bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4224: Sox2 control breast cancer stem-like cells in a breast carcinoma cell line
Authors: Leis Olatz , Erika Lopez-Arribillaga , David Chrastek , Martin Angel G
Published: February 2011

Many solid tumors, including breast cancer, exhibit a functional hierarchy of cells of which only a small subpopulation of stem-like cells can give rise to the differentiated cells that make the bulk of the tumor. Recently, a method for the culture of undifferentiated human mammary epithelial cells in non-adherent “mammospheres” has been described. Mammospheres are enriched in stem/progenitor cells giving rise to all three mammary cell types. Although gene profiling was used to compare sphere cells to differentiated cells, no specific markers were described.

Three main transcription factors: Oct4, Sox2 and Nanog, have probed responsible for maintaining the undifferentiated state of stem cells, particularly embryonic stem cells. Ectopic introduction of these factors into differentiated cells is capable of reprogramming these cells into embryonic stem-like cells (called iPS, induced pluripotent stem cells). It has been shown recently that one of the main regulators of oncogenic transformation, the p53 tumor supressor, controls the induction of pluripotency, thus linking the process of reprogramming with the process of tumorigenesis.

By characterizing factors that control mammosphere formation in the breast carcinoma cell line MCF7 we have identified Sox2 as a gene that is up-regulated upon sphere formation. Besides, ectopic expression of Sox2 is capable of inducing augmented sphere formation, suggesting that expression of Sox2 is a key regulator of breast cancer stem cell phenotype. Up-regulation of Sox2 expression is transcriptionally controlled at the level of the RR1 Sox2 promoter enhancer which is also involved in regulation of Sox2 expression in pluripotent stem cell lines.

The recent observation that the tumor suppressor p53 pathway acts as a barrier for induced pluripotency reprogramming has lead to the speculation that cancer stem cells may arise through an altered reprogramming-like mechanism. Our data suggests that Sox2 expression may be such an event.

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 4224.
Abstract 423: Suppression of endometrial cancer cell migration, invasion, and colony formation by the putative tumor suppressor gene MIIP
Authors: Wang Yingmei , Ji Ping , Yang Da , Hu Limei , Cogdell David , Broaddus Russell , Xue Fengxia , Zhang Wei
Published: February 2011

Objective: Endometrial carcinoma (EC) is the leading cancer of the female genital tract in the United States and the fourth most common cancer among women after breast, lung, and colorectal cancer. Augmented cell motility, invasion, and proliferation are hallmarks of aggressive EC. We aim to determine whether the recently discovered migration and invasion inhibitory protein (MIIP) gene functions as an inhibitor for these hallmarks in endometrial cancer cells.

Methods: We measured MIIP expression by Western blot and quantitative RT-PCR. The effect of MIIP on cell motility and invasion was measured by wound healing and Boyden Chamber transwell assay. The potential anti-tumor function of MIIP was measured by colony formation assay. Whole-genome transcriptome was determined in MIIP overexpressing cells and KEGG pathway analysis was done to determine the key pathways regulated by MIIP.

Results: The levels of MIIP mRNA and protein expression were decreased in high-grade EC compared with normal EC tissues. MIIP protein is detectable at low level in the HEC1A cell line but undetectable in the AN3CA and HEC1B cell lines. Knockdown of MIIP expression with siRNA in HEC1A cells significantly increased cell migration when compared with control siRNA treated cells (71.17±3.82 vs. 30.12±1.94, P<0.01). Elevated expression of the MIIP significantly decreased cell migration in AN3CA and HEC1B cells compared with control empty vector-transfected cells (AN3CA: 26.67±1.86 vs. 69.83±2.93, P<0.01; HEC1B: 21.83±2.04 vs. 89.83±5.56, P<0.01). The invasion of the MIIP-knocked-down HEC1A cells was significantly increased compared with control (50.67±1.86 vs.5.83±1.47, P<0.01). Consistently, cell invasion was significantly decreased in MIIP overexpressed cells compared with control HEC1B cells (18.33±2.33 vs.43.17±4.07, P<0.01). Overexpression of MIIP in EC cell lines led to reduced levels of matrix metalloprotease 2 and 9. Further, transfection with the MIIP expression vector significantly reduced colony formation by 73.6% in HEC1A compared with empty vector-transfected cells (52.25±3.30 vs.13.75±2.87, P<0.01). Gene expression profiling of MIIP overexpressed AN3CA and HEC1B cells and their control cells followed by KEGG pathway analysis showed that MIIP expression regulates TP53 signaling, Jak-STAT signaling, and PI3K/Akt pathways.

Conclusions: MIIP may function as a tumor suppressor gene for endometrial cancer and loss of MIIP expression may contribute to increased cell growth and migration/invasion activities in endometrial cancer 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 423.
Abstract 4231: Epigenetic regulation and tumorigenicity of ovarian cancer cells
Authors: Meng Fanliang , Sun Guiqin , Li Zhong , Zou Changping , Brewer Molly A.
Published: February 2011

Objective: The objective of this study was to investigate changes in CD133 expression and in tumorigenesis that occur in ovarian cancer cell lines treated with drugs that alter gene methylation.

