bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5011: Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia
Authors: Lendvai Agnes , Johannes Frank , Grimm Christina , Eijsink Jasper JH , Wardenaar Rene , Volders Haukeline H. , Klip Harry G. , Hollema Harry , Jansen Ritsert C. , Schuuring Ed , van der Zee Ate GJ , Wisman G. Bea A.
Published: August 2012

Introduction: Cervical cancer is the second most common cancer in women worldwide and development of cervical cancer goes through different well-defined premalignant stages. Epigenetic modifications, such as aberrant DNA promoter methylation is frequently observed in cervical cancer. Identification of hypermethylated regions that allow discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia or worse (CIN2+) by DNA methylation state may improve current population-based screening programs for cervical cancer. In this study, the DNA methylome of CIN3 lesions were characterized using genome-wide methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methods and results: Methyl-DNA ImmunoPrecipitation (MeDIP) assay combined with DNA microarray hybridization was used to compare DNA methylation patterns of epithelial cells derived from CIN3 lesions with normal cervical epithelium. In total, 80 hypermethylated differentially methylated regions (DMRs) were identified that significantly distinguished CIN3 lesions from normal cervice. Selected DMRs (n=9) were evaluated by methylation-specific PCR (MSP) on additional tissue specimens including normal cervix, CIN2/3 and cervical cancer samples and showed hypermethylation in up to 94.7% in CIN2/3 and 100% in cervical cancers. Quantitative MSP (QMSP) for two candidate biomarkers was applied to explore the test performance in a large series of cervical scrapings. Frequency and relative level of DNA methylation were significantly different between normal and cancer samples (p<0.05). In cervical scrapings from patients referred with an abnormal Pap smear, frequency and relative level of DNA methylation were also related with increasing severity of the underlying CIN lesion (p<0.01) In addition, ROC analysis showed that the methylation level was discriminative between low-grade CIN lesions and CIN2+ (p<0.05). Conclusion: Our study demonstrates specific DNA methylation patterns in high-grade CIN lesions through the newly identified discriminative hypermethylated DMRs and represents candidate biomarkers for early detection of high-grade CIN. This study was supported by the Dutch Cancer Society (NKB) (project-number RUG 2004-3161) and by OncoMethylome Sciences S.A., Liège, Belgium. CG was funded by the German Ministry of Education and Research (BMBF) within the NGFN-PLUS project PKT-01GS08111.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5011. doi:1538-7445.AM2012-5011
Abstract 5016: Epigenetics in renal cell carcinoma
Authors: Ramakrishnan Swathi , Sotomayor Paula , Lehet Kristin , Pili Roberto
Published: August 2012

In 2011, an estimated 60,920 men and women will be diagnosed of which 13,120 will die of cancer of the kidney and renal pelvis. The current standard line of therapy includes surgery [partial or radical nephrectomy] or the use of molecular targeted agents such as antiangiogenic agents or immunotherapy. The common mutations found in renal cell carcinomas [RCC] are Von-Hippau Lindau [VHL] gene in clear cell RCC, c-met gene in papillary RCC type I, Fumarate hydratase [FH] gene in papillary type II or the Brit Hogg Dube’ [BHD] gene in chromophobe RCC. However there have been several studies indicating that some of the above genes like VHL can be silenced through promoter methylation as well. Hence, there has been an increasing focus on epigenetic modifications that can occur in RCCs, because of the possibility of reversing these epigenetic marks. There have been considerable reports on the use of agents such as histone deacetylase [HDAC] inhibitors or DNA methyl transferase inhibitors in different RCC cell lines or clinical trials. In this study, we looked at the HDAC expression and activity in matched tumor and non-tumor renal cell carcinoma tissue obtained from tissue procurement services at RPCI. Class I HDACs [HDAC 1, 2 and 3] and two members of class II HDACs [HDAC 4 and 5] are overexpressed in tumors compared to their non-tumor counterparts. In contrast, a class II member HDAC 6 level is lower in the tumor tissues relative to their non-tumor counterparts which corresponded to higher levels to acetylated α-tubulin in those tumors. However, these HDAC expression levels did not tend to have a correlation with grade or stage. We also established xenografts, that are implanted orthotopically and efforts are underway to obtain relatively faster growing tumors subcutaneously. Ongoing studies include defining the relation between expression and activity of HDACs in these tissues and the interaction and impact of other proteins with Class I and II HDACs [such as ER-α, Class III HDACs, VHL] in the context of common active pathways in tumors such as hypoxia [results of which will be available at the time of the meeting]. Further work also includes to look into other histone as well as DNA modifications, as well as enzymes that cause these modifications, in the matched tissues and to explore the potential use of histone or DNA modifying agents as part of RCC treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5016. doi:1538-7445.AM2012-5016
Abstract 5017: DNA methylation array screening in salivary gland adenoid cystic carcinoma
Authors: Shao Chunbo , Tan Marietta , Bishop Justin A. , Vikani Ami R. , Ha Patrick K.
Published: August 2012

