bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4228: A functional role of OCT4 for prostate tumor initiation
Authors: Linn Douglas E. , Yang Xi , Sun Feng , Xie Yingqiu , Chumsri Saranya , Burger Angelika , Qiu Yun
Published: February 2011

We have established several drug resistant prostate cancer cell lines by long term culturing hormone refractory CWR-R1 cells in medium containing chemotherapeutic drugs. These cells displayed a significant increase in side-population cells due to overexpression of drug efflux pumps including ABCG2/BCRP and MDR1. To further characterize drug resistant CWR-R1 prostate cancer cell lines, we performed microarray analysis on two drug resistant cell lines in comparison with the drug sensitive parental line. Our Ingenuity pathway analysis suggested that OCT4/POU5F1, the key transcription factor for regulating pluripotency in embryonic stem cells, was upregulated in the drug-resistant lines compared to the parental cells, which was accompanied with transcriptional activation of a set of OCT4 known target genes. Upregulation of OCT4 in drug resistant cells was validated by RT-PCR and sequencing of PCR products as well as confirmed by western blot and specific shRNA knockdown. Analysis of the regulatory region of OCT4 gene revealed that drug-resistant cell lines had reduced methylation compared to the parental line. Furthermore, these drug resistant cells exhibited a significant increase in transformation activity in vitro and tumorigenicity in vivo.

Subcutaneous inoculation of as few as ten drug-resistant cells could initiate tumor formation in SCID mice, whereas no detectable tumors were observed from the parental line under similar conditions. Our data suggest that these drug resistant cells may be enriched for tumor initiating cells. Knock-down OCT4 expression in drug-resistant cells by the specific shRNA resulted in a considerable decrease in growth both in vitro and in vivo. Our data suggest that OCT4 may play an essential role in promoting tumor initiation of prostate 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 4228.
Abstract 4229: Wound repair promotes ovarian cancer stem cell self-renewal
Authors: Chefetz-Menaker Ilana , Lebowitz Noah , Aldo Paolomi , Alvero Ayesha , Holmberg Jennie , Mor Gil
Published: February 2011

Background: In normal tissues following injury, there is an expansion of tissue specific stem cells prior to their differentiation to initiate injury repair. Once the tissue is repaired, the stem cells return to a quiescent state. Tumor tissue, might follow similar characteristics as in normal tissues, but the control of the expansion process may be significantly altered. Recently, ovarian cancer stem cells (ovCSC) were isolated from ovarian cancer tissue and ascites using CD44/CK18 as markers. We hypothesize that these cells play a critical role in tumor repair and renewal. To test this hypothesis, we used an in vitro wound/healing system.

Methods: Cells were plated in an Essen ImageLock plate and wounds were made using an Essen WoundMaker. This sytem allows the generation of consistent wound locations and widths and precise imaging of the wound area. The healing process is monitored using an insitu imaging system (IncuCyte, Essen Instruments, NH) that records and quantifies the repair process in real time. Levels of cancer stem cell markers, CD44, Oct-4, and β-catenin were determined by Western blot analysis or Real Time PCR. Levels of cytokines and chemokines were determined using Luminex Technology.

Results: Initially we tested whether an in vitro wound/healing system can be used as a renewal model. Interestingly, ovCSCs showed a well coordinated and organized response to the wound. First, the cells created a “straight line” to replace the irregular edge of the wound. Afterwards, the cells proliferated towards the wound, repairing the wound until confluence was reached. Analysis of cancer stem cell markers by Western blot revealed that the cells which repaired the wound maintained CD44, CK18, Oct4, and β-catenin, previously shown as ovCSC markers. Additionally, the process of repair was characterized by significant and differential up-regulation in IL-6, GRO-α, and MCP-1 expression and secretion. Interestingly, the CD44 expression was increased in wounded wells when compared to control. Moreover, using specific primers for CD44 variant isoforms we found that wound healing promotes expression of different CD44 forms.

Conclusions: In this study we demonstrate ovCSCs have the capacity to repair an in vitro wound and that this process is accompanied by increased CD44 expression and cytokine secretion. This increased capacity of self renewal can lead to the accumulation of a large pool of progenitor cells, which has the potential to individually spread to other areas of the body as metastatic 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 4229.
Abstract 424: Invasion and metastasis of human colon cancer cells is prevented by aldose reductase inhibition
Authors: Srivastava Satish K. , Tammali Ravinder , Rychahou Piotr G. , Evers Mark B. , Awasthi Sanjay , Ramana Kota V.
Published: February 2011

