bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 78: Epigenetic regulation of the metastatic progression of non-small cell lung cancer by the histone H3K9 methyltransferase SETDB1
Authors: Tzeng Tsai-Yu
Published: June 2012

Aberrant histone methylation is contributes to tumor development and progression by histone methyltransferase. SETDB1 is an authentic H3K9 methyltransferase that contributes to the epigenetic silencing of target genes in cancer cells, but the role of SETDB1 function in mediating tumor metastasis has not been reported. We show here that H3K9Me2 and SETDB1 are down-regulated in metastatic lung cancer cell lines. Ectopic expression of SETDB1 in highly invasive lung cancer cells suppressed cancer cell migration, invasion and filopodia formation. In contrast, knockdown of endogenous SETDB1 in non-invasive lung cancer cells by RNA interference increases cell migration and invasion in vitro. Mechanistic investigations suggested that SETDB1 is specifically down-regulated by miR-29b in metastatic lung cancer cells. Ectopic miR-29b expression in non-invasion lung cancer cells increases cell invasion ability. These results indicate that SETDB1 contributes to the repressive role and concomitant dysregulation of epigenetic pathways in lung 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 78. doi:10.1158/1538-7445.AM2011-78
Abstract 784: Prevalence, phenotype and prognostic significance of IL-17-producing cells infiltratinghuman colorectal cancers
Authors: Amicarella Francesca , Zlobec Inti , Muraro Manuele Giuseppe , Han Junyi , Huber Xaver , Zuber Markus , Adamina Michel , Oertli Daniel , Lugli Alessandro , Terracciano Luigi , Zajac Paul , Spagnoli Giulio Cesare , Iezzi Giandomenica
Published: June 2012

Background: Lymphocytic infiltration is known to be associated with a favourable prognosis in human colorectal cancer (CRC). In particular, the presence of CD8+ T cells and, unexpectedly, of Foxp3+ regulatory T cells, has been found to be associated with improved patient survival. Recent evidence suggests that IL-17 and T helper (Th) 17 cells might also have an impact on anti-tumour immune responses. We have investigated prevalence, phenotype and prognostic significance of IL-17-producing cells in human CRC.

Material and Methods: IL-17 expression was evaluated by immunohistochemistry on a tissue micro-array (TMA) including 1420 cases of primary CRC with full clinico-pathological data. Furthermore, gene expression levels were assessed on CRC tissues by quantitative PCR.

Finally, in order to characterize the phenotype of IL-17-positive cells, expression of IL-17, in combination with that of specific surface molecules, was analyzed on freshly excised CRC specimens by flow cytometry.

Results: Frequencies of IL-17-producing cells, as well as IL-17 gene expression levels were significantly increased in tumour tissues as compared to autologous normal mucosa. IL-17-producing cells isolated from clinical specimens were exclusively comprised within the lymphocyte population and expressed CD4, but not CD8, and surprisingly, Foxp3 molecules. Accordingly, mRNA levels of genes encoding for cytokines favouring IL-17 acquisition by Foxp3+ T cells, including IL-6, IL-1beta, TGF-beta and IL-23, were found more elevated in CRC tissues as comparing to corresponding healthy mucosa.

High infiltration by IL-17 producing cells significantly correlated with low T and N stages, and, most importantly, with prolonged survival time in mismatch repair (MMR)- proficient, but not-deficient CRC. Moreover, the simultaneous CRC-infiltration by IL-17+ and Foxp3+ cells was significantly associated with improved survival in both MMR-proficient and -deficient tumours.

Conclusions: Our data suggest that IL-17 produced by tumour-infiltrating either CD4+ or Foxp3+ cells may promote a benign clinical outcome in CRC.

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 784. doi:10.1158/1538-7445.AM2011-784
Abstract 811: Co-inhibition of cyclooxygenase-2 and inducible nitric oxide synthase prevents N-nitrosomethylbenzylamine-induced esophageal squamous cell carcinoma in rats
Authors: Guan Huaji , Yan Fei , Wang Yufen , Battista Carter , Huang Xin , Stoner Gary D. , Nines Ronald G. , Edmonds Alexandria M. , Chen Tong
Published: June 2012

