bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3307: The multitasking of Sox2: Maintaining the stemness and inducing the malignant phenotype of gliomas
Authors: Alonso Marta M. , Manterola Lorea , Diez-Valle Ricardo , Cortes-Santiago Nahir , Liu Dan , Rubio Angel , Garcia-Foncillas Jesus , Idoate Miguel Angel , Fueyo Juan , Gomez-Manzano Candelaria
Published: June 2012

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, displays an enrichment gene expression related to a stem cell signature. Sox2, a major stemness factor involved in pluripotency and in the maintenance of neural stem cell identity, has been described as frequently overexpressed in poorly differentiated gliomas. However, the role that this molecule plays in brain tumors remains under investigation. In this study, we sought to ascertain the Sox2 potential function in the control of self-renewal of brain tumor stem cells (BTSCs) and the malignant phenotype of gliomas. Our results showed Sox2 expression in human surgical malignant gliomas. In addition, Sox2 expression was significantly higher in the BTSC lines than in established glioma cell lines. Downmodulation of Sox2 in BTSCs by using siRNA resulted in the loss of the self-renewal properties of this cell population, and modification in the pattern of expression of markers related with stem cells properties and differentiation. These results are of major importance since, in the TCGA, we observed Sox2 overexpression in more than 80% of the samples (n=188), gene amplification of SOX2 in 18% of the individuals, and hypomethylation of the promoter in about 90% of the samples when compared with normal tissue, suggesting the relevance of this transcription factor in the malignant phenotype of GBMs. In this regard, our results demonstrated that SOX2 plays a prominent role in the invasive and migratory capabilities of both BTSCs and established glioma cell lines. Thus, ectopic expression of Sox2 was sufficient to induce invasion of glioma cells and knockdown experiments demonstrated that Sox2 is essential for maintaining the invasive properties of glioma cells. Therefore, these results identify a multi-functional aspect of Sox2 as both, a stemness promoting molecule and an essential pro-invasive protein, elucidating the possibility of targeting Sox2 for glioma therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2011-3307
Abstract 3312: Protein activation pathway analysis of glioblastoma stem cells reveals potential novel biomarkers
Authors: Federici Giulia , Signore Michele , Martino Simona Di , Pelacchi Federica , De Maria Ruggero , Liotta Lance A. , Petricoin Emanuel F. , Ricci-Vitiani Lucia
Published: June 2012

The cancer stem cell (CSC) hypothesis postulates that within the tumor mass there exists cells that possess self-renewal characteristics and are the tumorigenic and chemoresistant component of the growing lesion. Currently, there are no therapies specifically targeting this population and thus, elucidation of the undermining molecular mechanisms responsible for these attributes are critical for all tumor types in order to identify therapeutics that target the deranged pathways within the CSCs. GBM is an incurable malignancy of the neural glia and it has been demonstrated the presence of CSCs within it. Indeed it has been shown that there is a population of cells expressing CD133, a putative CSC marker, that resides in the tumor and may lead to its progression. We studied a set of thirty CD133+ GBM cancer stem cell lines obtained from 30 GBM patients and performed a Reverse-phase Protein Microarray (RPMA) analysis to investigate the activation state of more than 100 key signaling proteins involved in proliferation, inflammation, adhesion, survival and migration. This screening allowed the characterization and functional mapping of activated intracellular signaling networks within the GBM cancer stem cell lines and identified 8 clear subgroups based on molecular signatures of functional signaling activation. Unsupervised and supervised/statistical analysis of the differentially activated signaling architectures revealed the incidence of several phosphorylated proteins that underpinned these subclasses, such as members of the growth factor receptor tyrosine kinase family (e.g. EGFR, IGFR, VEGFR and PDGFR); proteins that control cell cycle division (Histone H3, PLK1 and Aurora) and members of calcium signaling/energy metabolism (S100A7, AMPK and PKC family), amongst others. It is hoped that ongoing and further characterization of the signaling architecture of a diverse number of GBM CSCs will help establish the key therapeutic targets that could be validated for causal significance and graduate to the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3312. doi:10.1158/1538-7445.AM2011-3312
Abstract 3316: An alpha-secretase inhibitor decreases glioma stem cell growth by inhibiting the Notch pathway and LMW Cyclin E
Authors: Floyd Desiree H. , Kefas Benjamin , Seleverstov Olexsandr , Dominguez Charli , Comeau Laurey , Purow Benjamin W.
Published: June 2012

