Kun Yang

Aldehyde Dehydrogenase in Combination with CD133 Defines Angiogenic Ovarian Cancer Stem Cells That Portend Poor Patient Survival

Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors, but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH(+) cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors, compared with ALDH(-) cells, validating ALDH as a marker of ovarian CSC in cell lines. Notably, as few as 1,000 ALDH(+) cells isolated directly from CD133(-) human ovarian tumors were sufficient to generate tumors in immunocompromised mice, whereas 50,000 ALDH(-) cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines, we observed even greater growth in the ALDH(+)CD133(+) cells compared with ALDH(+)CD133(-) cells, suggesting a further enrichment of ovarian CSC in ALDH(+)CD133(+) cells. Strikingly, as few as 11 ALDH(+)CD133(+) cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC, ovarian CSC exhibited increased angiogenic capacity compared with bulk tumor cells. Finally, the presence of ALDH(+)CD133(+) cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together, our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs.

Human ovarian carcinoma–associated mesenchymal stem cells regulate cancer stem cells and tumorigenesis via altered BMP production

Accumulating evidence suggests that mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment; however, controversy exists regarding their role in solid tumors. In this study, we identified and confirmed the presence of carcinoma-associated MSCs (CA-MSCs) in the majority of human ovarian tumor samples that we analyzed. These CA-MSCs had a normal morphologic appearance, a normal karyotype, and were nontumorigenic. CA-MSCs were multipotent with capacity for differentiating into adipose, cartilage, and bone. When combined with tumor cells in vivo, CA-MSCs promoted tumor growth more effectively than did control MSCs. In vitro and in vivo studies suggested that CA-MSCs promoted tumor growth by increasing the number of cancer stem cells. Although CA-MSCs expressed traditional MSCs markers, they had an expression profile distinct from that of MSCs from healthy individuals, including increased expression of BMP2, BMP4, and BMP6. Importantly, BMP2 treatment in vitro mimicked the effects of CA-MSCs on cancer stem cells, while inhibiting BMP signaling in vitro and in vivo partly abrogated MSC-promoted tumor growth. Taken together, our data suggest that MSCs in the ovarian tumor microenvironment have an expression profile that promotes tumorigenesis and that BMP inhibition may be an effective therapeutic approach for ovarian cancer.

Metformin targets ovarian cancer stem cells in vitro and in vivo.

PURPOSEnStudies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC.nnnMETHODSnThe impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts.nnnRESULTSnMetformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis.nnnCONCLUSIONSnMetformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.

VEGFR3 Inhibition Chemosensitizes Ovarian Cancer Stemlike Cells through Down-Regulation of BRCA1 and BRCA2

In ovarian cancer, loss of BRCA gene expression in tumors is associated with improved response to chemotherapy and increased survival. A means to pharmacologically downregulate BRCA gene expression could improve the outcomes of patients with BRCA wild-type tumors. We report that vascular endothelial growth factor receptor 3 (VEGFR3) inhibition in ovarian cancer cells is associated with decreased levels of both BRCA1 and BRCA2. Inhibition of VEGFR3 in ovarian tumor cells was associated with growth arrest. CD133(+) ovarian cancer stemlike cells were preferentially susceptible to VEGFR3-mediated growth inhibition. VEGFR3 inhibition-mediated down-regulation of BRCA gene expression reversed chemotherapy resistance and restored chemosensitivity in resistant cell lines in which a BRCA2 mutation had reverted to wild type. Finally, we demonstrate that tumor-associated macrophages are a primary source of VEGF-C in the tumor microenvironment. Our studies suggest that VEGFR3 inhibition may be a pharmacologic means to downregulate BRCA genes and improve the outcomes of patients with BRCA wild-type tumors.

Identifying an ovarian cancer cell hierarchy regulated by bone morphogenetic protein 2