Worldwide colorectal cancer (CRC) is the third most common cause of cancer and is the second leading cause of cancer deaths in the USA. Although therapeutic options for patients with CRC have increased substantially, including earlier diagnosis, improved surgery as well as chemo- and radiation therapies, ∼61% of patients have metastatic disease and of these, 90% die within 5 years of diagnosis. Hence better therapeutic interventions to manage the metastatic process in CRC patients are mandatory. We have recently shown that inhibition of aldose reductase (AR), an enzyme that catalyzes reduction of lipid aldehydes and their glutathione conjugates, prevents human colon cancer cell growth in culture as well as in nude mice xenografts by inhibiting the NF-kB-dependent activation of inflammatory and carcinogenic markers. We have now investigated the effect of AR inhibition in metastatic spread of human CRC cells in nude mice and the molecular mechanisms by which AR inhibition prevents colon cancer cell invasion, migration, adhesion and metastasis. Our results indicate that AR inhibition prevents growth factors-induced migration and invasion of HT-29 CRC cells in a dose-dependent manner. AR inhibition also significantly inhibited adhesion of CRC cells to endothelial cells and inhibited the expression of ICAM-1, VCAM-1 and VE-cadherin. Further, we injected KM20 cells, transfected with a plasmid containing green fluorescent protein (GFP), into the spleen of athymic nude mice to establish liver metastases. The mice were treated with AR inhibitor or siRNA for 30 days. Our results indicate that AR inhibition or ablation prevents in vivo invasion and metastasis of CRC cells by reducing levels of AR, MMP2, cyclin D1, and CD34 and suppresses the activation of NF-kB. Since AR inhibitor fidarestat aggressively prevents human tumor growth and metastasis in nude mice and cellular models, and has already undergone phase-iii clinical studies for diabetic neuropathy, it is an excellent candidate for clinical application in preventing colon cancer tumorigenesis and metastasis.

Funding Support: NIH/NCI CA129383 (SKS).

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 424.
Abstract 4242: TRP53 DELETION INDUCES PROLONGED SURVIVAL OF BIPOTENT LIVER STEM CELLS AND THE EVOLUTION OF BI-DIFFERENTIATED HCC/CC
Authors: Lechel André , Katz Sarah-Fee , Begus-Nahrmann Yvonne , Hartmann Daniel , Schirmacher Peter , Rudolph K. Lenhard
Published: February 2011

Background:

Defects in the p53-signaling checkpoint have been described in 30-60% of human hepatocellular carcinoma (HCC) and are associated with less differentiated tumors and poor prognosis. The contribution of p53 deletion by itself to hepatocarcinogenesis is not known. Furthermore it is not clear, if liver stem cells play a role in the onset of liver tumor formation.

Methods:

We generated mice with liver specific TRP53 deletion (TRP53−/−) by crossing conditional TRP53 KO mice with AlfpCRE transgenic mice (CRE expression under the albumin-alphafetoprotein-enhancer-promoter). In vitro cultures were established from the developing liver tumors and analyzed for potential tumor stem cells using xenotransplantation. Endogenous liver stem/progenitor cells were analysed using FACS purification and in vitro culturing of purified liver stem cells (CD13+CD133+CD49f+).

Results:

Mice with liver specific TRP53 deletion developed liver tumors with a mixed differentiation (hepatocellular and cholangiocellular carcinoma). Purification and xenotransplantation of subpopulations of tumor cells from primary tumor cell cultures (using following surface marker: CD13, CD90, CD133, or cKit) revealed no differences in tumor forming capacity in NMRI nu/nu mice. Moreover, tumor formation was seen after xenotransplantation of very small tumor cell numbers (2-cells) indicating that the tumors have an inherent high tumor forming capacity.

An analysis of the colony formation capacity of endogenous liver stem/progenitor cells revealed a prolonged survival of bipotent liver stem cells in aged TRP53−/− mice compared to TRP53+/− or TRP53+/+ mice. The last two groups showed a complete loss of bipotent liver stem cells by 8 month of age, whereas bipotent liver stem cells were detected with a frequency of 0,4% in liver of 8-10 month old TRP53−/− mice.

Conclusion:

This study provides the first experimental evidence that TRP53 deletion leads to a prolonged survival of bipotent liver stem cells and the formation of bi-differentiated liver tumors. These data indicate that the p53-dependent tumor suppressor functions include the restriction of proliferation and survival of bipotent stem cells in the liver.

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 4242.
Abstract 4247: MicroRNA profile of the human colonic crypt base region is predictive of colon cancer
Authors: Haghighat Sepehr Sedigh , Viswanathan Vignesh , Opdenaker Lynn , Gonye Greg , Zhang Tao , Boman Bruce M. , Petrelli Nicholas J.
Published: February 2011

