bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2887: From fruit extract to structure activity relationship studies: Alpha-mangostin promotes G1 cell cycle arrest in 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. , Handa Harleen , Mukhtar Hasan
Published: February 2011

The angosteen fruit (Garcinia mangostana) is native to southeast Asia and has been receiving increased attention for its 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 alpha-mangostin being the most abundant. Here we report on the properties of dietary xanthones for cancer chemoprevention using prostate cancer (PCa) as a model disease. In a separate abstract we provide promising results suggesting that a standardized mangosteen fruit extract promotes cell cycle arrest and decreases tumor formation in an athymic nude mouse model. To gain an understanding of the pharmacology of xanthones in cancer inhibition we selected alpha-mangostin as the lead compound and evaluated it against fifty different protein kinases deregulated in cancer in a cell free kinase assay. A kinase that is often deregulated in a variety of cancers including prostate is cyclinD1/CDK4 where alpha-mangostin was found to significantly inhibit the activity. Using two different cells lines (PC3 and CWR22) we have evidence suggesting alpha-mangostin promoted G1 cell cycle arrest by flow cytometry and western blot analysis of cyclins and CDKs with observed effects in the range of 5-10 μM. Further confirmatory studies were performed by western blot analysis. A preliminary pharmacokinetic analysis of mice administered 1 mg of alpha-mangostin intraperitoneally resulted in a plasma concentration of 7.3 μM - within the concentration of in vitro mechanistic studies. Next, using an athymic nude mouse model implanted with CWR22Rv1 cells we randomized mice to receive intraperitoneally the alpha-mangostin (>95%) or placebo two times weekly with tumors measured twice weekly. At the conclusion of the study the treatment group had a significantly smaller tumor with an average tumor volume of (400 mm3) compared to the placebo group (1225 mm3) representing a 69% smaller tumor in the treatment group. Based on these promising results we used purified xanthones from the mangosteen fruit and selected 9 xanthones for structure activity relationship studies against cyclinD1/CDK4 using cell free biochemical assays and molecular modeling. Collectively these data indicate that the purified xanthone alpha-mangostin could be a promising agent for PCa prevention.

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 2887.
Abstract 2890: Effects of calcium and vitamin D on markers of vitamin D metabolism in normal colon mucosa: A randomized clinical trial
Authors: Ahearn Thomas , McCullough Marjorie L. , Flanders W. Dana , Long Qi , Fung Jessica , Ofori-Addo Anita , Dawadi Shuva , Bostick Roberd M.
Published: February 2011

Background: In colon cancer cell lines and rodent models calcium and vitamin D favorably modulate cell proliferation, differentiation, and apoptosis and affect and are affected by local expression of the vitamin D receptor (VDR), CYP27B1, and CYP24A1; however, the latter has not been investigated in humans. To evaluate the effects of supplemental calcium and vitamin D3 on VDR, CYP27B1, and CYP24A1 expression in the normal colorectal epithelium, we conducted a randomized, double-blinded, placebo-controlled 2×2 factorial clinical trial.

Methods: Ninety-two men and women with at least one pathology-confirmed sporadic colorectal adenoma were treated with calcium 2 g/day or vitamin D3 800 IU/day, alone or in combination vs. placebo over six months. VDR, CYP27B1, and CYP24A1 were detected in biopsies of normal-appearing colorectal mucosa using standardized automated immunohistochemistry, and their overall expression and distributions within the colon crypts were quantified by novel image analysis methods. The relative treatment effects were calculated and assessed using a MIXED effects model as implemented by SAS v9.2.

Results: Relative to the placebo group, in the calcium + vitamin D3 group VDR expression increased 19% (p=0.13) along the full length of crypts, 19% (p=0.09) in the upper 40% (differentiation zone) of crypts, and 31 % (p=0.13) in the lower 60% (proliferation zone) of crypts. In the vitamin D3 only group CYP27B1 expression increased 259% (p=0.04) along the full length of crypts, 211% (p=0.04) in the upper 40% of crypts, and 210% (p=0.04) in the lower 60% of crypts. CYP24A1 expression along the full length of crypts decreased 21% (p=0.51) in the calcium group, and increased 65% (p=0.22) and 51% (p=0.37) in the vitamin D3 and calcium + vitamin D3 groups, respectively.

Discussion: These preliminary results 1) suggest that, as hypothesized, calcium and vitamin D3, individually or in combination may modulate VDR, CYP27B1, and CYP24A1 expression in the normal-appearing human colorectal epithelium, which may promote normal patterns of cell proliferation, differentiation, and apoptosis; 2) provide further evidence to the potential importance of vitamin D metabolism and its autocrine/paracrine function in colorectal mucosa; 3) provide support for the efficacy of calcium and vitamin D3 in reducing risk for colorectal neoplasms; and 4) present the potential of the VDR and CYP27B1 and CYP24A1 as potential modifiable biomarkers of risk for colorectal neoplasms.