Materials and Methods: We selected two common drugs 5-aza-2′-deoxycytidine, a demethylating agent and Trichostain A(TSA), a histone deacetylase inhibitor and investigated three ovarian cancer cell lines(CD133+ cell lines A2780 and CD133- cell lines Hey and SKOV3) to determine their effect on ovarian cells in vitro and in vivo.

Results: We demonstrated that epigenetic modification did not significantly alter cell viability but suppressed spheroid formation of cancer cells lines. FACS results indicated that the percentage of CD133+ cells increased following treatment with 5uM 5-aza-2′-deoxycytidine and 300nM trichostatin A after 72° incubation. Xenograft assay of the NOD/SCIDmice results demonstrated that both drugs reduced spheroid formation and blocked tumorigenesis.

Conclusions: We concluded that 5-aza-2′-deoxycytidine and TSA can significantly suppress both spheroid formation and tumor formation and could be provide a new strategy for the treatment of resistant 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 4231.
Abstract 4232: Tumor-associated macrophages are responsible for EGF-R triggered upregulation of the Sox-2 signaling pathway in CSCs, which enhance tumorigenicity and tumor metastasis
Authors: Xiang Rong , Liao Debbie , Chen Mei Ting , Chuang Tsung-Hsien , Reisfeld Ralph A. , Luo Yun Ping
Published: February 2011

The cancer stem cell (CSC) hypothesis has gained significant recognition as the descriptor of tumorigenesis. The Sox-2 signaling pathway has an important role in the maintenance of CSCs. Tumor-associated macrophages (TAMs) play an ambiguous role in this process. Here, we demonstrate that the expression of Sox-2 was up-regulated in CSC of 4T07 breast cancer cells, effects induced and enhanced by co-culture of tumor cells with TAMs. In fact, after co-culture, 4T07 tumor cells develop an increased number of CSCs with up-regulated expression of stem cell specific markers (Scal-1 and ABCG2), as well as partner genes of Sox-2, i. e. Oct-4 and Nanog. Down regulation of Sox-2 effectively blocks the ability of TAMs to maintain the phenotype of CSC among 4T07 cancer cells, and enhances tumor development and its pulmonary metastasis in a syngeneic Balb/c mouse model. Most importantly, in a mechanism study, we found that the Sox-2 up-regulation was induced by EGF-R activation in CSC that was triggered by EGF, Macrophage Conditioned Medium (MCM) or by co-culture with TAMs. These effects can all be blocked by inhibitors of EGF-R. Our results suggest that TAMs in the tumor microenvironment(TME) provide a niche which is critical in the development and maintenance of CSCs in breast carcinoma. This critical effect of TAMs on CSC development was achieved by up-regulation of the Sox-2 signaling pathway in CSC. This, in turn, was induced by marked activation of EGF-R achieved through interactions between CSCs and TAMs. In summary, over expression of Sox-2 in CSC of 4T07 breast cancer cells was enhanced by activation of EGF-R, which was induced by TAMs in TME. This series of events is responsible for maintaining the phenotype of CSCs which enhances tumor development and pulmonary metastasis in vivo.

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 4232.
Abstract 4235: Notch ligand-dependent cancer stem cell niche in glioblastoma
Authors: Zhu Thant S. , Hervey-Jumper Shawn , DiMeco Francesco , Vescovi Angelo L. , Heth Jason A. , Muraszko Karin M. , Fan Xing
Published: February 2011

Recent data shows that endothelial cells function as a stem cell niche to promote CD133-positive cancer stem-like cells (CSLCs) self-renewal in glioblastoma (GBM). However, the mechanism by which endothelial cells function as a stem cell niche is largely unknown. To test if endothelial cells and differentiated tumor cells could function as a niche by providing Notch ligands to CSLCs, first we co-cultured GBM neurospheres with primary human endothelial cells (PHECs) which express Notch ligand Jagged/Delta. We found that PHECs promote GBM neurosphere growth, CD133 expression and clonogenicity in vitro, whereas knocking down Notch ligand expression in PHECs abrogates PHEC-induced GBM growth. We also found that the CD133-negative GBM cells express a higher level of Jagged/Delta and glial differentiation marker GFAP compared with CD133-positive population. When GBM neurospheres were forced to differentiate and grow as mono-layer attached cells, CD133-positive population was reduced, whereas expression of GFAP (glial marker), GalC (oligodendrocyte marker), Tuj1 (neuronal marker) and Notch ligands were induced. Furthermore, treatment of GBM neurospheres with a Jagged peptide increases GBM growth in a dosage dependent fashion. In addition, Notch ligands are highly expressed in the blood vessels of GBM primary tumors and intracranial xenografts in mouse, and CSLCs accumulate around the blood vessels within the tumors. Finally, we found that knocking down Notch ligands in PHECs inhibits GBM xenograft propagation in mice through reducing CD133-positive population. In summary, we show that Notch activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells replicating the classical process of lateral inhibition and by stromal niche signals, suggesting that targeting both CSLCs and their niche may provide a profound strategy to deplete GBM CSLCs.

