bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 254: Genome-wide shRNA screening identifies candidate proteins modulating the extrinsic apoptotic pathway
Authors: Kang Zhigang , Cao Liang
Published: August 2012

Apoptosis is a highly regulated process critical for embryonic development, and tissue and organ homeostasis. Deregulation of apoptosis is a common occurrence in cancer. Emerging oncology therapeutic agents designed to engage apoptosis are currently undergoing clinical investigations. To identify unknown proteins that modulate the sensitivity to death receptor DR5 targeted agent drozitumab and to uncover predictive biomarkers, we perform a genome-wide screening of 18,000 genes by using pooled short hairpin RNA libraries, followed by the next generation sequencing for candidate identification. We have identified and confirmed candidate shRNA constructs and target genes that resulted in selective desensitizing of the cells to drozitumab. The roles and mechanisms of action of the newly identified proteins in modulating the extrinsic apoptotic pathway will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 254. doi:1538-7445.AM2012-254
Abstract 2542: EXO1 is required for SN1 methylator-induced DNA damage response
Authors: Izumchenko Eugene G. , Brown Kevin D.
Published: August 2012

SN1 methylating agents are therapeutics that methylate the O6 position of guanine (O6meG) forcing mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes O6meG:T mispairs and activates DNA damage response (DDR). Exonuclease I (EXO1) functions during MMR by excising the damaged strand; however, how MMR mechanistically contributes to DDR activation and if EXO1 is required to activate MMR-dependent response remains unknown. Here we show that Exo1 knockdown in mouse embryonic fibroblasts (MEFs) results in decreased G2/M arrest and checkpoint response (ie, Cdc2/Chk1 phosphorylation), and increased cell proliferation and viability following exposure to the SN1 methylator MNNG. Moreover, ectopic expression of full-length human hEXO1 restored normal response to MNNG. We next examined gamma-H2AX foci formation as a surrogate marker of MMR-dependent DNA repair. 12h after MNNG treatment, 40% of MEFs scored positive for multiple gamma-H2AX foci and this population further increased with prolonged treatment. In contrast, Exo1 depleted MEFs displayed a significantly diminished number of gamma-H2AX foci-positive cells following MNNG. Increased association of Msh2/Msh6 with chromatin was observed in MNNG treated MEFs; however, Exo1 depletion resulted in no upregulation of MMR proteins within the chromatin fraction. Furthermore, MNNG-induced Msh2/Chk1 association was disrupted upon Exo1 depletion. Taken together, we conclude that Exo1 is required to activate DDR following SN1 methylator exposure. Data gathered suggests that Exo1 functions in activating MMR-dependent DNA repair, promoting assembly of MMR complexes with alkylated chromatin, and enhancing the association of MMR components with critical checkpoint mediators. Experiments designed to determine if Exo1 functions in a structural (ie, scaffolding) or catalytic (ie, nucleolytic) manner during SN1 methylator-induced DDR are currently underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2542. doi:1538-7445.AM2012-2542
Abstract 2543: A dual-fluorescent reporter system facilitates identification of thiol compounds that suppress oxidative frameshift mutations
Authors: Chang Christina L. , Li I-Chen , Chiu Chien-Yuan , Chi Jhih-Ying , Jian Siao-Ru , Wu Chang-Lin
Published: August 2012

DNA mismatch repair (MMR) system corrects replicative errors, especially in microsatellites, and minimizes mutations caused by DNA-damaging agents. Patients with chronic and cancer-associated inflammation display microsatellite instability (MSI), indicative of an inactivated MMR system. MMR-deficient patients also display frameshift mutations in coding microsatellites of many genes, including oncogenes and tumor suppressors. We previously reported that oxidative stress, which occurs in the inflammatory settings, not only inactivates the MMR function but also increases accumulation of frameshift mutations. However, it remains unclear whether oxidative frameshift mutations are preventable due to the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing RFP harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogeneic human colorectal cancer cell lines. By flow cytometry, the new reporter sensitively detected a low level of H2O2-induced frameshift mutations in MMR-proficient cells. Without a functional MMR system, 0.5 mM H2O2 increased frameshift mutations by approximately 10 folds in the cells. In MMR-deficient cells, our reporter revealed that 5 mM glutathione or N-acetylcysteine suppressed oxidative frameshift mutations by approximately 75% or 90% respectively, without affecting cell viability. In contrast, 0.5 mM ascorbic acid augmented H2O2-induced frameshift mutations by two folds, whereas 1 mM aspirin had no effects. Glutathione and N-acetylcysteine also partially suppressed oxidative frameshift mutations in coding microsatellites of endogenous hMSH6 and CHK1 genes, determined by a fluorescinated PCR-based assay. The new utility of these thiol compounds is potentially valuable for preventing oxidative frameshift mutations in the inflammatory settings. The dual-fluorescent reporter system with improved sensitivity will facilitate identification of additional compounds that modulate frameshift mutations relevant to cancer initiation and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2543. doi:1538-7445.AM2012-2543
Abstract 2549: Chromosomal instability and DNA repair status during folic acid deficiency in breast cancer cells
Authors: Wyatt Michael D. , Brady Katie , Litvinchuk Alexandra , Noxon Virginia , Wang Xiao-Hong , Sobol Robert W.
Published: August 2012