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 2890.
Abstract 2891: Control of nasopharyngeal carcinoma through serologic screening and clinical examination of at-risk subjects
Authors: Ji Mingfang , Shen Yahua , Cheng Weiming , Yu Yuanlong , Su Nianhua , Ng Sp , Ng Mh
Published: February 2011

Background. Nasopharyngeal Carcinoma (NPC) could be successfully treated by radiotherapy when the tumor is localized to the nasopharynx, while further progression to the adjacent tissues is attended by poor treatment outcome. However, 93% of the patients presented to outpatients department are diagnosed with the advanced cancer, while 86% of the localized tumors are asymptomatic, detected by clinical examination of apparently healthy subjects (Ji et al., submitted). Epstein Barr Virus (EBV) antibody levels of NPC patients are raised and maintained at high levels for up to 10 years before clinical onset (Ji et al., 2007). This suggests that repeated serologic screening could serve to monitor the tumor as it enters and transits such preclinical window and clinical examination of that risk subjects could detect the tumor as it is clinically manifested.

Objective: An early NPC detection program was instituted in Zhongshan City in Southern China since December 2007. The objective is to detect NPC while the tumor is confined to the nasopharynx.

Method. Subjects enrolled in this program are screened annually for levels of 3 EBV antibodies, namely IgA EBNA1, IgG EBNA1 and IgG zta, simultaneously. Clinical examination and endoscopy are performed for at-risk subjects who had elevated levels of 3 antibodies or IgA EBNA1 rod≥ 3.

Results: As of July 2009, 1373 adults were enrolled, 11 were assessed as high risk for NPC and 31 with elevated levels of one of 3 antibody. Five NPC cases were detected during clinical examination and all were from among those who had high levels of all the 3 EBV antibodies. Three of these cases showed no or a minimum of symptoms and were diagnosed with localized NPC (T1N0M0). The other 2 were overt cases and diagnosed with advanced loco-regional diseases (T2N1M0 and T3N1M0)

Conclusions: It is possible to control NPC by serologic screening and clinical follow-up of the at-risk subjects.

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 2891.
Abstract 2895: Effects of supplemental calcium and vitamin D3 on serum growth factors in sporadic colorectal adenoma patients in a randomized clinical trial
Authors: Owen Joy R. , Ahearn Thomas , Liang Bill , Fedriko Veronika , Woodard Jill , Bostick Roberd M.
Published: February 2011

Fluctuations in energy homeostasis via perturbation of the intricate balance between nutritional intake, physical/energy expenditure, and genetics are thought to play a role in risk for colorectal neoplasms. Nutritional supplementation with calcium and vitamin D3 may ameliorate risk for colorectal neoplasms and help lead to the development of treatable, non-invasive biomarkers of risk for the disease. Basic science findings indicate that calcium and vitamin D affect the expression of growth factors, such as Insulin-like Growth Factor (IGF-1), but there have been no reports of investigations on the effects of calcium and vitamin D3 on growth factors in humans. We investigated the effects of calcium 2.0 g and/or vitamin D3 800 I.U. daily over six months on serum levels of IGF-1 and Insulin-like Growth Factor Binding Protein (IGFBP-3) in a pilot, randomized, double-blind, placebo-controlled, 2×2 factorial chemoprevention trial (n = 88) in sporadic colorectal adenoma patients. In the calcium relative to the placebo group, both IGF-1 (p = 0.08) and IGFBP-3 levels (p = 0.03) increased by 10%, whereas there was no evidence for treatment effects in the vitamin D3 group. In the calcium plus vitamin D3 relative to the placebo group, IGF-1 increased by 9% (p = 0.12) but there was no evidence for a change in IGFBP-3. These findings tentatively suggest that dietary supplementation with calcium and/or vitamin D3 may affect signal transduction pathways mediating the release of growth factors into the systemic circulation in humans and provide support for developing treatable, non-invasive biomarkers of risk for colorectal neoplasms.

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 2895.
Abstract 2897: Profiling lipoxygenase metabolism in specific steps of colorectal tumorigenesis
Authors: Shureiqi Imad , Chen Dongning , Day R. Sue , Zuo Xiangsheng , Ross William , Moy Ofir , Morris Jeffrey Scott , Xiao Lianchun , Newman Robert A. , Yang Peiying , Lippman Scott M.
Published: February 2011