Objective: DNA methylation alterations, with either gain or loss of CpG methylation in the promoter region, can alter the expression of tumor suppressor genes or oncogenes. This contributes to the tumorigenesis of human cancers, including salivary adenoid cystic carcinoma (ACC). In this study, we used a DNA methylation array (Illumina HumanMethylation27 BeadChip, San Diego, CA) to screen for novel oncogene and tumor suppressor gene (TSG) candidates under the control of promoter methylation in ACC. Methods: The Illumina HumanMethylation27 BeadChip Array was used to analyze the DNA methylation patterns of ∼14.000 genes at 27,578 CpG sites in 21 ACC and 13 normal salivary gland tissues. With our basic screening algorithm, we narrowed the list down to ∼100 tumor suppressor gene candidates (hypermethylated in tumor) and oncogene candidates (hypomethylated in tumor). Next, the differential methylation levels in the promoter region of candidates were validated by bisulfite genomic sequencing in four primary ACC and four normal salivary gland tissues. Candidate genes that showed differential methylation were then confirmed by analyzing gene re-expression in seven cell lines treated with 5-Aza deoxycytidine, a global demethylating agent. Furthermore, the expression levels of the candidate genes were examined by quantitative reverse transcription PCR (qRT-PCR) in a separate ACC cohort. Lastly, the DNA methylation levels of promising candidate genes were validated by quantitative methylation specific PCR (qMSP) in a larger ACC cohort. Results: With the methylation array, we initially identified 28 oncogene candidates which displayed relative hypomethylation and 64 TSG candidates that showed relative hypermethylation in ACC compared to normal tissues. After the above mentioned additional validation steps were performed, we settled on two promising candidates, PDE9A and TP53TG3, that were significantly hypomethylated and overexpressed in primary ACC tumors compared to normal salivary gland tissue. Furthermore, the expression of PDE9A and TP53TG3 demonstrated consistent upregulation after 5-aza-cytidine treatment of cancer cell lines, suggesting that promoter methylation does play in a role in controlling gene expression. Conclusions: The Illumina HumanMethylation27 BeadChip array is a reliable and efficient tool for analyzing global gene methylation. It is useful in screening for novel oncogene and TSG candidates controlled by promoter methylation in salivary gland ACC. PDE9A and TP53TG3 were identified as promising novel oncogene candidates in salivary gland ACC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5017. doi:1538-7445.AM2012-5017
Abstract 5019: Reprogramming the cancer epigenome by “metabolic transduction”
Authors: Byun Jung S. , Gardner Kevin
Published: August 2012

This study focuses on the key concept of “metabolic transduction” as an important general mediator of cellular reprogramming in epithelial cancers. As a result of multiple studies on epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition in diverse epithelial cancers, the role of developmental plasticity during epithelial differentiation has emerged as a new area of research. Recent data from our lab suggest that metabolites and intermediates in cellular metabolism can act as secondary messengers to dynamically change and globally reprogram epigenetic signatures during normal development, tissue repair, and cancer. We refer to this process as metabolic transduction. Central to this concept is the role played by the C-terminal binding proteins, CTBP1 and CTBP2. Dimeric combination of these factors act as metabolic sensors that function, in the presence of the high energy intermediate NADH, to target multiple classes of chromatin modifying factors, including histone deacetylase, histone methyl-transferases, histone demethylases, DNA methyl-transferases to specific locales throughout the genome. Therefore, CTBP has the potential to mediate widespread changes in the epigenetic landscape and influence broad programs of cellular behavior. Genome-wide profiling studies by ChIP-seq reveal that CTBP controls a network of genes linked to cellular events that influence cellular re-programing or “plasticity” including stem cell transcriptional programs, angiogenesis, and EMT. These studies combined with immuno-histochemical profiling of patient samples indicates that CTBP plays an important role in driving progression of more aggressive forms of breast cancer including Triple Negative Breast Cancer and the basal-like subtypes. Recent studies in our group suggest a role for CTBP in enabling plasticity of cellular fate in malignant cells. Breast cancer cell lines capable of spontaneous de-differentiation into side populations with tumor initiation cell characteristic, such as Sum149PT, are highly enriched in CTBP and are dominated by CTBP controlled pathways. This association has significance in diverse types of cancer, since other transformed cell lines capable of spontaneous de-differentiation into side population, including the endometrial carcinoma cell line HEC-1A, show similar enrichment and dependency on CTBP. These striking similarities suggest that CTBP may represent a driver of a tumor cell plasticity, implicating this tumor cell feature as a new hallmark of epithelial cancers. Therefore, strategies to target CTBP, and cellular reprogramming in general, may provide new opportunities for therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5019. doi:1538-7445.AM2012-5019
Abstract 5021: Native accessible ChIP: A novel approach to interrogate protein-DNA interactions in accessible chromatin
Authors: Okino Steven T. , Meng Xiangdong , Kong Michelle , Wang Yan
Published: August 2012