The Notch pathway is deregulated in glioblastoma. Previous work suggests the inhibition of this pathway will have therapeutic benefits. The only inhibitors of Notch available block the gamma-secretase enzymatic complex necessary for processing Notch into the active form. However, Notch is also cleaved by alpha-secretase outside the plasma membrane, with the sheddases ADAM10 and 17. INCB3619 is a potent inhibitor of both ADAM10 and 17, and has been shown to inhibit growth of MCF-7 breast tumor cells when used with lapatinib. In this work, INCB3619 was used to inhibit endogenous Notch cleavage and downstream CBF-1 reporter activity in 0308 and 0822 human glioma stem cell lines. A microarray analysis of INCB3619 vs. DMSO treatment of 0308 cells identified many downregulated Notch pathway targets, but also identified new targets of INCB3619, such as CHI3L1/YKL40, an important prognostic indicator of many inflammatory diseases. Treatment of 0308 and 0822 glioma stem lines with INCB3619 inhibited their growth in culture due to a cell cycle arrest in G1 and a decrease in S phase cells. Growth inhibition by INCB3619 can be rescued with transfection of the active form of two Notch family members, NICD1 and 2. The growth inhibition of INCB3619 treated glioma stem cells is also stems from a shift in Cyclin E expression from a hyperactive low-molecular-weight form to the usual high-molecular-weight form. Also, INCB3619 induces higher expression of both p53 and p21, which can inhibit Cyclin E. Unlike with INCB3619, The treatment of glioma stem cells with DAPT, a well-known and potent gamma secretase inhibitor that cleaves Notch downstream of ADAM10 and 17, does not have a noticeable effect on Cyclin E processing or p21/p53 expression. INCB3619 therefore decreases glioma stem cell growth partly through a mechanism of Notch inhibition and also through inhibition of production of the hyperactive low-molecular-weight form of Cyclin E.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2011-3316
Abstract 3317: Single-cell invasion and chemoresistance of slowly proliferating brain-tumor stem cells
Authors: Siebzehnrubl Florian A. , Silver Daniel J. , Deleyrolle Loic P. , Suslov Oleg , Tugertimur Bugra , Yachnis Antony T. , Brabletz Simone , Brabletz Thomas , Reynolds Brent A. , Steindler Dennis A.
Published: June 2012

Despite intense efforts in basic research and clinical medicine, glioblastoma (GBM) remains one of the most lethal types of cancer. In particular, tumor recurrence after surgical resection, aggressive chemotherapy, and targeted radiation remains an insurmountable obstacle. Recurrence may be attributed to residual cancer stem cells that re-initiate tumor growth after primary clinical intervention. Here, we provide new evidence for a subpopulation of relatively quiescent and chemoresistant cancer stem cells that invade deeply into the parenchyma. We have identified a critical transcription factor that is upregulated in these glioma stem cells, which regulates epithelial-mesenchymal transition (EMT) in other tumors. Knockdown of this transcription factor results in an increased sensitivity to chemotherapeutic agents and reduced tumor cell invasion. We hypothesize that single cell invasion of malignant gliomas is regulated by similar molecular pathways as EMT and metastasis in solid tissue tumors. These pathways allow slowly proliferating glioma stem cells to leave the site of the primary tumor, invade deeply into the surrounding parenchyma and, by virtue of their resistance to chemotherapeutic agents, evade all types of classical brain tumor therapy. This offers a potential novel model for GBM recurrence, and a potential new therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2011-3317
Abstract 3318: A distinct subpopulation within CD133 positive brain tumor cells shares characteristics with endothelial progenitor cells
Authors: Kim Seung-Ki , Choi Seung Ah , Wang Kyu-Chang , Phi Ji Hoon , Lee Ji Yeoun , Cho Byung-Kyu , Park Chul-Kee , Park Sung-Hye
Published: June 2012

The cell surface marker CD133 was proposed as a brain tumor stem cell marker. However there have been substantial controversies regarding the necessity and role of CD133 in tumorigenesis. This study aimed to characterize CD133 positive cells in brain tumors.

Fresh human brain tumor specimens and whole blood were collected from the same patients [medulloblastoma (N=6), glioblastoma (N=4), ependymoma (N=1), and atypical teratoid/rhabdoid tumor (ATRT, N=2)]. Dual FACS staining for CD133/CD34, functional assay of CD133 positive cells from different origin for tumorigenesis and angiogenesis were done. We also investigated the in vivo tumorigenic potential and histological characteristics of four distinct groups on the basis of expression of CD133/CD34 markers (CD133+, CD133+/CD34+, CD133+/CD34-, and CD133-). CD133 positive brain tumor cells coexpressed significantly higher positivity for CD34 (67.97 ± 5.34 % in CD133 (+) cells vs. 11.96 ± 1.48 % in CD133 (−) cells, P<0.001). CD133 positive brain tumor cells formed neurosphere-like spheroids and differentiated into multiple nervous system lineages contrary to CD133 positive blood cells. Furthermore they showed biological characteristics of endothelial cells including vWF expression, LDL uptake and tube formation in vitro, unlike CD133 negative brain tumors cells. Pathologic analysis of the brains implanted with CD133+ glioma cells showed large, highly proliferative and vascular tumors with widespread necrosis and hemorrhage. Notably pure BTSC (CD133+/CD34-) or EPC (CD133+/CD34+) fraction alone did not generate tumor.

