bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4226: A luminal epithelial cell line isolated from PTEN−/−;P53−/− mouse protospheres forms adenocarcinoma in vivo
Authors: Hynes Paul G. , Abou-Kheir Wassim G. , Liu Yen-Nien , Martin Philip , Kelly Kathleen
Published: February 2011

Pten deletion in the mouse prostate epithelium results in the expansion of a prostatic stem/progenitor cell subpopulation, which co-fractionates with basal epithelial cells. Progenitor cells in the luminal layer of Pten null prostates also have been suggested as tumor initiating cells. Here we describe the isolation of a protosphere-derived, androgen independent cell line with a luminal epithelial phenotype that displays properties of prostate progenitor cells and forms adenocarcinoma in-vivo. The cell line was generated from Rosa26-CreERPtenfl/flP53fl/fl prostate cells. Cells were plated to form protospheres, which originate from stem/progenitor cells, and the deletion of Pten and p53 was induced by tamoxifen. After spheres were serially passaged for 3 generations, a monolayer cell line capable of growth in minimal serum-free media was established. A clonal line also was generated from the parental cell line. In order to determine the differentiated cell phenotype of the parental and the clonal cell lines, cells were stained over multiple generations for markers of epithelial (CK5, CK8, CK14) and neuroendocrine (beta 3 tubulin) cell lineages. The cell line is entirely CK8 positive with a minor component of CK8/CK14 double positive cells. Cells are capable of forming protospheres both in Matrigel and in suspension, a property of stem/progenitor cells. In addition, based on lentiviral reporter assays, a minor population of cells in the parental and the clonal cell line expressed Nanog, Oct4 and Notch dependent activity. Interestingly, Nanog and Oct4 positive cells showed higher sphere forming ability as compared to negative cells. In order to investigate their tumorigenic potential, the parental and the cloned lines were injected subcutaneously into athymic nude mice. Visible tumor formation was observed after 2 weeks, and histological analysis revealed adenocarcinoma and poorly differentiated carcinoma. In addition to CK8+ cells within the tumors, there were rare CK5+ and neuroendocrine cells, suggesting plasticity in the differentiation potential. In conclusion, we have isolated a prostate luminal epithelial cell line and derivative clones with a defined background (Pten and p53 null) that have stem-like and tumor initiating properties in vitro and in vivo. Future experiments are aimed at deciphering the molecular mechanisms regulating growth and differentiation downstream of Pten and p53 loss.

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 4226.
Abstract 4233: Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers
Authors: Siu Michelle KY , Wong Esther SY , Kong Daniel SH , Wong Oscar GW , Tam Kar Fai , Ngan Hextan YS , Le Xiao Feng , Cheung Annie NY
Published: February 2011

Introduction: Ovarian cancer is the most lethal of all gynecological malignacies due to its lack of symptoms at early stages. In addition, widespread intraperitoneal metastases and malignant ascites frequently develop. Therefore, it is very important to identify novel prognostic and therapeutic targets for this cancer. Many researchers believe that only a small subset of cancer cells are endowed with stem cell properties, which are responsible for tumor growth, metastatic progression and recurrence. Nanog is one of core transcription factors essential for the maintaining self-renewal and pluripotency in stem cells. This study investigated the clinical significance, functional roles and putative downstream targets of Nanog in ovarian cancer. Methods and Results: Differential protein expression profile of Nanog in clinical samples, including benign (n=6), borderline (n=7), carcinomas (n=97) and metastatic foci (n=24) were determined by immunohistochemistry. Differential mRNA expression pattern of Nanog in 3 normal ovarian epithelial cell lines, 16 ovarian cancer cell lines was assessed by quantitative real-time polymerase chain reaction. Subcellular localization of Nanog was performed by Western blotting. Nanog was found to be localized in the nucleus of ovarian cancers. High Nanog expression in ovarian cancers was significantly associated with high grade tumors, serous histological type and chemoresistance (p<0.05). Importantly, Nanog was found to be highly expressed in ovarian cancer cell lines with metastasis-associated property and in clinical samples of metastatic foci. Elevated Nanog expression was associated with poor overall and disease-free survival in the univariate analysis. Nanog expression became one of the independent prognostic factors in the multivariate analysis for overall but not disease-free survival. By Transwell migration and invasion assays, stable knockdown of Nanog in ovarian cancer cell line, OVCAR-3, impeded cell migration and invasion. Stable knockdown of Nanog also increased mRNA expression of E-cadherin (an essential epithelial mesenchymal transition marker, thus related to metastasis) and FOXO1A (an important gene related to chemoresistance). Conclusion: Nanog was associated with progression of ovarian tumors, patients’ overall survival and chemoresistance, suggesting that Nanog can be one of the potential prognostic markers and therapeutic molecular targets in ovarian cancer.