Significance Significant controversy persists regarding a hierarchical vs. stochastic model of cancer. Using a microfluidic single-cell culture device, we define for the first time, to our knowledge, the differentiation capacity of primary human ovarian cancer cells. We demonstrate that ovarian cancer follows a hierarchical model with rare stochastic events. Defining the differentiation capacity allowed us to explain apparently paradoxical actions of bone morphologenetic protein 2 (BMP2); BMP2 suppresses growth in vitro by suppressing bulk cell proliferation, but promotes growth in vivo by promoting cancer stem-like cell (CSC) expansion. This work supports BMP2 signaling as a critical therapeutic target regulating ovarian CSC growth. Whether human cancer follows a hierarchical or stochastic model of differentiation is controversial. Furthermore, the factors that regulate cancer stem-like cell (CSC) differentiation potential are largely unknown. We used a novel microfluidic single-cell culture method to directly observe the differentiation capacity of four heterogeneous ovarian cancer cell populations defined by the expression of the CSC markers aldehyde dehydrogenase (ALDH) and CD133. We evaluated 3,692 progeny from 2,833 cells. We found that only ALDH+CD133+ cells could generate all four ALDH+/−CD133+/− cell populations and identified a clear branched differentiation hierarchy. We also observed a single putative stochastic event. Within the hierarchy of cells, bone morphologenetic protein 2 (BMP2) is preferentially expressed in ALDH−CD133− cells. BMP2 promotes ALDH+CD133+ cell expansion while suppressing the proliferation of ALDH−CD133− cells. As such, BMP2 suppressed bulk cancer cell growth in vitro but increased tumor initiation rates, tumor growth, and chemotherapy resistance in vivo whereas BMP2 knockdown reduced CSC numbers, in vivo growth, and chemoresistance. These data suggest a hierarchical differentiation pattern in which BMP2 acts as a feedback mechanism promoting ovarian CSC expansion and suppressing progenitor proliferation. These results explain why BMP2 suppresses growth in vitro and promotes growth in vivo. Together, our results support BMP2 as a therapeutic target in ovarian cancer.

Endothelin receptor-A is required for the recruitment of antitumor T cells and modulates chemotherapy induction of cancer stem cells

Background: The endothelin receptor-A (ETRA) plays an important role in tumor cell migration, metastasis, and proliferation. The endothelin receptor B (ETRB) plays a critical role in angiogenesis and the inhibition of anti-tumor immune cell recruitment. Thus dual blockade of ETRA and ETRB could have significant anti-tumor effects. Results: Dual ETRA/ETRB blockade with macitentan (or the combination of the ETRA and ETRB antagonists BQ123 and BQ788) did not enhance antitumor immune cell recruitment. In vitro studies demonstrate that ETRA inhibition prevents the induction of ICAM1 necessary for immune cell recruitment. When used as a single agent against human tumor xenografts, macitentan demonstrated non-significant anti-tumor activity. However, when used in combination with chemotherapy, macitentan specifically reduced tumor growth in cell lines with CD133+ cancer stem cells. We found that ETRA is primarily expressed on CD133+ CSC in both cell lines and primary human tumor cells. ETRA inhibition of CSC prevented chemotherapy induced increases in tumor stem cells. Furthermore, ETRA inhibition in combination with chemotherapy reduced the formation of tumor spheres. Methods: We tested the dual ETRA/ETRB antagonist macitentan in conjunction with (1) an anti-tumor vaccine and (2) chemotherapy, in order to assess the impact of dual ETRA/ETRB blockade on anti-tumor immune cell infiltration and ovarian tumor growth. In vitro murine and human cell line, tumor sphere assays and tumor xenograft models were utilized to evaluate the effect of ETRA/ETRB blockade on cell proliferation, immune cell infiltration and cancer stem cell populations. Conclusions: These studies indicate a critical role for ETRA in the regulation of immune cell recruitment and in the CSC resistance to chemotherapy.

DR6 as a Diagnostic and Predictive Biomarker in Adult Sarcoma

Background The Death Receptor 6 (DR6) protein is elevated in the serum of ovarian cancer patients. We tested DR6 serum protein levels as a diagnostic/predictive biomarker in several epithelial tumors and sarcomas. Methods DR6 gene expression profiles were screened in publically available arrays of solid tumors. A quantitative immunofluorescent western blot analysis was developed to test the serum of healthy controls and patients with sarcoma, uterine carcinosarcoma, bladder, liver, and pancreatic carcinomas. Change in DR6 serum levels was used to assay the ability of DR6 to predict the response to therapy of sarcoma patients. Results DR6 mRNA is highly expressed in all tumor types assayed. Western blot analysis of serum DR6 protein demonstrated high reproducibility (ru200a=u200a0.97). Compared to healthy donor controls, DR6 serum levels were not elevated in patients with uterine carcinosarcoma, bladder, liver, or pancreatic cancers. Serum DR6 protein levels from adult sarcoma patients were significantly elevated (p<0.001). This was most evident for patients with synovial sarcoma. Change in serum DR6 levels during therapy correlated with clinical benefit from therapy (sensitivity 75%, and positive predictive value 87%). Conclusion DR6 may be a clinically useful diagnostic and predictive serum biomarker for some adult sarcoma subtypes. Impact Diagnosis of sarcoma can be difficult and can lead to improper management of these cancers. DR6 serum protein may be a tool to aid in the diagnosis of some sarcomatous tumors to improve treatment planning. For patients with advanced disease, rising DR6 levels predict non-response to therapy and may expedite therapeutic decision making and reduce reliance on radiologic imaging.