Hyperactivation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) has been observed separately in esophageal squamous cell carcinoma (ESCC) in rats that is induced by N-nitrosomethylbenzylamine (NMBA), a powerful carcinogen found in some human diets. In approach to discover new prevention strategies for ESCC, celecoxib and S,S′-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), selective inhibitors respectively to COX-2 and iNOS, were evaluated in the NMBA-rat preclinical esophageal cancer model. F344 rats were treated with NMBA (0.30mg/kg b.w.) three times per week for five weeks. Seventy-two hours after the last NMBA treatment, rats were fed with control diet with or without different doses of celecoxib and PBIT. At week 35, rats were sacrificed and esophageal tumors were counted. Celecoxib (1000 ppm) or PBIT (100 ppm) alone, and a combination treatment with celecoxib (500 ppm) and PBIT (50 ppm), significantly decreased the NMBA-induced tumor multiplicity (from 4.73 ± 0.45 to 2.00 ± 0.25, 1.89 ± 0.23, and 1.61 ± 0.27, respectively), which were accompanied by the inhibition of transcription, translation and kinase activity of both COX-2 and iNOS in all these treatments. By using this COX-2/iNOS co-inhibition strategy, celecoxib/PBIT combination inhibited cancerous esophagus cell proliferation by immunohistochemistry staining of PCNA, and decreased c-Myc expression in NMBA-induced papillomas by Western blot analysis. In addition, the histopathological examination of rat esophagus showed that celecoxib/PBIT combination significantly prevented NMBA-induced tumor transformation (from 8.373% to 1.765%). In conclusion, our results indicated that co-inhibition of COX-2 and iNOS by Celecoxib/PBIT combination prevented NMBA-induced ESCC in rats, which represents a new means of esophageal cancer prevention. (Supported by NIH/NCI RO1 CA131073-01A1).

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 811. doi:10.1158/1538-7445.AM2011-811
Abstract 818: Modulation of gene expression and cell cycle signaling pathways in Iressa-treated urinary bladder cancer in rats
Authors: Lu Yan , Liu Pengyuan , Grubbs Clinton , Lubet Ronald , You Ming
Published: June 2012

The EGFR inhibitor Iressa has demonstrated strong preventive efficacy in the N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) model of bladder cancer in the rat. To explore its antitumor mechanism, we implemented a systems biology approach to characterize gene expression and signaling pathways in rat urinary bladder cancers treated with Iressa. Eleven bladder tumors from control rats, seven tumors from rats treated with Iressa and seven normal bladder epithelia were profiled using Affymetrix Rat Exon 1.0 ST Arrays. We identified 713 down-regulated and 641 up-regulated genes in comparing bladder tumors vs. normal bladder epithelia. In addition, 178 genes were down-regulated and 96 genes were up-regulated when comparing control tumors vs. Iressa-treated tumors. Two coexpression modules that were significantly correlated with tumor status and treatment status were identified (r = 0.70, P = 2.80 × 10−15 (bladder tumor vs. normal bladder epithelium) and r = 0.63, P = 2.00 × 10−42 (Iressa-treated tumor vs. control tumor), respectively). Both tumor module and treatment module were enriched for genes involved in cell cycle processes. Twenty-four and twenty-one highly connected hub genes likely to be key drivers in cell cycle were identified in the tumor module and treatment module, respectively. Analysis of microRNA genes on the array chips demonstrated that tumor module and treatment module were significantly associated with expression levels of let-7c (r = 0.54, P = 3.70 × 10−8 and r = 0.73, P = 1.50 × 10−65 respectively). These results suggest that let-7c down-regulation and its regulated cell cycle pathway may play an integral role in governing bladder tumor suppression or collaborative oncogenesis and that Iressa exhibits its preventive efficacy on bladder tumorigenesis by up-regulating let-7 and inhibiting the cell cycle. The identified hub genes may also serve as pharmacodynamic or efficacy biomarkers in clinical trials of chemoprevention in human bladder 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 818. doi:10.1158/1538-7445.AM2011-818
Abstract 82: Expression of MUC17, a marker of pancreatic cancer, is under the control of epigenetic modifications
Authors: Kitamoto Sho , Yamada Norishige , Yokoyama Seiya , Houjou Izumi , Higashi Michiyo , Goto Masamichi , Batra Surinder K. , Yonezawa Suguru
Published: June 2012

MUC17 glycoprotein is a membrane-associated mucin that is mainly expressed in the digestive tract. It has been reported that aberrant MUC17 expression is observed in pancreatic ductal adenocarcinomas (PDACs) and correlated with the malignancy potential.

In the present study, we provided the first report of the MUC17 gene expression through epigenetic regulation such as promoter methylation, histone modification and microRNA (miRNA) expression. To investigate DNA methylation status in the 5’flanking region of MUC17, MassARRAY analysis was performed on 10 cell lines. This method permits the high-throughput identification of the methylation sites and their semi-quantitative measurement. Near the transcriptional start site (−179 to +52), a high level of CpG methylation was observed in MUC17-negative/low cells (e.g., Panc-1), whereas that of MUC17-positive cells (e.g., AsPC-1) was low. We also examined the relationship between DNA methylation and histone modification in MUC17 expression by ChIP assay, resulting the presence of dimethyl-H3-K9 in cells with no or low MUC17 was confirmed, whereas histone H3-K9 was more highly acetylated in MUC17-positive cells than in cells with no or low MUC17. These results suggest that the combination of DNA methylation and histone H3-K9 modification contribute to MUC17 expression. Futhermore, in the result of miRNA microarray analysis, we revealed that the five potential miRNAs are existed for the post-transcriptional regulation of MUC17.

