bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2872: Effects of ethanol and folate on peripubertal mouse mammary gland
Authors: Masso-Welch Patricia A. , Tobias Menachem E. , Kumar Shyam C. Vasantha , Tamburlin Judith , Koury Stephen T.
Published: February 2011

Alcohol consumption has been clearly linked with increased breast cancer risk in women, with risks incurred even at low levels of consumption. The proposed mechanisms whereby ethanol exerts its effects include decreased folate levels and diminished DNA repair capacity, coupled with acetaldehyde-generated DNA damage. Based on these proposed mechanisms, we hypothesized that ethanol would have increased deleterious effects during periods of rapid mammary gland epithelial proliferation, such as peripuberty. We also hypothesized that folate deficiency alone might mimic the effects of ethanol, and that folate deficiency would exacerbate the effects of ethanol. CD2/F1 female mice (N=8 per group) were pair-fed control and ethanol-containing liquid diets +/− 0.2g/L folate supplementation from 5 weeks until 9 weeks of age. Mice were sacrificed after this 4 week feeding regimen, and mammary glands were harvested for preparation of whole mounts and paraffin sections. Folate levels of liver and kidney were assessed to verify folate status. Analysis of mammary gland whole mounts revealed that mammary gland structure was significantly altered in folate-replete mice fed ethanol, compared to control-fed mice (no ethanol/ folate-replete). These ethanol/folate replete mice showed an increased total number of ductal branches, due to an increased number of terminal short branches; no effect was seen on the number of terminal end buds or primitive alveolar buds. Systematic histological analysis of paraffin sections using NIH Image J software revealed that diets containing ethanol in the presence of folate significantly increased the total number of ducts, ductal epithelial area, and number of ducts with epithelial multilayering. These results demonstrate that folate deficiency alone does not mimic the effects of ethanol, and that folate deficiency in the presence of ethanol blocks proliferative effects of ethanol on the mammary ductal tree. Future studies are examining the effects of peripubertal ethanol and folate exposure on gene expression in the mouse mammary gland.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2872.
Abstract 2873: Elucidation of dietary omega-6/omega-3 ratio effects on chemopreventive activity using mammographic density as a surrogate marker in rats
Authors: Zhu Zongjian , McGinley John N. , Jiang Weiqin , Manni Andrea , El-Bayoumy Karam , Thompson Henry J.
Published: February 2011

Although beneficial effects of omega-3 fatty acids in breast cancer prevention have been reported, the optimal dietary omega-6/omega-3 (n-6/n-3) ratio for chemoprevention has not been established. Although n-6 and n-3 fatty acids are essential for the normal development and maintenance of biological function, both classes of fatty acid share metabolizing enzymes and thus have the potential to antagonize each other's metabolism. Therefore, experiments were undertaken to elucidate the ratio(s) of dietary n-6/n-3 that is optimal for chemoprevention in the absence of adverse effects on normal development using mammographic density as a surrogate marker for breast cancer risk. Female Sprague Dawley rats (n=9/group, 32 days of age) were randomized to one of 7 dietary formulations that varied in their ratio of n-6/n-3 as follows: 25:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:25. The percent of dietary calories from fat was 30% across all 7 formulations. Mammary gland density was determined via digital analyses of abdominal-inguinal mammary gland whole mounts stained with alum carmine. Density was calculated using either area or integrated optical density and expressed as the percent of the mammary gland fat pad occupied by mammary epithelium. Mammographic density was unaffected in the high n-6 arm of the study; whereas, it was significantly reduced in the high omega-3 arm (16%, p<0.03). Efforts are ongoing to determine: 1) whether reduced density is accounted for by effects on cell cycle flux or induction of a pro-apoptotic environment, 2) the extent to which these effects are mediated by altered signaling via activated protein kinase B (Akt), and 3) the mechanism(s) by which the n-6/n-3 ratio alters eicosanoid-mediated bioactivity. Supported by grant KG081632 from the Susan G. Komen Foundation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2873.
Abstract 2879: Proteome changes in mouse prostate induced by oral administration of penta-O-galloyl-beta-D-glucose (PGG) suggest targets for cancer chemoprevention
Authors: Zhang Jinhui , Shaik Ahamd , Nhkata Katai , Wang Lei , Kim Kwan Hyun , Liao Joshua D. , Xing Chengguo , Kim Sung-Hoon , Lü Junxuan
Published: February 2011