Recently we developed novel technology for isolating accessible chromatin. When cells are permeabilized and treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin can be easily isolated and characterized. These DNA fragments, when analyzed by next-generation sequencing, provide a global map of accessible chromatin. We now report that the accessible chromatin isolated from cells is still associated with its cognate DNA-binding proteins and histones. Based on this, we developed a novel chromatin immunoprecipitation (ChIP) assay that does not require protein-DNA cross-linking or DNA shearing. Using this method, termed NA-ChIP (for native accessible ChIP), we demonstrate that RNA polymerase II (RNAPII), TBP and H3K4me3 are all associated with active promoters, but are rarely detected at silenced promoters. Comparison of NA-ChIP and a standard ChIP assay shows that immunoprecipitation (IP) efficiency is similar, but IP specificity is dramatically different. To measure IP specificity we compared the ratio of active promoter DNA precipitated with RNAPII (signal) relative to inactive promoter DNA precipitated (noise). We find that NA-ChIP is highly specific with a signal-to-noise ratio >500; standard ChIP is less specific with a signal-to-noise ratio of about 9. We conclude that NA-ChIP offers significant advantages over standard ChIP, including dramatically reduced background and an easier workflow.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5021. doi:1538-7445.AM2012-5021
Abstract 5026: Loss of microRNA-143/145 disturbs cell growth of cancers by impairing the MDM2-p53 feedback loop
Authors: Chen Wantao , Zhang Jianjun , Sun Qiang
Published: August 2012

BACKGROUND: Dysregulated microRNAs (miRNAs) play an important role in many malignant tumors, including epithelial cancers, but little is known about the molecular mechanisms and clinical significances of miRNAs dysregulation. The aim is to identify and determine the miR-143 and miR-145 how to specifically be involved in controlling MDM2 expression in a tumor tissue-specific manner. METHODS: Using real-time PCR, the expression of miR-143, miR-145 and MDM2 were evaluated and analyzed in epithelial cancer cell lines and oral cancer specimens. The different PCR-derived fragments and mutagenesis from the MDM2 mRNA 3′ UTR were inserted into the pGL3 vector to generate the luciferase reporters and the reporter activities were assayed by luciferase assay kits. The effects of overexpression of miR-143 and miR-145 on MDM2 and p53 levels were analyzed by western blot. The effects of miR-143 and miR-145 on cell proliferation and cell cycle were determined by the EdU assay and flow cytometry analysis. The effects of miR-143 and miR-145 on tumorigenicity in vivo were investigated with HNSCC cell xenograft models and the cell apoptotic rate as well as mechanism were tested by TUNEL and apoptosis-protein expression in the tumor tissues. RESULTS: The results showed that both miR-143 and miR-145, which belong to the same miRNA cluster, can negatively modulate expression of their target gene, MDM2. The miR-143 and miR-145 is post-transcriptionally activated by up-regulated p53, thereby generating a short miRNAs-MDM2-p53 feedback loop. Re-expression of these miRNAs suppresses cellular growth and triggers the apoptosis of epithelial cancer, in vitro and in vivo, by enhancing p53 activity via MDM2 turnover. Moreover, the miRNA-dependent MDM2 turnover contributes to the equilibrium of repeated p53 pulses in response to DNA damage stress. CONCLUSIONS: These findings suggest that MDM2 dysregulation caused by down-regulation of miR-143 and miR-145 contributes to epithelial cancer development and palys a key role in regulating cellular proliferation and apoptosis. Recruitment of miR-143 and miR-145 may be a reasonable strategy for treatment of epithelial cancers. KEYWORDS: miRNA-143, miRNA-145, MDM2, p53, epithelial cancers. ACKNOWLEDGMENTS: This study was supported by the National Natural Science Foundation of China (Grant No. 30973343 and 81101515), Projects of the Shanghai Science and Technology Committee (Grant No. 08JC1414400 and 10XD1402500), the Shanghai Leading Academic Discipline Project (S30206). *The first two authors contributed equally to this work.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5026. doi:1538-7445.AM2012-5026
Abstract 503: Investigating the role of Tks5 and invadopodia in prostate tumor progression
Authors: Burger Karen L. , Learman Brian S. , Boucherle Amy K. , Seals Darren F.
Published: August 2012

Much of the morbidity and mortality in prostate cancer patients occurs strictly in response to metastatic disease. While ongoing efforts are examining the therapeutic value of Src kinase inhibitors, the mechanism by which Src activates invasive cell behavior within the context of this disease are not well-understood. Invadopodia are cytoskeletal structures formed by cancer cells that enable binding to, proteolytic degradation of, and invasion through the extracellular matrix. We and others have previously demonstrated that the Src tyrosine kinase substrate and adaptor protein Tks5 is important for invadopodia formation and function. Here we demonstrate that Src is activated and Tks5 up-regulated in prostate tumor specimens and invasive cell lines. Remarkably, overexpression of Tks5 alone is sufficient to form invadopodia structures and enhance invadopodia-associated gelatin degradation and invasion in the LNCaP cell line. We further show that this increase in invasive behavior likely depends on Src kinase activity and Tks5 phosphorylation. Our results predict a role for Src-Tks5 signaling in prostate tumor progression. This hypothesis will be tested in future studies using (i) mouse xenografts to monitor tumor development from Tks5-modified prostate cancer cell lines, and (ii) human prostate tumor tissue sections to measure Tks5 staining relative to tumor grade.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 503. doi:1538-7445.AM2012-503
Abstract 5034: Healthy and cancerous serum RNA profiling by the novel RNA extraction reagent and highly sensitive DNA chip
Authors: Takizawa Satoko , Ichikawa Makiko , Sudo Hiroko , Ueda Yoji , Akiyama Hideo
Published: August 2012