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 4235.
Abstract 4237: Foxo3a is essential for survival of leukemia-initiating cells in chronic myeloid leukemia
Authors: Naka Kazuhito , Hoshii Takayuki , Muraguchi Teruyuki , Tadokoro Yuko , Ooshio Takako , Motoyama Noboru , Oshima Masanobu , Hirao Atsushi
Published: February 2011

Chronic myelogenous leukemia (CML) is caused by a defined genetic abnormality, BCR-ABL, a constitutively active protein tyrosine kinase. Although the introduction of imatinib, a small molecule inhibitor of ABL, represented a breakthrough in the treatment of CML, major part of patients treated in chronic-phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor of the production of differentiated leukemic cells, but does not deplete leukemia-initiating cells (LICs). To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, we here evaluated molecular mechanisms on survival of the CML LICs.

We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin-Sca-1+ (KLS+) population in BCR-ABL+ CML cells, as previously reported. Unexpectedly, phosphorylation levels of Akt in CML LICs appeared to be lower than that in non-LICs, KLS- cells, despite it is widely believed that BCR-ABL induces activation of Akt signal. Since Forkhead O (Foxo) transcription factors, which are important downstream targets of PI3K-Akt signaling, are essential for the maintenance of self-renewal capacity of normal HSCs, we focused on analysis of Foxo3a in CML. Consistent with Akt phosphorylation status, we found that cells with nuclear localization of Foxo3a were enriched in the CML LICs, whereas non-LICs showed the cytoplasmic localization, suggesting that Foxo3a is activated via Akt inactivation in the CML LICs, but not in majority of CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at 3rd transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at 3rd transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-3rd transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. Furthermore, deficiency of Foxo3a led to enhanced efficiency in elimination of CML LICs in combination with imatinib treatment. These results demonstrate a critical role of Foxo pathway in survival of CML LICs, and provide a novel therapeutics approach for CML patients by suppression of FOXO signaling.

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 4237.
Abstract 4244: Dysadherin can enhance tumorigenesis through conferring properties of stem-like cells to hepatocellular carcinoma cells
Authors: Park Jeong-Ran , Kim Ran-Ju , Lee Yoo-Kyung , Kim Soo-Rim , Roh Kyung-Jin , Nam Jeong-Seok
Published: February 2011

Hepatocellular carcinoma (HCC) is associated with a high potential for metastasis and disease recurrence, even after surgical resection. The cancer stem cell (CSC) hypothesis proposes that CSCs are responsible for chemo-resistance, recurrence and metastasis. Dysadherin is a prognostic indicator of metastasis and poor survival in many different cancer types. In this study, we investigated the possible link between dysadherin and CSC in HCC. The transfection of dysadherin cDNA into the liver cancer cell line PLC/PRF/5 enhanced the properties of CSC, including anti-apoptosis, sphere-forming ability, side population (SP) phenotype and tumor initiation ability in vivo. Furthermore, knockdown of dysadherin in the liver cancer cell line SK-Hep1 suppressed its stem cell-like properties. In particular, SP phenotype is mediated by the ATP-binding cassette (ABC) transporter proteins which are responsible for multidrug resistance (MDR) that is a major obstacle in cancer chemotherapy. Dysadherin was also shown to regulate ABCB1 expression. Taken together, these results show that new roles for the dysadherin in cancer progression that was in part dependent on CSC dynamics. Therefore, these findings suggest that dysadherin may be a potential molecular prognostic marker of HCC and may aid in the development of more effective therapies.

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 4244.
Abstract 4248: NANOG depletion decreases cell proliferation rate in colorectal carcinoma cells and tumor growth in nude mice
Authors: Espinoza Luis A. , Jessup John M.
Published: February 2011

Nanog is a critical transcription factor involved in maintaining embryonic stem cells in a pluripotent, dedifferentiated state and expression of NANOG or its nearly identical retrogene NANOGP8 is increased in human gastric, hepatocellular, and colon cancer. Our postulate is that NANOG/NANOGP8 is critical for the functions of putative CSC in colorectal carcinoma (CRC): self-renewal, pluripotency, spherogenicity, tumorigenicity and resistance to chemotherapy. Five human CRC lines are being used in this study (HCC 2998, KM-12c, Clone A, MIP-101, CX-1). CX-1 cells are more aggressive than the other CRC because they form more spheroids from single cells and are highly metastatic after intrasplenic compared to the other CRC that are tumorigenic but weakly metastatic. qRT-PCR analysis of spheroids of CX-1 and Clone A cultured in suspension in serum free medium indicated that NANOG transcript levels were increased by 43- and 12-fold, respectively, relative to monolayer cultures while the relative increase in transcript levels of several cancer stem cell markers such as EPCAM, ALDH1A1, ALDH3A1, CD44, CD133, and CD166 was less than 5-fold except for POU5F1 and SOX2 in Clone A, which were 5- and10-fold respectively. NANOG promoter activity was low in monolayer culture in CRC stable transfectants with a GFP NANOG promoter, but consistently increased as CRC detached and formed spheroids in serum free media. Stable transfection with lentiviral NANOG shRNA decreased NANOG transcript levels and spherogenicity of Clone A and CX1 cells compared to vector controls in a single cell spherogenicity assay in serum-free medium. NANOG downregulation also induced a S phase cell cycle arrest with inhibition of ATM and WEE1 expression with associated reduction in phosphorylation of other key cell-cycle checkpoint proteins. Finally, marked tumor growth retardation was observed in NOD/SCID mice injected subcutaneously with NANOG shRNA transfected cells compared to parental or vector controls. NANOG shRNA transfected cells were also more resistant to treatment with Topotecan, a camptothecin. In summary, our data suggest that expression of NANOG/NANOGP8 occurs in human CRC and may be critical for such functions of putative CSC as spherogenicity, cell proliferation, tumor formation and resistance to chemotherapy