1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose (PGG), a naturally occurring hydrolyzable gallotannin, has been shown by us and others to exhibit in vivo anti-cancer effects against prostate and breast cancer xenograft or lung cancer allograft when administrated by i.p injection or oral gavage. However, little is known of its safety for chronic chemoprevention use and virtually nothing is known of its in vivo target proteins. In the present study, we treated C57B6 mice with daily gavage of PGG from 7 weeks of age to 14 weeks of age with dosages of 1 and 2 mg per mouse (40-50 and 80-100mg/kg body weight). The dosage choices were made in reference to the efficacious anti-cancer dose of 20 mg/kg body weight. General health, body weights and food intake of mice were monitored. At necropsy, the organ weights were measured. Proteomic patterns in the prostate were profiled by 2D LC-MALDI-TOF/TOF analysis with iTRAQ labeling. Our results show that neither dose affected feed intake and body weight gain, but the 2 mg dose led to a statistically significant but very minor decrease of the weight of prostate and thymus. Proteome profiling by LC-MALDI-TOF/TOF identified 810 proteins from mouse prostates and 623 of them were identified with >95% confidence. Expression of 56 proteins was significantly modulated by either 1mg or 2mg PGG daily treatment, four different kinds of dose responses of proteins modulated by PGG were observed. Protein quantization results were validated by another mass spectrometry, ESI-Q-TOF. Selected proteins were further validated by Western blots and mRNA level by real-time PCR.

The proteome profiles modulated by PGG include suppression of oncoproteins such as fatty acid synthase and induction of tumor suppressor proteins such as glutathione S-transferase M. Overall, the data show protein changes induced by tolerated dosages of PGG in the normal prostate are conducive for cancer chemoprevention. The chemopreventive efficacy of PGG is being evaluated in a primary carcinogenesis model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2879.
Abstract 2884: Standardized mangosteen fruit extract promotes cell cycle arrest and apoptosis of PC3 cells and inhibits tumor formation in athymic nude mice
Authors: Johnson Jeremy J. , Syed Deeba N. , Adhami Vaqar , Rasmussen John T. , Siddiqui Imtiaz A. , Mukhtar Hasan
Published: February 2011

The purple mangosteen fruit (Garcinia mangostana) is native to southeast Asia and has been reported to contain a variety of health promoting properties that include antioxidant, anti-inflammatory, anit-microbial and anti-diabetic properties. The principle constituents isolated from the mangosteen fruit are the xanthones with well over 50 unique entities isolated to date. Here we report on the properties of a mangosteen fruit extract for cancer chemoprevention and/or chemotherapy using prostate cancer (PCa) as a model disease. Using a mangosteen fruit extract (MFE) a significant decrease in cell viability was observed against four different prostate cancer cell lines (LNCaP, CWR22, PC3, DU145). Several analytical standards were used to identify dietary xanthones by HPLC with alpha-mangostin found to be the most abundant xanthone of MFE representing approximately 59% of the extract. Next, we investigated the ability of MFE to promote cell cycle arrest by flow cytometry and observed a significant increase in cells in G1 phase when treated with 7.5 μg/mL of MFE after 24 hours. Further confirmatory studies were performed by western blot analysis of PC3 cells treated with MFE. To understand the possible interaction between xanthones and cyclins/cdks in a cell free system a biochemical based assay was performed and found to inhibit cyclinD1/CDK4 as well as other kinases. To determine the potential of MFE in an in vivo model we investigated the ability of MFE to inhibit colony formation of CWR22 and PC3 cells. MFE was found to completely inhibit colony formation of CWR22 cells at a concentration of 5 µg/mL. Next, using an athymic nude mouse model implanted with CWR22 cells mice were randomized to receive intraperitoneally the standardized mangosteen fruit extract (MFE) or placebo two times weekly with tumors measured twice weekly until tumors reached a volume of 1200 mm3. At the conclusion of the study the treatment group had a significantly smaller tumor with an average tumor volume of (143 mm3) compared to the placebo group (1135 mm3) representing an 87.5% smaller tumor in the treatment group. Based on these promising results we have evidence suggesting that MFE may be a promising agent for the prevention and/or treatment of prostate cancer. Further studies are needed to determine the potential of purified dietary xanthones from the mangosteen fruit for cancer chemoprevention and chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2884.
Abstract 2885: Retinoic acid suppressed HEY cell-induced xenograft tumor possibly through reduction of CD133+ cancer stem cells
Authors: Li Zhong
Published: February 2011

Ovarian cancer is the most lethal gynecologic cancer with the higher recurrence. Increasing evidence indicates that cancer stem cells plays pivotal role in tumorigenesis and chemoresistance. Retinoic acid (RA), a synthetic derivative of vitamin A, has been used for treatment and chemoprevention of ovarian cancer. But it is not clear if RA affects ovarian cancer stem cells. In this paper, we found that all-trans retinoic acid (RA) treated ovarian cancer cell HEY resulted in the significant smaller xenograft tumor in SCID/NOD mice than untreated HEY cells. To understand if RA inhibition of tumor is involved in dysfunction of cancer stem cells, we detected cancer stem cells in HEY-induced tumors. CD133 positive cells in ovarian cancer were reported to have stem-like features. We found that RA-pretreated HEY cell-induced tumors had less of CD133 positive cells (3.0%) than RA-untreated ones (5.5%). To determine if RA affects status of CD133+ cells, we first cultured HEY cells in spheroid condition. HEY cells formed spheroid in suspend culture in which the percentage of CD133+ cells was increased in analyses by flow cytometry and histochemical staining. A single spheroid of Hey cells was peritoneally injected into mice and induced ovarian tumors. Consequently, treatment of RA in initiating spheroid culture attenuated HEY spheroid formation. In analyzing the molecular mechanism of RA in tumor suppression, we found that RA-treated HEY cells increased phosphorylated PTEN and level of p53 and CRABP II, and reduced expression of CD133 in Hey cells in early period (24-48h). PTEN, a tumor suppressor gene and a PI3K functional antagonist, is widely involved in normal and cancer cell proliferation, migration, drug resistance and stem cell maintenance. We further overexpressed PTEN in HEY cells that dramatically suppressed spheroid formation, suggesting that RA-induced early PTEN activation, increases of p53 and CRABP II may be involved in change of malignancy of HEY cells. These results demonstrate that RA treatment of ovarian cancer cells would alternate the cell features that compromise the change of Cd133+ positive stem-like cells which are important in tumorigenesis.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2885.
Abstract 290: PDZ protein PDZK1 associates with PTEN and inhibits PTEN phosphorylation
Authors: Zheng Junfang , Yang Longyan , Xiong Ying , Wang Ying , Hu Jia , Zhou Yunying , Zhang Hong , He Junqi
Published: February 2011