Dysregulation of crypt cell proliferation and differentiation has been implicated in the development of colorectal cancer (CRC). It is believed that human colonic crypt renewal involves programmed cellular proliferation initiated by a few (6-8) stem cells residing at or near the bottom of the crypt, and that stem cell (SC) overpopulation may be the key to CRC initiation. As part of our investigation of regulatory factors involved in the stem cell origin of CRC, we investigated the role that microRNAs (miRNAs) might play in colon carcinogenesis. miRNAs are RNAs 20-24 nucleotides long that were recently shown to modulate many cellular signaling pathways through post-transcriptional regulation of messenger RNA (mRNA) levels and thus protein synthesis. Our previous microarray analysis (368 gene chip) showed increased expression of 37 miRNAs in CRCs compared to normal colonic epithelium. Here we further evaluated miRNA expression in CRC versus purified colonic epithelium by quantitative PCR (QPCR). Total RNA was immediately isolated from tissue by the TrizolTM method. RNA was transcribed into first-strand complementary DNA (cDNA) through reverse transcription (RT). cDNA was then amplified through QPCR. Statistical analysis and plots of expression data for miRNAs were done to find miRNA genes that are differentially expressed. We found, using QPCR, five miRNAs to be differentially expressed in CRCs versus normal colonic epithelium including mir-25 and mir-198. Profiling of purified colonic crypt subregions showed that a distinct signature involving 16 differentially expressed miRNAs characterizes the crypt bottom. This miRNA signature of the crypt bottom was found to be predictive of CRC. Out of the sixteen, three miRNAs (mir-206, mir- 7 and mir-25) were independently capable to distinguish colorectal tissue from normal colonic epithelia (p<0.01). Because this subset of miRNAs is differentially expressed in the crypt bottom, where SC resides, they may be involved in regulating SC population dynamics and contribute to the SC origin of CRC. Further identification and characterization of miRNAs expressed specifically in SC and changes in their expression in CRCs will provide important information to help understand mechanisms of colon tumorigenesis.

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 4247.
Abstract 425: BIRD0796, p38δ inhibitor, inhibits cholangiocarcinoma cell EGI-1 migration in a p53-dependent manner
Authors: Dachuan Huang , Chii Wong Jing , Huimin Bernice Wong , Kiat Ong Choon , Aikseng Ooi , Tean Teh Bin
Published: February 2011

Cholangiocarcinoma (CC) is a devastating cancer, which affects any area of the bile ducts. The new cases of CC have increased in the Western countries. Besides, CC is also an endemic in several Southeast Asia countries such as Thailand and Myanmar. CC is notoriously challenging to diagnose and treat. Currently, only surgical resection is associated with improvement of 5-year survival rate. However, distant metastases, extensive regional lymph node metastasis and vascular encasement or invasion preclude surgical resection. Our previous report showed that the p38δ gene expression was up-regulated in CC tumor sample as well as in CC cell lines. In this study, we further found that inhibition of p38δ by BIRD0796, a p38 δ inhibitor impaired migration ability in CC cell line EGI-1 in vitro (p = 0.006232). The result was further confirmed with p38δ siRNA knowdown (p = 0.0005) and plasmid over-expression experiments (p = 0.003). Interestingly, BIRD0796 did not exhibit similar inhibition effect on migration in TGB cells (p > 0.05). Analysis of p53 gene sequences revealed that there is a frame shift mutation (c.525 526insGCGC) in TGB cells but not any of functional mutation in EGI-1 cells. It indicates that the migration inhibition effect of BIRD0796 in CC metastasis may dependent on p53. Our results suggest that BIRD0796 may be a potential chemotherapeutic compound for CC.

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 425.
Abstract 4251: Murine hepatocellular carcinoma stem cells express pluri-potency-associated transcription factors and are sensitive to immune-mediated apoptosis
Authors: de Kluyver Rachel L. , Stauffer Jim , Brooks Alan , Shanker Anil , Sayers Tom
Published: February 2011

Worldwide the incidence of hepatocellular carcinoma (HCC) resulting from hepatitis C infection and chronic inflammation is increasing. Poor prognosis HCC is characterized by over-expression of Ep-CAM and alpha-fetoprotein (AFP) which define a population of tumor-initiating cells with stem/progenitor features. Current evidence supports the view that cancer stem cells (CSCs) constitute the primary pathogenic component of both metastases and minimal residual disease, and that elimination of CSCs is required for cure. Typically cell surface markers have been used to identify CSCs, however cell surface phenotype alone does not identify CSC in all tumors from the same tissue or in all patients. Therefore there is a need to define and test more reliable markers of CSCs. Oct-4, Sox-2 and Nanog transcription factors are key regulatory factors in maintenance of pluripotency and have been demonstrated to be robust intracellular biomarkers for tissue and cancer stem cells. A GFP-based reporter driven by a promoter responsive to Nanog or Oct-4 was used to mark pluripotent tumor cells from in vitro cultured, cancer cell lines (breast cancer- 4T1.2), and from autochthonous hepatocellular carcinoma induced by hydrodynamics-based gene transfer of constituitively active Akt and β-Catenin. This tumorigenesis model allows tumor initiation by delivery of clinically relevant oncogenes whose sporadic distribution reflects spontaneous cancer. The molecular profile of primary Akt/β-Cat-induced murine HCC recapitulates that observed in poor prognosis Ep-CAM+/AFP+ human HCC. Further, in vitro functional analyses suggest that Ep-CAM and CD133 (surface markers) and Nanog and Oct-4 (transcription factors required for maintenance and regulation of pluripotency in undifferentiated cells) are CSC markers in this model of HCC. Directly ex vivo, murine HCC stem cells express abundant CD1d and MHC class II in addition to TNF-R2, lymphotoxin-β receptor (LTβR) and DR5. The functional significance of CD1d, MHC class II, TNF-R2, LTβR and DR5 expression will be assessed in future studies using in vitro apoptosis and cytotoxicity assays and transplantation at orthotypic sites prior to adoptive transfer of tumor-specific T cells. Studies of freshly isolated CSCs have demonstrated that a variety of mechanisms combine to render CSC resistant to conventional chemo- and radiotherapies. This study has combined in vitro and in vivo approaches to address whether CSC can be rendered sensitive to T cell-mediated apoptosis, and will provide valuable proof-of-principle data to the merits of CSCs as targets for curative anti-cancer 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 4251.
Abstract 4252: Mechanistic and clinical implications of epigenetic modulation in liver cancer stem cells
Authors: Marquardt Jens U. , Raggi Chiara , Andersen Jesper B. , Seo Deakwan , Avital Itzhak , Holczbauer Agnes , Factor Valentina M. , Thorgeirsson Snorri S.
Published: February 2011