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 4233.
Abstract 4236: Characterization of maternal embryonic leucine zipper kinase in cancer stem/progenitor cells
Authors: Okemoto-Nakamura Yuko , Kornblum Harley I. , Nakano Ichiro
Published: February 2011

Glioblastoma multiforme (GBM) is the most frequent tumor in the adult brain and remains a highly lethal diseases. Recent studies have suggested that lack of targeted therapies for brain tumor stem cells (BTSC) is one of the reasons for the limited efficacy of the current therapies. Identification of the molecular target in BTSC is crucial to the development of GBM therapy.

Maternal embryonic leucine zipper kinase (MELK) is a member of Snf1/AMP serine/threonine kinase family and abundantly expressed in multiple cancers, including GBM. Previously, we demonstrated that the RNA expression of MELK is elevated in patient-derived tumor stem-like cells in GBM. Knockdown of MELK of tumor stem-like cells by siRNA resulted in apoptosis in vitro. In contrast, knockdown of MELK did not induce significant apoptosis in normal neural progenitors derived from mouse brains, raising a possibility that MELK is likely required for survival of tumor stem cells but not the normal counterpart. However, the difference in the mechanism of MELK action between tumor and normal stem cells is unknown. Here we investigated to discriminate the signaling pathways that regulate survival and proliferation of BTSC and normal neural stem cells.

Immunohistochemistry showed that MELK is abundantly expressed in surgical specimens of GBM and is mainly localized in the nuclei. Immunocytochemistry with patient-derived GBM spheres demonstrated that MELK is preferentially expressed by GBM stem-like cells but not in the differentiated tumor cells. We also found that MELK is phosphorylated at serine and threonine residues in GBM stem-like cells. To determine the signaling pathways that regulate MELK phophorylation in GBM stem cells, we treated GBM stem-like cells with a variety of the kinase inhibitors. Interestingly, a mitogen-activated protein (MAP) kinase inhibitor reduced the MELK protein expression in GBM stem-like cells but not in the normal counterpart. The MAP kinase pathway was up-regulated in GBM stem-like cells in comparison to the normal progenitors derived from human fetal brains, further supporting the significance of this pathway in GBM stem cells. Collectively, our data indicate that MAP kinase may regulate MELK phosphorylation and activation specifically in GBM stem cells but not in the normal neural stem cells.

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 4236.
Abstract 4243: HMGA1: A driver of the stem cell phenotype in intestinal cyrpt cells and colon cancer
Authors: Belton Amy M. , Gabrovsky Alexander , Iacobuzio-Donahue Christine , Almeida-Porada Graça , Colletti Evan , Huso David , Smith-Resar Linda
Published: February 2011

Although the HMGA1 gene is highly expressed in human embryonic stem cells and high-grade, poorly differentiated cancers, its function in these settings has not been clearly elucidated. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which are members of a family of non-histone, chromatin remodeling proteins. These proteins bind to the minor groove of AT-rich regions in the B-form of DNA, which leads to the recruitment of transcriptional factors that modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis and in aggressive human cancers arising from different embryologic origins, including cancers of hematopoietic, lung, breast, prostate, and colon cells. Forced overexpression of HMGA1a or HMGA1b induces a transformed phenotype in cultured cells and inhibiting HMGA1 blocks transformation in human cancer cell lines. Because HMGA1 functions in transcriptional regulation, we hypothesize that it induces neoplastic transformation by dysregulating specific molecular pathways. To investigate the pathways disrupted by HMGA1, we are studying our HMGA1a transgenic mice as a model system. As previously reported, these mice develop aggressive T-cell leukemia with complete penetrance and females also develop uterine cancer. At necropsy, we also discovered polyps involving the small and large intestines. Histopathologically, the transgenic intestines are characterized by a thickened mucosal surface, hyperproliferative changes, and hamartomatous polyps. Immunohistochemical analysis reveals an increase in the population of intestinal cells expressing the colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr-5) in transgenic compared to wildtype mice. These data support our observation of an expansion in the crypt progenitor/intestinal stem cell populations. Our preliminary results also show that inhibiting HMGA1 expression in colon cancer cell lines interferes with anchorage-independent cell growth and tumorigenesis, indicating that HMGA1 is required for these transformation phenotypes in colon cancer. In addition, we found that HMGA1 is enriched in >70% of primary tumors with high-grade colon cancer. These findings suggest that HMGA1 is important in maintaining the stem cell niche in the intestine. Moreover, HMGA1 could promote neoplastic transformation and tumor progression in the intestine by driving a stem-like phenotype in colonic cells.