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumour suppressor is a lipid and protein phosphatase that inhibits phosphoinositide 3-kinase (PI3K)-dependent signaling by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The carboxyl terminus (CT) of PTEN and PTEN post-translational modification by phosphorylation were reported to regulate PTEN signaling. However, the modes of regulation of PTEN signaling are not completely understood. The CT of PTEN has a typical PDZ domain-binding motif that interacts with several PDZ domain-containing proteins, which regulates PTEN signaling and function. To screen novel PTEN binding proteins, the PTEN CT was used to overlay against the lysates of rabbit tissue, including kidney, heart and brain. The molecular weight of positive band was similar with that of PDZ-domain containing 1 (PDZK1), a scaffolding protein. We further investigated this PTEN-associated protein by using GST pull-down in combination with mass spectrometry-based proteomics assays. PDZK1 was identified as one of the binding partners of PTEN. PTEN associated with PDZK1 via its PDZ domain 2 and 3, with binding affinity of 20 nM and 25 nM respectively, and the last four amino acids of the CT of PTEN were the key determinants of this interaction. Mutation of any one of the four amino acids to alanine abolished the association of PTEN with PDZK1-PDZ2, PDZK1-PDZ3 or PDZK1 full length, indicating that the PDZK1-PTEN association is stringent. Moreover, full-length PTEN was robustly associated with PDZK1, as determined by co-immunoprecipitation experiments in transiently transfected COS-7 cells, and mutation of the last amino acid valine to alanine also led to the abolishment of the interaction between PTEN full length and PDZK1. Immunofluorescence results showed that PTEN full length and PDZK1 can colocalize at cytoplasm of BHK cells. By interacting with PTEN, PDZK1 could inhibit the phosphorylation of PTEN and Akt activation induced by the stimulation of insulin in a dose-dependent manner, indicating PDZK1 can enhance the PTEN protein activity via their interaction. Taken together, these are the first results that present a interaction between PDZK1 and PTEN. PDZK1 interacts with PTEN to inhibit PTEN phosphorylation, leading to an increase in PTEN activity at inhibiting Akt activation. Our results suggest a regulatory role of PDZ domain binding on PTEN function by controlling its phosphorylation status.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 290.
Abstract 2903: High grade dysplastic polyps: Does size matter
Authors: Tobi Martin , Easler Jeffrey , Antaki Fadi , Levi Edi , Rambus Mary Ann , Lawson Michael
Published: February 2011

Adenomas with high-grade dysplasia (HGD) are recognized to be harbingers of future neoplastic lesions and colonoscopy is repeated in a year. There is little data regarding the neoplastic potential of the colon bearing such lesions. We postulated that lesions >1cm, in contrast to their smaller counterparts, would progress through an adenoma-carcinoma sequence. Conversely, smaller sized lesions might have a greater propensity for recurrence since there may be a priori more biological aggressiveness. Methods: We used the Adnab-9 monoclonal anti-adenoma risk marker to investigate high risk patients with HGD adenomas. Immunohistochemistry on normal-appearing colonic mucosa, ELISA of effluent acquired during colonoscopy and fecal Western blotting and ELISA, the stool collected prospectively was performed. Results: 23 patients with HGD adenomas, the majority found on the index colonoscopy were evaluated (table). A trend existed for increased NSAID intake in the large HGD group 5/7 (71%) compared to 2/7 (29%) in the small (p=0.28) and for increased numbers of metachronous adenomas in the small HGD group. More had symptoms in the large HGD group (67%) compared to 40% in small (NSS). No small HGD lesion was associated with a field effect (FE) in contrast to 100% in those patients with large lesion HGD (p<0.01). Concurrence with location of FE with synchronous adenomas was 5/6 (83%) and 2/5 (40%) with metachronous polyps in those with large HGD (p=0.24).

