shown to inhibit tumor growth and metastasis in preclinical tumor models including an ovarian cancer xenograft model and is currently under evaluation in a Phase 1 clinical trial. We have shown previously that inhibition of FAK preferentially targets cancer stem cells in breast cancer models. We extended these studies and report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs) in ovarian cancer models. VS-4718 and VS-6063 were first evaluated in several orthogonal cancer stem cell assays in vitro. Treatment of OVCAR-8 or TOV-21G human ovarian cancer cells with these FAK inhibitors in matrigel reduced the percentage of Aldefluor+ CSCs and attenuated tumorsphere forming efficiency. Accordingly, in a side population (SP) assay assessing CSCs by Hoechst dye exclusion, these FAK inhibitors reduced SP CSCs in the OVCAR-5 ovarian cancer cell line. Similar effects were observed with the breast cancer cell lines SUM159 and MDA-MB-231. In direct contrast, the standard-of-care cytotoxic agents, paclitaxel and carboplatin, increased the percentage of CSCs, indicating that these agents do not effectively target CSCs. Importantly, combination of VS-6063 or VS-4718 with cytotoxic agents attenuated the enrichment of CSCs caused by cytotoxic agents as measured by the Aldefluor assay and in vivo tumor initiating potential. To assess drug effects on tumor-initiating capability, the clearest arbiter of CSC activity, TOV-21G cells were pre-treated in vitro with various compounds and 1,000 of the resulting cells were injected into immunodeficient mice. VS-4718 alone or VS-4718 in combination with paclitaxel prevented tumor initiation, while paclitaxel alone did not reduce tumor initiation, suggesting that treatment with VS-4718 could effectively eliminate tumor initiating CSCs. Furthermore, ex vivo treatment of primary human ovarian cancer tissue with VS-4718 or VS-6063 also reduced the percentage of CSCs as assessed by CD24hi/CD117hi CSC markers and the Aldefluor assay. In addition to targeting CSCs, we also observed that FAK inhibitors enhanced the activity of paclitaxel to reduce bulk tumor cells both in vitro and in vivo. In summary, our data indicate that the FAK inhibitors VS-6063 and VS-4718 preferentially target cancer stem cells and enhance the activity of paclitaxel. These results provide rationale for the clinical development of Verastem's FAK inhibitors for the treatment of ovarian cancer. Citation Format: Vihren N. Kolev, Quentin G. Wright, Christian M. Vidal, Winnie F. Tam, Mahesh V. Pavdal, Qunli Xu, Jonathan A. Pachter. FAK inhibitors VS-6063 and VS-4718 preferentially target ovarian cancer stem cells. [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 A39.

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.

Background: Although metformin, a first-line drug for treating diabetes, may play an important role in inhibition of epithelial ovarian cancer cell growth and cancer stem cells (CSCs), metformin at low dose showed less effect on proliferation of ovarian cancer cells. In this study, we evaluated the effect of metformin at low dose on ovarian CSCs in order to understand the molecular mechanisms underlying ovarian tumorigenesis. Methods: The inhibitory effects of metformin at los dose on proliferation and population of ovarian cancer cells including SKOV3 and A2780 were assessed by cell proliferation assay and flow cytometry. Quantitative real-time PCR assays on expression of Bcl-2, Survivin and Bax were performed to determine the effect of the metformin at low dose on epithelial-mesenchymal transition (EMT) of cancer cells and CSCs. Tumor sphere formation assay was also performed to evaluate the effect of metformin on spheres forming ability of CSCs. The therapeutic efficacy and the anti-CSC effects of metformin at low dose were investigated by using both SKOV3 cells and primary tumor xenografts. In addition, the CSC frequency and EMT in tumor xenograft models were also assessed by flow cytometry and quantitative real-time PCR. Results: Metformin at low dose did not affect proliferation of ovarian cancer cells, however, it inhibited the population of the CD44+/CD117+ selectively, neither CD133+ nor ALDH+ cells. It suppressed expression of snail2, twist and vimentin significantly in cancer cells and CD44+/CD117+ CSCs in vitro. Low dose of metformin reduced survivin expression in CSCs leading to arrest of cell cycle. Metformin at low concentration inhibited the secondary and the tertiary tumor sphere formation, decreased the growth of either SKOV3 or primary ovarian tumor xenograft, enhanced the anticancer effect of the cisplatin, and lowered the proportion of the CD44+/CD117+ CSCs in the xenograft tissue. Metformin was also associated with a reduction of snail2, twist, and vimentin in CD44+/CD117+ ovarian CSCs in vivo. Conclusions: Our results implicate that metformin at low dose inhibits selectively CD44+/CD117+ ovarian CSCs through inhibition of EMT and potentiates the effect of the cisplatin. Citation Format: Gary G. Xiao, Rongrong Zhang, Ping Zhang, Hong Wang, Dongming Hou, Wentao Li, Chenwei Li. Inhibitory effects of metformin on epithelial-mesenchymal transition of CD44+/CD117+ ovarian cancer stem cells. [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 4734.