Lipoxygenases (LOXs) are key enzymes for the oxidative metabolism of polyunsaturated fatty acids into products that influence cell signaling, structure, and metabolism. Previous data on comparative levels of various LOX products in human tumorigenesis are limited primarily to retrospectively collected surgical samples from cancer patients. Therefore, we examined LOX product profiles by liquid chromatography/tandem mass spectrometry in the major steps of colorectal tumorigenesis (normal, polyp, and cancer) in a clinical study of 125 subjects (49 with a normal colon, 36 with colorectal polyps, and 40 with colorectal cancer) who underwent prospective colorectal biopsies to control for various potential confounding factors (e.g. diet, medications). Mean 13-hydroxyoctadecadienoic acid (13-HODE) levels were significantly higher in normal colons (mean = 36.11 ng/mg protein, 95% confidence interval [CI]: 31.56-40.67) than in paired colorectal cancer mucosa (mean = 27.01 ng/mg protein, 95% CI: 22.00-32.02; P = 0.0002) and in normal colons (mean = 37.15 ng/mg protein, 95% CI: 31.95-42.34) than in paired colorectal polyp mucosa (mean = 28.07 ng/mg protein, 95% CI: 23.66-32.48; P < 0.001). Mean 13-HODE levels, however, were similar between the left (mean = 37.15 ng/mg protein, 95% CI: 31.95-42.35) and right normal colons (mean = 32.46 ng/mg protein, 95% CI: 27.95-36.98; P = 0.09). No significant differences with regard to 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, or leukotriene B4 (LTB4) levels were detected between normal, polyp and cancer mucosa. Downregulation of 15-LOX-1 expression, the rate limiting enzyme for 13-HODE production, in human colorectal neoplastic mucosa was confirmed by quantitative real time PCR in a subset colorectal cancer patients for whom samples with adequate RNA quality were available. This study establishes that reduced 13-HODE levels are a specific alteration in the LOX product profile associated with human colorectal tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2897.
Abstract 2899: Multiple-dose phase 1 study of 3,3′-diindolylmethane (DIM): Pharmacokinetics and biological effects
Authors: Reed Gregory A. , Sunega Jean M. , Latinis Kevin , Gray John , Crowell James A. , Perloff Marjorie
Published: February 2011

We have completed our examination of the safety, tolerability, pharmacokinetics, and effects of twice daily doses of 100 mg and 200 mg of 3, 3’-diindolylmethane (DIM) for four weeks. Healthy adult subjects self-administered an absorption-enhanced formulation of DIM (BR-DIM). Eight subjects received the low dose, and six subjects received the high dose of BR-DIM. Based on medical histories, physical examinations and a battery of blood and urine tests we concluded that BR-DIM in this dosing regimen was very well tolerated, with no significant adverse effects. The pharmacokinetics of DIM were determined at each dose for both the first dose and last dose of BR-DIM in these subjects. The first dose Cmax and AUC values obtained for the two doses were similar to the results of our previous single ascending dose study of BR-DIM. Surprisingly, after four weeks of twice daily dosing, the mean Cmax of DIM fell by 29% and 73% and the mean AUC fell by 46% and 71% in the low and high dose groups, respectively. Multiple daily dosing with BR-DIM clearly altered expression either of specific transporters or of enzymes that biotransform DIM, resulting in a marked decrease in bioavailability. This decreased systemic exposure, however, must be considered in light of the observed induction of CYP1A2, one of our primary biomarkers of effect. Mean CYP1A2 activity increased by 250% at the low dose and by 181% at the high dose, suggesting that effective concentrations of DIM or an active metabolite were achieved in the liver, and that this resulted in induction of hepatic CYP1A2. No effects of BR-DIM were seen on activities of CYPs 2C9, 2D6, or 3A4. The lack of systemic exposure to DIM could result either from induced hepatic metabolism of DIM or from increased biliary secretion of DIM. This proposal for the altered pharmacokinetics of DIM, allowing hepatic effects but not extrahepatic effects, is consistent with the very modest effects of BR-DIM we observed on peripheral blood lymphocyte GST activity, and suggests that the lymphocyte activity may not be an accurate surrogate for hepatic effects of DIM. The lack of significant change in the 2-hydroxyestrone:16α-hydroxyestrone ratio, judged by us to be a key biomarker of efficacy in our previous trial with the DIM precursor indole-3-carbinol, raises the possibility that the change in that ratio is based to a significant extent on extrahepatic hydroxylation of estrone, rather than modulation of hepatic estrogen metabolism. Supported by NCI N01 CN-35008-38 and P20RR021940.

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 2899.
Abstract 2900: Risk of recurrence of colorectal neoplasia among type 2 diabetes mellitus patients randomized to placebo in the Polyp Prevention Clinical Trials
Authors: Acevedo Alejandro , Cruz-Correa Marcia R. , Diaz Yaritza , Grau Maria , Baron John
Published: February 2011

Introduction: Diabetes Mellitus (DM) has been reported to be associated with an increased risk of colorectal cancer is several case-control studies. However, very limited data have examined the risk of recurrence of colorectal neoplasia (CRN) among individuals with type 2 DM. This study examined the risk of recurrence of CRN (either adenomas or carcinoma) among individuals with type 2 DM compared to those without type 2 DM.

Methods: Retrospective cohort study evaluated subjects recruited into several randomized clinical trials (RCTs) (Antioxidant Polyp Prevention Study, the Calcium Polyp Prevention study, and the Aspirin/Folate Prevention Study). Diagnosis of type 2 DM was established by previous medical diagnosis and/or anti-diabetic medication use. Participants had to have undergone colonoscopy to the cecum, with removal of all polyps, within three to four months before study entry. Also, they must have had colonoscopies during follow-up study periods (as outlined for each protocol). Logistic regression was employed to estimate the association between type 2 DM and CRN using STATA 10.0.

