bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4823: Body mass index (BMI), change in BMI, and postmenopausal breast cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)
Authors: Sue Laura Y. , Genkinger Jeanine M. , Schairer Catherine , Ziegler Regina G.
Published: February 2011

Mid-life body mass index (BMI) has been consistently and positively associated with risk of postmenopausal breast cancer, but BMI at younger ages has been inversely associated with risk. Few studies have looked at the timing of adult BMI change and risk. Our analysis of adult BMI, BMI change, and postmenopausal breast cancer risk is, to our knowledge, the largest study to date. Our study population included 72,007 women, 55-74 years old at baseline, in the PLCO cohort: 3,677 postmenopausal breast cancer cases were ascertained from 1993 to 2007 (median follow-up, 9.9 y; interdecile range, 6.2 -12.9 y). Cox proportional hazards models were used to estimate multivariate relative risks (RRs) and 95% confidence intervals (95% CIs). BMI at age 20 and postmenopausal breast cancer risk were consistently inversely associated across use of menopausal hormone therapy (MHT) (Ptrend = 0.01). As the positive association between BMI at study baseline and breast cancer risk appeared to be limited to women who had never used MHT, we present these results among women who never used MHT. Postmenopausal breast cancer risk increased steadily across categories of increasing BMI at study baseline (Ptrend <0.0001) regardless of BMI at age 20. Furthermore, being overweight or obese (>25 kg/m2) at both study baseline and at age 20 had a protective effect on postmenopausal breast cancer risk, compared to being overweight or obese at study baseline but having a normal BMI (<25 kg/m2) at age 20. The RR of postmenopausal breast cancer for a BMI gain >5 kg/m2 between age 20 and age 50 was 1.82 (95% CI, 1.45-2.30), compared to women with no change in BMI (+/− 1 kg/m2). The RR for the same BMI gain from age 50 to age at baseline was 1.56 (95% CI, 1.29-1.90), compared to women with no change in BMI. In addition, in women who gained >5 kg/m2 between age 20 and age at baseline, postmenopausal breast cancer risk nearly doubled (RR, 1.93; 95% CI, 1.46-2.62), compared to the combined group of women with no change in BMI or with BMI loss (<-1 kg/m2). Among women who had never used MHT, BMI gain before and after age 50 was associated with increased postmenopausal breast cancer risk. Our study suggests that, with respect to postmenopausal breast cancer, healthy weight maintenance throughout adulthood is important and that BMI gain from age 20 to age 50 may play a particularly important role in increasing risk.

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 4823.
Abstract 4830: Insulin-like growth factors and kidney cancer risk in men
Authors: Major Jacqueline M. , Pollak Michael N. , Snyder Kirk , Virtamo Jarmo , Albanes Demetrius
Published: February 2011

Context: Causes of kidney cancer are not fully understood. Incidence is highest in those ages 50 to 70, and almost twice as high in men as in women. Smokers are twice as likely as non-smokers to develop renal cell carcinoma and about four times as likely to develop cancer of the renal pelvis. Insulin-like growth factor-I (IGF-I) has been shown to increase kidney microvascular growth and glomerular filtration rate, and its administration increases renal function in animal models of chronic renal failure, and has been proposed as a possible therapeutic agent (Hirschberg et al, 1998). However, the possible roles of IGF in the development of kidney cancer have not been well-studied. Objective: To examine the relation of serum levels of IGF-I and insulin-like growth factor binding protein 3 (IGFBP-3) to kidney cancer risk. Methods: We conducted a case-cohort study nested within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of 29,133 Finnish male smokers who were 50-69 years of age and not diagnosed with cancer at study entry. Serum concentrations of IGF-I, IGFBP-3 were measured in blood samples collected in 1985 to 1988. One hundred men were identified who had a diagnosis of kidney cancer >5 years after blood collection through the end of 1997. Self-reported information on lifestyle and medical history was collected and weight and height were measured at baseline. Multivariable logistic regression models were used to estimate the relative risk of kidney cancer associated with IGF levels. Results: A history of hypertension (known to be increased in those who are older, overweight, or heavier smokers) was more common among cases than among noncases. Men with IGF-I levels >108 ng/mL were 63% less likely to develop kidney cancer than men with IGF-I levels ≤108 ng/mL (OR=0.37; 95% CI=0.20-0.69). IGFBP-3 levels did not alter the association between IGF-I and kidney cancer risk. Further, no association was observed between IGFBP-3 levels and the development of kidney cancer. Conclusions: Low serum IGF-I levels in this cohort of older middle-aged male smokers are associated with increased kidney cancer risk, independent of IGFBP-3, age, anthropometry, lifestyle and medical history. Further research is needed to confirm the findings and examine the association in women and non-smokers.