Our data suggest the presence of a distinct subpopulation of CD133 positive cells isolated from human brain tumors, with characteristics of EPCs. Therefore, interpreting the results of experiments carried out with “CD133-positive BTSCs” should be extrapolated with caution.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3318. doi:10.1158/1538-7445.AM2011-3318
Abstract 3329: Magnetic isolation and rare cell gene expression analysis of breast cancer stem cells
Authors: Hardt Olaf , Wild Stefan , Hofmann Kay , Luo Shujun , Wiencek Yvonne , Kantelhardt Eva , Smith Geoffrey P. , Schroth Gary P. , Bosio Andreas , Dittmer Jürgen
Published: June 2012

Solid tumors consist of heterogeneous cell types including a subpopulation of stem like cells. Starting from bulk tumor mass, the analysis of these cancer stem cells (CSCs) is hampered by their rare frequency and their characteristics remain hidden.

To address this issue, we have developed a method for the semi-automated dissociation of human tumor tissue yielding a viable single cell suspension. Subsequently, a magnetic cell sorting (MACS) strategy was established allowing for the isolation of CD24-/CD44+ breast CSCs. Using these methods, we isolated breast CSCs from an invasive ductal carcinoma at a purity of 94%. Due to the limited starting material, a few hundred CSCs as well as bulk tumor cells were used for global PCR based RNA amplification. The amplified cDNA was sheared, size selected, and processed for high throughput gene expression analysis on the Illumina Genome Analyzer system. Samples were prepared in triplicate and over 10 million individual purity-filtered reads were analyzed per sample. In addition, Agilent whole genome microarrays were used for validation of expression differences.

Analysis of the sequence tags allowed us to identify novel putative markers overexpressed in breast CSCs. GeneOntology analysis was used to determine functional groups of genes which are differentially regulated between CSCs and bulk tumor cells. We found a strong overrepresentation of genes connected to TGF-beta as well as Wnt/GSK3 signaling supporting the correlation of the CSC phenotype with epithelial-mesenchymal-transition (EMT). Other differentially expressed genes are known to be involved in pluripotency, cell migration, and apoptosis further indicating a stem cell-like fate.Our proof of principle experiment demonstrates the advantage of isolating rare cell populations for subsequent molecular analysis by high throughput sequencing. The analysis of purified CSCs revealed new insights on the regulation, maintenance, and biology that would not have been observed when assessing only the bulk tumor mass. Analyzing the genetic set up of defined tumor subpopulations by Next Generation Sequencing can improve our understanding of cancer and could pioneer targeted diagnostic and therapeutic approaches.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2011-3329
Abstract 3342: Overexpression of transcription factor Fra-1 drives murine 4TO7 breast cancer stem cells out of dormancy and render them sensitive to chemotherapy
Authors: Lu Dan , Chen Si , Liao Debbie , Liu Ze , Xiang Rong , Reisfeld Ralph
Published: June 2012

Fra-1 is a member of the Fos family of transcription factors, which is highly expressed in multiple tumors and plays important roles in the transformation of malignant cells. Prior studies clearly illustrated some of the mechanisms of Fra-1 involvement in tumor proliferation and metastasis. However, the function of Fra-1 in cancer stem cells (CSCs) still remains to be resolved. We either overexpressed or silenced Fra-1 in murine 4TO7 breast cancer cells, and investigated its effects on regulation of the cell cycle, maintenance of side populations (SP) and determination of sensitivity to chemotherapeutic drugs both in vitro and in vivo. Overexpression of Fra-1 in 4TO7 cells significantly decreased the population of G0/G1-phase cells by DNA content analysis of fixed cells with propidium iodide (PI), as well as the intracellular flow cytometry (FCM) analysis of Ki-67 expression in fixed and permeabilized 4TO7 cells. We also performed the Hoechst 33342 dye exclusion assay, and detected a remarkable decrease of SP cells, which was further confirmed by intracellular staining of cancer stem cell marker Sca-1. Importantly, overexpression of Fra-1 correlated with a significant increase in chemosensitivity to either Doxorubicin (Dox) or cyclophosphamide (CTX) treatment both in vitro and in vivo. In contrast, extinction of Fra-1 increased the proportion of both G0/G1-phase cells and SP cells. These events in turn increased resistance of 4TO7 cells to chemotherapy both in vitro and in vivo. Together, our findings indicate that up- or down- regulation of Fra-1 in murine 4TO7 breast cancer cells can drive CSCs out of or back into dormancy, which may subsequently render them sensitive or resistant to chemotherapeutic drugs. In conclusion, we describe here a novel function of transcription factor Fra-1 in breast CSCs, which may hold important implications for future diagnosis and treatment of breast cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2011-3342
Abstract 3343: Preferential dormancy of Oct4-expressing breast cancer-initiating cells in bone marrow: Role for gap junctional intercellular communication
Authors: Patel Shyam A.
Published: June 2012