Results: 2,915 individuals (2068 women; 85.3% White; mean age 59.7 ± 9.3 years) were enrolled into any of the three RCTs. 882 (30.3%) subjects were assigned to placebo vs. 2033 (69.7%) to active drug; mean follow-up time was 35.0 ± 3.9 months from randomization to first colonoscopy. A total of 208 subjects had type 2 DM (mean age 62.9 ± 7.6 yrs, 75.5% females), of which 61(29.3%) were assigned to the placebo arm. Colorectal adenomas developed in 44.5% (27/61) and 55.1% (410/744) of DM and non-DM individuals, respectively (p=0.40) assigned to the placebo arm. While advanced colorectal adenomas (≥ 1 cm in size or advanced histology) developed in 9.8% (9/61) and 9.3% (69/744) of DM and non-DM individuals, respectively (p=0.89). Colorectal cancer developed in 1.64% (1/61) of DM patients vs. 0.4% (3/744) of non-DM patients (p=0.19). There was no statistically significant association between location of CRN, number/size of adenomas, or tumor stage and DM.

Discussion: Recurrence of colorectal adenomas was not increased among patients with DM compared to non-DM patients. Similarly, occurrence of colorectal advanced lesions was not increased in DM patients compared to non-DM patients. A small number of subjects with DM and short follow-up time may have limited the power of the study to detect differences in CRN.

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 2900.
Abstract 2914: Mice deficient in Smurf2 exhibit increased spontaneous tumorigenesis
Authors: Zhang Hong , Ramkumar Charusheila
Published: February 2011

Smurf2, a HECT family E3 ubiquitin ligase has been shown to ubiquitinate and target for degradation of several proteins that promote tumorigenesis, including TGF-β receptors, Smad2/3, β-catenin, and Mad2. Furthermore, we have previously found that Smurf2 can induce senescence in early passage cells in culture. As senescence has been postulated to act as a tumor suppression mechanism, we hypothesize that Smurf2 functions as a tumor suppressor. To test this hypothesis, we generated a Smurf2-deficienct mouse strain using a gene trapping strategy, in which transcription of Smurf2 is compromised. The homozygous Smurf2-deficient mice were obtained with the expected Mendelian ratio after intercrossing of heterozygous mice, and they exhibited no obvious developmental defects. Analysis of mouse embryonic fibroblasts from these Smurf2-deficient mice revealed that they exhibited delayed senescence entry and consequently had an increased tendency to become immortalized in culture. Cohorts of Smurf2-deficient and wild-type mice were generated and monitored for spontaneous tumor development. In this ongoing study, 37% (13 out of 35) of the Smurf2-deficient mice, but none of the wild-type mice showed signs of morbidity after 14-month of age. The moribund Smurf2-deficient mice were found to have enlarged spleen, pale kidneys and liver, and/or enlarged lymph nodes. Histopathology analysis indicated that most of the tumors are lymphomas. In addition, adenocarcinoma of the small intestine was found in one Smurf2-deficient mouse. Collectively, our data suggest that Smurf2 is a tumor suppressor. Preliminary studies of the underlying molecular mechanism of tumorigenesis in Smurf2-deficient mice will be presented.

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 2914.
Abstract 2919: XBP1 and the unfolded protein response in antiestrogen resistance in breast cancer
Authors: Shajahan Ayesha N. , Riggins Rebecca B. , Zwart Alan , Hickman F. Edward , Clarke Robert
Published: February 2011

Most of the 178,000 newly diagnosed cases of invasive breast cancer in 2010 will be estrogen receptor positive α positive (ER+). For these patients, endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole (LET), is the least toxic and most effective means to manage their hormone-dependent breast cancer. While 5 years of Tamoxifen (TAM) produces a 26% proportional reduction in mortality, advanced ER+ breast cancer largely remains an incurable disease and resistance to endocrine therapy remains a significant clinical problem. Previously, we have shown that antiestrogen resistant breast cancer cells over-express X-Box Binding Protein 1 (XBP1), a transcription factor that belongs to the basic region/leucine zipper (bZIP) family. XBP1(U) and XBP1(S) variants result from an unconventional splicing of the XBP1 mRNA by IRE1α. High ratio of XBP1(S):XBP1(U) mRNA in breast tumors is associated with poor response to TAM in patients. IRE1α, ATF6 and PERK are endoplasmic reticulum proteins that sense cellular stress in the unfolded protein response (UPR) - an adaptive signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen. Initially a compensatory mechanism allowing cells to recover normal endoplasmic reticulum function, prolonged UPR may induce cell death; this is often dependent upon which arm of the UPR predominates. XBP1 is an obligate component in both the IRE1α and ATF6 arms of the UPR. Here we show that antiestrogen resistant breast cancer cells survive by activating pro-survival autophagy through XBP1-mediated UPR. MCF7/LCC9 [Faslodex resistant; TAM cross-resistant; high level of XBP1(S)] cells showed an increase in UPR signaling as detected by increased expression of BiP/GRP78. MCF-7/XBP1 or T47D/XBP1 stable cells that over-express XBP1(S) showed increased level of basal and Faslodex-induced autophagy as measured by cleaved LC3BII protein fragment, GFP-LC3 activity, and reduced expression of p62/SQSTM1. Thus, our results highlight a novel cell fate signaling through the UPR in breast cancer cells that leads to antiestrogen resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2919.
Abstract 2938: A novel OX40 agonist licenses systemic anti-tumor immunity to overcome local immune suppression induced by well-established tumors
Authors: Pardee Angela D. , Epstein Alan L. , Storkus Walter J.
Published: February 2011