Folic acid deficiency is thought to be procarcinogenic because it promotes the incorporation of genomic uracil. Base excision repair (BER) normally prevents genomic instability by removing procarcinogenic base lesions. However, BER might paradoxically contribute to folate deficient-carcinogenesis if the pathway is initiated but cannot be completed, resulting in the accumulation of BER intermediates (DNA strand breaks). DNA polymerase ≤ (POLα) performs two key steps in BER and so we utilized human metastatic breast cancer cells in which the expression of POLα has been eliminated or transgenically expressed to determine the influence of POLα on chromosomal stability. We found that folic acid deficiency induced premature chromatin condensation (PCC) and strand breaks in POLα deficient cells, demonstrating that folic acid deficiency and BER deficiency induce chromosomal instability. We also examined the expression and localization of PARP1, a key component of BER and strand break repair. PARP1 was not cleaved during folic acid deficiency, suggesting that apoptosis was not occurring under these conditions. More interestingly, PARP1 was dramatically re-localized within the nucleus in POLα deficient cells. We are currently examining the effect of PARP inhibitors and the activation of PARP under these conditions. Collectively, the data suggests that folic acid deficiency in combination with deficiency in POLα is capable of inducing gross chromosomal instability that invokes DSB repair pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2549. doi:1538-7445.AM2012-2549
Abstract 255: Identification of a novel TRAIL sensitizer in TRAIL-resistant cancer cells
Authors: Park So Jung , Jo Yoon Kyung , Ha Ye Jin , Kim Jin Cheon , Cho Dong-Hyung
Published: August 2012

The Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL), a TNF-family member is a highly promising agent for cancer therapy as it selectively induces apoptosis in various cancer cells but not in normal cells. However, many primary tumors are resistant to TRAIL-induced apoptosis and the resistant mechanisms are not fully understood. To identify novel TRAIL signaling regulatory molecules, we screened a siRNA library that target 719 whole human protein kinases in TRAIL-resistant lung cancer cell line (A549 cell). As a screening result, we identified a member of NIMA-related kinases as a new regulator of TRAIL-induced cell death. NEK family play key role in controlling mitosis but the role in TRAIL signaling was not elucidated. Down-regulation of NEK by siRNA dramatically increased TRAIL-induced apoptosis in TRAIL resistant cancer cells including A549 cells. However it was not effective on both etoposide and TNF/CHX-mediated apoptosis. In addition, inhibition of NEK highly activated caspase-3 in response to TRAIL. On the other hand, over-expression of NEK reduced TRAIL-induced apoptosis in TRAIL sensitive cells. Our findings suggest that NEK is a novel negative modulator of TRAIL-induced apoptosis in cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 255. doi:1538-7445.AM2012-255
Abstract 2550: Expanded simple tandem repeat (ESTR) mutations in offspring of benzo(a)pyrene exposed DNA repair deficient male mice
Authors: Godschalk Roger , Verhofstad Nicole , Linschooten Joost , Benthem Jan Van , Steeg Harry Van , Yauk Carole , Schooten Frederik-Jan Van
Published: August 2012