Breast cancer dormancy in the bone marrow is a clinical problem that is supported by cancer resurgence after years of disease-free survival. This study seeks to explain why, despite aggressive treatment with anti-cancer agents, breast cancer cells (BCCs) can survive for later resurgence. The potential role of stem cell biology in malignancy has been supported by evidence of chemotherapy resistance among tumors and the idea that stem cells and tumor cells share common signaling pathways. Initial screening of primary tissues and BCC lines has demonstrated that the pluripotency marker Oct4 is expressed at low frequency. Thus, in this study, we selected Oct4-expressing BCCs in transfectants with GFP reporter vector under the control of the Oct4 5’ regulatory region. Based on Oct4 expression, we have isolated two subsets. The high expressers show self-renewing properties, long doubling times, increased proportion of cells in G0 phase, sphere formation at >97% efficiency, and serial passaging ability in nude mice. Based on these findings, we designate the high Oct4 expressers as breast cancer-initiating cells (BC-ICs). Functionally, the BC-ICs show preference for forming gap junctional intercellular communication (GJIC) with bone marrow stroma and resist treatment with carboplatin. In summary, the studies provide evidence for the existence of a population of cells within breast cancers that sustains tumor survival and might be the subset of BCCs that adapts dormancy in the bone marrow. These studies are highly significant to subclinical tumor survival and eventual resurgence of breast cancer at metastatic sites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2011-3343
Abstract 3345: Enhancer of zeste homolog 2 (EZH2) promotes expansion of breast tumor initiation cells through activation of Notch1-jagged signaling
Authors: Gonzalez Maria E. , Li Xin , Kruger Heather , Toy Kathy , Kleer Celina G.
Published: June 2012

EZH2 is an oncogene which has been recently shown to regulate the differentiation of tissue specific stem cells. However the role of EZH2 on mammary stem cells is unknown. Notch1 signaling has been implicated in cell-fate determination of human mammary stem cells. We hypothesized that EZH2 may contribute to initiation and expansion of mammary stem cells through activation of Notch1 signaling.

Methods: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammary cells and a lentivirus-mediated shRNA interference targeting EZH2 in SUM149 breast cancer cells. Mammary stem cells were identified using ALDH1+ and CD44+/CD24- by FACS. The effect of EZH2 on the tumorigenic ability of ALDH-1 positive and negative SUM149 cells was studied in a xenograft mouse model.

Results: We found preferential expression of EZH2 in the stem cell population of normal primary breast cells compared to non stem cells. In MCF10A cells, EZH2 overexpression, (EZ-OV), was predominantly increased in the ALDH-1+ population. When we tested the role of EZH2 in the stem cell population we found that EZH2-OV increased the stem cell population of MCF10A cells while EZH2-KD decreased it in SUM149 cells. Microarray analysis identified Notch1-Jagged1 signaling as the most significantly downregulated targets of EZH2 in ALDH+ vs. ALDH- SUM149 cells. Consistently, EZH2-OV in MCF10A and primary breast cells increased Notch1 mRNA specifically in the ALDH1+ population. EZH2-OV increased Notch1 protein level and promoter activity in MCF10A cells. Mechanistically, Notch1 inhibition by GSI reversed the effect of EZH2-OV in the stem cell population of MCF10A cells. Our in vivo studies demonstrated that EZH2 KD in SUM149 cells decreased the growth of ALDH-1+ cells in xenograft experiments. Consistently, EZH2 KD tumors exhibited decreased Notch1 and Jagged1 protein levels.

Conclusion: Collectively, these results indicate that EZH2 regulates mammary stem cells and requires, at least in part the activation of Notch1 signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3345. doi:10.1158/1538-7445.AM2011-3345
Abstract 3348: Naïve rat umbilical cord matrix stem cells abrogate mammary tumor growth through markedly enhanced tumor immune responses
Authors: Kawabata Atsushi , Ohta Naomi , Seiler Garret , Pyle Marla , Troyer Deryl , Tamura Masaaki
Published: June 2012