Immune modulating reagents that trigger the T cell costimulatory molecules GITR and OX40 are being evaluated as anti-cancer agents in early-stage clinical trials. However, little pre-clinical information is available regarding the efficacy and mechanism(s) of action for these agents in the setting of advanced, well-established disease. To further characterize the molecular, cellular, and treatment-associated consequences of GITR and OX40 engagement, novel agonistic reagents directed against murine GITR and OX40 (ligand-Fc fusion proteins) were recently constructed and characterized in vitro. We now show that the growth of well-established, day 17 sarcomas is significantly inhibited or ablated by a short course of either treatment, with OX40L-Fc demonstrating superior anti-tumor efficacy over GITRL-Fc at comparable doses. Despite diminished frequencies of CD4+Foxp3+ regulatory T cells (Treg) in the tumor microenvironment (TME) 3 days post-treatment, the presence of myeloid-derived suppressor cells (MDSC) and inhibitory cytokines such as IL-10 and TGFβ appears unaltered and effector T cell production of the Type-1 cytokine IFNγ remains low. Treatment with either GITR or OX40 agonists, however, induces a coordinate increase in CCR7+ dendritic cells in the tumor-draining lymph nodes (TDLN), followed approximately 7 days after treatment by profound expansion of effector T cells expressing the Type-1 chemokine receptor CXCR3. By day 10 post-treatment, particularly with the OX40 agonist, T cells displaying a recently-activated effector phenotype accumulate in the tumor microenvironment and secrete high levels of IFNγ. Collectively, these data suggests a model in which the first level effect of costimulatory treatment is to direct CCR7+ dendritic cells from the TME to the draining lymph nodes. A secondary, systemic response reflects the expansion of highly-reactive CXCR3+ Type-1 T cells in the TDLN and their subsequent trafficking into the TME. The therapeutic efficacy demonstrated in this model by OX40L-Fc, and to a lesser extent GITRL-Fc, strongly supports the translation of such modalities into human clinical trials, while the mechanistic insight gained from these studies suggests that successful immunotherapies will license both local, and perhaps more importantly, systemic Type-1 T cell immunity.

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 2938.
Abstract 294: Protein interactions of PTEN and its cancer-associated G20E mutant compared by SILAC-based parallel affinity purification and mass spectrometry
Authors: Gunaratne Jayantha , Goh Mei Xian , Foon Swa Lee , Lee Fen Yee , Sanford Emma , Wong Loke Meng , Hogue Kelly , Blackstock Walter , Okumura Koichi
Published: February 2011

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the phosphatidyl-inositol-3-kinase (PI3K) pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves PTEN's lipid phosphatase-dependent and -independent activities, the mechanism leading to the phosphatase independent function of PTEN is poorly understood. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here we use a cancer associated mutant, G20E, to gain insight on the phosphatase independent function of PTEN by investigating protein-protein interactions using stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and mass spectrometry (MS) has been developed to allow prioritizing interactors acquired from SILAC pull-downs and the comparison of the interactions between wild-type and G20E PTEN. Liquid chromatography-MS analysis of tryptic digested SILAC-PAP pull-downs revealed known and new interactors. Hierarchical clustering of the heat map generated from the proteins with elevated SILAC ratios from PAP-MS analysis show three distinct clusters: 1) wild-type specific interactors, 2) those unique to the G20E mutant and 3) proteins common to wild-type and mutant. Pathway analyses indicate that the new interactors can be subdivided into apoptosis, cell migration, polarity and the Ca2+ signaling network. These finding contribute to a better understanding of the novel mechanisms of PTEN's phosphatase dependent and independent functions.