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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnRecent 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.nnCitation 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.

Abstract 3735: A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like cells

Background: The high relapse rate in ovarian cancer patients is highly consistent with a cancer stem cell (CSC) model in which rare, inherently chemoresistant CSC are capable of proliferating and differentiating to cause disease. We recently demonstrated ovarian CSC (OvCSC) can be defined by Aldehyde Dehydrogenase (ALDH) enzymatic activity. ALDH expression is increased in chemotherapy resistant OvCSC and ALDH KO can reverse chemotherapy resistance. While ALDH inhibitors exist, they are not isoform specific. We therefore identified and screened numerous putative ALDH inhibitors, based on molecular homology to the known ALDH inhibitor DEAB, and assessed their impact on OvCSC. Methods and Results: While most ALDH inhibitors, including DEAB, had no impact on CSC, we identified a novel compound, 673A which specifically depletes OvCSC in vitro. Supporting functional depletion of OvCSC, treatment of primary tumor ascites resulted in 4-20 fold decrease tumor sphere formation. ALDH inhibition is highly synergistic with cisplatin both in vitro and in vivo as assessed by cell growth curves and tumor growth. Pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Unlike non-CSC depleting ALDH inhibitors, 673A is a pan-ALDH1A isoform specific inhibitor with IC50 Conclusions: We have identified a novel ALDH inhibitor, 673A, which selective inhibits three ALDH1A isoforms. 673A mediated ALDH1A isoform inhibition triggers programmed cell necrosis of OvCSC. Our data suggest that pan ALDH1A1 targeted therapy this may be a powerful new therapeutic approach to target OvCSC and bypass their resistance to apoptosis inducing drugs. Future studies will elucidate the importance of mitochondria metabolic pathways (Glycolysis, OXPHOS, and TCA cycle) in ALDH inhibition-induced necroptosis. Citation Format: Ilana Chefetz-Menaker, Kun Yang, Ron Buckanovich. A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like 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 3735.

Abstract 3731: Targeting ovarian cancer stem cells using a novel ALDH inhibitor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnBackground: Cancer cells with stem cell-like properties (CSC) are resistant to chemotherapy and are believed to be the source of cancer recurrence. Aldehyde dehydrogenase enzymatic activity (ALDH), alone and in combination with the stem cell marker CD133, can be used to define a hierarchy of Ovarian Cancer Stem Cells (OvCSC). ALDH is functionally important for stem cells, regulating motile cellular metabolic functions and the biosynthesis of retinoic acid. ALDH agonists have been demonstrated to enhance ‘stemness’ in some tissues. We therefore sought to determine the impact of ALDH antagonist on OvCSC.nnMethods and Results: We screened a variety of novel derivatives with confirmed or potential ALDH activity based on the molecular homology to the known ALDH inhibitor DEAB. Using the ALDEFLUOR assay and A2780 cancer cell line as a source of ALDH+CD133+ cells, we identified a lead compound 673 which effectively inhibits ALDH activity. Interestingly this compound also potently depletes CD133+ ovarian cancer cells. Supporting functional depletion of OvCSC, treatment of primary human ovarian tumor ascites cells with 673 resulted in 4-20 fold decrease tumor sphere formation. 673 treatment is highly synergistic with cisplatin both in-vitro and in-vivo as assessed by cell growth curves and tumor growth. Importantly, pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Finally, 673 is non-toxic to normal stem cells.nnConclusions: We have identified a novel ALDH inhibitor, 673, which depletes the ovarian cancer cells expressing CSC markers. CSC marker independent functional assays, with both cell lines and primary human ovarian cancer cells, indicate 673 is depleting OvCSC activity. Thus this may be a powerful new therapeutic approach to target CSC. Future studies will elucidate the molecular mechanism that underlies 673 OvCSC depletion activities as well as to identify a specific ALDH isoforms targeted in ovarian cancer.nnCitation Format: Ilana Chefetz, Kun Yang, Scott D. Larsen, Ronald J. Buckanovich. Targeting ovarian cancer stem cells using a novel ALDH inhibitor. [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 3731. doi:10.1158/1538-7445.AM2013-3731