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 4830.
Abstract 4834: Energy-dependent AMPK association with the ULK1-mTORC1 complex regulates autophagy
Authors: Lee Jong Woo , Park Sungman , Yoshinori Takahashi , Wang Hong Gang
Published: February 2011

Autophagy is an intracellular catabolic process through which bulky cytosolic components are sequestered for lysosomal degradation. Recent evidence suggests that mTOR suppresses autophagy through direct interaction with the ULK1-Atg13-FIP200 complex. However, the mechanism by which mTOR is controlled to activate ULK1 activity for autophagy induction is not yet well elucidated. Using tandem affinity purification (TAP) assay, we screened for ULK1 binding proteins and identified several additional ULK1 binding proteins that mediate the pro-autophagic activity of ULK1. Here we report AMP-activated protein kinase (AMPK), which is known to be a highly-conserved cellular energy sensor that is activated under conditions of low intracellular ATP, as a new ULK1 binding protein. We found that AMPK binds to the PS domain of ULK1 in an energy-dependent manner, whereas mTOR associates with ULK1 through raptor interaction with the kinase domain of ULK1 independently of nutrient availability. Mechanistically, AMPK inhibits the mTOR activity by recruiting 14-3-3 to the mTORC1 through phosphorylation of raptor in response to energy stress. Importantly, activation of AMPK by 5-amino-4-imidazolecarboxamide ribose (AICAR) is able to suppress the mTORC1 and induce autophagy in TSC2-deficient cells with wild-type raptor but not the mutant raptor (S722A/S792A) lacking AMPK phosphorylation sites. Our study indicates, therefore, that AMPK-mediated raptor phosphorylation plays an important role in the activation of ULK1 to induce autophagy under energy stress conditions.

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 4834.
Abstract 4835: Bif-1 induces the fission of Golgi membranes to generate Atg9-containing vesicles for autophagosome formation
Authors: Takahashi Yoshinori , Meyerkord Cheryl L. , Gonsar Kristin , Hori Tsukasa , Wang Hong-Gang
Published: February 2011

Dysregulation of autophagy, an intracellular bulk degradation system, is strongly associated with the pathogenesis of a number of diseases, including cancer. Despite the identification of 31 genes that are essential for autophagy, the molecular machineries that regulate the generation and expansion of autophagosomal membranes are not fully understood. We have previously reported that Bif-1 interacts with Beclin 1 through UVRAG and that loss of Bif-1 suppresses the formation of autophagosomes during starvation. Here, we provide further evidence that Bif-1 acts with the PI3KC3 complex II (Beclin 1-UVRAG-PI3KC3; PI3KC3C2) to regulate the formation of autophagosomes by regulating the trafficking of the membrane-integrated, autophagy-related (Atg) protein, Atg9, by inducing the fission of Golgi membranes during the induction of autophagy. In response to starvation, Bif-1 co-accumulates with Atg9 in cytosolic foci that are positive for Golgi complex-derived membranes. Loss of Bif-1 or inhibition of the PI3KC3C2 not only suppresses the fragmentation of Golgi membranes and the Atg9-containing vesicles that are induced by nutrient starvation or rapamycin treatment, but also results in the accumulation of Atg9 at the Golgi complex. Importantly, Bif-1 mutants, which lack membrane binding and/or bending activity by deletion of the helix 0, helix 1 insertion, or both regions of the Bif-1 N-BAR domain, fail to restore rapamycin-induced formation of Atg9 foci and autophagosomes in Bif-1-deficient cells. Furthermore, immunoelectron microscopic analyses reveal that Atg9 localizes not only to autophagosomal membranes but also crescent-shaped isolation membrane-like structures during starvation. These results suggest that Bif-1, together with the PI3KC3C2, plays a key role in the fission of Golgi membranes, which generates Atg9-containing vesicles for the formation and expansion of autophagosomal membranes.

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 4835.
Abstract 4836: RB/E2F1 in the crossroad of autophagy and apoptosis
Authors: Jiang Hong , Martin Vanesa , Gomez-Manzano Candelaria , Johnson David G. , Alonso Marta , White Erin J. , Xu Jing , McDonnell Timothy J. , Shinojima Naoki , Fueyo Juan
Published: February 2011

