High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo.Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy

Abstract 5007: WT1 promotes tumor growth and vascularization by regulating matrix metalloproteinase 9 in Ewing's sarcoma xenografts

Abstract WT1, a zinc-finger transcription factor, is expressed in tumors of varied origins including sarcomas. High level WT1 expression often correlates with poor patient survival. The role of WT1 in sarcoma biology and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. The aim of this study is to investigate the effect of WT1 expression on sarcoma growth and angiogenesis. We transfected the WT1-null Ewing's sarcoma cell line SK-ES-1 with one of two major WT1 isoforms, and used shRNA to silence WT1 expression in the WT1-positive Ewing's sarcoma cell line MHH-ES. Cells were injected into the flanks of immune deficient mice and tumor growth was monitored. Tumors arising fromWT1-expressing SK-ES-1 cells grew faster than control, while tumors arising from WT1-silenced MHH-ES cells grew more slowly than control. Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31 and the pericyte marker αSMA. In xenografted SK-ES-1 cells, quantification of CD31 positive area in WT1 expressing tumors showed 60 -70 % more compared to the controls. In contrast, CD31 expression is abolished in tumors arising from WT1-silenced MHH-ES cells. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays showed that WT1 can upregulate MMP9 promoter activity in transiently transfected NIH3T3 cells. Also, immunohistochemical analysis showed increased MMP9 expression in WT1-expressing tumors. Finally, imunohistochemical analysis of primary Ewing's sarcoma samples showed correlated expression of WT1, VEGF, and MMP9. Taken together, our data suggest that WT1 promotes tumor growth and angiogenesis, perhaps via upregulation of MMP9. Increased tumor angiogenesis may lead to more aggressive tumors and a worse outcome for patients, explaining the effect of WT1 on prognosis. 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 5007. doi:10.1158/1538-7445.AM2011-500

Abstract 5281: Regulation of tumor growth and angiogenesis by Wilms tumor 1(WT1)

Abstract Angiogenesis is required for the growth and metastasis of cancers. Although hypoxia is the major driver of angiogenesis, there is clear variation in angiogenesis between tumor types, and even between different tumors of the same histology, suggesting that there may be other factors that regulate tumor angiogenesis. One molecule that has been implicated in angiogenesis is a transcription factor known as WT1. WT1 was originally identified as a tumor suppressor gene, is overexpressed in tumors of varied origins including sarcomas, carcinomas, and leukemias, and high level WT1 expression often correlates with poor patient survival. The role of WT1 in tumorigenesis and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. Here, we show that expression of WT1 promotes clonogenic growth in soft agar and a migratory phenotype in vitro. Tumors arising fromWT1-expressing SK-ES-1 (SKWT1A and SKWT1D) cells grew faster than control (SKNC), while tumors arising from WT1-silenced MHH-ES (MHshRNA) cells grew more slowly than control (MHNC). Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31. Quantification of CD31 positive area in tumors grown from SKWT1A and SKWT1D showed 70 −80 % more staining compared to the controls. In contrast, CD31 expression is abolished in tumors arising from MHshRNA cells. Also, immunohistochemical (IHC) analysis showed enhanced expression of WT1 in tumor sections correlates with increased expression of pro-angiogenic molecules such as VEGF, MMP9, angiopoietin-1 (Ang1), and Tie2, further supporting our hypothesis that WT1 plays a major role in regulating tumor angiogenesis. Interestingly, MHHshRNA tumors exhibited decreased expression of VEGF, MMP9, Ang1, and Tie2 proteins when compared to the MHHNC tumors. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays demonstrated that WT1 can regulate the activity of the MMP9 promoter, and chromatin immunoprecipitation assays showed that WT1 can bind directly to the MMP9 promoter. Finally, IHC analysis of primary Ewing's sarcoma samples demonstrate that, as was seen in cell lines and xenografts, WT1, VEGF, CD31 and MMP9 expression levels in primary Ewing sarcoma tissues are correlated with each other. In summary, our data identifies MMP9 as a novel WT1 target gene and demonstrates the WT1 expression directly regulates tumor growth through an effect on angiogenesis and supports the notion that development of therapeutic strategies that target WT1 will provide effective treatment options for WT1-expressing tumors. 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 5281. doi:1538-7445.AM2012-528