Germ line mutations can be transmitted to the offspring and may thus affect an infinite number of generations thereafter. To study germ line mutations, expanded simple tandem repeats (ESTR) can be used as a sensitive tool, because they are known to be highly unstable and therefore exhibit high mutation rates. This facilitates the assessment of germ line mutations in a relatively small number of offspring following exposures that may pose a genetic risk to the germ line. We used two ESTR loci Ms6-hm and Hm-2 in families of benzo(a)pyrene (BaP) exposed and unexposed male mice, proficient or deficient for global genome repair (Xpc-/-). Male mice (C57BL/6) were exposed to BaP during 6 weeks (13 mg/kg bw, 3 times per week) and were crossed with female mice (Balb/C) 6 weeks after the last exposure to obtain offspring and to ensure the analysis of mutations originating from spermatogonial stem cells. The overall ESTR mutation rate at both ESTR loci, irrespective of the DNA repair capacity, was higher in offspring of benzo(a)pyrene exposed mice than in control mice (9.4% vs. 4.5%, P=0.054). This effect was predominantly seen in BaP exposed DNA repair deficient Xpc-/- mice; mutation frequencies were 12.5% in the exposed group (12 out of 96 offspring carried a mutant allele) and 3.4% in the unexposed group (3 out of 87), respectively (P=0.024). In DNA repair proficient mice, BaP was unable to increase the mutation frequency transmitted to the offspring (5.8% vs. 5.5% for offspring of exposed and unexposed fathers). These results indicate that analysis of tandem repetitive sequences is a sensitive method for the detection of germline mutations transmitted to the offspring and that the environmental mutagen BaP is able to induce germ line mutations in DNA repair deficient mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2550. doi:1538-7445.AM2012-2550
Abstract 2551: New understanding of cancer induction by ionizing radiation
Authors: Burns Fredric J. , Tang Moon-Shong (Eric) , Wu Feng
Published: August 2012

Ionizing radiation is a unique carcinogen that provides an opportunity to vary the location of biological damage within a cell nucleus. For example, electron and x-ray beams deposit dose uniformly and randomly, while energetic heavy ions deposit dose in densely packed straight lines along the ion paths. Cancer yields for these 2 radiations vary as follows: upward curvature with dose rate sparing for random and linearity and no dose rate sparing for heavy ions. Dose rate sparing has long been assumed to be based on biological repair, but it has never been explained why dose responses should differ between these radiation or why repair should be ineffective for heavy ions. To understand this better, consider how the distribution of a typical molecular lesion, DNA double strand breaks (DSBs), varies with the type of radiation. For heavy ions at a specific energy, DSBs are expected to be lined up at random intervals along each track, their number being proportional to average track length in each nucleus, i.e. DSBs per nucleus are proportional to radiation dose (D). Increased charge on the heavy ion causes increased lineal energy transfer (L), i.e. DSBs are also proportional to L. Similarly for heavy ions fusions between DSBs would also be proportional to D and L. The comparable scenario for randomly deposited radiation differs in that the DSBs are distributed randomly with the nuclei. Their number remains proportional to radiation dose, but now fusions would exhibit proportionality to dose-squared as a direct result of the randomness of the DSBs. Moreover randomness eliminates simultaneity. Consequently DSBs can be eliminated before second ones occur close enough to fuse, which automatically gives rise to dose rate sparing. DSB fusion yields based on the above considerations would have the form Yfusions = CfLD + BfD2 with no dose rate sparing for the first term and positive dose rate sparing for the second term. If fusions are the initial lesion of radiation carcinogenesis, then Ycancer = CcaLD + BcaD2. Extensive cancer data in rat skin based on radiations with very different L values (56Fe (L = 150 keV/µ), 40Ar (L = 125 keV/ µ), 29Ne (L = 26 keV/ µ), 1H (L = 0.34 keV/ µ).) and doses are presented that confirm the existence of independent linear and dose-squared terms and that dose rate sparing only applies to the dose-squared term. The data support the proposition that DSB fusions could be the initial lesion of radiation carcinogenesis in the rat skin model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2551. doi:1538-7445.AM2012-2551
Abstract 2556: Stat1 plays a major role in skin tumor promotion by chrysarobin but not TPA
Authors: Bozeman Ronald , DiGiovanni John
Published: August 2012