Proteins, metabolites and DNA are already known as components of serum or plasma biomarkers, however RNA has not been a strong biomarker candidate because of its instability. Exosomes that are small vesicles secreted by various cells are recently reported to play important roles in intercellular communications by transferring proteins, DNA and also RNA to distant cells through circulatory system. Surprisingly, the exosomal RNA in serum preserves its integrity and thus holds a potential to be a new blood biomarkers. In this report, we show the exhaustive analysis of miRNA and mRNA in serum by DNA chip for the highly purified RNA extracted with a novel reagent. Serum contains various types of nucleic acids, mainly small RNA, mRNA, and also short DNA fragment. We suppose that the contamination of DNA to the extracted RNA often causes a discrepancy between the DNA chip analysis and qRT-PCR validation. The novel reagent was able to extract RNA from serum without contamination of short DNA fragment, resulting in better RNA quantification and decreasing DNA-related noise outputs. Using this novel RNA extraction reagent and the highly sensitive DNA chip “3D-Gene,” we analyzed healthy and cancerous serum miRNA profiles. Over 500 miRNAs are detected in healthy and cancerous sera reproducibly, and some miRNAs were detected specifically in cancerous sera, such as breast, gastric, cervix cancer. In addition, we detected over 19,000 mRNAs from amplified RNA which were similarly extracted from healthy serum and detected by the DNA chip. This indicates that not only serum miRNA but also serum mRNA have a potential capability to be a biomarker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5034. doi:1538-7445.AM2012-5034
Abstract 5039: Comparative analysis of miRNA in hormonal refractory prostate cancer
Authors: Lee Min Jin , Cha Youn Jin , Kang Suki , Ham Wonsik , Choi Young Deuk , Cho Nam Hoon
Published: August 2012

Background: Nucleotides 2-7 within mature miRNAs, short RNA molecule of between 19 and 22, create the ‘seed region’ that specifies the specific mRNA. To date, miRNAs have been linked to the etiology, progression and prognosis of cancer. The gain or loss of specific miRNAs can function as an oncogene or tumor suppressor. Design: By using recently developed miRNA 14K array (Agilent), we designed to compare miRNA expression between HRPCa cell line (PC3) and sensitive cell line (LNCaP). As validation for target mRNA following bioinformatics, candidates were applied on the neoadjuvant bicalutamide treated radical prostatectomized specimens using immunohistochemistry. Results: Amongst 57 upregulated miRNAs (ratio >2.0), oncomirs-miRNAs that are amplified or overexpressed in PC3, includes miR-31, -27a, -23a, -9, -100, -29a, -24, let 7i, -21, -221, 29b, -222, -181a. Probable candidates for target mRNAs to each oncomir are tumor suppressor gene family, including p27, PTEN, apoptosis related genes (DAXX, PDCD4, caspase recruitment domain 10/11), ECM adhesion genes (Integrin β4, ADAMTS 7/8), SOCS 6. In 23 downregulated miRNAs (ratio>2.0), tumor suppressor miRNAs that are downregualted or deleted from chromosomes acting as tumor suppressor genes, include miR-141, -200 a/b/c, -148a, -429, -34a, -195, -99a, -203, -125b-2. Possible candidates for target mRNAs are oncogeneic family are EMT-related genes including ZEB1, cell cycle related genes (Wee1, cyclin E1, CDK6), prostate specific proteins (STEAP1, prostein), RTK family (AXL, Rab-38, Rab-9A), transcriptional factor promotion (EIF2, SOX6), mitogen activator (MAP p38), secretory protein (SDF2). Among the top-listed candidates, STEAP1, prostein, ZEB1, and cyclin E1 were relatively overexpressed in the refractory PCa to compare with regressing cancer cells. Conclusion: Complicated networks of miRNA play substantial and crucial roles in the regulation of hormonal refractory prostate cancer, predominantly by virtue of STEAP1, prostein, ZEB1, cyclin E1 overexpression, and suppression of apoptosis-related genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5039. doi:1538-7445.AM2012-5039
Abstract 5194: Heterogenous expression of chemokine receptors in primary patient-derived GBM lines; association of CXCR3, CXCR4, and CXCR7 with a slow cycling sub-population
Authors: Pham Kien , Liu Che , Luo Defang , Reynolds Brent A. , Harrison Jeffrey K.
Published: August 2012