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 4243.
Abstract 4261: Inhibition of Bmi-1 suppressed the tumorigenicity of oral cancer-derived ALDH1-positive cells
Authors: Shih-Hwa Chiou
Published: February 2011

Bmi-1, a member of the Polycomb family of transcriptional repressors, is essential for maintaining the self-renewal abilities of adult stem cells. Bmi-1 has been demonstrated to play a role in tumorigenesis in head and neck squamous cell carcinomas (HNSCCs). A recent study has further suggested that ALDH1 may be considered to be a putative marker for HNSCC-derived cancer stem cells. However, the role that Bmi-1 plays in HNSCC-derived ALDH1-positive cells (HNSCC- ALDH1+) has yet to be determined. In this study, we demonstrated that HNSCC- ALDH1+ cells possess tumor initiating properties, are capable of self-renewal, and express higher levels of Bmi-1 as compared to HNSCC-ALDH1− cells. To further explore the functional role of Bmi-1 in HNSCC-ALDH1+ cells, we used a lentiviral vector expressing shRNA to knock down Bmi-1 expression (sh-Bmi-1) in HNSCC- ALDH1+ cells. Silencing of Bmi-1 significantly enhanced the sensitivity of HNSCC- ALDH1+ cells to chemoradiation and increased the degree of chemoradiation- mediated apoptosis that occurred. Importantly, knockdown of Bmi-1 increased the effectiveness of radiotherapy and led to the inhibition of tumor growth in nude mice transplanted with HNSCC-ALDH1+ cells. Kaplan-Meier survival analysis indicated that the mean survival rate of HNSCC-ALDH1+ tumor-bearing immunocompromised mice treated with radiotherapy was significantly improved by treatment with sh-Bmi-1 as well. In summary, these results suggest that Bmi-1 is a potential target for increasing the sensitivity of HNSCC cancer stem cells to chemoradiotherapy.

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 4261.
Abstract 4271: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells
Authors: Li Yanyan , Zhang Tao , Korkaya Hasan , Liu Suling , Lee Hsiu-Fang , Newman Bryan , Yu Yanke , Clouthier Shawn , Schwartz Steven , Wicha Max , Sun Duxin
Published: February 2011

Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells

The existence of cancer stem cells (CSCs) in breast cancer has profound implications for cancer prevention. The purpose of this study was to evaluate sulforaphane, a naturally occurring dietary compound from broccoli/broccoli sprouts, for its efficacy to inhibit breast CSCs as well as to elucidate the potential mechanisms of its effect. Aldefluor and mammosphere formation assays were used to assess the effect of sulforaphane on breast CSCs in vitro. A NOD/SCID xenograft model was employed to determine the efficacy of sulforaphane in targeting breast CSCs in vivo. The potential mechanism of sulforaphane was investigated utilizing Western blotting and β-catenin reporter assays. The results showed that sulforaphane (1∼5 µM) decreased aldehyde dehydrogenase (ALDH)-positive cell population by 65%∼80% in human breast cancer cell line (P < 0.01), and reduced the size and number of primary mammospheres by 8∼125-fold and 50%∼75% (P < 0.01), respectively. Daily injection with 50 mg/kg sulforaphane for two weeks reduced ALDH-positive cells by more than 50% in tumors (P = 0.003). Furthermore, sulforaphane eliminated breast CSCs in vivo, thereby abrogating tumor re-growth in secondary mice (P < 0.01). Western blotting showed that sulforaphane down-regulated the Wnt/β-catenin self-renewal pathway. These data suggest that sulforaphane targets breast CSCs through the down-regulation of the Wnt/β-catenin pathway as a potential mechanism. These findings provide a scientific rationale for use of sulforaphane and potentially broccoli/broccoli sprout dietary interventions for breast cancer chemoprevention.

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 4271.
Abstract 4280: Color-coded in vivo imaging of tumor stem cells and co-implanted non-stem cancer cells
Authors: Suetsugu Atsushi , Osawa Yosuke , Nagaki Mashito , Moriwaki Hisataka , Bouvet Michael , Hoffman Robert M.
Published: February 2011

We have developed color-coded simultaneous imaging of tumor growth, metastasis and drug resistance of co-implanted cancer stem cells (CSC) and non stem cancer cells (NSCC). In this study, CD 133+ Huh-7 human hepatoma cells were considered as liver cancer CSC, and CD133− Huh-7 cells were considered liver cancer NSCC. CD133+ cells were isolated by magnetic bead sorting. CSC were labeled with GFP and NSCC were labeled with RFP using retroviruses. The same number of GFP CSC and the RFP NSCC were mixed and co-cultured or injected subcutaneously or in the spleen of nude mice. CSC had higher proliferative potential compared with NSCC in vitro. Dual-color imaging demonstrated that CSC were more tumorigenic and more resistant to chemotherapy in vivo than NSCC. To compare the effect of CSC and NSCC on stromal cells or blood vessels in tumors, non-colored CSC or NSCC along with RFP stromal cells were implanted into nestin driven (ND)-GFP transgenic nude mice which express ND-GFP in nascent blood vessels. Extensive RFP-expressing stromal and GFP-expressing nascent blood vessels cells were imaged in the tumors formed from CSC. Tumors formed from NSCCs had less GFP-expressing nascent blood vessels and RFP-expressing stromal cells suggesting that more stromal cells and blood vessels were induced by CSC than NSCC. Thus, liver cancer CSC have more malignant characteristics such as higher proliferation, more resistance to chemotherapy, and induction of stromal cells and blood vessels compared with NSCC. Color-coded imaging of CSC and NSCC is a powerful tool to investigate the characteristics and roles of CSC and NSCC in tumor progression.