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 294.
Abstract 2940: Altered hematopoiesis and lymphomagenesis through loss of chromatin-associated protein KIF4
Authors: Mazumdar Manjari
Published: February 2011

Hematopoiesis, the process of blood cell formation is largely dependent on specialized chromatin environment for stem cell self-renewal and multi-lineage differentiation. The kinesin motor protein KIF4 associates with chromatin during interphase, performs essential functions in mitosis and its loss causes hypercondensation of mitotic chromosomes leading to genomic instability. We have demonstrated that KIF4 is a modulator of interphase chromatin architecture. In KIF4-null mouse ES cells, heterochromatin blocks are lost, pericentormeric heterochromatin regions are decondensed in S phase and chromatin is globally decondensed. The global chromatin decondensation has functional consequences as indicated by mis-regulation of significant number of genes detected by microarray expression analysis. To understand the molecular mechanism of KIF4 function in vivo, we have developed KIF4 knockout (KIF4 KO) mouse. Here we show that in the absence of KIF4 animals develop splenomegaly, substantial populations of mice have thymic and splenic hyperplasia and ∼40% of heterozygous female mice developed Follicular B cell lymphoma similar to human Non-Hodgkin lymphoma. Flow sorting (FACS) analysis of young mice indicated that loss of KIF4 causes defects in hematopietic development and differentiation. Lymphocytes from animals lacking KIF4 exhibit chromatin defects, aneuploidy, DNA damage and slow progression of lymphocytes through cell cycle. More importantly, we find that there is an accumulation of mature B cells in the spleen in the absence of KIF4. The increased survival of germinal center B cells in the absence of KIF4 may cause the observed hyperplasia and B cell neoplasm. The mechanism of survival and expansion of lymphocytes in spite of genomic instability could be linked to the defects in chromatin organization and changes in transcriptional potential. We propose KIF4 may act as a modulator of chromatin function to regulate cell fate decision during lymphocyte development and a guardian of genome stability to prevent hematopoietic neoplasm. Hematologic malignancy via deletion of a chromatin-associated molecular motor protein is a novel paradigm both in the fields of molecular motors and cancer biology.

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 2940.
Abstract 2942: The effect of AR expression level on the chromatin binding of the receptor
Authors: Urbanucci Alfonso , Sahu Biswajyoti , Seppälä Janne , Kukkola Saara , Waltering Kati , Lähdesmäki Harri , Jänne Olli , Visakorpi Tapio
Published: February 2011

Treatment of castration resistant prostate cancer remains to be a challenge. Androgen receptor (AR) is overexpressed and still active at this stage of the disease. Our goal is to identify AR downstream genes and to study the effect of AR overexpression on the chromatin binding.

We established, by stable transfection of wtAR into LNCaP cell, two cell lines that over express moderate (LNCaP-ARmo: 2-3 fold) and high (LNCaP-ARhi: 4-5 fold) receptor amounts of protein. We cultured the cell lines in androgen depleted medium for 4 days and subsequently treated them for 2 hours with vehicle or 1nM and 100nM of 5α-dihydrotestosterone (DHT). Subsequently, we used chromosome immunoprecipitation and deep sequencing (ChIP-seq) to identify AR binding. Expression profiling was done by Illumina array. The data analysis is ongoing. We first studied the well known AR target gene PSA. Our data indicated that in depleted medium there is not apparent difference in the loading of the promoter and enhancer of the PSA regulatory region in different cell lines. Instead, the promoter and the enhancer are more loaded in LNCaP-ARmo/−ARhi than in control cell line upon DHT stimulation. Our preliminary data show that there is a positive correlation between AR-levels and whole genome-wide binding upon stimulation with 1 nM DHT. Whereas, in 100 nM DHT, which is a saturating concentration of the ligand, there is a negative correlation. In the 1 nM DHT, AR seems to bind to different genomic loci in LNCaP-ARhi or ARmo than in control cells. In conclusions, the amount of AR has an effect on the chromatin binding of the receptor. Cells overexpressing AR seems to be more androgen-responsive.

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 2942.
Abstract 295: PKCε phosphorylates and modulates the cell membrane translocation of RhoA
Authors: Su Ti-Zhi , Straight Samuel , Bao Li-Wei , Cavey Greg , Pan Quintin
Published: February 2011

Accumulating evidence has clearly defined the importance of PKCε and small Rho GTPases in tumor metastasis. Previous studies in our laboratory have demonstrated that PKCε signals through RhoA to modulate tumor cell invasion and motility in head and neck squamous cell carcinoma. In this study, we further delineated the link between PKCε and RhoA. Recombinant PKCε directly phosphorylated recombinant RhoA using an in vitro kinase assay. Phosphopeptide mapping with LC-MSE identified T127 and S188 as confident phosphorylation sites and T19 and S26 as tentative phosphorylation sites on RhoA. Interestingly, recombinant PKCε was able to bind to recombinant RhoA in the absence of PKC activators and ATP. This observation demonstrates that the binding interaction between PKCε and RhoA does not require an active PKCε conformation and thus, the interaction between these proteins may be more complex than a typical substrate-kinase event. N-terminus fluorophore-tagged PKCε (CherryFP or CFP) and RhoA (GFP or YFP) were co-expressed in HEK293 cells to examine the interaction between these two proteins in live-cells. Live-cell fluorescence microscopy followed by FRET stoichiometric analysis confirmed the interaction between PKCε and RhoA in the cytoplasm of non-stimulated cells. Stimulation of the cells with PMA to activate PKCε resulted in the translocation of PKCε and RhoA from the cytoplasm to the cell membrane and increased the binding of PKCε to RhoA at the cell membrane by three-fold as measured by FRET. Taken together, our results demonstrate, for the first time, that PKCε phosphorylates, co-localizes, and regulates the cell membrane translocation of RhoA.