WHO class IV glioblastoma multiforme (GBM) is one of the most malignant and aggressive forms of primary brain tumors and despite multimodal treatments patients have modest survival rates. The poor prognosis and therapy resistance of glioblastoma are believed to be a consequence of a subpopulation of glioma stem-like cells. As such, targeting functions associated with this cell population is likely to yield new therapeutic strategies. Roles for CXCR3, CXCR4, and CXCR7 in glioblastoma are well documented although previously published studies involved primarily the characterization of glioma cell lines grown in the presence of serum. Our studies were undertaken to understand the roles of chemokine receptors CXCR4 and CXCR7, and their ligands CXCL11 and CXCL12, in modulating glioblastoma stem-like cell functions. Tumors xenografts developed from patient-derived primary GBM cells cultured as gliomaspheres in serum-free conditions expressed CXCR4, CXCR7, CXCL11, and CXCL12 in a manner similar to expression patterns of these genes in human GBM tissues. Flow cytometry analysis of CXCR4 and CXCR7 on various patient-derived primary GBM lines indicated marked heterogenous expression of these receptors. Variable percentages of CXCR4+, CXCR7+, and CXCR4+/CXCR7+ (double positive) populations were evident. In contrast, chemokine receptors CCR3 and CXCR3 were co-expressed on a small but similar percentage of cells in all cell lines. RT-PCR indicated that all lines expressed CXCL11 while none expressed CXCL12. A slow cycling sub-population of cells, which express stem-like cell markers and exhibit high tumorigenic frequencies, were enriched for CXCR3, CXCR4, and CXCR7. CXCR4+ and CXCR7+ subpopulations were capable of forming spheres in serum-free culture conditions and exhibited plasticity in regenerating the cellular heterogeneity of their parental phenotype. In addition, both CXCR4+ and CXCR7+ cells were able to initiate the formation of intracranial tumors. In vitro functional studies showed that while CXCL12, but not CXCL11, stimulated the migration of GBM cell lines, both of these ligands were able to promote short-term proliferation. Collectively, the results suggest that CXCR4 and CXCR7 may regulate the stem-like properties of GBM cells. The high degree of heterogeneity in CXCR4 and CXCR7 expression both within and between glioblastomas suggests that personalized treatment of glioblastoma patients using selective antagonists that target CXCR4 and CXCR7 may need to consider the complex expression patterns and specific functions of these receptors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5194. doi:1538-7445.AM2012-5194
Abstract 5198: Elucidation of dynamic prostate epithelial hierarchy: Insights into transcriptional and epigenetic regulatory mechanisms
Authors: Rane Jayant K. , Pellacani Davide , Droop Alastair P. , Ylipää Antti , Stower Michael J. , Simms Matthew S. , Mann Vincet M. , Visakorpi Tapio , Collins Anne T. , Maitland Norman J.
Published: August 2012

The aim of present investigation is to elucidate the complex stem cell dynamics within prostate cancer, which can be utilized to design novel diagnostic and therapeutic strategies for the management of prostate cancer. In order to determine precise transcriptional and microRNA regulatory mechanisms modulating stem cell self-renewal and differentiation, unique cellular assays have been developed in our lab that utilize homogeneous fractionated cell populations enriched from primary patient prostate cultures. Using a prospective bioinformatic analysis of gene expression data from Birnie et. al., 2008, we have identified LCN2, CEACAM6, and S100p as candidate genes for regulation of prostate stem cell differentiation. Their over-expression in differentiated cells, as compared to stem cells, was validated in respective cells enriched from cultures obtained from BPH, cancer and castration resistant prostate cancer samples and from primary human prostate cancer xenografts. Interestingly, the analysis of 25,000 published human Affymetrix microarray chips revealed that LCN2, CEACAM6, and S100p have a more similar expression pattern than that of any other genes in the entire human genome, suggesting that they may have common function and are co-regulated. Indeed, the promoter analysis showed that the promoters (1kb from TSS) of all these genes have common binding sites for 40 transcription factors with very high affinity and P < 0.001. Most of these transcription factors have a well-documented role in cell differentiation (RA, AR, and NANOG) and prostate carcinogenesis (NF-kB). Significant up-regulation in the expression of these genes in prostate cell lines after treatment with all-trans retinoic acid and androgen analogue R1881 further suggested the role of AR and RA in prostate differentiation. Along with transcriptional regulation, Agilent v3 miRNA microarray data revealed obviously distinct miRNA expression profiles in stem and differentiated prostate epithelial cells, confirming crucial role of miRNA in main taining epithelial hierarchies, in prostate. We anticipate that evaluation of integrative transcriptional (LCN2-CEACAM6-S100p)-microRNA regulatory network, with further functional studies, will comprehensively establish a detailed knowledge base for potential regulatory mechanisms involved in prostate stem cell and prostate cancer stem cell differentiation. These insights will be valuable to formulate efficient ‘differentiation therapy’ for the management of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5198. doi:1538-7445.AM2012-5198
Abstract 5199: Tumor vascularization by endothelial differentiation of endometrial cancer stem cells
Authors: Nakamura Mitsuhiro , Kyo Satoru , Zhang Xivzhi , Takakura Masahiro , Maida Yoshiko , Mizumoto Yasunari , Bono Yukiko , Sasagawa Toshiyuki , Inoue Masaki
Published: August 2012