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 4280.
Abstract 4281: Oncostatin M renders epithelial cell adhesion molecule-positive liver cancer stem cells sensitive to 5-fluorouracil by inducing hepatocytic differentiation
Authors: Yamashita Taro , Honda Masao , Nio Kouki , Kaneko Shuichi
Published: February 2011

Recent evidence suggests that a certain type of hepatocellular carcinoma (HCC) is hierarchically organized by a subset of cells with stem cell features (cancer stem cells: CSCs). Although normal stem cells and cancer stem cells (CSCs) are considered to share similar self-renewal programs, it is uncertain whether differentiation signaling is kept maintained in both types of cells. In this study, we investigated the effect of oncostatin M (OSM), an interleukin 6-related cytokine known to induce the differentiation of hepatoblasts into hepatocytes, on liver CSCs. Recombinant human OSM was used for both in vitro and in vivo experiments. Epithelial cell adhesion molecule (EpCAM) was used for the isolation of liver CSCs. Gene and protein expression was evaluated by quantitative reverse transcription-PCR, immunohistochemistry, and intracellular fluorescence-activated cell sorting. Apoptosis was evaluated by Annexin V and activated-caspase 3 staining. Tumor initiating capability was examined by the subcutaneous tumor formation in NOD/SCID mice. OSM receptor expression was detected in the majority of EpCAM-positive (EpCAM+) HCC with stem/progenitor cell features. OSM treatment (100 ng/ml) resulted in the induction of hepatocytic differentiation of EpCAM+ liver CSCs derived from hepatocellular carcinoma (HCC) cell lines and primary HCCs, in terms of decrease of alpha-fetoprotein and cytokeratin 19 production and increase of albumin production. Furthermore, OSM treated cells exhibited reduced expression of stemness-related genes including Bmi1 and POU5F1. While pretreatment of HCC cell lines with OSM before transplantation in vitro resulted in the decreased tumorigenic capacity, the OSM treatment after transplantation in vivo showed small tumor suppressing effects irrespective of hepatocytic differentiation of the tumor. Intriguingly, when the OSM treatment was combined with chemotherapeutic reagent 5-fluorouracil to eradicate non-CSCs, we observed the dramatic increase of apoptotic cells in vitro and the suppression of tumorigenesis in vivo compared with either OSM or 5-fluorouracil treatment alone. In conclusion, our data suggest that OSM can be effectively utilized for the differentiation and active cell division of dormant EpCAM+ liver CSCs, and the combination of OSM and conventional chemotherapy such as 5-fluorouracil efficiently eliminates HCC by targeting both CSCs and non-CSCs.

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 4281.
Abstract 4289: Early study on LGR5/GPR49 molecule as a potential colon cancer stem cell target for the antibody conjugated drug treatment
Authors: Mao Weiguang , Tien Janet , Goldenberg David , Stawicki Scott , Vij Rajesh , Xia Xiaoling , Hongo Jo-Anne , Ross Sarajane , Kozak Kathy , Firestein Ron , Wu Yan , Spencer Susan , Polakis Paul
Published: February 2011

LGR5 (GPR49) is a Wnt pathway downstream target gene. It has already been confirmed that LGR5 gene is up regulated by APC or β-catenin mutation. LGR5 has recently been identified as a biomarker on the human and murine intestinal or colon stem cells. Our gene expression data shows that LGR5 is highly expressed in colon cancer, with minimal expression in normal tissue. Our strategy in this project is to eliminate tumor stem cells by drug conjugated antibody. Our goal is to find a tumor stem cell marker and develop antibody to target the tumor stem cells. Here we developed and characterized both anti-LGR5 phage and monoclonal antibodies by multi molecular techniques. We finally focus on one phage antibody (YW353) and three monoclonal antibodies (2H6, 3G12 and 8E11) as therapeutic drugs for further study. We evaluated antibody drug conjugate (ADC) in vitro and in vivo with different animal tumor models derived from cell lines and human tumor explants. Good efficacy was observed in these animal models following a single dose of 5mg/kg. Conclusion: LGR5 is an attractive colon cancer target for the administration of anti-LGR5 ADC in human cancer. More in vivo models will be tested to confirm the efficacy and evaluate safety.