Conclusions: Small HGD lesions appear to progress by pathways beyond the purview of the adenoma carcinoma sequence characterized by the significant lack of a field effect. They arise in a colon that may be destined for greater than usual risk for CRN even than that seen with larger HGD lesions. Larger prospective studies should be undertaken to confirm these preliminary findings and determine the tumorigenic pathway but in the interim surveillance for smaller lesions should not be limited to a single 1 year of follow-up.ParameterLarge HGDSmall HGDAll HGDN131023Age x±sd (years)65.5±10.064.7±8.765.2±9.3%Race AA53.93043.5BMI30.3±7.127.9±6.229.3±6.6%Left-sided76.97073.9%TA/TVA/VA69.2/23.1/7.760/40/063/32.7/4.3Size x±sd (mm)21.5±13.76.7±3.115.1±12.8Follow-up yrs3.6±2.15.3±2.7(p=0.15)4.2±2.4Synchronous adenomas1.8±3.3 6/14 46.2%1.4±1.5 5/10 50%1.5±2.7 11/24 45.8%Metachronous adenomas1.3±1; 7/10 70%2.1±2.34* 4/7 57% (p=0.6)1.3±1.7 11/17 64.7%%Ad-9 WB+12.528.620FecalAdnab-92/8 (25%)2/7(28.6%)4/15(26.7%)Effluent Elisa+4/9(44.4%)2/7(28.6%)p=.66/16(38%)%Field Effect6/6 (100)**0/5 (0%)6/11(55.5%)**OR 0.007:CI 0.0001-.415;p=0.0022; *p=0.3 Note: F/U time NSS

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2903.
Abstract 2904: Lyophilized black raspberry extract modulates interleukin 8 expression in human cervical cancer: An insight into phytochemical-based chemoprevention?
Authors: Backes Floor J. , Zhang Zhaoxia , Seamon Leigh G. , Nagel Christa I. , Cohn David E. , Knobloch Thomas J. , Weghorst Christopher M.
Published: February 2011

Introduction

Cervical cancer is the 3rd most common cancer globally. The long latency from dysplasia to invasive cancer makes it amenable to prevention strategies. Phytochemicals, including those in lyophilized black raspberries (LBRs), are of great interest due to their growth inhibitory and anti-tumorigenic effects in preclinical models. Previously we showed that LBR extracts inhibited the proliferation of human cervical cancer cells in vitro in a dose- and time-dependent manner. Angiogenesis is an important part of carcinogenesis. We hypothesized that LBRs modulate the expression of an angiogenesis associated signaling cascade.

Methods

Human cervical cancer cells (SiHa) were treated with vehicle control (.02% DMSO) or LBR extract (200 µg/mL) for 1 or 3 days (d). Cells were harvested at 1d or 3d and RNA was extracted. Gene expression profiling was performed using a TaqMan Gene Signature Angiogenesis Array, which simultaneously screens 94 genes involved in angiogenesis. Genes demonstrating ≥2-fold change in expression following geNorm transformation were used for Ingenuity Pathway Analysis (IPA) network modeling. Biomarkers modulated in a manner favorable for cancer prevention were validated by real time RT-PCR.

Results

Short-term LBR exposure of SiHa cells in vitro resulted in inhibition of cell proliferation, and reduced expression of IL8 and MMP2 genes detected using the Angiogenesis Array. IPA network modeling predicted that MMP2 and NFkB would be part of the IL8 signaling cascade and this is supported in the literature. Validation with real time RT-PCR showed a 24-fold (1d) and 28-fold (3d) reduction in IL8 expression following LBR exposure. However, MMP2 and NFkB did not demonstrate a significant differential expression at either time point.

Conclusion

Short-term exposure of cervical cancer cells to LBR results in a striking reduction of IL8 gene expression. Although MMP2 and NFkB are associated with known IL8 signaling mechanisms, they are not modulated by LBR extract in this in vitro model. Therefore, the growth inhibitory activity and transcriptional repression of IL8 following LBR extract exposure proceed via an MMP2-NFkB-independent cascade. Other signaling mechanisms within the IL8 signaling cascade (HIF, AP1, STAT3) may be involved and will be further explored. Since IL8 plays an important role in tumor growth, metastasis, and angiogenesis, it supports the potential for phytonutrient-based chemoprevention of cervical cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2904.
Abstract 2906: Curcumin reduces aberrant crypt foci (ACF) number but does not alter prostaglandin E2 (PGE2) or 5-hydroxyeicosanopentaneic acid (5-HETE) concentration in ACF or in normal human colorectal mucosa
Authors: Carroll Robert E. , Turgeon D K. , Vareed Shaiju , Kakarala Madhuri , Tuck Melissa , McLarin Chrisitne , Benya Richard V. , Meyskens Frank , Brenner Dean E.
Published: February 2011

Curcumin, a dietary polyphenol derivative of turmeric, has potent cancer preventive activity in in vivo colon carcinogenesis models. To determine whether curcumin has pharmacodynamic effects on the human colon, we administered curcumin at doses of 2 g or 4 g to separate groups of 20 healthy human smokers who had 8 or more aberrant crypt foci on screening colonoscopy. We removed 3 aberrant crypt foci and 4 flat mucosal biopsies under magnifying endoscopy prior to and after 28 days of daily curcumin dosing. We enrolled 41 subjects (66% African Americans, 27% Caucasian, mean age 55+/−6.5 years). PGE2 and 5-HETE concentrations on tissue biopsies (assayed with liquid chromatography tandem mass spectroscopy) are shown in the Table.