Evidence for existence of cancer stem cells (CSCs) has been reported in several malignant tumors. CSCs have properties of self-renewal, tumorigenesis and multilineage differentiation. Many studies about CSCs have focused on self-renewal, tumorigenesis. However, it is unclear about multilineage differentiation. Our previous study showed CD133 was a CSC marker and a critical prognostic marker in endometrial cancer. In that study, we demonstrated that CD133+ cells were capable of generating both CD133+ and CD133- cells, whereas CD133 cells did not generate CD133+ cells, indicating that CD133+ CSCs have a differential ability. The present study investigated endothelial differentiation of endometrial cancer cells. We have established endometrial cancer cells from normal human endometrial epithelial cells by introduction of HPV16 E6/E7, hTERT and K-ras genes (EM-E6/E7/hTERT/K-ras cells). Histological findings of tumors formed from EM-E6/E7/hTERT/K-ras cells showed that tumors were composed of solid and vascular regions. Vascular regions were stained human-specific anti-CD31 antibody, suggesting that EM-E6/E7/hTERT/K-Ras cells are capable of differentiating to endothelial cells. Subcutaneous xenografts from clinical samples in endometrial cancer also demonstrated that human endometrial tumors contained human vessels labeled by human-specific anti-CD31 antibody. Endothelial tube formation assay (in vitro angiogenesis) revealed that isolated CD133+ endometrial cancer cells could show tube formation while CD133- cells could not, indicating that CD133+ endometrial cancer cells had an ability of endothelial differentiation. Furthermore, western blot analysis showed high expression of VEGFR1 in CD133+ cells. We demonstrated a new function of CSCs in endometrial cancer cells. These findings shed light on the establishment of new target therapy to endometrial cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5199. doi:1538-7445.AM2012-5199
Abstract 5202: Bone marrow-derived epithelial cells contribute to chronic skin inflammation and skin tumor formation in the mouse
Authors: Park Heuijoon , Readio Nyssa , Jin Guang , Asfaha Samuel , Singh Anupama , Singh Ashok , Yang Xiangdong , Patterson Kelly S. , Trempus Carol , Wang Timothy C. , Morris Rebecca J.
Published: August 2012

Although a stem cell origin for tumors was postulated nearly 200 years ago, a critical role of keratin-15 expressing hair follicle stem cells in skin tumorigenesis has only recently been established. In addition to tissue stem cells, bone marrow cells can play a reparative function in damaged organs and contribute to cancer development as recently demonstrated in a gastric cancer model of Helicobacter felis-induced chronic inflammation. We report here that bone marrow derived cells (BMDCs) are an exogenous source of epithelial cells in tumor development in the multistage skin carcinogenesis model following gender mismatched allogeneic bone marrow transplantation. First, clusters of BMDCs were detected in the epithelium of 37.78% papilloma samples with a genetic marker (EGFP) indicating their bone marrow donor origin. Second, clusters of keratin immunoreactive BMDCs were located primarily in the basal epithelium of the papillomas. Third, a subset of BMDCs in the basal epithelium was found to be highly proliferative as measured by incorporation of BrdU and expression of Ki67. Furthermore, a significant contribution of BMDCs was identified in 53.06% of ulcer-associated dysplastic skin samples by immunostaining and confirmed by presence of a Y chromosome. Moreover, an enhanced contribution of bone marrow-derived epithelial cells was observed in the dysplastic epidermis. Therefore, these results demonstrate that a subset of BMDCs participates as a new epithelial cell source in chronically damaged skin lesions including papillomas and ulcers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5202. doi:1538-7445.AM2012-5202
Abstract 5205: Iron metabolism informs glioblastoma stem cell maintenance
Authors: Schonberg David L. , Rich Jeremy N.
Published: August 2012

Glioblastoma is a highly lethal cancer that is characterized by florid angiogenesis, invasion into normal brain, and therapeutic resistance. Glioblastomas display cellular hierarchies with a self-renewing, tumorigenic glioblastoma stem cell (GSC) at the apex. The significance of GSCs in tumor biology has been supported by work from our group and others showing that GSCs are relatively resistant to conventional therapies, promote tumor angiogenesis, and are highly invasive. By comparing GSCs to matched differentiated tumor bulk, we have identified a number of molecular targets that function in maintenance of the cellular hierarchy and tumor growth. We have now identified increased iron metabolism as a characteristic of GSCs. Normal glial progenitors accumulate iron to increase migration and proliferation. Brain tumors accumulate iron and iron regulators are associated with poor survival. As hypoxia induces iron regulators and maintains the cellular hierarchy, we are investigating iron regulation in GSCs. Ectopic delivery of iron to GSCs increases tumorsphere growth while disrupting iron metabolism in GSCs using shRNA reduces cell growth and decreases the expression of angiogenic growth factors. Collectively, these results suggest that iron metabolism is important for GSC maintenance and tumorigenic potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5205. doi:1538-7445.AM2012-5205
Abstract 5207: The chemokine 25 and chemokine receptor 9 axis suppresses colon cancer invasion and metastasis
Authors: Chen Huanhuan , Tucci Serena , Gümüs Zeynep , Shen Xiling , Lipkin Steven M.
Published: August 2012