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 4289.
Abstract 4294: Differentiated colorectal cancer cells protect tumor-initiating cells from irinotecan
Authors: Emmink Benjamin L. , van Houdt Winan J. , Vries Robert G. , Hoogwater Frederik J.H. , Jimenez Connie R. , Clevers Hans , Rinkes Inne H.M. Borel , Kranenburg Onno
Published: February 2011

Normal colon tissue stem cells drive tissue turnover by generating daughter stem cells and a rapidly proliferating pool of progenitor cells that give rise to differentiated cell types with specialized functions. This functional hierarchy is preserved in colon tumors in which a tumor-initiating cell (or cancer stem cell) compartment drives the formation of proliferating and differentiated cell types with reduced tumorigenic potential. Normal colon stem cells possess a variety of resistance mechanisms that allow them to endure a lifetime of genotoxic insults. By inheriting these traits, tumor-initiating cells (TIC) are thought to be endowed with an intrinsic resistance to chemotherapy. However, the relationship between chemo-resistance and tumor-initiating potential in colon tumors is poorly understood.

We studied this relationship using a series colonosphere cultures isolated from freshly resected primary colon tumors and liver metastases. These cultures are highly enriched for TIC, based on their high clone and tumor-forming potential and expression of the stem cell marker ALDH1. The TIC-enriched cultures display resistance to the toposomerase I inhibitor irinotecan. However, drug resistance is mediated by a small subpopulation of non-tumorigenic differentiated ALDH1-negative cells expressing the drug efflux pump ABCB1. ABCB1 is localized to the brush border of cells at the colonosphere periphery and of cells forming intra-spheroid tubules. In human colorectal liver metastases ABCB1 is predominantly localized to the luminal side of differentiated tubule-forming cells. These ABCB1 drug-resistant cells are proliferation-competent but unable to initiate tumor formation. Conversely, tumor-initiating cells are ABCB1-negative and drug-sensitive, but could generate non-tumorigenic drug-resistant offspring in vitro and in tumor xenografts.

Our work shows that colorectal tumor-initiating cells do not display intrinsic resistance to irinotecan, most likely because they are derived from a normal stem cell population that does not express ABC transporters. Tumor-initiating cells generate a protective shield of drug-resistant ABCB1-positive offspring. Furthermore, the preservation of tumor-initiating capacity following irinotecan exposure requires the cooperative action of drug-sensitive tumor-initiating cells and their drug-resistant differentiated offspring.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 4294.
Abstract 4295: The combination of a novel anti-delta-like 4 ligand (DLL4) antibody with irinotecan inhibits growth of Kras-mutated colorectal tumors
Authors: Fischer Marcus , Yen Wan-Ching , Lewicki John , Gurney Austin , Hoey Timothy
Published: February 2011

Kras mutations in colorectal cancer (CRC) are associated with clinical resistance to treatment with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies. It has been suggested that cancer stem cells drive tumor growth and progression and are preferentially resistant to many current therapies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway which is known to mediate stem cell self-renewal and vascular development. We have recently demonstrated that inhibition of human DLL4 in the tumor reduced cancer stem cell frequency, whereas inhibition of mouse DLL4 resulted in hyperproliferation of the tumor vasculature (Cell Stem Cell, 5:168-177, 2009). The goal of the present study is to extend the utility of anti-DLL4 in treatment of mutant Kras CRC. Consistent with clinical findings, mutant Kras colorectal xenograft tumors were generally insensitive to EGFR-targeted monoclonal antibody cetuximab and addition of anti-DLL4 did not further enhance cetuximab-mediated anti-tumor effect. In contrast, anti-DLL4 plus irinotecan was equally efficacious against both wild type and mutant Kras colon tumors. Gene expression analysis showed that anti-DLL4 affected vascular-related genes in the stroma and Notch target genes in the tumor and stroma. Further analysis of mutant Kras tumors indicated that anti-DLL4/ irinotecan combination produced a 5-fold decrease in colon cancer stem cell frequency compared to control antibody treated group. In conclusion, our findings provide a rationale for targeting cancer stem cells and tumor vasculature through inhibition of the DLL4/Notch signaling for treatment of CRC patients with Kras mutation.

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 4295.
Abstract 4296A: Myeloproliferative disease induced by leukemogenic Ptpn11 (Shp-2) phosphatase arises from hematopoietic stem cells
Authors: Xu Dan , Wang Siying , Yu Wen-Mei , Chan Gordon , Araki Toshiyuki , Bunting Kevin D. , Neel Benjamin G. , Qu Cheng-Kui
Published: February 2011

Germline and somatic gain-of-function (GOF) mutations in the tyrosine phosphatase PTPN11 (SHP-2) are associated with juvenile myelomonocytic leukemia (JMML), a clonal myeloproliferative disease (MPD) of early childhood. However, the mechanism by which PTPN11 mutations induce this disease is not fully understood. Here we report that the germline mutation Ptpn11D61G increases mouse hematopoietic stem cell (HSC) activity, as evidenced by accelerated cycling, increased stem cell pool, enhanced self-renewal, and elevated short-term and long-term repopulating capabilities. More importantly, the MPD is reproduced in primary and secondary recipient mice transplanted with Ptpn11D61G mutant marrow cells or sorted Lineage- Sca-1+c-Kit+ cells, indicative of the long-term stem cell origin of the disease. The leukemogenic effect of Ptpn11D61G mutation appears to be attributed to enhancing cytokine signaling. Furthermore, HSC homeostasis disrupted by Ptpn11D61G mutation is partially restored by deletion of Gab2, an important interacting protein/target of SHP-2. MPD phenotypes are markedly ameliorated in Ptpn11D61G/+/Gab2−/− double mutant mice. Together, these studies suggest that SHP-2 associated JMML arises from HSCs and that targeting SHP-2/Gab2 mediated signaling in stem cells may be useful for the treatment of this disease.