Epigenetic mechanisms play critical roles in both stem cell and cancer biology. Epigenetic modulation directs stem cells towards pluripotency and promotes differentiation of more mature derivatives. We hypothesized, that if similar mechanisms are relevant to the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSCs isolation and rigorous evaluation.

Five hepatoma cell lines were treated with Zebularine (ZEB), an effective DNMT1-inhibitor. Putative CSC were isolated using the “side population” (SP) approach. FACS-sorted SP and non-SP fractions were examined for tumorigenic capacity and differences in gene expression. SP-derived signatures were integrated with HCC microarray database and tested for a prognostic value.

ZEB-treatment significantly reduced the SP size while remarkably increasing the proportion of cells with CSC properties as judged by a superior tumor-initiation capacity relative to the untreated SP cells. This was further confirmed in serial transplantation as well as direct cell tracking experiments using genetically labeled SP and non-SP cells. Furthermore, integrative transcriptome analysis of the ZEB-enriched CSC populations identified common stemness traits although various oncogenic pathways (e.g. EGFR, SRC and MYC) were activated in individual hepatoma cell lines. Moreover, each individual as well as the common CSC signatures were associated with poorly differentiated tumors, high recurrence rate and poor survival in liver as well as lung cancers.

In conclusion, we demonstrate that modulation of the liver cancer epigenome is a useful tool to increase the representation of highly tumorigenic cells with CSC properties within the SP fraction. The integrative analysis of the CSC transcriptome revealed a pernicious interaction of common CSC stemness genes with variety of known oncogenic pathways in liver cancer. The significance of CSC- enriched gene signature for the prognosis of liver and other cancers provides a rationale for therapeutic targeting 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 4252.
Abstract 4258: Role of β2SP in asymmetric division and differentiation of intestine epithelial cells
Authors: Li Ying , Jogunoori Wilma , Yao Wenguo , Cao Hong , Mishra Bibhuti , Mishra Lopa
Published: February 2011

The adult intestinal epithelium of the gastrointestinal tract has a well-defined organizational structure. The epithelium can be divided into two regions, a functional region that houses differentiated cells (villi) and a proliferative region (crypts of Lieberkühn) that represents the epithelial stem cell niche. In the small intestine, finger-like projections protrude from the floor of the intestine, forming villi. Multiple proliferative crypts surround each villus base. All epithelial cells that populate the villus originate from these crypts. Multipotent epithelial stem cells reside in the crypts and give rise to four principal epithelial lineages: absorptive enterocytes, mucin secreting-goblet cells, peptide hormone secreting enteroendocrine cells, and Paneth cells. Three of these cell lineages differentiate as they migrate up and out of the crypts onto the adjacent villus. The fourth lineage, Paneth cells, differentiates with a downward migration to reside at the base of the crypt. It is believed that these different cell types are generated by asymmetric cell divisions of stem/progenitor cells. However, the molecular mechanisms of how stem cells differentiate into four different cell types are only partially understood. Aim: in the current study, we evaluated the role of β2SP (β2 spectrin) in the differentiation and asymmetric division of stem/progenitor cells in normal intestine epithelium. Results: Our preliminary data showed that the number of secreting-goblet cells and peptide hormone secreting enteroendocrine cells was reduced in β2SP+/− mice compared to the wt mice. The number of cells undergo asymmetric division was also 3 folds lower in β2SP+/− mice than in wt mice. In in vitro studies, polarization of Par3 and PKCζ was impaired in β2SP−/− MEF cells. The coimmunoprecipitation assay demonstrated that β2SP directly bound to Par3 and actin in normal intestine epithelial cells, indicating that β2SP was involved in the spindle orientation and positioning during the asymmetric division of intestine epithelial cells. Conclusion: β2SP interacts with Par3 and regulates spindle orientation and polarization in the process of cell division, thereby contributing to the differentiation and asymmetric division of intestinal stem/progenitor 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 4258.
Abstract 4260: Activation of cancer stem cells in hepatic Pten deficient mice requires liver injury
Authors: Galicia Vivian A. , Kanel Gary , He Lina , Vendryes Christopher , Stiles Bangyan L.
Published: February 2011