Our findings indicate that MUC17 is regulated tightly through epigenetic manners including DNA methylation, histone modification and miRNA regulation and also suggest the possible functional importance of MUC17 in both physiological and pathological conditions. Furthermore, for the clinical aspect, the DNA hypomethylation was also observed in patients with PDAC. This may be of importance for the diagnosis of carcinogenic risk and the prediction of outcomes for 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 82. doi:10.1158/1538-7445.AM2011-82
Abstract 820: The synthetic triterpenoids, CDDO-methyl ester and CDDO-ethyl amide, and the histone deacetylase inhibitor vorinostat delay carcinogenesis in a transgenic model of estrogen receptor-negative breast cancer
Authors: Tran Kim M. , Sporn Michael B. , Liby Karen
Published: June 2012

The incidence of estrogen receptor (ER)-positive breast cancer has declined over the last several years; however, there has been no change in the incidence of ER-negative breast cancer. Novel drugs and drug combinations are needed for the chemoprevention of ER-negative breast cancer. We show that the methyl ester or ethyl amide derivatives of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-ME and CDDO-EA, respectively) and the histone deacetylase inhibitor Vorinostat (suberoylanilide hydroxamic acid (SAHA)) delay the formation of ER-negative mammary tumors in MMTV-Polyoma Middle T (PyMT) mice. Female PyMT mice were fed control diet, CDDO-ME (50mg/kg diet), CDDO-EA (400mg/kg diet), and SAHA (250mg/kg diet), or the combinations of each triterpenoid and SAHA, beginning at 4 weeks of age. To detect initial tumor development, mice were palpated twice a week. CDDO-ME, CDDO-EA, and SAHA each significantly delayed the development of ER-negative tumors, by an average of 4.3 weeks (p < 0.001), 1.2 weeks (p < 0.05), and 2 weeks (p < 0.001), respectively, compared to the control group. The combination of either CDDO-ME or CDDO-EA with SAHA was significantly more potent than the individual drugs at delaying tumor development, with an average of a 5.75 (p < 0.001) and 5.83 (p < 0.001) week delay, respectively, compared with the control group. Additionally, CDDO-ME and SAHA significantly improved survival, with an average improvement of 5.2 weeks (p < 0.001) and 1.5 weeks (p < 0.05), respectively, compared to the control group. As a single agent, CDDO-EA had no significant effect on survival; however, current studies suggest that the combination of CDDO-EA and SAHA may synergistically improve survival in PyMT mice. To elucidate the mechanisms responsible for tumor delay and improvement in survival, primary epithelial cells were isolated from mammary tumors of female PyMT mice. Treatment with CDDO-ME at nanomolar concentrations resulted in a significant repression of the biomarker cyclin D1 and the induction of the cell cycle regulator p21 at the protein level. Current studies are investigating additional mechanisms of action of CDDO-ME, CDDO-EA, and SAHA and will be presented. This study shows that as single agents, CDDO-ME, CDDO-EA, and SAHA delay ER-negative mammary tumorigenesis and also synergize when used in combination for prevention.

This work was supported by the Breast Cancer Research Foundation.

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 820. doi:10.1158/1538-7445.AM2011-820
Abstract 839: A novel Gemini vitamin D analog represses a stem cell marker CD44 and its variant CD44v6 and inhibits CD44-Met signaling in breast cancer
Authors: So Jae Young , Smolarek Amanda K. , Maehr Hubert , Uskokovic Millan , Cai Li , Liu Fang , Suh Nanjoo
Published: June 2012

CD44, a transmembrane glycoprotein that is involved in cell-cell interaction and cell adhesion, has been recognized as a key cell-surface marker for breast cancer stem cells. The CD44 positive breast cancer cells are enriched in residual breast tumors after conventional therapies, suggesting CD44 as an important therapeutic target to block the recurrence of breast cancer. To investigate CD44 expression during tumor progression, we used the CD44 overexpressing MCF10DCIS.com cell line as a model for breast cancer progression. We injected MCF10DCIS.com cells into immunodeficient mice and analyzed the resulting tumors after 1, 2, 3, 4 and 5 weeks from cell injection. MCF10DCIS.com cells formed ductal carcinoma in situ like lesion by week 2 and invasive tumor lesions by week 3. CD44 proteins were highly expressed in the membrane of epithelial cells in mammary tumors, suggesting that human MCF10DCIS.com cell line might be a useful model for studying therapeutic agents targeting CD44 positive breast cancer cells. We previously reported that a Gemini vitamin D analog, BXL0124, significantly down-regulated the expression level of CD44 in MCF10DCIS.com cells in vitro and in DCIS xenograft tumors. In the present study, we demonstrated that BXL0124 strongly repressed the protein expression level of two alternative splicing variants of CD44, CD44v3 and CD44v6, in MCF10DCIS.com xenografted tumors. Because these CD44 variants are closely associated with breast cancer growth and invasion, the repression of CD44 variants by BXL0124 in MCF10DCIS.com xenograft tumors may contribute to the inhibitory effect of BXL0124 on tumor growth and invasion. We further investigated the effect of BXL0124 on the Met receptor, which is known to interact with CD44v6 for its downstream signaling transduction to control cancer invasion and metastasis. The treatment with BXL0124 decreased the protein expression level of CD44v6 and inhibited hepatocyte growth factor (HGF)-induced Met signaling in cultured MCF10DCIS.com cells. In the CD44 promoter assay, mutation of p53 binding site in the CD44 promoter abolished the repression of CD44 transactivation by BXL0124, indicating that p53 is necessary for the repression of CD44 by BXL0124. In conclusion, BXL0124 repressed CD44 expression in a p53-dependent manner and inhibited CD44-Met signaling in MCF10DCIS.com cells. Our study suggests the Gemini vitamin D analog, BXL0124, is a potentially useful agent in inhibiting breast cancer by targeting CD44 expressing cancer stem cells.