Chemokines help regulate tumor cell invasion and metastasis. Here, we show that the Chemokine 25/Chemokine Receptor 9 (CCL25/CCR9) axis suppresses colon cancer (CRC) invasion and metastasis. CCR9 protein expression levels are highest in colon adenomas, and progressively decrease in invasive and metastatic CRCs. CCR9 is expressed in both primary tumor cell cultures and colon cancer initiating cell lines (CCIC) derived from early stage (I/II) CRCs. CCL25 stimulates cell proliferation through a network of transcription factors downstream of AKT signaling. In vivo, systemically injected CCR9+ early stage CCIC spontaneously form orthotopic gastrointestinal xenograft tumors. Blocking CCR9 signaling prevents CRC tumor formation in the native gastrointestinal CCL25+ microenvironment, while increasing extra-intestinal tumor multiplicity. NOTCH signaling, which promotes CRC metastasis, increases extra-intestinal tumor multiplicity by stimulating CCR proteosomal degradation. These data provide insights into the mechanisms that regulate CRC progression, invasion and metastasis and describe a novel in vivo experimental system to study these processes. Our findings also raise a safety concern for ongoing Inflammatory Bowel Disease clinical trials using CCR9 antagonists.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5207. doi:1538-7445.AM2012-5207
Abstract 5212: Irradiation-induced uPAR promotes stemness via Wnt/β-catenin signaling in medulloblastoma cells
Authors: Asuthkar Swapna , Gondi Christopher S. , Nalla Arun Kumar , Velpula Kiran Kumar , Gorantla Bharathi , Rao Jasti S.
Published: August 2012

Urokinase plasminogen activator receptor (uPAR) is known to promote invasion, migration and metastasis in cancer cells. In this study, we show that ionizing radiation (IR) induced uPAR has a role in Wnt/β-catenin signaling and mediates induction of cancer stem cells (CSC)-like properties in medulloblastoma cell lines UW228 and D283. We observed that IR induced the expression of uPAR, CSC markers such as CD44 and Msi-1, and activates Wnt/β-catenin signaling molecules. Overexpression of uPAR after IR treatment led to the activation of Wnt signaling, which was demonstrated by an increase in nuclear translocation of β-catenin and β-catenin-Lef/Tcf-mediated transactivation, thereby promoting cancer stemness. Quercetin, a potent Wnt/β-catenin inhibitor suppressed uPAR and uPAR-mediated Wnt/β-catenin activation. Treatment with shRNA specific for uPAR (pU) suppressed the β-catenin-Tcf/Lef-mediated transactivation both in vitro and in vivo. Further, we show that uPAR is physically associated with the Wnt effector molecule β-catenin using immunocytochemistry and immunohistochemistry; these results were confirmed by immunoprecipitation analysis. Most interestingly, we demonstrate for the first time that the localization of uPAR in the nucleus is associated with transcription factors (TF) and their specific response elements. The association of uPAR with β-catenin-Tcf/Lef complex and various other TF involved in neurogenesis during embryonic development and cancer demonstrates the receptor's possible role at generating CSC-like properties in primitive neuroectodermal tumor (PNET) cells of medulloblastoma. Considering all of the data, we conclude that uPAR is a potent activator of stemness, and the targeting of uPAR in combination with radiation has significant therapeutic implications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5212. doi:1538-7445.AM2012-5212
Abstract 5240: Breast cancer in the metastatic niche: A role for stress-induced dormancy
Authors: Taylor Donald P. , Wells Alan
Published: August 2012

Breast cancer dormancy in the metastatic site allows tumors to avoid clinical detection and evade treatment leading to recurrence after years or decades, with devastating mortality rates. How the aggressive carcinoma cells that initially underwent a cancer-associated Epithelial to Mesenchymal Transition (EMT) from the primary tumor to disseminate and maintain a long-term dormant state in an ectopic site is unknown. We have found that normal parenchymal cells of certain metastatic organs cause a partial reversion of the EMT to a more epithelial phenotype (Mesenchymal to Epithelial Reversion Transition, or MErT). Compared to carcinoma cells in the primary breast tumor, MErT is characterized by increased E-cadherin expression, MAPK and AKT signaling, and chemoresistance. While this phenotype should reflect low proliferation or quiescence, the MErT cells in vitro have heretofore maintained a highly proliferative state. Herein we demonstrate that a pre-stressed hepatic microenvironment allows for EMT cells to increase their MErT phenotype and to become dormant. Our research strategy is to modulate the initial liver stress through biochemical challenges applied to primary rat hepatocytes co-cultured with hepatic resident cells and a human immortalized breast cancer cell line (MDA-MB-231). Biochemical challenges (e.g. TGFβ, TNFα, IL-6, EGF, HGF, and DAMPS) are applied in varying concentrations and incubation periods in order to pre-stress the hepatic culture. In order to maintain hepatic metabolic function we implement a novel 3D perfused micro-well bioreactor. As a secondary aim we explore whether E-cadherin expression is required for metastatic seeding through splenetic injections in immune compromised mice. We aim to uncover novel targets that are not strictly against the cancer cells, but rather, against shared molecular pathways across cancer, parenchymal, and non-parenchymal cells within the hepatic niche. Sustaining and perhaps enhancing the ectopic site resilience may yield therapies directed toward maintaining dormant carcinomas so that secondary EMTs will be blocked and cancer recurrence greatly reduced.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5240. doi:1538-7445.AM2012-5240
Abstract 525: Toll-like receptor-4 mediated cutaneous immune responses augment ultraviolet radiation-induced DNA damage and tumor development
Authors: Ahmad Israr , Guroji Purushotham , Jimenez Hugo , Simanyi Eva , Nagar Anusuiya , Nagar Priyamvada , Yusuf Nabiha
Published: August 2012