Cancer stem cells have been identified in solid and aqueous tumors and are defined as “altered” stem cells with the capacity to form tumors. We used the liver specific Pten (phosphatase and tensin homologue deleted on chromosome ten) deletion murine model to investigate the role of hepatic cancer stem cells in vivo. Proliferation of hepatic progenitor cells in other rodent models has been observed as a consequence of chronic liver injury. Pten-null mice demonstrate escalating levels of hepatic injury markers from 6-12M of age. In addition, TUNEL analysis revealed that hepatocytes from mutant mice are undergoing extensive apoptosis relative to control mice. We hypothesize that hepatocyte cell death induced by lipotoxicity promotes progenitor cell proliferation to replace liver parenchyma. We show here that activation of hepatic progenitor cells (oval cells) in this model occurs progressively. In mutant mice hepatic injury is followed by elevated expression of hepatic progenitor cell markers Cytokeratin 19 (CK 19), Epithelial cell adhesion molecule (EpCAM), α-fetoprotein (AFP) as well as activation of the Wnt/β-Catenin signaling pathway. Furthermore, we report that liver specific Pten-null mice develop both cholangiocellular carcinoma (CC) and hepatocellular carcinoma (HCC) by 12-15 months of age. Collectively these results suggest that liver tumors in this model arise from hepatic progenitor cells. Our studies also demonstrate that when hepatic injury is attenuated tumor onset is significantly delayed, highlighting the relevance of hepatic injury to progenitor cell induced carcinogenesis. In addition, we show that reactive oxygen species (ROS) is the likely molecular mediator for hepatocyte cell death. This study presents in vivo evidence for the contribution of hepatic injury to progenitor cell mediated liver tumorigenesis.

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 4260.
Abstract 4262: Characterization and regulation of a CXCR4-expressing subpopulation in head and neck cancer stem cells
Authors: Kiang Alan , Abhold Eric L. , Blair Katherine J. , Yu Michael A. , Haas Martin , Brumund Kevin , Weisman Robert A. , Ongkeko Weg M.
Published: February 2011

The cancer stem cell (CSC) hypothesis proposes that only the CSCs within a tumor possess the ability to self-renew and to generate new tumors. Under this model, CSCs are presumed responsible for cellular invasion and metastasis, as well as the recurrence of cancers following chemotherapy. In head and neck squamous cell carcinoma (HNSCC), cells which are CD44 positive and lineage negative (CD44+/Lin-) have been shown to possess properties of CSCs. These cells form tumors in NOD/SCID mice at a much greater efficiency compared to CD44-/Lin- cells, and express the proto-oncogene BMI-1. More recently, it has been shown in pancreatic cancer that a distinct subpopulation of CSCs expressing the chemokine receptor CXCR4 is essential for tumor metastasis. Here we demonstrate the existence of a highly invasive CXCR4+ subpopulation in HNSCC cancer stem cells and elucidate the pathways through which this subpopulation could be regulated.

For this study putative CSC cultures derived from primary HNSCC tissue were used from which CXCR4-expressing subpopulations were isolated. A matrigel invasion assay revealed that the CXCR4+ fraction of CSCs was significantly more invasive compared to the CXCR4- fraction. We also surveyed the mRNA expression of these two fractions by qRT-PCR, and found that expression of the proteinases MMP2 and MMP9 was markedly higher in CXCR4+ cells. Interestingly, treatment with nicotine increased the expression of CXCR4 and its ligand SDF-1 in the parental CSC culture, suggesting a possible mechanism by which CXCR4 could be regulated within the CSC population. Nicotine, a major component of tobacco, is known to have proliferative, anti-apoptotic, and angiogenic effects, and has been shown to promote epithelial-to-mesenchymal transition in a variety of human cancers.

Mortality from HNSCC remains high due primarily to cancer invasion of vital structures and development of distant metastases. Our findings shed light on the mechanisms by which CXCR4 confers an invasive and metastatic CSC phenotype. In addition, the observed effects of nicotine could provide novel insights towards the regulation of the CXCR4/SDF-1 axis in HNSCC, for which tobacco use has been identified as the leading risk factor. Continued efforts in mapping the molecular mechanisms surrounding CXCR4-mediated invasion and metastasis of CSCs could lead to new and effective treatment strategies for HNSCC.