Mesenchymal stem cells derived from the umbilical cord matrix (UCMSC) have great potential for therapeutic use in multiple diseases including cancer. We have demonstrated that un-engineered naïve human and rat UCMSC attenuate growth of several organ type tumors (Ayuzawa et al., Cancer Lett.2009; Ganta et al., Cancer Res, 2009, Doi et al., Cytotherapy 2010). Since UCMSC are easy to prepare in relatively large quantities and are poorly immunogenic in allogeneic transplantation, they are potentially useful in cancer therapy. However, the mechanism by which UCMSC attenuate tumor growth has not been studied rigorously. Hence, the objectives of this study were to examine the ability of UCMSC to control the growth of mammary tumors and to determine possible mechanisms by which UCMSC attenuate the tumor growth. Intratumoral injection of rat UCMSC markedly attenuated tumor growth of orthotopic Mat B III autografts in female F344 rat mammary gland. Histopathology of the tumors in the rat UCMSC group indicated that a large number of lymphocytes had infiltrated into the peritumoral area and were occasionally observed in the intratumoral area. Immunohistochemistry revealed that although CD3+ T lymphocytes were located mainly in the peritumoral area rather than in the intratumoral area, CD20+ cells were rarely seen suggesting that the majority of infiltrated lymphocytes in the rat UCMSC treated tumors were T cells. In addition, the treatment of rat UCMSC increased the CD 8+ cell infiltration throughout tumor tissue. CD68+ cells were scarcely observed in the tumors of the PBS control group but not in the rat UCMSC treated group. Transwell culture system-based in vitro migration assay revealed that rat UCMSC significantly enhanced migration of peripheral blood mononuclear cells. These results suggest that naïve rat UCMSC attenuated mammary tumor growth at least in part by markedly enhanced host immune responses against tumors. This research demonstrates that naïve rat UCMSC can be used as powerful anti-cancer therapeutic cells for breast cancer treatment. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU CVM Dean's Fund, NIH P20 RR017686, p20 RR1556, R21CA135599 and Kansas Bioscience Authority research fund.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3348. doi:10.1158/1538-7445.AM2011-3348
Abstract 3363: Iron deficiency suppress EMT through downregulation of N-cadherin in esophageal cancer
Authors: Nishitani Seishi , Noma Kazuhiro , Watanabe Shinichiro , Onishi Teppei , Ohara Toshiaki , Tomono Yasuko , Hashimoto Yuuri , Tazawa Hiroshi , Fujiwara Toshiyoshi
Published: June 2012

[Introduction] Epithelial-Mesenchymal-transition(EMT) plays a great roles in cancer metastasis and invasion.Mecanism of EMT remains controversial, but it is recognized that some protein change their expression, including E-cadherin and N-cadherin.It is reported that downregulation of N-cadherin reduce cancer metastasis and invasiveness.

Iron metabolism and its relationship with cancer cell were studied for a long time. Iron overload is known to induce some kinds of cancer, which suggests that prevention of iron overload is a therapeutic strategy of cancer prevention. Iron deficiency is also known to suppress the tumor growth in vivo study. But the efficacy is not superior to the ordinary chemotherapy, it's not a standard therapeutic strategy of cancer. Recently, it is reported that iro metabolism is essential for EMT, and iron defficiency downregulate EMT change. In this study, we demonstrated that iron deficiency downregulate the expression of the EMT protein, N-cadherin, and suppressed the ability of metastasis and invasiveness. This result suggests that Iron controlled treatment can be novel therapeutic agent for cancer under EMT. [Matherials and Methods] TE-1,TE-4,TE-8 and TE-10 esophageal squamous cell carcinoma, OE19 and OE33 esophageal adenocarcinoma cell lines were used in this study. In vitro study, cell viability was determined by XTT assay. Iron chelator deferasirox was prepared in vitro study to make iron deficient condition. To determine that iron deficiency suppresses EMT and invasiveness of cancer cell line, western blotting analysis, the three-dimensional spheroid model analysis and migration assay were done. In vivo study, Normal diet and Iron deficient diet were fed for 3 weeks before implantation of TE-4 cells in athymic male nude mice(n=8/group). After implantation, each diet was fed continually. [Results]Deferasirox reduces proliferation of all cell line in a concentration manner. According to western blotting analysis, deferasirox induces reduction of N-cadherin protein in TE-4 and TE-10 cell lines. N-cadherin downregulation declines invasiveness of TE-4 and TE-10 cell lines in the three-dimensional spheroid model analysis and migration assay. In vivo study, tumor growth was suppressed in iron deficient diet group (tumor volume: normal diet vs iron deficient diet = 30.7±1.1 vs 10.9±2.2) [Conclusion]Iron controlled treatment can be novel therapeutic agent for invasiveness of cancer under EMT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2011-3363
Abstract 3373: Aberrant expression of TG2 reprograms mammary epithelial cells by inducing mesenchymal transition (EMT) and stem cell-like characteristics
Authors: Kumar Anupam , Xu Jia , Hui Gao , y Dihu , Reuben James , Mehta Kapil
Published: June 2012

The “Incurable” nature of metastatic breast cancer (MBC) poses a major clinical challenge in successful treatment of breast cancer. Despite significant advances in early detection and disease management, the prognosis of MBC patients remains poor with an estimated 5-year survival rate of 26%. Understanding the molecular pathways/factors responsible for the progression of MBC is critical for improved clinical outcomes. We and others previously found that metastatic breast cancer cells express high basal levels of the proinfflamtory protein tissue transglutaminase (TG2) and that its expression is associated with metastatic and drug-resistant phenotypes in breast cancer cells. However, mechanisms that underlie TG2-mediated metastatic transformation of breast cells remained elusive.