Curcumin concentrations assayed by HPLC in the colonic mucosa were undetectable (<10μg/gm tissue weight) while curcumin conjugate conjuges were detected in the majority of post treatment tissue samples. Curcumin does not reduce PGE2 or 5-HETE in colorectal mucosa after 28 days of administration. A daily dose of 4 g reduces ACF in colorectal mucosa. These data suggest that curcumin may reduce carcinogenesis in human colorectal mucosa but not via inhibition of arachidonic acid metabolism.‡pg/μg protein mean±SD ‡ mean±SEM p<0.05 PGE-2 ACFPGE-2Nl5-HETE ACF5-HETE NlACFPre-Treatment-2g1.2±1.8‡2.1±3.21.4±0.92.3±1.315.7±1.3Post-Treatment-2g1.6±1.82.8±3.71.4±1.02.4±1.215.0±1.5Pre-Treatment-4g3.4±4.82.7±1.92.3±1.32.4±1.517.8±2.0Post-Treatment-4g3.7±3.22.6±2.92.4±1.22.2±1.611.1±2.8†

(Support: NCI N01-35610, F. Meyskens PI Univ California Irvine)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2906.
Abstract 2912: F-box protein 10: A novel NF-κB–dependent antiapoptotic protein regulates TRAIL-induced apoptosis
Authors: Ge Rongbin , Wang Zongwei , Zeng Qing , Xu Xiaoyin , Olumi Aria F.
Published: February 2011

Introduction and Objective

Tumor necrosis factor-related apoptosis—inducing ligand (TRAIL) induces selective apoptotic death of human cancer cells while sparing normal human cells. Although TRAIL holds great promise as a potential anti—cancer agent, some tumors develop resistance to TRAIL. Previously, we have shown that the AP—1 family member, c—Fos, is an important modulator of apoptosis. Although FBXL10 has been implicated to regulate an AP—1 family protein, c—Jun, its role in mediating apoptotic pathways has not been previously investigated. Here, we report that FBXL10 is a novel NF—kB dependent anti—apoptotic molecule, which directly binds and represses c—Fos, another AP—1 family member, rather than c—Jun. Repression of c—Fos leads to resistance of cancer cells to TRAIL—induced apoptosis.

Methods

RT—PCR, Western blot and immunofluorescence assays were applied to evaluate protein expression level. RNAi, Chromatin immunoprecipitation (ChIP), EMSA, hydrodynamic—based transfection, oligonucleotide decoy technologies, plasmid reconstruction and site—directed mutation were performed to analyze the interaction between FBXL10, c—Fos and NF—kB. Orthotopic prostate xenografts were carried out for in—vivo analyses.

Results

FBXL10 was suppressed and c—Fos was upregulated in TRAIL sensitive cancer cells and xenografts after treatment with TRAIL. However, in TRAIL resistant cancer cells and xenografts, FBXL10 and c—Fos were not affected. Expression of FBXL11, a FBXL10 homologous protein and c—Jun, was not affected by TRAIL. Silencing of FBXL10 sensitized resistant cells to TRAIL. Conversely, over—expression of FBXL10 repressed TRAIL—induced apoptosis. To behave as an anti—apoptotic molecule, we found that FBXL10 directly binds and represses c—Fos promoter in vitro and in vivo. In addition, FBXL10 regulates c—FLIP, another anti—apoptotic molecule, by a c—Fos dependent pathway. We also found that expression of FBXL10 is NF—kB—dependent, and TRAIL or proteasome inhibitors down—regulate FBXL10 via inhibiting NF—kB signaling. Using ChIP and EMSA assays, we found that NF—kB—p65 directly binds the FBXL10 promoter in vivo and in vitro, and promotes expression of FBXL10.

Conclusions

Differentiating molecular mechanisms between TRAIL sensitive and TRAIL resistant cancer cells will improve the efficacy of apoptotic therapies for cancer patients. In this study, we demonstrates for the first time, that FBXL10 plays an anti—apoptotic role and indicates a novel NF—kB/FBXL10/c—Fos/c—FLIP signaling pathway in TRAIL—mediated apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2912.
Abstract 2921: Antitumorigenic effects by ERβ in three different human colorectal cancer cell lines
Authors: Edvardsson Karin , Strom Anders , Gustafsson Jan-Ake , Williams Cecilia
Published: February 2011

The estrogen receptor beta (ERβ) is the predominant ER in the human colonic epithelium. Levels of ERβ are reduced in colorectal cancer compared to normal colon tissue. In a previous study, we generated ERβ-expressing colon cancer cells and showed that ERβ inhibits proliferation as well as colon cancer xenograft growth, as a consequence of ERβ mediated inhibition of cell-cycle pathways.