Autophagy is a protective mechanism that renders cells viable in stressful conditions. Mounting evidence suggests that this cellular process is also a tumor suppressor pathway. We hypothesized that retinoblastoma protein (RB), a key tumor suppressor, induces autophagy. To test this hypothesis, we first transduced RB into RB-defective human cancer cells: sarcoma osteogenic (Saos-2) cells, hepatoma (Hep3B) cells, and brain tumor stem cells (MDNSC23). The ectopic RB induced autophagy as demonstrated by significantly increased percentage of cells with acidic vesicular organelles (from less than 10% to more than 30%, P < 0.05), and the lipidation of LC3-I leading to the formation of autophagosomes/autolysosomes that were visualized by EGFP-LC3 punctation (from less than 20% to more than 80%, P = 0.003). These double-membraned vesicles were clearly shown by ultrastructural study via transmission electron microscopy. However, when Beclin 1 was silenced with siRNA in Saos-2 cells, the RB-mediated autophagy was blocked. In addition, study of autophagy flux with Saos-2 cells expressing double-tagged mRFP-EGFP-LC3 fusion protein revealed that RB stimulated the formation and maturation of autophagosomes. Consistently, RB activators p16INK4a and p27/kip1 caused autophagy in an RB-dependent manner. Importantly, we found RB mutants Delta-22 and R661W deficient for binding E2F failed to induce autophagy. Moreover, E2F1 overexpression antagonized RB-mediated autophagy, leading to apoptosis in Saos-2 cells. In agreement with the above observations, silencing E2F1 with siRNA resulted in autophagy in U-87 MG cells; and autophagy levels increased from 4% in wild-type MEFs to 40% in E2F1 knockout MEFs (P = 0.002). Collectively, our data reveal that RB/E2F1 plays a key role in the decision of a cell to undergo autophagy or 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 4836.
Abstract 4838: Alpha-methyltryptophan, a blocker of the amino acid transporter SLC6A14, induces autophagy and apoptosis in SLC6A14-positive breast cancer and colon cancer cell lines
Authors: Karunakaran Senthil , Schoenlein Patricia V. , Thangaraju Muthusamy , Prasad Puttur D. , Ganapathy Vadivel
Published: February 2011

SLC6A14 (also known as ATB0,+) is a Na+/Cl− -coupled amino acid transporter that transports 18 of the 20 proteinogenic amino acids, including all essential amino acids. Normal tissues express this transporter at low levels, whereas tumor tissues (colon, breast, and cervix) upregulate this transporter several-fold. The unique functional features of SLC6A14 (broad substrate specificity and multiple driving forces) make it an ideal transporter for tumor cells to maintain amino acid nutrition necessary for enhanced cell proliferation. We speculate that SLC6A14 could not only be an ideal tumor-specific delivery system for amino acid-based anti-cancer drugs but also be a direct drug target for cancer chemotherapy. Recently we identified alpha-methyltryptophan (ALT) as a blocker of SLC6A14. Here we examine the potential of this blocker as an anti-cancer agent in SLC6A14-positive breast and colon cancer cells. Treatment of MCF7 (a breast cancer cell line), HCT116 and LS174T (colon cancer cell lines) with ALT causes amino acid starvation as evidenced by the upregulation of asparagine synthetase and CHOP. These three cell lines are SLC6A14-positive. In contrast, ALT treatment of breast cancer cell line MDA-MB231, which is SLC6A14-negative, does not show any evidence of amino acid starvation. Similarly, ALT treatment has no effect on normal breast and colon epithelial cell lines (MCF10A and CCD841, respectively). These cell lines also do not express SLC6A14. These data show that ALT-induced amino acid starvation is mediated specifically through the blockade of SLC6A14. Since nutrient signaling is also associated with changes in mTOR pathway, we examined the influence of ALT treatment on phosphorylation of S6 and S6kinase. Blockade of SLC6A14 with ALT in SLC6A14-positive cancer cell lines leads to decreased mTOR signaling as evidenced by decreased phosphorylation of S6 and S6kinase. Again, such changes are not seen in SLC6A14-negative cancer or normal cell lines. The initial response of the SLC6A14-positive cancer cell lines to ALT treatment is induction of autophagy. ALT-induced autophagy is documented in these cells by light and electron microscopic examination as well as by biochemical means. Inhibition of autophagy with 3-methyladenine under these conditions leads to apoptosis. We conclude that SLC6A14 is a potential drug target for cancer chemotherapy and that ALT could serve as a lead compound for the future design of high-affinity blockers of SLC6A14.

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 4838.
Abstract 4840: 2-Deoxy-D-glucose induces autophagy through endoplasmic reticulum stress rather than by lowering ATP
Authors: Xi Haibin , Kurtoglu Metin , Liu Huaping , Wangpaichitr Medhi , You Min , Savaraj Niramol , Lampidis Theodore J.
Published: February 2011