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 4262.
Abstract 4263: Epithelial-mesenchymal transition mediator, S100A4, in maintenance of head and neck cancer initiating cells
Authors: Yu Cheng-Chia , Chiou Shih-Hwa , Yu Yau-Hua , Lo Jeng-Fan
Published: February 2011

Background: Cancer initiating cells (CICs) represent a rare subpopulation of cells, which are responsible for tumor growth. Previously, we have identified and characterized the CICs from head and neck squamous cell carcinomas (HNSCCs), termed head and neck cancer initiating cells (HN-CICs). HN-CICs possess both the hallmarks of stem cells and malignant tumor cells. Herein, we determined the critical role of S100A4, a common mediator of epithelial-mesenchymal transition (EMT), in maintenance of stemness properties and cancerous phenotypes in HN-CICs.

Methods: Affymetrix microarray analysis was performed to identify S100A4 as a putative candidate on mediating the stemness properties of HN-CICs. Consequently, we elucidated the stemness properties and tumorigenicity of HN-CICs with S100A4 down-regulation or overexpression, respectively. Finally, immunohistochemical staining of S100A4 on HNSCC tumor tissues was examined.

Results: S100A4 transcripts and protein level were found significantly up regulated in the enriched HN-CICs. Down-regulation of S100A4 significantly reduced the self-renewal ability and stemness properties in HNSCCs. In addition, knockdown of S100A4 lessened tumorigenicity of HNSCCs both in vitro and in vivo. Conversely, over-expression of S100A4 enhanced the CSC properties in HNSCCs. Furthermore, expression of S100A4 was positively correlated with clinical grading, and survivals and recurrence, respectively (p<0.001, 0.05 and 0.01) according to the immunohistochemical staining. Finally, depletion of endogenous S100A4 resulted in down-regulation of Notch2 and PI3K/pAKT and up-regulation of PTEN in HNSCCs.

Conclusion: Our study reveals the crucial role of S100A4 signaling pathways in the maintenance of stemness properties and tumorigenicity of HN-CICs, and S100A4 might be a potential therapeutic target for HNSCC by eliminating HN-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 4263.
Abstract 4266: Potential role of reactive oxygen species in affecting tumorigenicity of glioblastoma stem cells
Authors: Wang Feng , Hammodi Naima , Yuan Shuqiang , Chen Gang , Feng Li , Kim Sangbae , Colman Howard , Lee Ju-Seog , Xu Ruihua , Huang Peng
Published: February 2011

Glioblastoma multiforme (GBM) is a common and highly aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of cancer stem cells, which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. In this study, we used two lines of stem-like cancer cells (GSC11 and GSC23) derived from the tumor tissues of two GBM patients by classical neurosphere culture system, and showed that addition of serum (FBS) to the medium induced an increase of ROS, leading to aberrant differentiation and eventual cell death. Although both GBM stem-like cell lines showed high percentage of CD133-positive cells (20% in GSC11 and 70% in GSC23), exposure to serum only caused a moderate decrease of CD133 expression at later time points. Several methods were used to further test the role of ROS in affecting the stemness of the GSC cells and their self-renewal capacity. Addition of 5% FBS or removal of the antioxidant supplements (B27) from the culture medium caused the spheroid GSC cells to attach to the culture surface, exhibit differentiation morphology, decrease expression of certain stem-cell markers, and eventually die within two weeks. Serum exposure also resulted in a decrease of the GSC ability to form colonies in soft agar assay and to grow tumors in a mouse xenograft model. Although an increase of general cellular ROS could be detected when GSC cells were exposed to FBS, the mitochondrial superoxide appeared to be prominent and likely to be a primary event, which led to a compensatory increase of the mitochondrial SOD2 expression and upregulation of glutathione synthesis. Interestingly, addition of the antioxidant N-Acetylcysteine to the FBS-containing medium partially blocked the FBS-induced aberrant differentiation and improved cell viability and self-renewal capacity, suggesting that ROS may play an important role in affecting stemness and differentiation status of the cancer stem cells. We postulate that during tumor development, only the progeny cells that acquire the ability to counteract the impact of ROS increase induced by serum may eventually proliferate and grow as the tumor bulk, and that proper redox-modulation may be a potential novel strategy to block tumor formation and possibly kill the stem-like 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 4266.
Abstract 4268: Breast cancer stem cells play a role in trastuzumab resistance in mouse xenograph models
Authors: Hasan Korkaya , Paulson Amanda K. , Zen Qin , Smithkort Hayley J. , Quraishi Ahmet A. , Patel Shivani , Liu Suling , Heath Amber , Clouthier Shawn G. , Wicha Max S.
Published: February 2011