To understand the significance of TG2 in MBC, we stably transfected TG2 in non-transformed breast mammary epithelial cells (MCF10A and MCF12A). Interestingly, TG2 expression induced marked changes in the morphology of cells from cobblestone looking cells to elongated mesenchymal like appearance. Study of various epithelial and mesenchymal markers revealed the loss of epithelial (e-cadherin and β-catenin) and gain of mesenchymal (vimentin and fibronectin) markers in TG2 expressing cells. Moreover, TG2 expression resulted in constitutive activation of Akt and NF-kB and upregulated transcription repressors like, Snail1, Zeb1, Zeb2 and twist1. Interestingly, TGF-β a known inducer of EMT, failed to induce EMT in MCF10A cells transfected with TG2 Sh-RNA. TG2 expression in MCF10A cells was associated with increased invasion, disrupted polarity, increased resistance to doxorubicin, and ability of cells to grow and form colony in soft-agar. Next we determined weather TG2-induced EMT promotes the stem cell phenotype. Study of breast stem cell markers revealed that TG2 expression is associated with dramatic increase in CD44high/CD24low cell subpopulation. Moreover, mammosphere-forming ability of both normal and cancerous breast epithelial cells was significantly augmented by TG2 expression. TG2-derived stem cells showed increase in self-renewal ability and they terminally differentiated into basal and/or luminal cell types in response to prolactin treatment. These results imply that aberrant expression of TG2 could reactivate a comprehensive program in differentiated mammary epithelial cells to endow them stem cell-like properties for successful colonization and macromatastasis. These results also supported the contention that TG2 could serve as a promising therapeutic target for treatment of MBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3373. doi:10.1158/1538-7445.AM2011-3373
Abstract 3379: Iron-deficient anemia promotes breast cancer growth and lung metastasis
Authors: Jian Jinlong , Yang Qing , Huang Xi
Published: June 2012

Young breast cancer patients <45 years old are at a higher risk of dying from the disease than older patients. Specific risk factors contributing to this high mortality rate have not been identified. Iron deficient anemia (IDA) is a common ailment in young pre-menopausal women both in developed and developing countries. In the present study, we hypothesize that IDA promotes breast cancer growth and metastasis. To test this hypothesis, Balb/c mice were fed three levels of iron diets, 3.5 ppm Fe as iron deficient, 35 ppm as normal low, and 350 ppm as normal high for 12 wks. Consistent with previous literature reports, iron levels in diets ranging from 35 to 350 ppm, which were considered normal, did not lead to significant differences in serum iron and transferrin saturation rates. However, an iron level at 3.5 ppm in the diet caused an IDA in mice. After subcutaneously inoculating 4T1 mammary cancer cells into the flanks of mice fed different iron diets, mice with IDA condition have significant higher tumor volumes as compared to those with normal iron status (2922 + 161 mm3 in 3.5 ppm Fe group vs 1636 + 86 mm3 in 35 ppm Fe group and 1453 + 90 mm3 in 350 ppm Fe group). Lung metastasis was also higher in IDA mice with 22.8 + 0.9 nodules per lung lobule in 3.5 ppm vs 13.8 + 0.8 in 35 ppm and 13.6 + 0.9 in 350 ppm Fe. To elucidate molecular mechanisms of IDA in tumor growth and metastasis, levels of epithelial mesenchymal transition (EMT) markers, such as snail, slug and zeb1, were found to be significantly higher in tumor tissues isolated from IDA mice. When investigating signal pathways regulating EMT, it was found that Notch pathway, including Notch 2, 3, 4, and Notch ligands such as Jagged1, 2, were all induced by IDA. In contrast to Notch, transforming growth factor (TGF)-β and wingless-int (WNT) signal pathways, known to stimulate EMT, were not affected, suggesting that IDA-induced EMT is Notch pathway-specific. Since anemia causes hypoxia, the expression of HIF-1α, a key transcription factor controlling hypoxic responses, was also induced by IDA. Similarly, Notch signal pathway and EMT markers were higher after transfecting HIF-1α cDNA into 4T1 cells. These data show that IDA-mediated breast cancer growth and lung metastasis is through HIF-1α accumulation and activation of Notch and EMT. Our study suggests that identification of IDA as a poor prognostic factor and its clinical improvement as a potential cancer therapy could be significant to the high mortality rate of young breast cancer patients. This work is supported by R21 CA132684.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3379. doi:10.1158/1538-7445.AM2011-3379
Abstract 3408: Ovarian cancer stem cell cytokine microenvironment
Authors: Levina Vera V. , Marrangoni Adele , Gorelik Elieser , Edwards Robert , Lokshin Anna
Published: June 2012

Objectives: Epithelial ovarian cancer (EOC) is an extremely aggressive disease associated with a lack of early symptoms, rapid progression to peritoneal metastases, and poor prognosis for patients. Currently available treatments are not effective in preventing or slowing metastatic spread, and the therapeutic resistance of EOC remains a major unresolved clinical and scientific problem.

Cancer stem cells (CSCs) represent the most aggressive part of the tumor. CSCs are drug resistant and are thought to be responsible for the failure of chemotherapy. Earlier, we demonstrated that chemotherapy can lead to selection of CSCs. We hypothesized that malignancy of CSCs was associated with an efficient cytokine network.