Based on emerging evidence that oncogenes direct increased glucose metabolism in tumor cells, the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG) appears to be a promising drug to selectively target cancer cells. 2-DG however, not only blocks glycolysis and reduces cellular ATP production but also interferes with N-linked glycosylation, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Both bioenergetic challenge and ER stress/UPR have been shown to activate autophagy, a bulk cellular degradation process that plays either a pro- or anti-death role. This led us to investigate the following, 1) which pathway that 2-DG interferes with is responsible for stimulating autophagy, and 2) the role of autophagy in modulating 2-DG-induced cellular responses and toxicity. In four cancer cell lines (1420, MDA-MB-435, SKBR3 and T98G) we find that 2-DG upregulates autophagy (LC3B II), increases the ER stress/UPR markers, Grp78 and CHOP, and lowers ATP levels. Addition of exogenous mannose reverses 2-DG-induced autophagy and expression of GRP78 and CHOP but does not recover the lowered levels of ATP. Additionally, when autophagy is either blocked or enhanced by 3-methyladenine (3-MA) or rapamycin, respectively, 3-MA increases whereas rapamycin reduces cancer cell sensitivity to 2-DG. In support of these results, knock down of autophagy-related gene 7 (Atg7) with small interfering RNAs (siRNAs) increases sensitivity to 2-DG in 1420 cells, which correlates with increases in the ER stress/UPR markers as well as the apoptotic marker, cleaved caspase 3. Overall, these results indicate that the major mechanism by which 2-DG stimulates autophagy is through ER stress/UPR, and autophagy protects cells from 2-DG-induced toxicity through relief of ER stress. Thus, combining autophagy inhibitors with 2-DG may be useful clinically.

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 4840.
Abstract 4842: Lysosomal-associated protein multispanning transmembrane 5 gene (LAPTM5) is associated with spontaneous regression of neuroblastomas
Authors: Inoue Jun , Misawa Akiko , Tanaka Yukichi , Ichinose Shizuko , Sugino Yuriko , Hosoi Hajime , Sugimoto Tohru , Imoto Issei , Inazawa Johji
Published: February 2011

Background; Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors.

Key findings; We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells.

Conclusions; We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.

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 4842.
Abstract 4851: BH-3 mimetics radiosensitizes human prostate cancer via modulating autophagy
Authors: Lian Jiqin , Wu Xiaoqing , Tang Wenhua , Karnak David , He Fengtian , Meng Yang , Lawrence Theodore S. , Xu Liang
Published: February 2011

Radioresistance markedly impair the efficacy of cancer therapy and involve anti-apoptotic signal transduction pathways that prevent the treatment-induced cell death. Anti-apoptotic Bcl-2 family proteins such as Bcl-xL, Bcl-2 and Mcl-1 are overexpressed in prostate cancer and contribute to prostate tumor initiation, progression and resistance to radiotherapy. (−)-Gossypol, a natural product from cottonseed, has recently been identified as a potent small molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 and is now in advanced clinical trials. Since Bcl-2 also inhibits autophagy and radiation induces autophagy, we hypothesize that (−)-gossypol may improve prostate cancer's response to radiotherapy by inducing autophagy in apoptosis-resistant prostate cancer cells with high Bcl-2.

(−)-Gossypol inhibits tumor cell growth and preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and resistant to apoptosis, both in vitro and in vivo, but not in cells with low Bcl-2. (−)-Gossypol radiosensitized AI-prostate cancer PC-3 and CL-1 cells in clonogenic assay. (−)-Gossypol increased autophagy induced by X-ray radiation in these cells. Orally administered (−)-gossypol achieved a much greater efficacy with long-term tumor regression when used in combination with ionizing radiation. Immunoprecipitation /pull-down assay showed that (−)-gossypol induced autophagy via blocking Bcl-2-Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1 and activating autophagic pathway signaling. Knockdown of the Beclin1 or atg5 expression blocked the autophagy induced by (−)-gossypol or radiation.

Taken together, our data demonstrate that (−)-Gossypol exerts its radiosensitizing activity through promoting radiation-induced autophagy, via modulating Bcl-2-Beclin1 interaction and atg5. (−)-Gossypol significantly enhances the anti-tumor activity of radiotherapy in vitro and in vivo, and represents a promising new regime for treatment of hormone-refractory human prostate cancer with overexpression of Bcl-2.

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 4851.
Abstract 4853: Reduced expression of DRAM2/TMEM77 in tumor cells interferes with cell death
Authors: Jang Ik-Soon , Kim Soohyun , Choi Jong Soon , Park Sun-Mi , Park Junsoo
Published: February 2011

Although the role of autophagy in tumorigenesis remains controversial, recent reports support the notion that inhibition of autophagy promotes tumor formation. Damageregulated autophagy regulator (DRAM) has been identified as an effector molecule that is critical for p53-mediated apoptosis, and we investigated whether there might be other DRAM-like molecules linking autophagy and apoptosis. In this study, we cloned a novel DRAM-homologous protein, DRAM2, and showed that the expression of DRAM2 is down-regulated in ovarian tumors. DRAM2 is mainly localized in the lysosome, and colocalizes with DRAM. While expression of DRAM or DRAM2 individually did not induce cell death, co-expression of DRAM2 with DRAM significantly induced cell death, while the silencing of endogenous DRAM2 attenuated cell death, suggesting that DRAM2 is involved in cell death. Thus, we propose that reduced expression of DRAM2 may contribute to enhanced cell survival in tumor cells.