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 295.
Abstract 2953: Evaluating gene expression in FFPE breast cancer tissues using DASL®
Authors: Burr Tom , Dixon Rick , Green Andy , Ellis Ian , Murray Cliff
Published: February 2011

The study of gene expression in conventionally processed tissues is hampered by degradation of mRNA. Expensive, low multiplex, quantitative PCR methods can be unreliable due to the limited template sizes. One way to overcome this problem is to use array-based methods. DASL® technology relies on random priming for production of cDNA, in concert with universal bead-arrays to allow the detection and relative quantitation of expression of specific gene subsets.

Using the Illumina DASL® Cancer Panel (500 cancer-associated genes on one array), we evaluated the expression of key genes in archival FFPE tissue samples from 80 breast cancer patients with well characterised pathological and clinical features. We first assessed transcript integrity in the samples on the basis of levels of mRNA encoding RPL13A, prior to running the Cancer Panel. A subset of genes of interest was then assessed by qPCR to confirm the relative levels observed using the DASL® assay. Finally the expression of the same subset of genes was evaluated at the protein level by immunohistochemistry.

We were able to predict, with good accuracy based upon RPL13A assays, those samples unsuitable for DASL® analysis. Furthermore, the results of DASL® analysis showed good correlation with protein levels, as measured by immunohistochemistry, for a number of key genes including ERBB2 (HER2) and ESR1 (ER). We conclude that DASL® represents a powerful tool for assessing expression of multiple genes in archival tissue.

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 2953.
Abstract 2954: Overexpression of specific genes in surgical margins of head and neck squamous cell carcinoma patients may predict a significantly increased risk of recurrence
Authors: de Carvalho Ana Carolina , Carvalho André L. , Soares Fernando A. , Kowalski Luiz Paulo , Maia Danielle C C , Rettori Marianna M. , Vettore André L.
Published: February 2011

Head and Neck Squamous Cell Carcinoma (HNSCC) is a high incidence and mortality disease. Despite significant improvements in therapeutic strategies, the overall survival rate in 5 years is around 50%. The presence of microscopic tumor in surgical margins is associated with treatment failure, locoregional recurrence and reduction of overall survival. For this reason, it is vital to ensure tumor-free surgical margins in these patients. Commonly, the extension of surgical resection is determined during the surgery through a low sensitivity histological exam of frozen tissue stained with Hematoxylin and Eosin. Since genetic alterations precede histological alterations, molecular markers may be useful to identify cells that have already started malignant transformation without having developed a histological phenotype that could be detected through conventional histopathological techniques. Also, novel molecular techniques make possible to obtain molecular data of surgical margins in a period of time compatible with the surgery, helping the surgeon in the resection of positive molecular margins in the same surgery and thereby reducing the risk of local recurrence. This study aimed to determine the gene expression pattern of specific markers in HNSCC surgical margins and verify the utility of these markers in detecting the presence of tumor cells in these tissues and predict tumor recurrence. Nine selected genes were evaluated and a panel of 5 genes showed a high specificity and good sensibility. Overexpression of at least one of these genes was found in 50% (23/46) of histologically free surgical margins analyzed and was associated with local recurrence. Patients with positive molecular margins were 7.3-fold more likely to have local recurrence. These preliminary results suggest that the approach used in this analysis is more sensible than traditional histopathological exams and could be useful for the intraoperative diagnosis of the presence of tumor cells in HNSCC surgical margins and as an informative tool to identify and monitor subsets of HNSCC patients with an increased risk of recurrence and consequently reduced survival.

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 2954.
Abstract 296: Protein kinase D regulates Rho GTPase activity through rhotekin
Authors: Pusapati Ganesh Varma , Rykx An , Vandoninck Sandy , van Lint Johan , Adler Guido , Seufferlein Thomas
Published: February 2011

The Protein Kinase D (PKD) family of serine / threonine kinases comprises PKD1,PKD2, and PKD3 and is grouped under the calcium / calmodulin - dependent protein kinase (CAMK) superfamily. PKDs play an important role in various biological processes including proliferation, adhesion, migration, cell shape, survival and apoptosis. Identification of the putative target substrates for this kinase family would provide a deeper insight into their role in the physiological and pathological processes. In the present study, we identified rhotekin (RTKN), an effector protein of small GTPase Rho, as a potential substrate of PKDs. Using the consensus PKD substrate sequence motif scan approach, we identified Ser435 in the C-terminal region of rhotekin as the potential phosphorylatable residue. We generated a phospho-specific antibody corresponding to Ser435 and could validate that Ser435 is indeed the critical amino acid targeted by PKD2 in vivo in HEK-293T cells. The phospho-mimecking mutant of rhotekin (Ser435Glu) when transfected into HEK-293T cells resulted in the increase of endogenous active RhoA GTPase levels, as determined from the Rho activation assay. Rho GTPases were shown to be an important upstream molecules regulating PKD activity in the process of cancer cell migration. However, our results demonstrate for the first time the role of PKDs in the regulation of RhoGTP activity levels. Considering the role of RhoGTPases in various cellular processes, our results provide a hint to the potential regulation of various cellular processes by PKDs through phosphorylation of rhotekin and thereby regulating the activity of RhoGTPases.