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 839. doi:10.1158/1538-7445.AM2011-839
Abstract 84: OGDHL is epigenetically silenced and suppresses growth in human cervical cancer
Authors: Sen Tanusree , Ravi Rajani , Wu T.-C. , Sidransky David , Hoque Mohammad Obaidul
Published: June 2012

Despite the significant benefits conferred by screening for cervical cancer, cervical cancer is responsible for 190,000 deaths annually worldwide and the leading cause of cancer mortality in women in developing countries. Thus, there is a great need for improvements in cervical screening and targeted therapies. Such improvements depend, however, on a better understanding of the molecular biology of cervical neoplasia.

The epigenetic silencing of tumor suppressor genes is a crucial event during carcinogenesis and metastasis. Recently, in a human genome-wide survey, we identified oxoglutarate dehydrogenase-like (OGDHL) gene as a cancer specific methylated gene located on chromosome 10q11.23. Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multienzyme OGDH complex (OGDHC) whose malfunction is associated with neurodegeneration. The essential role of this complex is in the degradation of glucose and glutamate. OGDHL is down-regulated by promoter hypermethylation in cervical cancer cell lines and cervical cancer specific methylation was observed with 100% specificity. These properties suggest an important tumor suppressor role for OGDHL, yet direct genetic evidence of a tumor suppressor function for OGDHL is lacking and the molecular mechanism(s), through which OGDHL exerts its tumor suppressor functions, has not been shown.

Here, we report that restoration of OGDHL expression in cervical cancer cells lacking endogenous OGDHL expression suppressed cell proliferation and invasive phenotypes in in vitro and colony formation in soft agar. On the other hand, knockdown of OGDHL expression in cervical cancer cells expressing endogenous OGDHL promoted cell proliferation, invasive phenotypes and several malignant behaviors. Mechanistic analysis indicated that OGDHL up-regulation increases the production of reactive oxygen species (ROS) leading to apoptosis through down-regulation of AKT signaling cascade. Conversely, silencing OGDHL stimulated the signaling pathway via increased phosphorylation of PI3K, AKT and ERK1/2; it was also associated with phosphorelation of NFκβ, a molecule when activated can cause the cells to develop survival advantage, resist cell death and resist chemotherapy drugs.

Taken together, these data suggest that inactivation of OGDHL, an isoform of the 2-oxoglutarate dehydrogenase, can contribute to cervical tumorigenesis via activation of the AKT signaling pathway and support the therapeutic potential for this protein.

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 84. doi:10.1158/1538-7445.AM2011-84
Abstract 860: Benzyl isothiocyanate inhibits oncogenic actions of leptin in human breast cancer cells by suppressing activation of signal transducer and activator of transcription 3
Authors: Kim Su-Hyeong , Nagalingam Arumugam , Saxena Neeraj K , Singh Shivendra V , Sharma Dipali
Published: June 2012

Obesity is a well-established risk factor for breast cancer progression and molecular effects of obesity are mediated by adipocytokine, leptin. Considering the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., gardencress), confers significant protection against mammary carcinogenesis in a transgenic mouse model (MMTV-neu). The BITC-mediated prevention of mammary cancer development in MMTV-neu mice correlated with reduced cell proliferation, increased apoptosis, and tumor infiltration of T cells. The present study provides first evidence for efficacy of BITC against oncogenic effects of letpin. BITC-treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 human breast cancer cells (an estrogen-responsive cell line with wild-type p53) at pharmacologic doses. Leptin-stimulated migration and invasion of these cells was also inhibited significantly in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC-treatment suppressed leptin-induced phosphorylation of signal transducer and activator of transcription 3 (Stat3) and cyclin D1 transactivation. BITC-treatment efficiently inhibited Stat3 recruitment to the cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a decrease in level of phospho-Stat3. These studies indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivastion. Taken together, these data show that BITC treatment can inhibit the oncogenic actions of leptin via blocking its downstream signaling molecule, Stat3.

In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. This work was supported in part by the USPHS grants NCI-RO1 CA129347 (to SVS), NIDDK-K01DK076742 and R03DK089130 (to NKS), NCI-R01CA131294 and NCI-R21 CA155686 (to DS).