The present study was conducted to investigate genome wide changes by ERβ in three human colon cancer cell lines (SW480, HT29 and HCT116). They have very low or no levels of ERβ expression and were transduced with lentivirus containing an ERβ expression cassette. Microarray studies showed that ERβ regulated 623, 643 and 124 genes for SW480, HT29 and HCT116, respectively.

Comparisons showed few genes in common for the three cell lines. However, over representation analysis showed that several themes, such as cell proliferation, cell communication and apoptosis were regulated in all of the cell lines.

By comparing our results with those from a ChIP-on-Chip assay in breast cancer cells, we found that a considerable proportion of regulated genes had an ERβ binding site nearby or within the gene.

Our preliminary conclusion is that ERβ appears to, at the gene expression level, have related anti-tumorigenic effects in different colon tumor cells, but different genes may be regulated in different cells types. Furthermore, the fact that several of the regulated genes possessed an ERβ DNA binding region nearby or within the gene indicates a direct regulation for many of these genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2921.
Abstract 2935: Modulation of immune checkpoint B7-H1/B7-DC/PD-1 interaction in combination with live attenuated Listeria monocytogenes expressing a tumor associated antigen effectively treats hepatic colorectal cancer metastases
Authors: Yoshimura Kiyoshi , Olino Kelly , Edil Barish , Wada Satoshi , Pan Xiaoyu , Meckel Kristen , Pardoll Drew , Schulick Richard
Published: February 2011

Introduction: The successful eradication of tumor deposits by immunotherapy requires the generation of an appropriate primary and memory immune effector immune response as well as the establishment of a tumor micro-environment within that organ to target, traffic and amplify the appropriate immune response. B7-H1 is expressed on dendritic cells (DCs), macrophages, T/B cells and on various cancer cell lines. B7-H1 when bound to its T-cell receptor, programmed death-1 (PD-1) induces a negative regulatory signal inhibiting T cell responses. These are particularly attractive targets of therapy in metastatic colorectal cancer as the cancer and the liver microenvironment express B7H1, while tumor infiltrating CD8+ T cells express PD-1. It appears that cancer is able to exploit the B7H1-PD-1 inhibitory mechanism thus making the blockade of this interaction capable of enhancing our already validated vaccine platform of doubly attenuated Listeria Monocytogenes (LM).

Methods and Results: Isolated hepatic metastases were generated in Balb/c mice and treated with intraperitoneal injections of 0.1 x LD50 of LM on postoperative days 3, 6, 9. In addition, intravenous injection of a mouse anti-mouse B7-H1 blocking antibody was given on days 4,7,10 and compared to mice given vaccine alone, antibody alone, and no treatment. We analyzed immune cell populations in the liver with flow cytometry to define the activity, specificity and kinetics. After blockade of B7-H1 we performed survival analysis and in vitro studies for T cell proliferation of activated T cells.

Results: Mice treated with LM and B7-H1 showed a 60% survival, LM alone showed 30% survival, while antibody alone and untreated mice did not survive (Figure 1). We first demonstrated B7-H1 expression on colon cancer cell line CT26. We then found CD8+ T cells and conventional dendritic cells (cDC) treated with LM, showed an up-regulation of B7-H1. However, these receptors upregulation were both abgrogated when combined with B7H1 blocking antibody. When B7H1 blocking antibody was used alone, there was a decrease in B7H1 expression but an increased expression on PD-1 on CD8+ T cells. T cell proliferation assay in vitro showed sustained T cell activity with B7-H1 blockade even in the presence of tumor.

Conclusion Blockade of B7-H1 is an effective method of overcoming immune evasion and improves the efficacy of LM vaccine in the treatment of metastatic colorectal cancer to the liver.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2935.
Abstract 2947: Knockdown of splicing factor SRp20 induces apoptosis in ovarian tumor cells
Authors: He Xiaolong , Arslan Ahmet D. , Ho Tsui-Ting , Beck William T.
Published: February 2011