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 4853.
Abstract 4855: Role of HDACi-induced autophagy in malignant peripheral nerve sheath tumors (MPNST)
Authors: Torres Keila E. , Lopez Gonzalo , Liu Juehui , Pollock Raphael E. , Lev Dina
Published: February 2011

Malignant peripheral nerve sheath tumors (MPNSTs) are rare tumors that remain incurable mainly due to their high metastatic potential. These biologically aggressive tumors account for 3-10% of soft tissue sarcoma (STS). Roughly 50-60% of MPNSTs develop within the background of an inherited disease known as neurofibromatosis type-1 (NF1), while the rest develop sporadically. Despite aggressive combined modality therapy, the 5 year survival rates range from 35% to 50%. Thus, the understanding of MPNST resistance and development of a more efficient and targeted therapeutic approachs is essential to improve the otherwise dismal prognosis for these patients. Histone deacetylase inhibitors (HDACi) are novel compounds with efficacious in vitro and in vivo anti-tumor effects in a variety of tumor types including STS. Human MPNST cells treated with HDACi exhibit significant growth arrest. However, no apoptosis is observed but rather a dramatic induction of autophagy as assayed by multiple approaches. Whether drug-induced autophagy's role is to protect or kill the cells remains to be determined. Autophagy blockade via siRNA (Beclin 1 and Atg 5) or pharmacological manipulation with bafilomycin A1 and chloroquine significantly enhances HDACi pro-apoptotic effects. Furthermore, in vivo studies revealed that single agent HDACi or chloroquine alone had a modest effect on tumor growth, whereas combined treatment resulted in marked growth inhibition. These findings suggest that autophagy may mediate a potential protective response in MPNST and strongly support further study of combined HDACi with autophagy blockers as a novel therapeutic approach for MPNST.

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 4855.
Abstract 486: Abscopal effect of low-LET ionizing radiation on Rel protein signal transduction
Authors: Veeraraghavan Jamunarani , Kiosseva Svetlana , Madhusoodhanan Rakhesh , Natarajan Mohan , Herman Terence S. , Aravindan Natarajan
Published: February 2011

Ascertaining the ionizing radiation (IR)-induced bystander response and identifying its mechanistic regulation would raise a paradigm shift in our understanding of the radiobiological effects. Recent evidence from our lab and others clearly prompted that the transcriptional regulator NFκB would play a key role in induced responses in non-targeted bystander cells. In this study, for the first time, we investigated the orchestration of NFκB signaling after IR in an organ (heart) distant from the exposure field. The C57/BL6 mice either mock irradiated, exposed to single dose IR (SDR: 2 or 10Gy) or fractionated IR (FIR, 2 Gy/day for 5 days) were sacrificed 24h post-irradiation. All IR exposures were limited to the lower abdomen area (1cm diameter), while the rest of the animal was protected by a specially designed cerrobend shield. EMSA analysis was used to examine the changes in NFκB DNA binding activity in the heart. Transcriptional activation of 88 mediators associated with NFκB signaling pathway was assessed using QPCR profiling. Phosphorylation of ERK1/2 and p38 that play a key role in NFκB signal transduction were examined using immunoblotting. Induced DNA fragmentation was measured using TdT nick end labeling. IR significantly induced NFκB DNA binding activity in the heart. Further more, real-time QPCR profiling showed a significant and differential modulation in the transcriptional responses of NFκB signaling pathway molecules. To that end, of the 88 genes analyzed, 28, 51 and 41 genes were upregulated after 2Gy, 10Gy and FIR, respectively. More importantly, at these exposures13 out of 28, 27 out of 51 and 25 out of 41 induced mediators of NFkB pathway were significantly upregulated. Conversely, lower abdominal IR exposure markedly suppressed 44, 20 and 30 genes after 2 Gy, 10Gy and FIR, respectively in the heart of the exposed animals. Compared to 2Gy, FIR significantly induced 26 genes, while 10Gy upregulated 27 genes. In addition, 6 and 15 genes were significantly upregulated after acute dose of 2 and 10Gy as opposed to fractionated dose. Consistently, immunoblotting analysis revealed a profound phosphorylation of both ERK1/2 and p38 in the heart. Interestingly, distant IR exposure significantly enhanced DNA fragmentation in the heart tissue. Taken together, these data clearly indicated an induced abscopal response in mice heart after clinically relevant doses of IR exposure. More importantly, these data implies that orchestration of NFκB signal transduction may play a key role in induced abscopal responses.

Funding Support: Presbyterian Health Foundation, OKC.

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 486.
Abstract 4862: Histone deacetylase inhibition induces DNA damage, slows down replication forks and activates dormant origins
Authors: Leo Elisabetta , Conti Chiara , Eichler Gabriel S. , Sordet Olivier , Martin Melvina M. , Fan Angela , Aladjem Mirit I. , Pommier Yves
Published: February 2011

Histone acetylation is often altered in cancers and histone deacetylases inhibitors (HDACi) are actively pursued as anti-cancer agents.