Methods: Ovarian CSCs and primary adherent epithelial cultures were established from ascites from ovarian cancer patient with stage III EOC. Ovarian CSCs were isolated using magnetic beads separation columns and human CD133 and CD44 Microbead kits from Miltenyl Biotec and were grown as tumor spheres. The High Content Screening (HCS) and High Content Analysis (HCA) platform from Thermo Scientific Cellomics was applied to study the CSC markers, embryonic markers, transcription factors, epithelial mesenchimal transition markers, cytokines, chemokines, growth and angiogenetic factor receptor expression and intracellular cytokine accumulation. Multiplex xMAP technology was used for the analysis of human cytokines and growth factors in cell culture medium. An in vivo SCID mouse model was used for the analysis of tumorigenity of bulk EOC cells and CSCs.

Results: We established primary EOC cell lines and CSCs from ascites of 3 EOC patients. We confirmed that cells growing in tumor spheres demonstrate all the properties of CSCs: they express high levels of CSCs markers and low levels of differentiated markers, they have capacity to self-renew and differentiate, and they are highly tumorigenic in SCID mice. The EMT and CSC phenotypes were both exhibited in ovarian CSCs. We found that ovarian CSCs have a specific cytokine microenvironment involving the up-regulation of multiple cytokine-receptor axes.

Conclusions: Our findings provide new knowledge regarding ovarian CSC biology. They elucidate the important role of the specific cytokine-receptor signaling axis for the self-renewal and proliferation of ovarian CSCs. We concluded that an optimal antitumor efficacy will be achieved by inhibiting both tumor cell populations: CSCs and bulk EOC cells. We suggest that combining two treatment modalities, e.g. RTK inhibitor that targets CSCs, and cisplatin that eradicates bulk tumor cells will represent a novel paradigm in EOC therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3408. doi:10.1158/1538-7445.AM2011-3408
Abstract 341: Overexpression of FGFR2 gene is associated with advanced stage squamous cell carcinoma of the lung
Authors: Kikuchi Ryutaro , Huang Cheng-long , Miyahara Ryo , Sonobe Makoto , Menju Toshi , Kobayashi Masashi , Ishikawa Masashi , Kitamura Jiro , Date Hiroshi
Published: June 2012

Introduction: Molecular oncology has revealed various types of genetic abnormality are involved in tumorigenesis of lung carcinomas. For example, mutations of epidermal growth factor receptor (EGFR) are associated with non-smoker patients with lung adenocarcinomas, and mutations of K-ras are associated with smoker patients with lung adenocarcinomas. However, few genetic abnormalities were reported in squamous cell carcinomas of the lung. In order to identify the specific genetic abnormality in squamous cell carcinoma of the lung, we performed a clinical study on the fibroblast growth factor receptor (FGFR) gene in patients with non-small cell lung cancers (NSCLCs). Method: One hundred and thirty eight NSCLC patients were studied. Quantitative real-time PCR was performed to evaluate gene expressions of all four members of human FGFR, including FGFR1, FGFR2, FGFR3, and FGFR4. In addition, gene status of EGFR (exon 19-22), K-ras (exon 1), and p53 (exon 5-8) was also investigated using PCR-SSCP confirmed by direct sequencing. Results: FGFR2 gene expression was significantly higher in smoker patients than in non-smoker patients (P=0.008). Regarding the tumor histology, FGFR2 gene expression was significantly higher in squamous cell carcinomas than in adenocarcinomas (P<0.001). Furthermore, among squamous cell carcinomas, FGFR2 gene expression was significantly higher in advanced stage tumors than in early stage tumors (P=0.034). Among squamous cell carcinomas, FGFR1 gene expression was also significantly higher in advanced stage tumors than in early stage tumors (P=0.031). In contrast, there was no these relationships in FGFR3 gene expression or FGFR4 gene expression. Regarding other gene status, FGFR2 gene expression was significantly higher in tumors with wild-type of EGFR than in tumors with mutant EGFR (P=0.004), and was likely to be higher in tumors with wild-type of K-ras than in tumors with mutant K-ras. Furthermore, FGFR1 gene expression was likely to be higher in tumors with wild-type of K-ras than in tumors with mutant K-ras (P=0.064). However, there was no difference in FGFR3 gene expression or FGFR4 gene expression according to EGFR gene status and K-ras gene status. In addition, there was no difference in gene expression of FGFR1, FGFR2, FGFR3, or FGFR4 according to p53 gene status. Conclusions: Overexpression of FGFR2 gene expression is associated with lung carcinomas with wild-type of EGFR and wild-type of K-ras, especially in advanced stage of squamous cell carcinomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 341. doi:10.1158/1538-7445.AM2011-341
Abstract 3417: Class III HDAC induces epithelial-to-mesenchymal transition in prostate cancer cells
Authors: Byles Vanessa , Zhu Lijia , Chmilewski Laura K. , Lovaas Jenna D. , Faller Douglas V. , Dai Yan
Published: June 2012