SRp20 is a member of the serine/arginine-rich (SR) protein family with multiple functions in RNA processing such as alternative splicing and polyadnylation. Our previous studies found that SRp20, together with another splicing factor, polypyrimidine tract-binding protein (PTB), is up-regulated in human ovarian tumors compared to normal ovarian surface epithelia (He, X et al, Clin Cancer Res, 10:4652). In order to determine whether overexpressed SRp20 is required for ovarian tumor cell growth, we studied the effects of SRp20 knockdown in several ovarian cancer cell lines. The knockdown of SRp20 expression was achieved by lentivirus-delivered, doxycycline (Dox)-induced siRNAs. We identified two effective siRNAs against SRp20, SRp20si1 and SRp20si2, which can suppress SRp20 expression by approximately 50% and 90%, respectively. In ovarian cancer cell lines A2780, SKOV3 and IGROV1, knockdown of SRp20 by either SRp20 siRNA caused substantial inhibition of cell growth and colony formation in soft agar. The degree of inhibition correlated with the extent of SRp20 suppression. Massive down-regulation of SRp20 expression by SRp20si2 led to almost complete loss of A2780's capability to form colonies in soft agar. Moreover, Hoechst 33342 staining revealed that the percentage of apoptotic cells was remarkably increased when SRp20 was knocked down ∼90% by SRp20si2. In contrast, moderate suppression (∼50%) of SRp20 by SRp20si1 did not trigger apoptosis. These results suggest that overexpression of SRp20 is required for ovarian cancer cell growth and survival. To determine which apoptotic pathway is activated by SRp20 knockdown, we examined the cleaved fragments of several caspases by immunoblotting and found that caspases 9, 3 and 7 but not caspases 2 and 8were cleaved in cells with SRp20 knocked down by SRp20si2. These results imply that the intrinsic apoptotic pathway is activated by massive SRp20 knockdown. In support of this, we also observed a substantial down-regulation of Bcl-2, a major anti-apoptotic regulator of the intrinsic apoptotic pathway, in the above cells. How SRp20 regulates Bcl-2 expression is unclear at present and currently under investigation. (Supported in part by CA40570 and CA138762 from NCI (to WTB), in part by OCRF (to XH), and in part by UIC).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2947.
Abstract 2948: Nicotine-induced expression of MUC4 in pancreatic cancer cells is mediated by E2F1 and STAT-1 upregulation: Relevance to smoking and pancreatic cancer
Authors: Kunigal Sateesh S. , Batra Surinder K. , Chellappan Srikumar P.
Published: February 2011

The membrane-bound mucins belong to an ever-increasing family of O-glycoproteins. Based on their structure and localization at the cell surface they are thought to play important biological roles in cell-cell and cell-matrix interactions and cell signaling. The human mucin gene MUC4 is overexpressed in pancreatic cancer and cancer cell lines, while remaining undetectable in the normal pancreas, indicating its potential role in pancreatic cancer pathogenesis. Smoking has been correlated with pancreatic cancer, even though the underlying molecular mechanisms are unknown. Attempts were made to elucidate the molecular mechanisms involved in the regulation of MUC4 gene expression by nicotine as well as cytokines. In this report, we used pancreatic cancer cell lines CD18 as well as PANC1 stimulated with nicotine to identify the signaling pathways and transcription factors regulating MUC4 expression. Through chromatin immunoprecipiation assay and real-time PCR, we found that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression. Additionally, the effect is abrogated by short interfering RNA (siRNA)-mediated inhibition of E2F1 and STAT-1 expression, supporting pivotal role played by E2F1 and STAT-1 in MUC4 regulation. The same molecules were responsible for MUC4 induction by nicotine in combination with IFN- and RA. Inhibiting the acetylacholinergic receptors with inhibitors reversed the effects of nicotine stimulation. Similarly, the induction was dependent on the PI3 kinase as well as Src signaling pathways. Additionally, nicotine treatment alone and in combination with IFN- and RA activates and relocalizes signal transducer and activator of transcription-1 (STAT-1) via phosphorylating Jak2 at the tyrosine residue to induce MUC4. These results suggest that nicotine alone and also in combination with IFN- and RA, upregulates the MUC4 expression involving JAK-STAT pathway. In conclusion, our findings identify novel mechanisms for MUC4 regulation in pancreatic cancer cells and will be helpful understanding the transcriptional regulation of MUC4 in response to exposure to nicotine and tobacco smoke.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2948.
Abstract 2949: The VEGF quadruplex-forming sequence inhibits cell growth of lung cancer
Authors: Thomas Shelia D. , Grant Campbell M. , Clarkson Cortney R. , Miller Donald M. , Sedoris Kara C.
Published: February 2011

Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGFq), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with VEGFq induces cell death by inhibiting gene expression. To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGFq or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Changes in cell proliferation were compared to levels of VEGF protein expression. Quadruplex formation was confirmed by circular dichroism spectroscopy. Our results demonstrate that VEGFq formed a stable parallel quadruplex structure, while the MutVEGF sequence did not form a quadruplex. Treatment of A549 cells with VEGFq caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay. No change in the growth of nontransformed fibroblast cells occurred in response to VEGFq indicating the effect is specific for malignant cells. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGFq or MutVEGF showed prominent uptake and nuclear localization of VEGFq not MutVEGF after 72 h. Inhibition of cell proliferation corresponded with decreased VEGF protein expression. No specific change in the cell cycle was measured after 72 h. These results demonstrate striking growth inhibition of A549 cells in response to the VEGF quadruplex-forming sequence related to inhibition of VEGF expression and suggest significant therapeutic implications for the treatment of non-small cell lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2949.
Abstract 2957: Characterization of oral tumors by massively parallel ligation sequencing
Authors: Laborde Rebecca R. , Tuch Brian B. , Olsen Kerry D. , Kasperbauer Jan L. , Moore Eric J. , Barker Melissa A. , De La Vega Francisco M. , Smith David I.
Published: February 2011