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 860. doi:10.1158/1538-7445.AM2011-860
Abstract 862: Expression of the fat-1 gene retards the growth of cervical cancer cells in vitro and in vivo
Authors: Lim Kyu , Song Kyoung-Sub , Shin Soyeon , Kim Nayeong , Jeong Soyeon , Heo Jun-Young , Park Ji-Hoon , Seo Kang-Sik , Kwon Hyun-Joo , Park Hae-Duck , Dai Yifan , Park Jong-Il , Park Seung-Kiel , Kweon Gi-Ryang , Yoon Wan-Hee , Hwang Byung-Doo , Jing Kaipeng
Published: June 2012

Omega 3-polyunsaturated fatty acids (ω3-PUFAs) are known to inhibit proliferation of cancer cells; in contrast, ω6-PUFAs promote the growth of cancer cells. The fat-1 gene of the Caenorhabditis elegans encodes a ω3-desaturase that catalyzes the conversion ω6-PUFAs to ω3-PUFAs and then increases the amount of ω3-PUFAs. The objective of this study is to examine the effect of fat-1 gene expression on HeLa and SiHa human cervical cancer cells. Fat-1 gene stable cell lines (f-HeLa and f-SiHa) were established from HeLa and SiHa cells, respectively. The expression of fat-1 gene significantly inhibited cervical cancer cell proliferation and f-HeLa cells showed an increase in the proportion of cells in G2/M phase comparing with the cells expressing the control vector (c-HeLa). In addition, when treating HeLa cells with docosahexaenoic acid (DHA), an enhanced proportion of cells in the G2/M phase was also observed, indicating that fat-1 gene inhibited cervical cancer cell proliferation by inducing a G2/M phase cell-cycle arrest. Furthermore, transwell migration assay for invasion showed a reduction of cell migration in the f-HeLa cells when compared with the c-HeLa cells. Finally, the growth of f-HeLa cells in vivo was significantly reduced comparing with the c-HeLa cells when inoculated into nude mice. Taken together, these results suggest that the expression of fat-1 gene prevents cervical tumor growth and indicate a cancer therapeutic approach of the ω3-PUFAs. Therefore, a stable cell line of fat-1 gene is useful to study the anti-cancer effects of ω3-PUFAs.

[This research was supproted by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (E00026 and R13-2007-020-01000-0)].

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 862. doi:10.1158/1538-7445.AM2011-862
Abstract 891: The association of single nucleotide polymorphisms in folate metabolism genes with hypermethylation patterns in smokers at high risk for lung cancer
Authors: Flores Kristina G. , Stidley Christine A. , Picchi Maria A. , Mackey Amanda J. , Byers Tim , Berwick Marianne , Belinsky Steven A. , Leng Shuguang
Published: June 2012

Hypermethylation of promoter regions serve as potential biomarkers for the early detection of lung cancer. Because folate metabolism is necessary for nucleotide synthesis and DNA methylation reactions, that when perturbed, may both contribute to the development of cancer, hypermethylation of promoter regions in folate metabolism genes may be particularly important to explore. Dietary folate was recently found to protect against gene promoter hypermethylation patterns. This study investigated the association of functional and haplotype tagging single nucleotide polymorphisms (SNPs) in folate metabolism genes with promoter hypermethylation in sputum cell DNA from a cohort of current and former smokers (n=937). This cohort was a subset of the Lovelace Smokers’ Cohort of approximately 2000 high risk current and former smokers who were non-Hispanic white and for whom a complete methylation panel was obtained. Methylation panel status from a 12 gene panel was dichotomized into high and low methylation based on a cutoff of the number of genes methylated (> = 3 genes for women and > = 4 genes for men were categorized as high). Lymphocyte DNA was interrogated on a 96 SNP Illumina Goldengate platform. A SNP in the cystathionine-beta synthase gene (CBS), that converts homocysteine to cystathionine, was found to be significantly associated with gene promoter hypermethylation. Analyses used logistic regression and adjusted for multiple comparisons. There was an interaction between gender and CBS (p<0.0001), so we conducted a stratified analysis by gender. Males with the variant allele had an increased risk of methylation (OR = 3.61; 95% CI: 1.71, 7.64); p<0.001) and females with the variant allele had a decreased risk for methylation (OR = 0.64; 95% CI: 0.40, 1.00; p=0.05). A 20% reduction in CBS gene transcription was seen in bronchial epithelial cells heterozygous for the CBS variant allele compared to cells without the variant allele but was not statistically significant. Epidemiological studies have shown that men have higher levels of homocysteine compared to women but the mechanisms regulating this difference are not known. This study shows that a SNP in the CBS gene is associated with methylation patterns in smokers at high risk to develop lung cancer and differs by gender. This work may further help to elucidate the differences in lung cancer risk by gender.