The epithelial-to-mesenchymal transition (EMT) is a crucial program for initiating invasion and metastasis in many epithelial tumors. EMT is characterized by loss of cell adhesion and increased cell mobility; however, the mechanism underlying this transition is not fully understood. Here we identified SIRT1, a class III histone deacetylase, as a critical regulator of EMT. We show that forced expression of SIRT1 disrupts the epithelial morphology in non-transformed prostate epithelial cells, concomitant with a decrease of epithelial marker E-cadherin, and an increase of mesenchymal protein N-cadherin, and fibronection expression, indicating EMT. In contrast, silencing SIRT1 expression in metastatic prostate tumor cells restores cell to cell adhesion and induces changes in morphology characteristic of epithelial cells, concomitant with an increase of E-cadherin and a decrease of N-cadherin and fibronection expressions. In addition, we show that SIRT1 represses the activity of the E-cadherin proximal promoter with an E-box dependent mechanism, indicating a connection between SIRT1 and the zinc-finger EMT transcription factor, which needs the E-box to suppress E-cadherin transcription. Moreover, we performed ChIP assays to show that SIRT1 and ZEB1 bind to the same DNA fragment on the E-cadherin promoter, and SIRT1 occupancy in the E-cadherin promoter requires ZEB1. In addition, we show that acetylated H3K9 levels were inversely related to occupancy of SIRT1 and ZEB1. Finally, our results show that SIRT1 knockdown reduces prostate cancer cell migration and invasion. Thus, in this study we identified SIRT1 as an important regulator of EMT and demonstrated a new transcriptional mechanism regulating E-cadherin expression by the cooperation of SIRT1 with ZEB1. These findings suggest that overexpressed SIRT1 in prostate cancer cells may be targeted to reverse EMT, and SIRT1 could be a potential target in the treatment of prostate cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2011-3417
Abstract 3421: Twist1 and clusterin regulate epithelial-mesenchymal transition
Authors: Shiota Masaki , Zoubeidi Amina , Kumano Masafumi , Naito Seiji , Nelson Colleen C. , Gleave Martin E.
Published: June 2012

INTRODUCTION AND OBJECTIVE: Clusterin (CLU) is a stress-activated, cytoprotective molecular chaperone, and is highly expressed in cells surviving apoptotic stimuli and in advanced and treatment-resistant cancers. In addition, CLU has recently been reported to be implicated in epithelial-mesenchymal transition (EMT). Similarly, oncogenic transcription factor Twist1 which binds to E-box (core sequence, 5’-CANNTG-3’) is known to be a master regulator of EMT. Although CLU is known to be transcriptionally activated by heat-shock factor 1, the mechanism CLU expression in EMT is not fully elucidated. This study begins to define mechanism of CLU expression and relevance in EMT of prostate cancer.

METHODS: Human prostate cancer PC-3 and LNCaP cells were used to examine whether the manipulation of Twist1 expression affected CLU expression using quantitative real-time PCR, Western blotting and luciferase reporter assay using CLU promoter region. Chromatin-immunoprecipitation (ChIP) assays were used to determine whether Twist1 binds to CLU promoter region. We also examined markers of EMT after treatment with TGF-β1 and manipulation of Twist1/CLU expression.

RESULTS: Twist1 overexpression was shown to upregulate CLU expression whereas Twist1 knockdown reduced CLU expression. ChIP assay and luciferase reporter assay revealed that Twist1 was bound to CLU promoter region and regulated CLU transcription. Treatment with TGF-β1, which is known to be an EMT-inducing cytokine, upregulated Twist1 expression followed by CLU expression. Furthermore, CLU knockdown ameliorated EMT induced by TGF-β1 and Twist1.

CONCLUSIONS: This study indicates that CLU regulates EMT induced by TGF-β1 and Twist1 in prostate cancer cells. Modulation of CLU expression may be a useful strategy for developing novel therapeutics regulating EMT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3421. doi:10.1158/1538-7445.AM2011-3421
Abstract 3448: Subtypes of medulloblastoma have distinct developmental origins
Authors: Gibson Paul , Tong Yiai , Robinson Giles , Poppleton Helen , Thompson Margaret C. , Currle David S. , Eden Christopher , Hogg Twala L. , Finkelstein David , Pounds Stanley , Patay Zoltan , Scoggins Matthew , Pei Yanxin , Ogg Robert , Lee Youngsoo , Zindy Frederique , Lindsey Janet C. , Boop Frederic A. , Gajjar Amar , Clifford Steven C. , Roussel Martine F. , McKinnon Peter J. , Ellison David W. , Wechsler-Reya Robert , Gutmann David H. , Gilbertson Richard J.
Published: June 2012

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype), arises outside the cerebellum from cells of the lower rhombic lip (LRL). We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the LRL and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused an aberrant accumulation of proliferating precursor cells within the LRL. These lesions persisted in the dorsal brainstem of all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that modeled faithfully the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2011-3448