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 4248.
Abstract 4249: Shape change induces nitric oxide and stemness in colorectal cancer cells
Authors: Jessup J. Milburn , Thorgeirsson Snorri S. , Espinoza Louis A.
Published: February 2011

Shape changes in cancer cells profoundly alter the expression of genes that affect malignant properties. We postulated that shape change may increase expression of NANOG, an embryonic stem cell transcription factor expressed in human colorectal carcinoma (CRC) that regulates pluripotency and proliferation. Since shape change also increases intracellular nitric oxide (iNO) expression, we further postulated that iNO modulates NANOG expression. Clone A and CX-1 human CRC lines were cultured in serum-containing medium in monolayer cultures or in spheroids in serum free medium in low attachment plates. NANOG promoter activity was assessed in stable transfectants expressing promoter-driven GFP. iNO concentrations were determined by fluorescence cytometry with DAF-FM diacetate while Nanog gene and protein expression was analyzed by qRT-PCR and densitometry of western blots, respectively. Spherogenicity was measured by the percentage of spheroids formed from single cells in serum-free medium. Monolayer cultures with flat, spreading cells had low NANOG gene and protein expression, promoter activity and iNO whereas spheroids displayed high NANOG gene expression, promoter activity and iNO. 7 day monolayer cultures initiated with 104 cells/ml (Sparse) grew horizontally compared to cultures initiated with 106 cells/ml (Dense) that grew vertically and formed colonies. Sparse cultures had low NANOG gene expression, low NANOG promoter activity and low iNO. Dense cultures had high NANOG gene expression, NANOG promoter activity and iNO. Since NO stimulates cGMP production, the effects of the NO synthase inhibitor L-NMMA, NOC (a NO donor), 8-Br-cGMP (cGMP agonist) and ODQ (cGMP inhibitor) were tested on CRC monolayers. Physiologic concentrations of NOC increased NANOG and Oct4 gene and protein expression 3-fold in highly metastatic CX-1 cells but not in weakly metastatic Clone A cells. NOC also increased CD44 in CX-1 but not Clone A. cGMP may regulate NANOG expression since 8-Br-cGMP increased NANOG and Oct4 protein expression up to 3-fold in CX-1 but not in Clone A and without affecting CD44 expression. ODQ inhibited NANOG expression. L-NMMA stimulated both NANOG protein expression and the spherogenicity of CX-1 but not Clone A. NOC and 8-Br-cGMP increased inducible NO synthase (NOS2) but not NOS3 gene expression 3-fold in both spheroids and monolayers while ODQ decreased NOS2 levels. Assays are in progress to test the effects of NOC and the cGMP modulators on spherogenicity. Shape change, iNO and cGMP enhance NANOG, NOS2 and CD44 expression to alter CRC cancer stem cell function. cGMP may be an important regulator of stemness genes.

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 4249.
Abstract 4254: The Wnt/ B-catenin pathway regulates colon cancer stem cells proliferation
Authors: Kanwar Shailender S. , Yu Yingjie , Nautiyal Jyoti , Patel Bhaumik , Majumdar Adhip P.N.
Published: February 2011