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 4296A.
Abstract 4304: Malignant melanoma cells with aggressive properties are common and not distinguishable by proposed markers for cancer stem cells
Authors: Prasmickaite Lina , Engesæter Birgit , Skrbo Nirma , Oliver Nina K. , Engebråten Olav , Suo Zhenhe , Aamdal Steinar , Fodstad Øystein , Mælandsmo Gunhild M.
Published: February 2011

In malignant melanoma, conflicting results have been reported regarding the presence of exclusive cells with enhanced aggressive properties, so called cancer stem cells (CSC), raising a doubt whether melanoma follows a CSC model and is hierarchically organized. By characterizing melanoma cells from short-term cell cultures, xenografts and patient biopsies, we aim to collect further evidence either to: i) support the existence of distinct CSC-like subpopulations, or ii) strengthen a notion about the common presence of cells demonstrating aggressive behavior and lack of cellular hierarchy in melanoma. By using individual-cell assays, we have shown that a large fraction (up to 60 %) of random single melanoma cells display high clonogenicity and self-renewal i.e. properties associated with tumorigenic potential. In search for a marker for such clonogenic/tumorigenic cells, we found that melanomas often harbor a large distinct subpopulation with elevated activity of Aldehyde Dehydrogenase (ALDH), a proposed marker for CSC-like cells in some hierarchically organized cancers. Interestingly, ALDH activity in melanoma patient biopsies seemed to correlate to the expression of melanoma-associated-antigen, HMW-MAA, which is linked to tumor progression. Furthermore, in vivo ALDH+ melanoma cells could convert to ALDH−, while the opposite conversion was rare, indicating a certain “cellular hierarchy” with respect to the ALDH phenotype. However, comparison of ALDH+ and ALDH− cells revealed that both subpopulations are highly clonogenic, tumorigenic and resistant to drugs proposed for melanoma therapy, DTIC and HGS-ETR2. This suggests that in malignant melanoma, ALDH, likewise earlier investigated “surface markers” of CSCs, does not distinguish cells with enhanced biological aggressiveness. In conclusion, melanoma cells exhibiting properties linked to aggressive phenotype are common and not restricted to subpopulations expressing proposed “CSC markers”, which contradicts the traditional view of CSCs.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 4304.
Abstract 4306: Functional role of CD133 in glioblastoma multiforme
Authors: Bandopadhyay Gagori , Grabowska Anna M. , Coyle Beth , Watson Susan A.
Published: February 2011

Introduction: Glioblastoma multiforme (GBM) is a cytologically malignant tumour of the central nervous system, associated with poor prognosis and fatal outcome (5 year survival, <6%). Such tumours are believed to be initiated and maintained by a subpopulation of cells, which resemble normal adult stem cells. Cancer stem cells (CSCs), may contribute to the chemo-/radio-resistance exhibited by these tumours and can be identified using the immunocytochemical marker CD133. This pentaspan membrane protein is associated with increased tumorigenicity, chemo-/radio-resistance and poor prognosis. In this study we investigated the functional role of CD133 in the progression of GBM.

Methods: CD133-specific siRNA and siPORT-Amine transfection reagent were used to achieve transient knockdown of CD133 in GBM cell lines. Gene and protein expression were measured over time using real-time PCR and FACS, respectively. GBM cell lines were cultured in 1% oxygen to induce hypoxia. Transient knockdown of hypoxia-inducible factors (HIF) was also achieved using HIF-specific siRNA, in hypoxic conditions. Biological functions of CD133 were assessed by performing wound-healing assay to investigate migration; MTT to measure the rate of proliferation; neurosphere formation potential to assess tumorigenicity; and etoposide drug challenge to assess chemo-resistance.

Results: CD133-specific siRNA successfully knocked down gene expression of CD133 (85% knockdown; p<0.0001 at Day 3 post transfection; n=3). Consequent downregulation in protein expression was also confirmed (p<0.05; n=1). CD133 knockdown was correlated to susceptibility to chemotherapeutic agents, migration, proliferation and tumorigenicity. Hypoxia upregulated CD133 expression by 4-fold (p<0.0001) compared to cells cultured in normoxia. Knockdown of hypoxia inducible factors resulted in the downregulation of CD133 in hypoxia, at 48hrs post transfection. In GBM cell line U251, HIF1-α and HIF2-α knockdown significantly downregulated CD133 gene expression (HIF1-α = 40% downregulation; HIF2-α = 60% downregulation; p<0.001); HIF2-α downregulated CD133 significantly more than HIF1-α (p<0.01), when compared to control negative siRNA.