SAHA (suberoylanilide hydroxamic acid) is the first HDACi approved for clinical use.

To monitor the effects of SAHA on DNA replication and genomic integrity in cancer cells, we detected broken DNA with COMET assays and analyzed phosphorylation of histone H2AX (gH2AX). Nuclear replication factories in single cells were visualized after pulse labeling with two thymidine analogs and confocal immunofluorescence microscopy. Nascent strand reverse transcriptase polymerase chain reaction (RT-PCR) in the human g-globin locus was used to assess the effects of SAHA on replication fork origin firing. In addition, cellular replicons were analyzed by genome-wide molecular combing. Our experiments prove that pharmacological concentrations of SAHA induce replication damage, as shown by single-cell and single-DNA molecule analyses. Molecular combing demonstrated a slow-down of replicons and activation of dormant replication origins in response to SAHA treatment. Similar results were obtained with siRNA-mediated HDAC3 depletion indicating the selectivity of the effect of SAHA. Activation of dormant origins at the g-globin locus control region was confirmed by nascent strand RT-PCR. Our findings demonstrate specific replication alterations and DNA damage in response to SAHA and emphasize the importance of chromatin acetylation for DNA replication in human cells.

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 4862.
Abstract 4863: The histone lysine-methyltransferase NSD1 and its functional role in non-small cell lung cancer (NSCLC)
Authors: Joseph Roy , Stunkel Walter , Phong Mark , Fong Siew Chinn , Li Lei , Campbell Robert M.
Published: February 2011

NSD-1 (Nuclear receptor-binding SET domain protein 1) belongs to the epigenetic family of histone methyltransferases (HMTs), which are frequently found to be mutated in overgrowth syndromes. In cancer, NSD-1 is found as part of fusion proteins (e.g., NUP98-NSD1), which play a major role in the etiology of AML. In contrast to data from hematological tumors, little is known regarding the role of NSD-1 in cancers from solid origin, except that high level expression of NSD-1 has been correlated with poor survival in breast cancer patients (Miller, 2005). NSD-1 over-expression as well as copy number gain for the NSD-1 gene has been described in a variety of tumor cell lines. We studied the role of NSD-1 in the NSCLC line NCI-H460 using an inducible knockdown system (pSLIK). We found that knockdown of NSD-1 (>70% reduction in target mRNA expression) led to a significant decrease in cell proliferation as well as nearly 100% inhibition of anchorage-independent growth, whereas an empty vector transfected stable cell line did not. NSD-1 knockdown induced apoptosis in a time-dependent manner as measured by flow cytometry-based Annexin V staining. Cell death was found to follow a PARP-dependent mechanism suggesting that apoptosis is a contributor to growth inhibition/cell death. By conducting immuno-blot experiments against a panel of histone modifications, we examined putative in vivo histone substrates of NSD1. Our data suggest that NSD1 knockdown in NCI-H460 cells leads to a decrease in histone H4K20 trimethylation (64% inhibition after 24h), but did not affect H3K36 trimethylation as previously reported for NSD1 fusion proteins in AML. Further studies of changes in additional histone modifications are ongoing. In summary, we describe for the first time a functional role for NSD-1 in a NSCLC cell line with implications for this HMT in solid tumor growth/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 4863.
Abstract 4866: The dynamic interaction of nuclear Smads with peri- and centromeric heterochromatin is involved in the modulation of histone modification and chromatin structure
Authors: Kim Tae-aug , Kim Jonghyun , Kim Saejung , Whang John , Misako Sato , Wakefield Lalage , Vladimir Larionov , Kim Seong-Jin , Niederhuber John
Published: February 2011

Smads are intracellular signaling transducers of TGF-β family ligands. Through TGF-β activation, phosphorylated R-Smads (Smad2/3) form active heteromeric complexes with co-Smad (Smad4) and translocate to the nucleus where they interact with chromatin to assemble the transcriptional machinery of various target genes. Evidence also indicates that Smads are involved in chromatin modulation via interactions with histone modifiers. The role of pre-existing nuclear Smads in chromatin modulation, however, has not been described, nor have the molecular mechanisms of their dynamic interaction with heterochromatin been elucidated. Here we report that nuclear Smads directly bind to specific sites on heterochromatin, altering chromatin architecture upon TGF-β activation. Subcellular fractionation indicates that both active and inactive nuclear Smad 2/3 remains in the biochemically insoluble fraction. We found that only non-phosphorylated Smad3 interacted with HP1α, a key component of heterochromatin under transcriptionally inactive conditions. The level of HP1α, but not HP1β and γ, was regulated in a TGF-β dependent manner in both Smad 2/3 KO MEFs and mouse mammary epithelial cells. Localization of the Smad3 in the pericentromeric heterochromatin and centromere regions of the human chromosomes suggests Smad3 may function as an organizer of chromatin architecture and chromosome assembly during mitosis. ChIP assay indicated that pericentromeric and centromeric heterochromatin bound Smads were dissociated by TGF-β activation and translocated to target gene promoter regions to activate gene transcription. Interestingly, TGF-β enhanced pericentromeric heterochromatin binding of Smad3 in a Smad3 overexpressed human breast cancer cell line, MCF10Ca1, but dramatically decreased in a cell line with deletion of MH2 domain in Smad3. Furthermore, deletion of MH2 domain markedly increased by more than 100 folds both H3 lys4 dimethylation and lys9 dimethylation in centromeric heterochromatin but diminished them in pericentromeric heterochromatin. This suggests that the MH2 domain of the Smad3 may play a critical role on modulation of heterochromatin structure in a manner that activates or represses genome wide gene transcription activity. In conclusion, these results indicate that nuclear Smads are involved in the modulation of chromatin structure through the regulation of HP1α and their direct interaction with peri- and centromeric heterochromatin. Therefore dysregulation of nuclear Smad expression may increase genomic instability.