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.

Ovarian cancer is the most lethal gynecologic malignancy and the 5th leading cause of cancer related deaths among women in the USA. Most patients eventually succumb to chemoresistant disease and the purpose of this study is to understand the mechanism of development of chemoresistance and disease relapse. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are resistant to cytotoxic chemotherapy and cause relapse. The tumor microenvironment can potentially provide an optimal cancer stem cell niche for cancer stem cell growth and maintenance. Cancer associated fibroblasts (CAFs) are one of the main constituents of the tumor microenvironment in ovarian tumors, promoting tumor progression and chemoresistance. We have studied the potential role of CAFs in maintaining CSC population and enhancing chemoresistance with an objective to develop effective approaches to overcome chemoresistance and tumor relapse. Co-culture of high grade serous ovarian cancer cells with CAFs resulted in increased resistance to carboplatin. ALDH1A1 is a well-established marker for ovarian cancer CSCs and the ALDH+ population was significantly increased upon co-culture with CAFs. Similarly, CAFs also enhanced spheroid formation of ovarian cancer cells seeded in ultralow adhesion plates in CSC medium. Interestingly, co-culturing ALDH- ovarian cancer cells with CAFs resulted in the induction of ALDH+ cells within 6 days. Analysis of the signaling pathways activated in ovarian cancer CSCs and the gene expression profiles of ovarian cancer CAFs indicated the potential role of Wnt signaling in the productive cross-talk between CAFs and ovarian cancer CSCs. Treatment with Wnt inhibitors abrogated the induction of CSCs by CAFs. By selectively silencing porcupine, a protein involved in Wnt ligand lipidation and secretion, we further confirmed that CAF derived Wnts are responsible for the induction of CSC. Studies are ongoing to identify the specific Wnt ligand involved in the cross-talk and downstream pathways activated during CSC induction and maintenance by CAFs. Our results indicate that CAF-derived Wnt ligands are instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse. Citation Format: Yiming Fang, Mohamed A. Abd El Aziz, Kartikeya Tiwari, Anirban K. Mitra. Cancer associated fibroblasts promote ovarian cancer chemoresistance by inducing cancer stem cells through Wnt signaling [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 4685.