It is becoming increasingly evident that a small sub-population of self-renewing, multipotent cells termed cancer stem cells (CSCs) is responsible for driving majority of the epithelial cancers including colon cancer. The observation that CSCs display resistance to conventional chemotherapeutics suggests that they may be responsible for the recurrence of cancers. One of the properties of CSCs is that they form floating spheroids (colonosphere) in a defined media. Different signaling pathways such as Wnt, Hedgehog, Notch and Bmi have been implicated in the regulation of colonosphere formation and stem cell self renewal. The current investigation was undertaken to examine the role of Wnt/B-catenin pathway in regulating the growth and maintenance of colonosphere. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT 116 (p53 null; K-ras mutant) and HT 29 (p53 mutant) were used. We have observed that colonospheres formed in vitro exhibited higher expression of colon CSCs markers ALDH1, LGR5, CD133, CD44 and Musashi, compared to the corresponding parental cancer cells, indicating the predominance of CSCs in colonosphere. Further support for this inference comes from the FACS analysis that show 95-97% of CD44 positive cells in colonospheres. Additionally, colonosphere showed increased levels of B-catenin, cyclin D1 and C-Myc and down-regulation of Axin-1 and phosphorylated B-catenin. Increased expression of B-catenin was associated with a marked transcriptional activation of TCF-LEF. To determine whether B-catenin plays a role in the maintenance and formation of colonosphere, we transfected the colon cancer cells with B-catenin siRNA and subsequently analyzed colonosphere formation. The results revealed a marked reduction in the colonosphere formation following down-regulation of B-catenin indicating its role in colonosphere formation. Further support for this interpretation comes from the observation that the upregulation of cMYC /cyclin D1 (the target genes of LEF/TCF) results in enhancement of the colonosphere formation. In conclusion, our data suggest that Wnt/B-catenin pathway plays a critical role in the growth of 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 4254.
Abstract 4255: Significance of mesenchymal stem cells in growth and metastasis of colon cancer
Authors: Shinagawa Kei , Kitadai Yasuhiko , Tanaka Miwako , Sumida Tomonori , Kodama Michiyo , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: February 2011

Background: Recently, mesenchymal stem cells (MSCs) are reported to migrate to tumor sites as well as sites of injury and to incorporate into tumor stroma, but the effects of interactions between MSCs and tumor cells and the mechanisms underlying these effects remain unclear. We examined the role of MSCs in the tumor micro-environment using an orthotopic colon cancer model and a model of liver metastasis from colon cancer. Materials and Methods: Human MSCs labeled with fluorescent dye were injected into tail veins of nude mice bearing KM12SM human colon cancer cells. KM12SM cells alone or KM12SM cells mixed with MSCs were transplanted into the cecal wall or spleen of nude mice. Differentiation of MSCs in primary colon tumors and metastatic liver tumors were also analyzed. Migration assay and invasion assay were performed to examine attraction between KM12SM cells and MSCs in vitro. Results: In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in significantly greater tumor weight than did transplantation of KM12SM cells alone (p < 0.05). The survival rate was significantly lower in the mixed-cell group, and liver metastasis was seen only in this group (p < 0.001). Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index (p < 0.01), significantly greater microvessel area (p < 0.01), and significantly lower apoptotic index (p < 0.001). Splenic injection of KM12SM cells mixed with MSCs resulted in a significantly greater number of liver metastases than did splenic injection of KM12SM cells alone (p < 0.01). MSCs incorporated into the stroma of primary and metastatic tumors expressed α-smooth muscle actin and platelet-derived growth factor receptor-β as carcinoma-associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors (p < 0.05, respectively). Conclusion: MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis and by inhibiting apoptosis of tumor 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 4255.
Abstract 4264: CD133/prominin-1 regulates the tumorigenicity of oral squamous carcinoma cells-derived cancer initiating cells
Authors: Chen Yu-Syuan , Lo Jeng-Fan , Yu Cheng-Chia
Published: February 2011

Background: Oral squamous carcinoma cell (OSCC) is a lethal cancer with clinical, pathological, phenotypical, and biological heterogeneity. Previously, we enriched a subpopulation of oral cancer derived cancer initiating cells (OC-CICs) by tumor sphere formation approach, and identified CD133 as a putative CICs marker. However, the function of CD133 in OC-CICs remains unclear. Herein, we determined the critical role of CD133, an important CICs marker, in the maintenance of stemness characteristics and tumorigenic phenotype in OC-TICs.

Methods: First, side population (SP) assay, a methodology to identify CICs in cancer cells, was performed to characterize the existence of OC-CICs. Next, we want to evaluate the expression of CD133 in OC-CIC isolated from SP cells transcriptionally and translationally. Lentiviral-mediated stable overexpression and down-regulation of CD133 expression in OSCCs and OC-CICs was established. Consequently, we investigated the effect of CD133 down-regulation or overexpression on stemness properties and tumorigenicity of OSCCs and OC-CICs, respectively.

Results: We identified that CD133 was significantly up-regulated in SP cells of OSCCs. Stable overexpression of CD133 enhanced the CICs properties in OSCCs. In opposite, down-regulation of CD133 significantly reduced the self-renewal ability, side population cells, and expression of stemness genes of OC-CICs. Additionally, down-regulation of CD133 enhanced the differentiation capability but inversely diminished “stemness” gene expression of OC-CICs. Finally, knockdown of CD133 in OSCC or OC-CICs lessened both in vitro malignant abilities including cell migration/cell invasion/anchorage independent growth and in vivo tumor growth by xenotransplantation assay.

Conclusion: We showed that SP cells with CICs properties existed in OSCCs. CD133 contributes to the maintenance of stemness and tumorigenicity of OC-CICs. Silencing CD133 might be a potential therapeutic target for OSCC by eliminating CICs.