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 4866.
Abstract 4867: High-throughput LanthaScreen® cellular assays for interrogating post-translational modifications of p53 and histones
Authors: Robers Matt , Machleidt Thomas , Hermanson Spencer , Wilkinson Jennifer , Wetter Justin , Bi Kun
Published: February 2011

Post-translational modifications such as phosphorylation, acetylation and ubquitination play important roles in regulating the structure and functions of histones and transcription factors such as p53, which in turn regulate essential gene expression. Dysregulation of these post-translational modifications has been linked to cancer and metabolic diseases. In particular, histone deacetylases (HDACs) have been pursued as valuable targets for various therapeutic interventions. However, detection of these enzymatic activities in a cell-based format has remained intractable using HTS-compatible technologies. To enable the drug discovery for these post-translational modification enzymes, we have developed and validated high-throughput compatible LanthaScreen® cellular assays for the analysis of histone and/or p53-specific acetylation, ubquitination and phoshorylation in cell backgrounds of interest. The cell cycle-dependent acetylation of histone H3 at Lys9 and the phosphorylation of histone H3 at Ser10 can be detected with terbium-labeled anti-Histone H3 acetyl-lys 9 and anti-phospho-Ser10 specific antibodies, respectively. In addition, poly-ubiquitination of Histone H2B can be measured with terbium-labeled anti-poly-ubiquitin antibody. We have also applied this technology and developed cellular assays for measuring DNA-damage induced phosphorylation at Ser15 and acetylation at Lys382 of p53. These assays were further validated with both small molecule inhibitor and siRNA against specific HDAC family members. Our results suggest that the deacetylation of Histone H3 Lys 9 is mediated by type I/II HDACs, whereas the deacetylation of p53 at Lys382 is mediated synergistically by both SIRT1 and type I/II HDAC activities. Together, these assays enable interrogation of multiple enzymatic activities responsible for post-translational modifications of challenging targets such as histones and p53.

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 4867.
Abstract 4868: Interaction between DNMT3B and polycomb repression complexes and their role in histone modification
Authors: Li Yajun , Van Emburgh Beth O. , Jin Bilian , Robertson Keith D.
Published: February 2011

Epigenetic modifications, such as DNA methylation and the histone code, play crucial roles in regulating gene expression during development. Aberrant DNA methylation patterns, characterized by global genomic hypomethylation and gene specific hypermethylation at CpG islands, are found in all cancer cells and contribute directly to tumor development. DNA methyltransferases are responsible for the de novo or heritable maintenance of DNA methylation. DNMT3A and DNMT3B de novo methyltransferases contribute largely to the methylation of retrotransposon sequences and satellite repeats in pericentromeric regions. It is still unclear how gene-specific epigenetic modification patterns are established and maintained in cancer cells. Recently, high-throughput studies reported an overlapping distribution of polycomb repression complexes regulating histone modifications and DNA methyltransferase mediated DNA methylation, suggesting that there are interactions among these epigenetic regulators. Our previous data from DNA methyltransferase knock out cell lines revealed that epigenetic modifiers and the histone code influence the propensity of a gene to become hypermethylated in cancer and that DNMT3B plays an important role in regulating PRC1 function. To gain insight into the mechanisms of how DNMT3B/DNA methylation effects the histone code, and vice versa, we studied the protein-protein interactions between PRC1 components and DNA methyltransferase 3B and their functional consequences. We expressed DNMT3B and each PRC component in both baculovirus and E. coli expression systems. Pull down assays showed that there were multiple interactions between PRC1 components (eg. CBX4 /hPc2) and DNMT3B. Protein-protein interaction domains were further mapped. We then used an in vitro ubiquitination system to study the role of DNMT3B in histone H2A ubiquitination and also studied the effects of these interactions on gene expression in human tumor cell lines. Our results suggest that the interplay between DNMT3B/DNA methylation and PRC1 is important for the function of each protein/complex and that aberrations in these interactions contribute to epigenetic dysregulation in cancer cells. (Supported by NCI grant R01CA114229)