Conclusion: CD133 knockdown alters biological properties of GBM cells. Furthermore, hypoxia increases CD133 expression in GBM cells, mediated by HIF-1α and HIF-2α. This of particular importance in view of the hypoxic nature of GBMs, which suggests that hypoxia aids survival of cancer stem cells, rendering these tumours resistant to therapy.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 4306.
Abstract 4312: Identification of putative stem cell markers of CD133 and CD44 in human ovarian cancer specimens and ovarian cancer cell lines
Authors: Sun Guiqin , Meng Fanliang , Li Zhong , Zou Changping , Brewer Molly A.
Published: February 2011

Objective: Ovarian cancer may arise from a subpopulation of CD133+ 44+ cells, which are self-renewing tumor-initiating cells.

Materials and Methods: We investigated the tumorigenicity of the cell line A2780 which is CD133high44low, the cell line A2780 with siRNA against CD133 (CD133low44low), and human ovarian cancer cells from ascites, from primary ovarian cancer, and from metastatic ovarian cancer (CD133 high/CD44high) harvested from patients at the time of surgical cytoreduction in ultra low attachment plates in serum free medium. Spheroids from each of these sources were used to test their tumorigenicity in SCID mice.

Results: Cells from A2780 cell line (CD133 high/CD44low) formed tumors in SCID mice in 2 months but silencing CD133 with siRNA CD133 (CD133 low/CD44low) inhibited tumorigenicity in SCID mice and retarded but did not prevent spheroid formation. Spheroid cultures of ascites, primary ovary, and metastasis (CD133 high/CD44high) are capable of inducing primary ovarian tumors and metastatic tumors in SCID mice in 2 months compared to 4-6 months in sorted, non-spheroid cells.

Conclusions: The data suggest that CD133 expression is necessary for tumorigenesis in the A2780 cell line but not for spheroid formation. Our data also suggests that spheroids which are CD133 high/CD44high from human tumor recapitulate ovarian cancer in women. These novel in vivo and vitro models may offer useful tools to develop therapies targeted to ovarian cancer.

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 4312.
Abstract 432: Expression of PITX2 is correlated with invasiveness in breast cancer cells and its downregulation reduces invasive potential
Authors: Pillai Sreeraj G. , Dasgupta Nupur , Fleming Timothy P. , Watson Mark A. , Aft Rebecca
Published: February 2011

Objective

We have previously found that PITX2 expression in the bone marrow (BM) of women newly diagnosed with breast cancer correlates with the development of distant disease. PITX2 is not expressed in the BM of normal volunteers, and thus the source of PITX2 is likely to be disseminated tumor cells which lodge in the bone marrow. PITX2 plays a well-defined role in development. This study was conducted to understand the role of expression of PITX2 in the breast cancer phenotype.

Experimental procedures

PITX2 has three different transcript variants. The RNA expression level of each transcript variant was determined by qRT-PCR using specific primers designed for each isoforms in a variety of breast cancer cell lines. PITX2 was stably silenced using shRNA lentiviral constructs against all isoforms of PITX2 (Sigma, St. Louis) and selecting with puromycin. Individual clones were selected and tested for PITX2 expression. Those cells with near total knockdown of the gene were used for matrigel invasion assays using manufacturer's protocols. Briefly, about 3 × 104 cells were suspended in growth factor free media and added to each well of invasion chamber and complete growth medium was added to the lower chamber as chemoattractant. The number of cells which invaded was counted at 24h and 48h after seeding. Control cells included empty vector, a non-targeting sequence, and shRNA against the unrelated gene, Beta 2- Microglobulin. The number of invasive cells were counted from five different fields from each well and tallied. Percentage invasion was calculated as the number of cells passed through the Matrigel to the total number of cells before harvest. All experiments were repeated three times.

Results

PITX2 isoforms A and B were found to be expressed in the invasive cell lines MDA MB 231 and C1A1 while none of the isoforms were expressed in non-invasive cell lines MCF7 and MCF10A. In matrigel invasion assay, the knockdown of PITX2 substantially reduced invasiveness compared to the controls. There was a 63.8 % reduction in invasion in PITX2 deficient cells at 24 hrs and 72.6 % reduction at 48 hrs compared to the parental MDA MB 231 cells.

ConclusionPITX2 is expressed in breast cancer cell lines which have the ability invade and is not expressed in cell lines which cannot invade. Silencing of PITX2 in breast cancer cell lines abrogates the ability of breast cancer cells to invade. These findings combined with our clinic data suggest that PITX2 plays an important role in the invasiveness of breast cancer.