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 4264.
Abstract 4272: Elimination of human lung cancer stem cells through targeting of the SCF-c-kit autocrine signaling loop
Authors: Levina Vera V. , Marrangoni Adele , Wang Tingting , Parikh Simul , Su Yunyun , Herberman Ronald , Lokshin Anna , Gorelik Elieser
Published: February 2011

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation and tumor regeneration after chemotherapy. Earlier we demonstrated that chemotherapy led to the selection of CSCs and that the high malignancy of lung CSCs was associated with an efficient cytokine network. Here we propose that blocking SCF-c-kit signaling will inhibit CSC proliferation and survival. CSCs were isolated from nonsmal cell lung cancer (NSCLC) cell lines as tumor spheres under CSC-selective conditions, and their stem/progenitor properties were confirmed. A Cellomics ArrayScan HCS Reader was applied to test the cell surface markers, the intracellular protein distribution and cell proliferation. CSCs in contrast to bulk cells expressed high levels of c-kit receptors and produced SCF. Proliferation of CSCs was stimulated by exogenous SCF and was inhibited by antibodies neutralizing SCF and by imatinib (Gleevec), a specific inhibitor of c-kit. Cisplatin treatment eliminated major of bulk cells but not CSCs, whereas imatinib or anti-SCF antibody destroyed CSCs. In combination cisplatin and imatinib or anti-SCF antibody prevented growth of both tumor cell subpopulations.

Our results elucidate the important role of SCF-c-kit signaling axis for the self-renewal and proliferation of lung CSCs. Blocking SCF-c-kit signaling by anti-SCF antibody or imatinib in combination with cisplatin could be more efficient in elimination of human tumor cells than each modality used separately.

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 4272.
Abstract 4273: DNA methyltransferase 3B mediates 5-aza-deoxycytidine hypersensitivity in cisplatin-sensitive and cisplatin-resistant pluripotent embryonal carcinoma cells
Authors: Beyrouthy Maroun J. , Garner Kristen , Hever Mary P. , Freemantle Sarah J. , Eastman Alan , Dmitrovsky Ethan , Spinella Michael J.
Published: February 2011

Testicular germ cell tumors (TGCTs) are the most common solid tumors of 15-35 year old men. TGCT patients are frequently cured with cytotoxic cisplatin-based therapy. However, TGCT patients refractory to cisplatin-based chemotherapy have a poor prognosis, as do those having a late relapse. Pluripotent embryonal carcinoma (EC) are the malignant counterparts to embryonic stem (ES) cells and are considered the stem cells of TGCTs. Here we show that human EC cells are highly sensitive to 5-aza-deoxycytidine (5-aza-CdR) as compared to a variety of somatic solid tumor cells. This is associated with markedly abundant expression of the pluripotency-associated DNA methyltransferase 3B, DNMT3B, as compared to somatic tumor cells. Knockdown of DNMT3B in EC cells resulted in substantial resistance to 5-aza-CdR, strongly implicating that 5-aza-CdR sensitivity is mechanistically linked to high levels of DNMT3B. Intriguingly, cisplatin resistant EC cells retain an exquisite sensitivity to low dose 5-aza-CdR treatment and pretreatment of 5-aza-CdR re-sensitizes these cells to cisplatin-mediated toxicity. These findings indicate that high expression of DNMT3B, a likely byproduct of their pluripotent and germ cell nature, sensitizes TGCT-derived EC cells to low dose 5-aza-CdR treatment. Decreased proliferation and survival with low nanomolar concentrations of 5-aza-CdR is associated with ATM activation, H2AX phosphorylation, increased expression of p21, and the induction of genes known to be methylated in TGCTs. Data pertaining to the mechanism of DNMT3B-dependent, 5-aza-CdR hypersensitivity in TGCTs will be presented. Efforts to extend this work to clinical samples are ongoing.

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 4273.
Abstract 4274: High aldehyde dehydrogenase activity identifies a chemotherapy-resistant population of ewing's sarcoma cells with a stem cell phenotype that retains sensitivity to EWS-FLI1 inhibition
Authors: Awad Ola , Yustein Jason T. , Shah Preeti , Gul Naheed , Rotenberg Julie B. , Toretsky Jeffrey , Loeb David
Published: February 2011

Ewing's sarcoma family tumors (ESFT) are the second most common bone tumors in children and young adults. Despite being quite responsive to chemotherapy, patients with localized disease have a 30% recurrence rate, and 80% of patients with metastatic disease die within 5 years of diagnosis. The expression of the EWS-FLI1 fusion protein as a result of chromosomal translocation (11; 22) (q24; q12) is crucial for initiation and maintenance of the tumor; however, the mechanism by which EWS-FLI1 mediates neoplastic transformation is poorly understood. RNA helicase A (RHA) physically interacts with EWS-FLI1, modulating its oncogenic activity. The cancer stem cell hypothesis proposes that tumors originate from and are maintained by a small subset of chemotherapy-resistant stem cells. Thus, identification of Ewing's sarcoma stem cells will lead to the development of targeted therapies that should improve the treatment of patients with this disease. We attempted to identify a population of Ewing's sarcoma cells with stem cell properties (self-renewal in vitro, clonogenic activity, and tumor initiating activity in immunodeficient mice) based on high aldehyde dehydrogenase (ALDHhigh) activity. The ALDHhigh population was enriched for stem cell activity (compared to the ALDHlow cells) as defined by these assays, and was resistant to standard chemotherapy drugs to treat sarcomas (doxorubicin and etoposide). Both populations expressed EWS-FLI1. We then investigated whether a novel small molecule, YK-4-279, which specifically targets the RHA/EWS-FLI1 interaction, is toxic to the chemotherapy-resistant ALDHhigh cells. We found that the ALDHhigh cells retain sensitivity YK-4-279. Our data demonstrate that ESFT cells that express high levels of ALDH have stem-like properties, and, combined with evidence that YK-4-279 inhibits the growth of ESFT xenografts in mice, provide support for a novel approach to ESFT therapy. This approach should improve the survival of patients with these tumors in the future.