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 296.
Abstract 2960: TRIM29 is a novel biomarker for basal-like breast cancer
Authors: Liu Jin , Bernard Philip
Published: February 2011

Basal-like breast cancer is a subtype of tumors associated with aggressive clinical behavior and poor prognosis. The genes responsible for their aggressive phenotype have not been well characterized and may provide an opportunity for targeted therapies. Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family. Microarray and RT-qPCR analyses shows TRIM29 overexpressed in Basal-like breast tumors and it is highly correlated to other basal tumor markers such as CK5/6, CK17, EGFR, and FOXC1. We recently observed using immunohistochemistry that TRIM29 predominantly localizes to the nucleus in basal tumor epithelial cells, in contrast to a cytoplasmic localization seen in ER-positive tumors. Thus, TRIM29 may act as a transcriptional regulatory factor involved in carcinogenesis. In order to investigate the role of TRIM29 in the initiation and progression of Basal-like breast cancer, we employed chromatin immunoprecipitation and massively parallel sequencing (ChIP-seq) to create a genome-wide profile of in vivo TRIM29-binding sites in MDA-MB-468 cells, a human breast cancer cell line. ChIP-seq analysis revealed 94 putative TRIM29 binding regions in the cancer genome of MDA-MB-468 cells with an estimated false discovery rate of less than 5%. One TRIM29 binding motif was found in the TRIM29 binding regions with a core consensus sequence of CTGTCTCC. We also identified some known cancer genes in close proximity to TRIM29 binding sites, including JUND, FBXO31, CCND3, EGFR, and FOXC1. In addition to mapping TRIM29-DNA binding sites, we used co-immunoprecipitation (co-IP) coupled with tandem mass spectrometry to identify proteins that interact with TRIM29 in MDA-MB-468 cells. Proteins that co-precipitated with TRIM29 included splicing factor proline/glutamine-rich (SFPQ, a transcriptional co-repressor), poly(A) binding protein (PABPC4); FUS and TAF15 RNA polymerase II (both are proto-oncoproteins and transcriptional activators), and A kinase anchor protein 13 (AKAP13, an adaptor protein involved in protein kinase A signaling pathway). Confirmation of the initial co-IP results was achieved through several independent techniques, including detection of binding partners in TRIM29 precipitates by Western blot and reciprocal co-IP using antibodies to partner proteins and detection of TRIM29 in the precipitates. Overall, results from our studies indicated that besides functioning as a transcription factor, TRIM29 may also participate in signal transduction pathways by forming protein complexes. Our studies have identified new biomarkers for basal-like breast cancers and revealed kinases that could potentially be targeted by biologic therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2960.
Abstract 2962: Role of Cyr61 in the progression of pancreatic cancer
Authors: Mehta Smita , Dhar Kakali , Haque Inamul , Majumder Monami , McGregor Douglas H. , Banerjee Sushanta K. , Banerjee Snigdha
Published: February 2011

Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth most common cause of cancer in men and fifth in women in the United States. The overall survival rate is very poor in PDAC due to late presentation, tumor aggressiveness and lack of effective treatment strategy. The objective of this study is to explore the role of Cyr61 in the progression of the PDAC. Cyr61/CCN1 is a secreted, cysteine-rich heparin-binding protein, a member of CCN family and also plays a role in cell proliferation, adhesion, migration and differentiation during development but very little is known about cancer biology. We studied Cyr61 mRNA expression and protein level in tissue samples (archival and tissue array samples) using In Situ hybridization and immunohistochemistry. Its expression was also studied in different cell lines using Northern Blotting, quantitative real time PCR and Western blot analysis. The studies revealed that Cyr61 mRNA and protein was highly expressed in PDAC as compared to normal and chronic pancreatitis sections. The expression profile of Cyr61 increases as the disease progressed from Grade I to Grade III. Interestingly, in some PDAC sections Cyr61 expression was undetected or barely detected. Cyr61 was highly expressed in Panc-1, an invasive cell line, as compared to less invasive cell lines (i.e., BxPC3 and Capan-1) where expression of Cyr61 is very minimal. In order to evaluate the functional role of Cyr61, we nullified Cyr61 in Panc-1 cells using retroviral-mediated shRNA. We determined the migration of these cells and the effect of the conditioned media of these cells on in vitro angiogenesis. We found that Cyr61 is essential for in vitro migration and induction of angiogenesis. Furthermore, these studies reveal that Cyr61 contributes in the development of drug resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2962.