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 432.
Abstract 4329: In vivo visualization of epithelial-mesenchymal transition in real time using a rapidly tuneable E-cadherin
Authors: Leong Hon Sing , Lizardo Michael M. , Nambiar Shruti C. , Ablack Amber , Kim Daero , Chambers Ann F. , Lewis John D.
Published: February 2011

The transformation of benign tumour cells to invasive, metastatic tumour cells involves the dynamic redeployment of factors involved in cell adhesion and motility collectively known as epithelial-mesenchymal transition (EMT). While the loss of E-cadherin localization at cell-cell junctions is a hallmark feature of EMT, the impact of E-cadherin re-expression and modulation in metastatic cancer cells at distinct steps of the metastatic cascade has not been explored. To address this, we developed an intravital imaging approach that allows us to visualize changes in behaviour of highly metastatic MDA-MB-231-LN breast cancer cells in real time using a dynamically tuneable form of E-cadherin. Here, we show that upon induction of E-cadherin in vivo, cell-cell adherens junctions begin to appear within 30 minutes, concurrent with the accumulation of plasma membrane-localized E-cadherin and the retraction of tumour cell protrusions and the induction of a rounded epithelial-like morphology. Furthermore, subsequent depletion of E-cadherin results in a rapid reversion to the mesenchymal morphology. These studies demonstrate that E-cadherin is sufficient to induce a rapid mesenchymal to epithelial transition in vivo, and that sustained E-cadherin expression is required to maintain this less invasive morphology.

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 4329.
Abstract 4345: Proteasome inhibitor, bortezomib, inhibits cell proliferation and induces apoptosis of medulloblastoma cells
Authors: Yang Fan , Jove Veronica , Chang Shirley , Hedvat Michael , Liu Xiangli , Yen Yun , Jove Richard
Published: February 2011

Medulloblastoma is the most common brain tumor in children. Proteasome inhibitors have been reported to inhibit cell proliferation and survival in a variety of tumor cells. Bortezomib (Velcade), an inhibitor of the 26S proteasome, has been approved by FDA to treat multiple myeloma. Here, we investigated effects of bortezomib on human medulloblastoma cells. Bortezomib inhibited cell proliferation and induced apoptosis of Daoy medulloblastoma cells in a time- and dose-dependent manner. Significantly, a relatively low dose of 10 nM bortezomib effectively inhibited the viability of Daoy cells within 48 h. Bortezomib increased the release of cytochrome C to cytosol, resulting in activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP). Caspase inhibitor (Z-VAD-FMK) could rescue tumor cells from the cytotoxicity of bortezomib on both cell proliferation and survival. Bortezomib increased expression of the pro-apoptotic protein, Bak, and cleavage of the anti-apoptotic protein, Mcl-1. In addition, bortezomib increased the expression of p21Cip1, a negative regulator of cell cycle progression. Bortezomib also induced the accumulation of phosphorylated I[[Unable to Display Character: ĸ]]Bα in Daoy cells, resulting in inhibition of NF[[Unable to Display Character: ĸ]]B signaling. MG-132, another proteasome inhibitor, had similar effects on Daoy cells. Moreover, bortezomib inhibited the growth of human medulloblastoma cells in a mouse xenograft model. Our data suggest that proteasome inhibitors are promising drugs for the treatment of pediatric medulloblastomas.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 4345.
Abstract 4354: Celecoxib reduces MGMT expression and potentiates the effect of temozolomide in childhood medulloblastoma
Authors: Baryawno Ninib , Milosevic Jelena , Wickström Malin , Sveinbjörnsson Baldur , Eksborg Staffan , Kogner Per , Johnsen John I.
Published: February 2011

Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects DNA from the biological effects of alkylating agents. MGMT has been implicated in resistance to alkylating drugs such as temozolomide used in the clinic against brain tumors. Temozolomide has been shown to be ineffective in tumors expressing MGMT and in medulloblastoma high expression of MGMT is correlated with poor prognosis. Therefore, the demand to find agents that deplete MGMT is warranted.

Methods: medulloblastoma cell cytotoxicity of chemotherapeutic drugs in combination with Celecoxib was examined in MGMT positive cell lines using fluorometric microculture cytotoxicity assay. In vivo, nude NMRI mice carrying MGMT positive D283 MED xenografts were treated with celecoxib in combination with temozolomide. For mechanistic studies, western blot and quantitative PCR were used.

Results: the clinically available COX-2 inhibitor Celecoxib induced a synergistic or an additive cytotoxic effect in combination with Temozolomide, Cyclophosphamide, Doxorubicin, Irinotecan, Vincristine, Rapamycin or CCI-779 in vitro. In vivo, Celecoxib synergistically potentiates the effect of Temozolomide through downregulation of MGMT expression.

Conclusions: this study shows that Celecoxib reduces the DNA repair capacity of MGMT in medulloblastoma and increases the susceptibility to temozolomide. These findings support the use of pharmacological MGMT depletion as a rational approach for intensification of temozolomide therapy in the treatment of medulloblastoma.

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 4354.