Grant Support: This work is supported by K01CA128823 NCI and R01CA097356 NCI

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 891. doi:10.1158/1538-7445.AM2011-891
Abstract 892: Survey of human cancer tissues for the presence of bovine leukemia virus
Authors: Buehring Gertrude Case , Jensen Hanne M. , Tesluk Henry
Published: June 2012

Bovine leukemia virus (BLV) is an oncogenic retrovirus that causes B cell leukemia/lymphoma in 5% of infected cattle. Infection is widespread in the USA: 24% of beef herds, 84% of dairy herds, and 100% of large dairy operations. BLV has been detected in T cells, B cells, endothelium, and mammary epithelium, and infected cells may be present in the blood and milk of cattle. Previously we found IgG antibodies to BLV in 39% of humans sampled, and BLV-related DNA in 60% (67/112) of malignant human breast tissues. Presence of BLV-related DNA in human mammary epithelium was significantly associated with breast cancer risk (odds ratio = 3.4, confidence interval = 1.77-6.44; P≪ .0002).

The purpose of this study was to test other types of human cancers for BLV-related DNA as a first step in exploring whether BLV might be involved in malignancies of tissues other than breast. Specimens were surgical pathology archival material collected from 1992-2003. We performed PCR-in-situ hybridization (PCR-ISH) on formalin-fixed tissue sections, targeting the highly conserved tax region, which codes for the oncogenic protein of BLV. This method is not subject to molecular contamination, and can localize the signal to a particular cell type in the intact tissue. Our survey included the following malignancies: stomach, colon, pancreas, prostate, seminoma, ovary, endometrium, sarcoma (1 liposarcoma, 3 fibrosarcoma, 4 fibrohistiocytoma, 1 Ewing sarcoma), lymphoma (10 B cell, 2 T cell, 3 Hodgkin), squamous cell skin carcinoma, lung, thyroid, and brain (3 astrocytoma, 4 meningioma, 3 glioblastoma).

BLV-related amplicons were detected in the following cancer types: stomach (5/10), colon (1/11), brain (2/10 [1 astrocytoma and 1 glioblastoma]), lymphoma (1/15 [T cell lymphoma]), sarcoma (1/9 [poorly differentiated high grade sarcoma]), and squamous cell skin carcinoma (2/10). No tissue type we tested showed a frequency of BLV-related DNA as great as we previously detected in breast cancer (60%). Sample sizes in this pilot survey were too small to provide an accurate estimate of overall frequency in the population or to make any conclusions about the association of BLV-related DNA with the malignancies studied. However, the data do provide some interesting leads for further research with an increased number of specimens and normal control tissue, especially for the cancer types exhibiting at least one positive specimen.

This research was supported by a seed grant to HM Jensen from the Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Davis.

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 892. doi:10.1158/1538-7445.AM2011-892
Abstract 897: Thymidine kinase 1, a novel biomarker specific to the plasma membrane of cancerous cell lines
Authors: Sharp Daniel W. , Estevez Dagoberto , Whitehurst Robert A. , Alegre Melissa M. , Germann Brit L. , Foster Joshua W. , Robison Richard A. , O'Neill Kim L.
Published: June 2012

Thymidine Kinase 1 (TK1), an important nucleotide salvage pathway enzyme, has been shown to be upregulated in most malignant cancers. Recent studies have shown that TK1 not only has diagnostic and prognostic potential, but could also be used as a molecular target for therapeutic treatments. Previous research from our lab has shown the expression of TK1 on the plasma membrane of lymphoma. This study provides further insight on the role of TK1 as a diagnostic target for several different tumor cell lines. This study analyzed the localization of TK1 in ten different cancer cell lines using flow cytometry (FCM). The lines studied were A375, a malignant melanoma, CAL27, a squamous cell carcinoma derived from tongue, MDA-MB-231, a breast adenocarcinoma, MDA-MB-435S, previously described as a ductal carcinoma, HT29, a colorectal adenocarcinoma, SW620, a colorectal adenocarcinoma, A549, a lung carcinoma, NCI-H460, a large cell lung carcinoma, DU 145, a prostrate carcinoma, and PC3, a prostate adenocarcinoma. FCM was carried out using a polyclonal antibody against human TK1 as the primary antibody and a FITC conjugated secondary antibody. A sample stained with only secondary antibody was used as a negative control for the experiment. Five replicates were analyzed for each cell line. TK1 was found to bind between 48% and 96%, depending on the cell line. Non-cancerous tonsil tissue and normal lymphocytes were used as controls, where TK1 surface binding was less than 20%. These data indicate the feasibility of TK1 as a possible molecular target for immunotherapy in many types of cancer. Further research is ongoing to determine specific immunotherapy treatment options, and to verify the expression of TK1 in other cancer cell lines.

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 897. doi:10.1158/1538-7445.AM2011-897
Abstract 922: Disruption of bone morphogenetic protein receptor 2 accelerates mammary carcinoma metastasis to the lung
Authors: Owens Philip , Pickup Michael W. , Novitskiy Sergey V. , Chytil Anna , Gorska Agnieszka E. , Aakre Mary , Moses Harold L.
Published: June 2012

Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor β (TGFβ) superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in cancers as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have both tumor suppressive and promoting effects. We sought to determine whether disruption of the Bone Morphogenetic Protein Receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen (PyVmT). Mice expressing PyVmT under the mammary specific MMTV promoter were combined with mice that have Doxycycline inducible expression of a dominant negative (DN) BMPR2. We did not observe any differences in tumor latency. However, when we measured pulmonary metastases, we found that mice expressing the BMPR2-DN had a five fold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN expressing tumor cells had higher rates of proliferation and epithelial to mesenchymal transition (EMT). We also identified unique changes in the tumor microenvironment, which included inflammatory cells and secreted chemokines/ cytokines that accompanied BMPR2-DN expressing tumors. We identified several molecules including ccl-9 (MIP1γ), which was elevated in BMPR2-DN expressing tumors. Ccl-9 has recently been shown to be an important chemokine in the recruitment of myeloid cells that promote metastatic progression. We discovered that tumor cells treated exogenously with BMP4 ligand repressed ccl-9 mRNA and that this repression was limited in cells expressing BMPR2-DN. We conclude that BMPR2 has tumor suppressive function in mammary epithelia and that disruption can accelerate mammary carcinoma metastases via paracrine stimulation of myeloid cells in the tumor microenvironment.

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 922. doi:10.1158/1538-7445.AM2011-922
Abstract 96: The functional role of DNA hypomethylation in liver cancer
Authors: Stefanska Barbara , Bhattacharyya Bishnu , Suderman Matthew , Huang Jian , Hallett Michael , Han Ze-Guang , Szyf Moshe
Published: June 2012

DNA hypomethylation, a process of losing methyl marks, may play an important role in cancer, especially through activating genes that promote cancer metastasis, the deadly aspect of cancer. We previously delineated using whole genome promoter methylation microarrays the landscape of hypomethylation in hepatocellular carcinoma which is one of the most common cancers worldwide. Our studies revealed that 50% of differentially methylated promoters in tumors are hypomethylated compared to adjacent normal tissue. In the present studies, using liver cancer cell lines as a model system, we evaluated the importance of the identified hypomethylated genes in liver cancer development and progression. We tested whether the hypomethylation observed in liver cancer is driven by MBD2, a protein reported previously to be implicated in DNA demethylation, which we found to be overexpressed in liver cancer patients.

Following siRNA MBD2 depletion in HepG2 hepatocellular carcinoma cell line, we used methylated DNA immunoprecipitation, chromatin immunoprecipitation (ChIP) and 60K custom microarrays covering 12K bp upstream and 10K bp downstream of transcription start site to determine methylation and MBD2 binding in 300 top genes hypomethylated and/or induced in liver cancer patients and/or HepG2 cells. The array data were validated by quantitative ChIP and/or pyrosequencing. For functional analyses, we used siRNA transfection, soft agar and invasion assays.

We discovered MBD2 binding peaks in 172 genes out of 300, mainly within their promoter regions. Using bioinformatic tools, we identified MBD2 binding motif and established putative transcription factors recognizing this sequence which may play a key role in recruiting MBD2 to target genes. 41% of those genes were hypermethylated in HepG2 cells after MBD2 depletion in a fragment corresponding to MBD2 binding site. In addition, expression analysis for some of the genes showed attenuation. In HepG2 cells, we depleted expression of several of the genes hypomethylated in cancer and regulated by MBD2 using siRNA and demonstrated a decrease in cell growth and invasive capacities.

Our results establish a role for MBD2 in coordinating inhibition of a panel of genes involved in cancer growth and metastasis.

This study was supported by a grant from the MDEIE program of the government of Quebec and the National Cancer Institute of Canada to MS.

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 96. doi:10.1158/1538-7445.AM2011-96
Abstract 968: Invadopodia formation and microparticle release are required for cancer cell extravasation in vivo
Authors: Leong Hon S. , Robertson Amy E. , Ablack Amber A. , Turley Eva A. , Chambers Ann F. , Lewis John D.
Published: June 2012

During the metastatic process, cancer cells must undergo trans-endothelial migration from the vessel lumen into underlying tissue in a process known as extravasation. Little is known about the dynamic mechanical aspects of this process. To address this, we performed real-time, sub-cellular resolution intravital imaging of human cancer cells during arrest, intravascular migration and extravasation using a shell-less avian embryo xenograft model. We find that extravasation occurs at endothelial junctions and that the majority of extravasation events occurred during the 12 hours subsequent to the intravenous injection of cancer cells. In the majority of extravasation events, long cytoplasmic extensions identified as invadopodia were observed prior to extravasation which breached underlying endothelium. We observed the release of microparticles during these invadopodial extension events prior to extravasation, and this resulted in a ∼40% reduction in cell volume post-extravasation. Based on this, we hypothesized that invadopodia are required for extravasation, and that cancer cell extravasation could be abrogated by inhibiting factors required for invadopodia function. We found that treatment with Src kinase inhibitors significantly reduced invadopodia formation in vivo and resulted in a ∼60% decrease in extravasation events when compared to vehicle treated controls. Furthermore, a higher proportion of cells in the Src kinase inhibitor-treated animals remained intravascular compared to vehicle control. In conclusion, we determined that 1) extravasation occurs at junctions between adjacent endothelial cells, 2) cancer cells form invadopodia that breach the endothelial layer prior to successful extravasation and 3) cancer cells undergoing extravasation release cancer microparticles into both the vessel lumen and tissue interstitium with a corresponding decrease in cell volume. Pharmacological inhibition of invadopodia by Src kinase inhibitors (Bosutinib, Dasatinib) reduces cancer cell extravasation, revealing a novel and potentially important mechanism of action against metastatic cancers.