One of the most significant advances in breast cancer therapeutics has been the development of HER2 targeted therapies for treatment of HER2 overexpressing breast cancer. The use of HER2 inhibitors such as trastuzumab have improved patient outcome in both the adjuvant and advanced setting. However, one-third of HER2-positive tumors do not respond to HER2 targeted agents and resistance may develop in patients with chronic exposure. Nearly 50% of patients who respond to HER2 targeted agents relapse within a year. The mechanism of resistance to HER2 targeted agents is not entirely clear. However, increasing evidence indicates that this resistance may be associated with loss of PTEN (phosphotase and tensin homolog), the gain of function of somatic mutations of PI3KA or truncation of the extracellular domain of HER2. We have recently demonstrated that trastuzumab reduces the cancer stem cell (CSC) population in trastuzumab sensitive but not trastuzumab resistant cell lines. In order to elucidate the mechanisms of trastuzumab resistance we engineered MCF7 cells to overexpress HER2, knockdown PTEN or both. HER2 overexpression or PTEN knockdown increased the proportion of CSC's as assessed by the Aldefluor assay and tumor initiation in NOD/SCID mice. Cell lines with both HER2 overexpression and PTEN knockdown contained a significantly higher proportion of Aldefluor positive (3.2 %) compared to either alteration alone (1.7%, 1.9% respectively and 0.5 % in parental cells). In addition, cells with both PTEN deletion and HER2 overexpression showed several fold increase in their invasive properties in vitro as compared cells with either alteration alone. Furthermore, cells with the combined alterations but not those with either alone were highly metastatic in NOD/SCID mice consistent with the demonstrated role of CSC's in mediating tumor metastasis. Trastuzumab treatment significantly reduced the Aldefluor positive population in HER2 overexpressing cells. In contrasts trastuzumab had no effect on the Aldefluor positive population in cells with both HER2 overexpression and PTEN knockdown. These studies suggest that PTEN deletion renders Her 2 overexpressing MCF7 breast cancers resistant to trastuzumab by regulating the CSC population. Strategies aimed at blocking downstream signaling through PI3K or Akt inhibition may effectively target this important cell population in trastuzumab resistant tumors.

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 4268.
Abstract 4275: BMI-1 promotes drug resistance and cellular invasion in head and neck cancer stem cells
Authors: Lew Daniel , Yi David , Wang-Rodriguez Jessica , Ahmad Zana K. , Kiang Alan , Abhold Eric L. , Haas Martin , Brumund Kevin , Weisman Robert A. , Ongkeko Weg M.
Published: February 2011

Recently, the growth and metastasis of tumors, including head and neck squamous cell carcinomas (HNSCC), have been attributed to a subpopulation within tumors known as cancer stem cells (CSC). Studies have shown that HNSCC cells that are CD44+ are putative CSC and have the capability to self-renew, differentiate and preferentially express BMI-1. The polycomb group protein BMI-1 is a transcriptional repressor that silences the genes responsible for the expression of the cell cycle inhibitor genes p16 and p19. The gene is essential for maintaining the self-renewal capability of both normal and CSC. Very recent studies have shown that BMI-1 may also be implicated in other critical roles in cancer cell behavior including its role in the invasiveness of cancer cells. In this study, we demonstrate that in HNSCC CSC, BMI-1 is critical for cellular invasion. Knockdown of BMI-1 in CD44+ cells results in significant inhibition of invasion while overexpression of BMI-1 in CD44- cells promotes invasion. Furthermore, we show that this enhaced invasion conferred by BMI-1 is through induction of epithelial-to-mesenchymal transition (EMT) genes and the down-regulation of E-cadherin. In addition, we present evidence that the proteinases MMP2 and MMP9 are induced upon overexpression of BMI-1.

CSC are also characterized by being chemoresistant, in part through their enhanced ability to extrude drugs because of their high expression of drug transporters. In this study, we demonstrate another mechanism for the resistance of CSC to DNA-damaging agents. We present evidence that another function of BMI-1 in CSC is its ability to regulate apoptosis. Knockdown of BMI1-1 in CSC using siRNA resulted in increased levels of cisplatin-induced apoptosis compared to cells transfected with scrambled siRNA. Our findings lend support to previous studies that have implicated BMI-1 in the regulation of pro-survival genes as well as the DNA damage response pathway.

Our findings have revealed that BMI-1 is not only essential for self-renewal and tumorigenesis, but also that it has critical functions in cancer stem cell drug resistance and invasion, and most likely, metastasis. Since BMI-1 controls many of the properties of CSC and is preferentially expressed in HNSCC CSC, this oncogene may serve not only as a tumor marker, but also as an ideal therapeutic target.

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 4275.
Abstract 4277: Prosective cloning of highly invasive differentiated breast cancer cells
Authors: Kim Jiyoung , Villadsen René , Fogh Louise , Fridriksdottir Agla J. , Rank Fritz , Timmermans-Wielenga Vera , Rønnov-Jessen Lone , Bissell Mina J. , Petersen Ole W.
Published: February 2011

Breast cancer consists of a variety of cell types with distinct phenotypes. Here, we examine the heterogeneity by a panel of normal human breast lineage markers from a recently described hierarchy. By use of multicolor imaging, we show that random heterogeneity is in fact patterned. Tumor cells exhibit traits of normal luminal-, and basal- progenitors, respectively. By combining fluorescence activated cell sorting and single cell cloning, we demonstrate that the patterned heterogeneity reflects a lineage hierarchy reminiscent of that seen in the normal breast. Distinct classes of tumor cells have differing functional abilities and behavior. Thus, multipotent basal-like tumor cells with stem cell properties give rise to lineage restricted tumor cells with luminal-like properties. Luminal-like tumor cells, in turn, are most aggressive as revealed by soft agar colony formation, invasiveness, and in vivo tumorigenecity. We therefore conclude that breast cancer heterogeneity, to some extend, arise as a consequence of normal-like lineage evolution. However, more importantly, the progeny of cancer stem cells remains deregulated raising serious doubts about the current concept of the cancer stem cell as the ultimate drug target.