UV (ultraviolet) B induced DNA damage plays a critical role in development of skin cancer. The molecular basis for this biologic activity resides at least in part from the demonstrated ability of this form of radiant energy to damage DNA, predominantly in the form of cyclobutane pyrimidine dimers (CPD). The purpose of this study was to determine whether toll-like receptor-4 (TLR4) contributed to UV-induced DNA damage responses and cutaneous tumor development. TLR4 deficient and wild type (WT) mice were subjected to a local UVB induced DNA damage regimen consisting of 90 mJ/cm2 UVB radiation. Wild type mice exhibited significant (p<0.05) DNA damage in the form of cyclobutane pyrimidine dimers (CPD), whereas TLR4 deficient mice developed significantly fewer CPD (p<0.05) in their skin and bone marrow dendritic cells (BMDC). The expression of DNA repair gene XPA was significantly less (p<0.05) in skin and BMDC from WT mice than TLR4 deficient mice after UVB exposure. When cytokine levels were compared in these two strains after UVB exposure, BMDC from UV-irradiated TLR4 deficient mice produced significantly more IL-12 and IL-23 cytokines (p<0.05) than BMDC from WT mice. Furthermore, we found that Toll like receptor-4 (TLR4) promoted UVB induced tumor development. We observed that UVB induced skin carcinogenesis was retarded in terms of tumor incidence, and tumor latency, in mice deficient in TLR4 compared to WT mice, whereas significantly greater (p<0.05) numbers of tumors occurred in WT mice. Further, we found that tumor-bearing WT mice produced more IL-17 and IL-10 (p<0.05), whereas TLR4 deficient mice produced more IFN-γ (p<0.05). CD4+CD25+ regulatory T-cells from WT mice produced more IL-10 (p<0.05) than regulatory T-cells from TLR4 deficient mice. There was a significant increase (p<0.05) in Foxp3 expression in skin of WT mice than TLR4 deficient mice. Thus, strategies to inhibit TLR4 may allow us to develop immunopreventive and immunotherapeutic approaches for management of UVB induced cutaneous DNA damage and skin cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 525. doi:1538-7445.AM2012-525
Abstract 5255: Methionine restriction limits prostate tumor cell proliferation
Authors: Allott Emma H. , Tang Xiaohu , Masko Elizabeth M. , Gaines Alexis , Abern Michael R. , Chi Jen-Tsan A. , Freedland Stephen J.
Published: August 2012

Introduction: Methionine restriction has been shown to have growth inhibitory effects in a number of tumor cell lines in vitro. Methionine restriction has previously been demonstrated to slow PC-3 prostate tumor cell growth in vivo, however not at dietary levels tolerable to humans. Up to 90% dietary methionine restriction has been demonstrated to be both tolerable and safe in a preliminary human clinical trial. We hypothesized that 80%-90% methionine restriction could limit prostate tumor cell growth in vitro and in vivo. Methods: We reduced methionine levels by 40%, 80%, 90% and 100% in prostate cancer cell lines in vitro, relative to standard RPMI medium. Cell proliferation and viability were measured using MTS and trypan blue assays, respectively. PC-3 cells were grafted into a xenograft nude mouse model and animals were randomized to standard chow, 80% or 90% methionine restricted diets. Plasma methionine levels were measured before and after the dietary intervention. Tumor volume was measured throughout the study and mice were sacrificed when tumors reached 1000 mm3. Results: Methionine restriction exerted a dose and time-dependent effect on PC-3 tumor cell proliferation and viability in vitro. Methionine restriction of 80% and 90% for a period of 6 days reduced PC-3 tumor cell line proliferation by 20% and 40%, respectively, as measured by MTS assay (p<0.0001). Following 6 days of 80% and 90% methionine restriction, PC-3 cell viability was reduced by 10% and 15%, respectively. We are currently awaiting results from our in vivo study which is underway. Discussion: Methionine restriction slows prostate tumor cell line growth and reduces cell line viability in vitro. Based upon these in vitro results we hypothesize that an 80% - 90% methionine restricted diet will slow tumor growth in vivo, and we await the results of this dietary intervention in mice. Methionine restriction at this clinically relevant level which is tolerable and safe to humans may have the potential to slow tumor growth in prostate cancer patients; however more preliminary studies are needed in order to establish whether 80%-90% methionine restriction actually does slow tumor growth in vivo and if so to understand the mechanisms by which this occurs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5255. doi:1538-7445.AM2012-5255