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 968. doi:10.1158/1538-7445.AM2011-968
Abstract 969: Molecular characterization of circulating tumor cells in breast cancer by liquid bead array hybridization assay
Authors: Markou Athina N. , Strati Areti , Malamos Nikos , Georgoulias Vassilis , Lianidou Evi
Published: June 2012

Introduction: The aim of our study was to develop and validate a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in Circulating Tumor Cells (CTCs), and application of this assay in peripheral blood samples of breast cancer patients.

Patients and method: We designed a novel multiplexed PCR-coupled liquid bead array that consist of the following steps: a) in silico designed gene-specific primers and capture probes for CK-19, HER-2, Mammaglobin (hMAM), MAGE-A3, TWIST-1 and PBGD (as a control gene), b) RNA isolation from immunomagnetically enriched CTCs, c) multiplex PCR, d) sequence hybridization array, e) detection in the Luminex platform. We performed extensive optimization experiments using the SKBR3 and MDA-MB-231 cell lines as positive controls, to maximize analytical sensitivity and specificity and we further validated the performance of this array in respect to cross reactivity and intra and inter-precision. Finally, we applied the developed methodology in peripheral blood samples of 64 patients with operable breast cancer, 25 patients with verified metastasis and 17 healthy individuals.

Results: The analytical performance of the developed array was evaluated in tumor cell lines in respect to analytical sensitivity and specificity. Cross reaction studies were performed for each gene target in the presence of all other targets. The assay is highly specific for each gene in complex multiplexed formats, since the discriminatory power of fluorescent bead sets is unique to specifically detect by sequence hybridization the presence of individual gene specific PCR products. The developed liquid bead-based array is highly sensitive, since it can detect the expression of each individual gene at one SKBR3 cell level. The validation of the array included within day and between-days precision studies. None of the genes tested was detected in the CTC fraction of healthy donors while in patients with verified metastasis, CK-19 was detected in 65%, HER-2 in 20%, MAGE-A3 in 30%, hMAM in 20% and TWIST-1 in 20%. In operable breast cancer patients, CK-19 was detected in 26.6%, HER-2 in 12.5%, TWIST-1 in 31.2%, MAGE-A3 in 18.7% while hMAM was detected in 10.9% of the patients.

Conclusions: Our results show that individual gene expression can be readily measured in CTCs using a bead-based platform. This may form an efficient basis for a multiplex approach to measure multiple genes (up to 100) in the same sample, thus saving sample volume and reducing the total cost and time of analysis. This is the first time that the Luminex technology is used for gene expression studies in CTCs.

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 969. doi:10.1158/1538-7445.AM2011-969
Abstract 978: A regulatory role for TAZ in mesenchymal differentiation of glioblastoma
Authors: L Bhat Krishna P. , Salazar Katrina , Vaillant Brian , Aldape Kenneth
Published: June 2012

Gene expression profiling studies have revealed that the mesenchymal signature (MES) is a predictor for poor survival and treatment resistance in glioblastoma (GBM) patients, whereas patients with a proneural (PN) signature perform better in the clinic. We found that the HIPPO pathway transcription co-factor TAZ, but not YAP is epigenetically silenced by CpG island hypermethylation in lower grade/PN tumors whereas its promoter is hypomethylated in GBM/MES tumors. Treatment of a demethylation agent (2′-deoxy-5-azacytidine) increased TAZ mRNA levels in glioma stem cells lacking this protein. Using bioinformatic approaches we found TAZ to be significantly connected to the MES gene signature network that was distinct from recently reported regulators of MES transition in GBM, viz., STAT3 and CEBPβ. Approximately 60% of the in silico inferred targets were confirmed to be direct targets of TAZ by ChIP-PCR. Silencing TAZ in GSCs decreased invasion and blocked tumorigenesis when implanted intracranially into mice. Conversely, overexpression of TAZ in GSCs that lack this protein resulted in increased MES gene expression, suppressed PN gene expression, increased invasion, differentiation toward an osteogenic and chondrocytic lineage and reduced tumor latency in the xenograft model. In primary NSCs, forced TAZ resulted in increased expression of mesenchymal markers such as fibronectin, and smooth muscle actin, whereas astrocytic or neuronal differentiation was suppressed. All aforementioned phenotypes were abolished when overexpressing a single point mutant version of TAZ (S51A) that lacks binding to its bona fide transcription partner, TEAD. Finally overexpression of TAZ in a PGDF-RCAS transgenic mouse model caused increased tumor grade and incidence and shift toward a MES phenotype. Our study identifies TAZ as important master regulator of PN to MES transition in gliomas and presents as an attractive therapeutic target for this devastating disease.

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 978. doi:10.1158/1538-7445.AM2011-978