Cancers of the head and neck rank as the 6th most commonly observed cancers worldwide with an average 5-year survival rate of only 50% for advanced disease. Nearly 95% of head and neck cancers are squamous cell carcinomas (SSC) and these are frequently highly invasive metastatic tumors. The newly developing technologies of Next Generation Sequencing have allowed for the most comprehensive genetic analysis of these cancers to date. We have taken 3 tumor/normal pairs from patients with oral squamous cell carcinoma and performed full transcriptome and mate-pair DNA sequencing analysis using the AB SOLiD system. These runs generated 129-256 million 50bp long sequence reads per sample, with 21 to 56 million reads per sample uniquely aligning to human genome sequence. Analysis of these samples has revealed pathways with aberrant expression in tumor versus normal tissue. Our results show up-regulation of some metalloproteinase genes, HGMA2, which is a prognostic marker for oral carcinoma, and extracellular matrix, adhesion, and motility gene classes in tumors. Our functional analysis also indicates extensive down-regulation, in the tumors, of genes functioning in epidermal development. We were also able to measure allelic specific expression for the 50% most highly expressed genes and detect examples of allelic imbalance linked to pathways with dysregulated gene expression. Additionally, the depth of sequencing that was achieved (5-16X coverage of RefSeq Exons) also allowed us to detect mutations occurring in the transcripts. We have also detected evidence of RNA editing associated with point mutations, which we are validating in a larger group of samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2957.
Abstract 297: Activating transcription factor 4 negatively regulates the mammalian target of rapamycin via Redd1 expression in response to 2-deoxyglucose
Authors: Jin Hyeon-Ok , Hong Seok-Il , Park In-Chul
Published: February 2011

Aerobic glycolysis is one of major metabolic changes found in cancer cells and is a valid target for cancer therapy. The nonmetabolizable glucose analogue, 2-deoxyglucose (2-DG) interferes with glucose metabolism and enhances chemotherapy/radiotherapy in cancer cells. We have investigated the mechanism of action by which 2-DG inhibits mammalian target of rapamycin (mTOR) activity. We found that 2-DG treatment led to decreased mTOR activity which was regulated by induction of Redd1 protein in both lung cancer cells and cervical cancer cells. We also found 2-DG treatment led to increased ATF4 protein expression and transcriptional activity. Suppression of ATF4 using siRNAs led to decrease of 2-DG induced Redd1 expression. Our data suggested that 2-DG induced Redd1 expression is regulated by the ATF4, leading to mTOR inhibition.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 297.
Abstract 2973: Poly(ADP)ribose inhibition forces the repair of topoisomerase I cleavage complexes through homologous recombination and non-homologous end joining
Authors: Zhang Yongwei , Regairaz Marie , Seiler Jennifer , Agama Keli , Doroshow James , Pommier Yves
Published: February 2011

Poly(ADP-Ribose) polymerase (PARP) inhibitors represent a promising class of novel anticancer agents. In the present study, we explored the strategy of combining the classical topoisomerase I (Top1) inhibitor camptothecin (CPT) and the novel PARP inhibitor ABT-888 in human cancer cells. ABT-888 potentiated cell killing by CPT. By colony forming assay, the survival ratio of CPT-treated colon cancer HT-29 cells was 52%, while the survival fraction were decreased to 35% when HT-29 cells were co-treated with CPT and ABT-888. By measuring γH2AX, a DNA double-strand break marker, we found that ABT-888 enhanced the γH2AX level induced by CPT. The enhanced γH2AX response in the presence of ABT-888 affected both replication-related γH2AX but also transcription-related γH2AX. In osteosarcoma U2OS cells, ABT-888 also enhanced γH2AX in both EdU (5-ethynyl-2′- deoxyuridine) positive and negative cells. Even in non-replicating human primary lymphocytes, ABT-888 enhanced CPT-induced γH2AX foci (1.3 ± 1.2 vs. 5.1 ± 3.1 γH2AX foci/per cell in the absence and presence of co-treatment with ABT-888, respectively). Proteasome inhibition with MG-132 blocked the γH2AX induction by CPT, and PARP inhibition could not overcome the block, which demonstrates that PARP acts downstream from the proteasome. Neutral Comet assays showed that the enhanced γH2AX response was related to DNA breaks. Alkaline elution assays revealed that ABT-888 increased CPT-induced DNA single-strand breaks (SSBs) but without a corresponding increase in DNA protein crosslinks indicating that ABT-888 was inducing DNA breaks in addition to the Top1-DNA cleavage complexes trapped by CPT. These additional DNA breaks were dependent on XPF/ERCC1 endonuclease. Furthermore, after removing CPT, reversal of γH2AX in the absence or presence of ABT-888 showed the same reversal rate, although there was more γH2AX in the presence of ABT-888, suggesting that PARP inhibition did not affect the repair of double-strand breaks. Moreover, PARP inhibition enhanced DNA-PK and ATM activation with increased formation of Rad51 foci. Therefore, we propose that PARP inhibition forces the cells to activate alternative endonucleolytic and recombination pathways in response to Top1-mediated DNA damage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2973.