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 4274.
Abstract 4282: The role of glycosphingolipids in the formation of cancer stem cells and drug resistance
Authors: Gupta Vineet , Patwardhan Gauri Anand , Gu Xin , Rowan Brian , Jazwinski Michal , Liu Yong Yu
Published: February 2011

Tumorigenesis and malignant progression of cancers rely on a small subset of cells known as cancer stem cells (CSCs). In addition to forming primary tumors, metastasis and relapse, CSCs always display resistance to cytotoxins and this resistant feature protects CSCs during the course of chemotherapy. Glucosylceramide synthase (GCS) is a limited enzyme catalyzing the first glycosylation in glycosphingolipids synthesis. Glycosphingolipids are likely to play an essential role in maintaining the stemness of stem cells, since that deletion of GCS induces apoptosis of embryonic stem cell and stops embryo development in GCS knockout mice. The stage-specific embryonic antigens (SSEA-3, SSEA-4) are glycosphingolipids and serve as common pluripotent markers for human embryonic stem cells. In the present study, we have found that GCS modulates the formation and maintenance of breast cancer stem cells. GCS overexpression was interrelated to the increase of breast cancer stem cells (BCSCs) and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cell. Analyzed by using flow cytometry and immunostaining, the BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold, and 3-fold in MCF-7/Dox and NCI-ADR/RES cell lines, as compared to MCF-7 cell lines, respectively. In BCSCs, GCS enzyme activity was 2-fold greater than other populations sorted from MCF-7/Dox. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs in MCF-7/Dox cells. We observed tumorigenesis of NCI/ADR-RE/GCS (GCS overexpressed) and NCI/ADR-RE/asGCS (GCS silenced) in athymic nude mice. Aggressive tumors were found in all mice inoculated with NCI/ADR-RES and NCI/ADR-RE/GCS cells that were BCSC-enriched; however, no tumor or metastasis was observed in mice injected with NCI-ADR/asGCS cells. Furthermore, MBO-asGCS treatment (1 mg/kg) significantly decreased the numbers of BCSCs isolated from tumors of NCI-ADR/RES, as compared with control groups. Furthermore, BCSCs after MBO-asGCS treatment were more sensitive to doxorubicin. These results, for the first time, demonstrate that glycosphingolipid is involved in the regulation of cancer stem cell formation. Silencing GCS eliminates BCSCs, that can reverses drug resistance as well as prevent tumor relapse.

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 4282.
Abstract 4298: Identification of subpopulation with more defined characteristics of cancer stem cells in endometrial cancer
Authors: Nakamura Mitsuhiro , Kyo Satoru , Zhang Xiuzhi , Takakura Masahiro , Hashimoto Manabu , Mizumoto Yasunari , Mori Noriko , Sasagawa Toshiyuki , Inoue Masaki
Published: February 2011

[Background] Cancer stem cells (CSCs) have been known to be the major source of tumor propagation and might be an attractive therapeutic target. Our previous study showed that CD133+ Ishikawa and MFE 280 endometrial cancer cells have the ability of tumorigenicity, self-renewal and differentiation capacity in vitro and in vivo, while CD133- cells did not. Furthermore, CD133+ cells showed more resistance to cisplatin- or paclitaxel-induced cytotoxicity than CD133- cells. These results indicate that CD133 is the CSC maker in endometrial cancer cells. In the present study, we sought to identify the subpopulation with more defined CSC characteristics in endometrial cancer.

[Methods] We examined the expression of CD133, CD44 and Hoechst 33342 side population (SP) marker by flow cytometry (FACS) in endometrial canecr cells. MIcroarray analysis was performed to investigate the molecules essential for the CSCs characteristics by 3D-Gene chip (TORAY).

[Results] FACS analysis demonstrated that in the CD133+ Ishikawa and MFE280 cells, about 50% cells showed CD44 expression, whereas about 20% cells expressed it in CD133- cells. Isolated CD44+/CD133+ cells showed more extensive proliferation and tumorigenicity, compared with CD44+/CD133-, CD44-/CD133+ and CD44-/CD133- cells, indicating that the CD44+/CD133+ endometrial cancer cells have the potential of more defined CSCs. On the other hand, the ratio of SP fraction was similar in CD133+ and CD133- cells. Microarray analysis showed elevated expression of MT1-MMP and VEGF in CSCs, compared to non-CSCs. Invasion assay revealed that CSCs have more invasive property than non-CSCs. The inhibition of MT1-MMP gene by siRNA significantly reduced the invasive ability of CSCs. These results suggest that MT1-MMP is one of the critical molecules that generate biological features of CSC and may be a potential therapeutic target in endometrial cancer.