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 4868.
Abstract 4878: Bioactive dietary supplements reactivate ER expression in ER-negative breast cancer cells by epigenetic mechanisms involving active chromatin modification
Authors: Meeran Syed M. , Li Yuanyuan , Ahmed Amiya , Tollefsbol Trygve O.
Published: February 2011

Breast cancer is the second leading cause of cancer-related death among women in the United States. One of the important classifications of breast tumors is based on the presence or absence of the estrogen receptor (ER). While the majority of breast cancers are ER-positive, approximately 30% are ER-negative. Patients with ER-positive breast cancer receive hormonal therapy using either tamoxifen or aromatase inhibitors, and have a better prognosis. However, treatment of patients with ER-negative tumor is challenging due to the poor response to hormonal therapies in the absence ER expression. Many studies have demonstrated the chemopreventive properties of green tea polyphenols (GTPs) and sulforaphane (SFN) through inhibition of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activity, respectively. In the present study, we have observed that GTPs and SFN dose-dependently inhibit the cellular proliferation in both ER-positive and ER-negative human breast carcinoma MCF-7 and MDA-MB-231 cells, respectively. Treatment of ER-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ER mRNA expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ER reactivation at 3 days in MDA-MB-231 cells. The reactivation of ER expression was consistently correlated with ER promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ER promoter revealed that GTPs and SFN decreased the level of dimethyl H3K9, but increased the acetylation by increasing the level of acetyl H3, acetyl H4 and acetyl H3K9. In addition, a combination of GTPs and SFN significantly inhibited DNMT and HDAC activity, which might have an important role in the reactivation of ER expression in MDA-MB-231 cells. The combination of GTPs- and SFN-mediated inhibition of DNMT activity and increase of acetylation underscores the importance of DNMTs and HDACs in maintaining the repressive environment at target genes such as ER. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that HDAC inhibitors combined with demethylating agents are a promising strategy for the epigenetic reactivation of ER expression for the effective treatment of ER-negative breast cancer.

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 4878.
Abstract 4880: CASZ1, a neuroblastoma tumor suppressor gene, is epigenetically silenced by EZH2
Authors: Wang Chunxi , Woo Chanwook , Liu Zhihui , Wei Jun , Song Young , Wang Lifeng , Khan Javed , Ge Kai , Thiele Carol J.
Published: February 2011

Casz1 is a C2H2 Zn-finger transcriptional factor that localizes to a region of LOH on Chr1p36.22 in tumors of Neuroblastoma patients who have a poor prognosis. Moreover, high CASZ1 expression is found in tumors of patients with a good prognosis while tumors from patients with a poor prognosis express low levels of CASZ1 (58 patients, p= 2.07×10-6). Studies indicate that CASZ1 functions as a tumor suppressor gene. Our finding that treatment of NB cells with HDAC inhibitors results in a 10-fold increase in CASZ1 expression suggests that epigenetic silencing contributes to the functional loss of CASZ1 expression from the remaining Chr1p36.22 allele. In studying the mechanism of CASZ1 epigenetic silencing, we found a bivalent chromatin modification region around the CASZ1 Transcription Start Site (TSS) that contains both a H3K27 methylation mark as well as an H3K4 methylation mark. The H3K27 methylation is thought to be a gene repressive marker, which is catalyzed by Polycomb repressor complex 2 (PRC2) proteins. PRC2 contains three core components, EZH2, EED and SUZ12, in which EZH2 (enhancer of zeste 2) is a methyltransferase catalyzing H3K27 methylation. Using Chromatin Immunoprecipitation (ChIP) we identified a 5-fold decrease in binding of EZH2, EED and SUZ12 to the bivalent region after HDAC inhibitor treatment. Consistent with decreased PRC2 binding is the finding of decreased tri-methylation of H3K27 and an increase in H3K4me3, a histone mark associated with transcriptionally active genes. The changes in histone modifications are associated with the activation of the CASZ1 gene. RNA silencing of EZH2 also leads to increases in CASZ1 expression. Consistent with this, CASZ1 expression is also up-regulated (3- fold) in EZH2 knockout MEFs compared to EZH2 wild type MEFs. This indicates that CASZ1 expression on the remaining allele is silenced by epigenetic modifications mediated by EZH2. High expression of EZH2 and EED are also found in tumor samples of neuroblastoma patients with a poor prognosis (58 patients, p=2.6×10-3 for EED and p=5.87×10-4 for EZH2). This is consistent with a model in which aberrant epigenetic gene silencing of the tumor suppressor gene CASZ1 contributes to neuroblastoma 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 4880.