WT1 regulates angiogenesis in Ewing Sarcoma

Angiogenesis is required for tumor growth. WT1, a protein that affects both mRNA transcription and splicing, has recently been shown to regulate expression of vascular endothelial growth factor (VEGF), one of the major mediators of angiogenesis. In the present study, we tested the hypothesis that WT1 is a key regulator of tumor angiogenesis in Ewing sarcoma. We expressed exogenous WT1 in the WT1-null Ewing sarcoma cell line, SK-ES-1, and we suppressed WT1 expression using shRNA in the WT1-positive Ewing sarcoma cell line, MHH-ES. Suppression of WT1 in MHH-ES cells impairs angiogenesis, while expression of WT1 in SK-ES-1 cells causes increased angiogenesis. Different WT1 isoforms result in vessels with distinct morphologies, and this correlates with preferential upregulation of particular VEGF isoforms. WT1-expressing tumors show increased expression of pro-angiogenic molecules such as VEGF, MMP9, Ang-1, and Tie-2, supporting the hypothesis that WT1 is a global regulator of angiogenesis. We also demonstrate that WT1 regulates the expression of a panel of pro-angiogenic molecules in Ewing sarcoma cell lines. Finally, we found that WT1 expression is correlated with VEGF expression, MMP9 expression, and microvessel density in samples of primary Ewing sarcoma. Thus, our results demonstrate that WT1 expression directly regulates tumor angiogenesis by controlling the expression of a panel of pro-angiogenic genes

TGF-β signaling pathway inactivation and cell cycle deregulation in the development of gastric cancer: Role of the β-spectrin, ELF

We have shown that loss of ELF, a stem cell adaptor protein, disrupts TGF-β signaling through Smad3 and Smad4 localization. Notably elf+/-/smad4+/- mice develop gastric cancer presenting this as an important model for analyzing molecular event in gastric carcinogenesis. To gain further insight into the functional role of ELF in gastric cancer suppression, we carried out a detailed characterization of cell cycle events leading to gastric tumorigenesis. elf-/- cells and elf+/-/smad4+/- mice demonstrate a marked alteration of cell cycle regulators, such as Cdk4, K-Ras, and p21. Levels of Cdk4 increased compared to normal controls, suggesting loss of ELF results in functional abnormalities in cell cycle regulation. We further demonstrate that the elf-/- MEFs show a disruption of G1/S cell cycle transition and a significant reduction in senescence. Thus, in response to ELF deficiency, the abnormalities of G1/S checkpoint and senescence contribute their increment of susceptibility to malignant transformation. © 2006

Inactivation of ELF/TGF-β signaling in human gastrointestinal cancer

TGF-β/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. ELF, a β-spectrin, plays a key role in the transmission of TGF-β-mediated transcriptional response through Smads. ELF was originally identified as a key protein involved in endodermal stem/progenitor cells committed to foregut lineage. Also, as a major dynamic adaptor and scaffolding protein, ELF is important for the generation of functionally distinct membranes, protein sorting and the development of polarized differentiated epithelial cells. Disruption of elf results in the loss of Smad3/Smad4 activation and, therefore, a disruption of the TGF-β pathway. These observations led us to pursue the function of ELF in gastrointestinal (GI) epithelial cell-cell adhesion and tumor suppression. Here, we show a significant loss of ELF and reduced Smad4 expression in human gastric cancer tissue samples. Also, of the six human gastric cancer cell lines examined, three show deficient ELF expression. Furthermore, we demonstrate the rescue of E-cadherin-dependent homophilic cell-cell adhesion by ectopic expression of full-length elf. Our results suggest that ELF has an essential role in tumor suppression in GI cancers. © 2005 Nature Publishing Group. All rights reserved

Passaging as sarcospheres enriches for ALDH^high cells.

<p>TC71 cells were grown under standard, adherent conditions. The cells with the highest 3% of ALDH activity were isolated and replated in Mesencult under nonadherent conditions. The resulting sarcospheres were disaggregated into single cells, sorted, and the ALDH^high cells replated under identical conditions. After 5 such passages, cells from disaggregated sarcospheres were analyzed by the Aldefluor assay. The initial gate, which encompassed only 3% of the starting cells, included 27% of the cells passaged as sarcospheres.</p

ALDH expression in primary human ESFT xenografts.

<p>Single cell suspensions were made from each of four early passage primary human ESFT xenografts. (A) The first step of purification was isolation of viable cells based on scatter and PI exclusion (enclosed in gate R0). Viable cells were stained with Aldefluor without (B) or with (C) DEAB. To exclude contamination with murine cells, single cell suspensions were incubated with an antibody against murine MHC after Aldefluor treatment without (D) or with (E) DEAB. FACS was used to eliminate the cells expressing mouse MHC (gate R2 in panels D and E), and cells were sorted for ALDH activity (F). In panel F, gate R3 is the ALDH^high cells and gate R4 is the ALDH^low cells. Three other xenografts were also analyzed by Aldefluor without (G, H, I) or with (J, K, L) DEAB.</p