[Conclusions] We demonstrated that CD44+/CD133+ cells might be more defined CSCs in endometrial cancer and identified important molecules essential for characterizing endometrial CSCs that might be a therapeutic target of this tumor type.

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 4298.
Abstract 43: Role of Ets-1 in the regulation of energy metabolism in cancer cells
Authors: Verschoor Meghan L. , Verschoor Chris P. , Singh Gurmit
Published: February 2011

Cancer cells predominantly utilize glycolysis for ATP production even in the presence of abundant oxygen, an environment that would normally result in energy production through oxidative phosphorylation. Although the molecular mechanism for this metabolic switch to aerobic glycolysis has not been fully elucidated, it is likely that mitochondrial damage to the electron transport chain and the resulting increased production of reactive oxygen species (ROS) are significant driving forces. In this study, we have investigated the role of the transcription factor Ets-1 in the regulation of mitochondrial function and metabolism. Microarray analysis of the effects of Ets-1 over-expression in 2008 ovarian cancer cells was conducted using the Affymetrix GeneChip® Human Gene 1.0ST Array. Our findings show that Ets-1 up-regulates key enzymes involved in glycolysis and associated feeder pathways, fatty acid metabolism, and antioxidant defense. Additionally, Ets-1 down-regulates genes involved in the citric acid cycle, electron transport chain, and mitochondrial proteins. These results were validated using real-time qRT-PCR. The mitochondrial activity of both ovarian cancer Ets-1 over-expressing and breast cancer Ets-1 down-regulating cells was evaluated by examining oxygen consumption using various electron transport chain inhibitors (Oxygraph 2k), growth in the absence of glucose, and sensitivity to the glycolytic inhibitor 2-DG. We have found that Ets-1 expression is directly correlated with cellular oxygen consumption whereby increased expression causes decreased oxygen consumption. Ets-1 over-expression also caused increased sensitivity to glycolytic inhibitors, as well as pronounced growth inhibition in a glucose-depleted culture environment. In the context of ROS production, Ets-1 up-regulation induces a significant reduction in H2O2 production, likely due to Ets-1-mediated up-regulation of antioxidants. Collectively, our findings show that Ets-1 is involved in the regulation of cellular metabolism and response to oxidative stress in cancer cells. (This work was supported by operating grants to GS provided by the Canadian Institutes of Health Research)

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 43.
Abstract 430: Serpin-A1 and A3 as potential pharmacodynamic biomarkers for NOV-002, a redox-modulating anti-cancer agent
Authors: Grek Christina L. , Townsend Danyelle M. , Uys Joachim D. , Pazoles Christopher J. , Tew Kenneth D.
Published: February 2011

NOV-002 is a formulation of disodium glutathione disulfide (GSSG) that is in Phase III clinical trials for the treatment of advanced non-small cell lung cancer and other oncology indications in combination with standard chemotherapeutic regimens. In addition to increased efficacy compared to chemotherapy alone (survival, tumor growth), NOV-002 has been shown to mitigate chemotherapy-induced hematological suppression. In animal studies it was found that, when administered after chemotherapy, NOV-002 stimulated proliferation of bone marrow progenitor cells and normalized peripheral blood levels of leukocytes and platelets. NOV-002's pharmacokinetic properties demonstrate first order absorption / elimination with a plasma half-life of ∼13 mins. The redox regulation of protein thiols persist in plasma for ∼4 hours. Using mass spectrometry, we identified that Serpin-A1 and -A3 were S-glutathionylated following NOV-002 administration to mice. Serpin-A1 and -A3 were also glutathionylated in a dose- and time- dependent S-glutathionylation following NOV-002 treatment of mouse or human plasma in vitro. There is evidence that members of the Serpin protein family can influence myeloproliferation and hematopoetic progenitor cell mobilization (Winkler et al., 2005; van Pel et al., 2006). Specifically, down regulation of serpins has been demonstrated in bone marrow during progenitor cell mobilization (van Pel et al., 2006), and this influences the marrow microenvironment and migratory behavior of hematopoetic precursor cells. Furthermore, glutathionylation of Serpins has been shown to inhibit their activity (Taygi et al., 1991 and 1992). Since our data shows that Serpins A1 and A3 are S-glutathionylated in plasma following NOV-002 treatment, there may be a mechanistic link between this effect and its in vivo myeloproliferative actions. Moreover, liberation of serpins into the peripheral circulation may also provide an indication that proteolytic pathways have been activated. Since the plasma half-life of S-glutathionylated proteins is ∼4 hours (Townsend et al., 2006), it may be possible to identify and develop these post-translationally modified blood proteins as pharmacodynamic biomarkers.

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 430.