Many antibiotics produced by bacteria also have antineoplastic activity. Examples of such bacterial compounds include actinomycin D, doxorubicin, mitoxantrone, bleomycin, mitomycin, etoposide, and others. Recently, it was identified that Xenorhabdus budapestnesis (Xbu) produces a secondary metabolite, fabclavine, that has antibiotic property. Here, for the first time we demonstrate that fabclavine exhibits potent cytotoxic activity against ovarian cancer cells and ovarian cancer stem cells and demonstrate its mechanism of action, Fabclavine was isolated and purified from cell-free supernatants of Xbu cultures and analyzed by mass spectrometry. Proliferation assays conducted on several ovarian cancer cell lines Ovcar3, OVCA433, ID8, and breast cancer cell lines MCF7 and 4T1 demonstrate that fabclavine inhibits the cancer cell proliferation at nanomolar (250-400nM) concentration. Fabclavine is ten times more potent than cisplatin in inhibiting cell proliferation of ovarian cancer cell line OVCA33 and breast cancer cell line MCF7. We also tested the effect of fabclavine in 3D culture of ovarian cancer cell lines. The results indicate, even in 3D culture, fabclavine killed 40-50% of the cells at 300nM concentration. In addition, fabclavine was also tested against ovarian cancer stem cells isolated from five ovarian cancer patients and grown in 3D culture. Fabclavine inhibited the growth of stem cells by 70-85%. In cancer cell lines fabclavine induced apoptosis in 20-30% cells after 24-hour treatment. Caspase activity assay for caspase3, caspase8, and caspase9 shows a significant increase in caspase3 and caspse8 activity with no change in caspase9 activity, suggesting an activation of the extrinsic apoptosis pathway in the fabclavine-treated cells. This observation was supported by the increase in phospho-FADD upon exposure to fabclavine. When conjugated to agarose beads, fabclavine efficiently precipitated two death receptors, DR4 and DR5, from ovarian and breast cancer cell extracts. These experiments indicate that fabclavine is a novel DR4/5 ligand and should therefore be considered as a death receptor-targeting agent for the treatment of ovarian and other tumors. We will present data on the comparison of DR4/5 binding to fabclavine versus its natural ligand, TRAIL, and will also present data from PDex and mouse xenograft models on the ex vivo and in vivo effects of fabclavine on the proliferation of ovarian tumors and cancer stem cells. Citation Format: Arvinder Kapur, Mayur Kajla, Susan Paskewitz, Allegra Cappuccini, Pooja Mehta, Geeta Mehta, Manish Patankar. A novel death receptor ligand fabclavine inhibits cancer and cancer stem cell proliferation by extrinsic apoptosis [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 A16.

The cancer stem cell (CSC) hypothesis proposes that in a heterogeneous tumor only a rare population of stem-like cells has the ability to initiate new tumors. These rare CSC are believed to be the source of resistance to therapy, recurrence, and metastasis. A better understanding of ovarian CSCs could lead to novel therapeutic approaches to specifically target CSCs. Unfortunately, the study of human CSCs is hampered by heterogeneity of patient's tumor samples, long requirements for tumor growth in vivo, and the need for tumor growth in immune-deficient mice. We have therefore characterized CSCs in a transgenic murine model of ovarian cancer. This mouse model with conditional deletion of Apc, Pten and p53 tumor suppressor genes develop advanced ovarian cancer similar to that seen patients. Study of CSC in a murine tumor model will provide a homogeneous, rapid means to systematically study CSCs in a native microenvironment. Using flow cytometry to characterize a cell line derived from this tumor model we identified the expression of several potential CSC surface markers including CD24, CD44, CD90, CD117, CD133 and ALDH. In vitro analysis demonstrates that cells defined by the expression of CD24 and CD133, but not the other CSC markers, have an increased tumor sphere forming capacity; a hallmark of CSC. We next assessed in vivo tumor initiation and growth rates. No preferential tumor initiating or growth capacity was observed for CD44+, CD90+, CD117+, or ALDH+ versus their negative counterparts. CD133+ cells demonstrated a trend for increased tumor initiation. CD24+ cells vs CD24- cells, had significantly greater tumor initiation and tumor growth capacity. As few as 200 CD24+ could initiate tumors. Consistent with a stem cell phenotype we have found that CD24+ cells, compared to CD24- cells, preferentially express stem cell markers Nanog and c-myc. CD24+ cells vs. CD24- cells also demonstrated preferential phosphorylation of STAT3. Suggesting an important role for STAT3 in CD24+ CSC, CD24+ cells were preferentially sensitive to either JAK2 inhibition (which prevents pSTAT3) or direct pSTAT3 inhibition. Finally, we found in vivo therapy with the JAK2 inhibitor TG101209 dramatically reduced tumor metastases, and combined with chemotherapy, prolonged overall survival. These findings indicate that CD24+ cells have a CSC phenotype and play a role in tumor migration and invasion in other organs. Moreover, these findings suggest that combination of chemotherapy and CSCs targeted therapies can potentially improve survival by inhibiting metastasis. This abstract is also presented as Poster B36. Citation Format: Daniela Burgos-Ojeda, Rong Wu, Kathleen Cho, Ronald Buckanovich. Targeting CD24+ ovarian cancer stem-like cells in a transgenic murine model of ovarian cancer restrics metastasis. [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 PR07.