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 4277.
Abstract 4285: C-Met signaling supports a stem-like transcriptional program and phenotype in glioblastoma
Authors: Li Yunqing , Lal Bachchu , Cázares Hugo Guerrero , Quinones-Hinjosa Alfredo , Eberhart Charles , Laterra John
Published: February 2011

Neoplastic stem-like cells that efficiently propagate tumor xenograft formation exist in solid cancers including glioblastoma. These cells are considered to be responsible for therapeutic resistance and tumor recurrence. The nature of these neoplastic stem-like cells and the molecular signaling pathways that support their phenotype are incompletely defined. The receptor tyrosine kinase c-Met and its ligand HGF promote the formation and growth of multiple cancers including the gliomas. We recently found that GBM-derived neurospheres that are enriched in tumor-initiating stem-like cells widely express activated c-Met. Using flow cytometry and quantitative reverse transcription-PCR, we found that neurosphere cell subpopulations expressing c-Met at high levels also express high levels of the neuroprogenitor/stem cell markers SoX2 and Nestin in comparison to low c-Met expressing cells. Also, CD133+ cells were found to express up to 10-fold higher levels of c-Met relative to CD133- cells. We hypothesize that c-Met might function to support GBM-derived stem-like cells. To test this hypothesis, we examined the effects of c-Met activation/inhibition on the growth and gene expression patterns of established human GBM neurosphere cell lines and low passage primary neurospheres derived from GBM xenograft lines. HGF statistically significantly enhanced cell growth rates and neurosphere forming capacity, a biological marker of the stem cell phenotype. Conversely, inhibiting c-Met kinase activity with SU11274 statistically significantly diminished neurosphere formation and depleted cultures of CD133+ cells. C-Met activation by HGF activated MAPK, AKT and Stat3 and induced expression of SoX2, KLF4 and c-Myc, transcription factors that support stem cell phenotypes. Together, these findings establish a link between c-Met expression/function and the neoplastic stem-like molecular fingerprint and phenotype in GBM-derived cells. The findings suggest that c-Met inhibitors may have a role in the development of therapeutic strategies for targeting tumor-initiating stem-like cells in malignant glioma and other solid cancers. Funded by CA129192 (NIH) and the Brain Tumor Funders Collaborative.

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 4285.
Abstract 4286: Modulation of T cell activation by malignant melanoma initiating cells
Authors: Schatton Tobias , Schütte Ute , Frank Natasha Y. , Zhan Qian , Hoerning Andre , Robles Susanne C. , Zhou Jun , Hodi F. Stephen , Spagnoli Giulio C. , Murphy George F. , Frank Markus H.
Published: February 2011

Highly immunogenic cancers such as malignant melanoma are capable of inexorable tumor growth despite the presence of antitumor immunity. This raises the possibility that only a restricted minority of tumorigenic malignant cells might possess the phenotypic and functional characteristics to modulate tumor-directed immune activation. Here we provide evidence supporting this hypothesis, by demonstrating that tumorigenic ABCB5+ malignant melanoma-initiating cells (MMICs) possess the capacity to preferentially inhibit interleukin (IL)-2-dependent T cell activation and to support, in a B7.2-dependent manner, regulatory T (Treg) cell induction. Compared to melanoma bulk populations, ABCB5+ MMICs expressed lower levels of the major histocompatibility complex (MHC) class I, showed aberrant positivity for MHC class II, and exhibited lower expression levels of the melanoma-associated antigens (MAAs) MART-1, ML-IAP, NY-ESO-1, and MAGE-A. In addition, tumorigenic ABCB5+ subpopulations preferentially expressed the costimulatory molecules B7.2 and PD-1 in both established melanoma xenografts and clinical tumor specimens in vivo. In immune activation assays, ABCB5+ melanoma cells inhibited mitogen-dependent human peripheral blood mononuclear cell (PBMC) proliferation and IL-2 production more efficiently than ABCB5− populations. Moreover, coculture with ABCB5+ MMICs increased, in a B7.2 signalling-dependent manner, CD4+CD25+FoxP3+ Treg cell abundance and IL-10 production by mitogen-activated PBMCs. Consistent with these findings, ABCB5+ melanoma subsets also preferentially inhibited IL-2 production and induced IL-10 secretion by cocultured patient-derived, syngeneic PBMCs. Our findings identify novel T cell-modulatory functions of ABCB5+ melanoma subpopulations and suggest specific roles for MMICs in the evasion of antitumor immunity and in cancer immunotherapeutic resistance.

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 4286.