The cellular and molecular mechanisms that underlie promotion by diverse tumor promoting agents remain to be fully elucidated. Studies using Stat1 knockout (KO) mice have shown that they were highly resistant to two-stage skin carcinogenesis using chrysarobin as the promoter. In contrast, Stat1 KO mice treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) as the tumor promoter exhibited no significant differences in tumor formation. To further investigate mechanism(s) whereby Stat1 mediates skin tumor promotion by chrysarobin Stat1 KO mice and wild-type (WT) controls were treated with the respective tumor promoters and Stat1-dependent signaling pathways were evaluated. Mice deficient in Stat1 displayed an attenuated induction of epidermal Cox-2 mRNA and protein as well as reduced PGE2 levels compared to WT controls following topical application of chrysarobin, whereas, mice treated with TPA exhibited no significant differences in Cox-2 levels or PGE2 levels. Other results also suggest that Stat1 may regulate various cytokine/chemokines involved in the inflammatory response following chrysarobin treatment. Preliminary experiments have also shown that Stat1 KO mice exhibit reduced induction of interferon regulatory factor-1 (IRF-1) following chrysarobin treatment, compared to WT controls. In contrast, treatment with TPA caused no significant differences in expression of IRF-1. Collectively, these findings suggest that chrysarobin may elicit a Stat1-dependent signaling response that is dependent on IRF-1, whereas TPA-mediated tumorigenesis is independent of this signaling pathway. These and other ongoing studies aimed at characterizing the role of Stat1 and IRF-1 signaling in chrysarobin-mediated tumor promotion will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2556. doi:1538-7445.AM2012-2556
Abstract 256: Involvement of ERK and ROS in CLEFMA-induced apoptosis in lung cancer cells
Authors: Yadav Vivek R. , Sahoo Kaustuv K. , Awasthi Vibhudutta
Published: August 2012

CLEFMA (4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid) is novel synthetic molecule with potent growth inhibitory activity in human cancer cells. Our microarray gene expression analysis suggested that it induces oxidative stress in lung adenocarcinoma H441 cells but not in normal lung fibroblasts. That oxidative stress is a triggering mechanism for both apoptotic and autophagic cell death has been supported by several recent reports. Compared to normal cells, the cancer cells are more vulnerable to oxidative stress-induced cell death because of their high metabolic activity, increased likelihood of electron escape during oxidative phosphorylation and overburdened antioxidant system. In this study, we tested our hypothesis that CLEFMA-induced apoptotic cell death was dependent on the accompanying increase in oxidative stress in H441cells. Methods: Inhibition of cell growth by CLEFMA (10 µM) was estimated by MTT or hexosaminidase assay. ROS production, cell cycle and apoptotic cell population were assessed by flow cytometry. The phosphorylation levels of extracellular signal-regulated kinase (ERK1/2), p38 and JNK1/2 were examined by Western blotting. N-acetylcysteine (NAC, 5 mM) and PD98059 (5 mM) were used as ROS scavenger and ERK1/2 inhibitors, respectively. The molecular markers of apoptosis were analyzed by RT-PCR and immunobloting. Results: NCI-DTP program confirmed anticancer activity of CLEFMA in 60 human cancer cell lines. CLEFMA-treated H441 cells showed typical molecular signatures of apoptosis- upregulated pro-apoptotic and downregulated anti-apoptotic genes. There was time-dependent activation of caspases 3/7, PARP cleavage and BAX; the expression of anti-apoptotic XIAP1 and BCL-xL was reduced, whereas pro-apoptotic p53 was increased. Since BID was not activated, it appears that CLEFMA works through intrinsic apoptosis pathway. CLEFMA also induced sustained activation of ERK, p38 and JNK. The pharmacologic inhibition of ERK, but not that of p38 and JNK, resulted in decreased anti-proliferative activity of CLEFMA. ERK inhibition with PD98059 abrogated CLEFMA-induced apoptosis. The sustained ERK activation was found to be associated with the inhibition of dual specificity phosphatase, DUSP6, expression. Treatment with ROS scavenger NAC abrogated CLEFMA-induced cell death, ERK activation and pro-apoptotic biomarkers, and reinstated DUSP6 expression. Conclusion: From the results of this study, we conclude that CLEFMA-induces apoptosis by enhancing oxidative stress in the cancer cells, and that the accompanying prolonged ERK activation is required for CLEFMA-induced H441 cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 256. doi:1538-7445.AM2012-256
Abstract 2562: Ultra sensitive immuno-array based multiplexed assay capable of profiling RTKs and signaling proteins, heterodimers, and growth factors for routine analysis of clinical samples with limited availability
Authors: Hoe Nicholas , Zhou Jinyao , Kirkland Richard , Nguyen Anthony , Roxas Gio , Kuy Crystal , Mateling Michael , Lee Tani , Magonova Katya , Kim Phillip , Liu Xinjun , Singh Sharat
Published: August 2012

Patient stratification based on target protein expressions or mutations alone has limitations in selecting efficacious therapeutic regimens for solid tumors. Information on the activation status of signaling pathway proteins as determined by their phosphorylation states and the level of expression, profiling of growth factors responsible for its activation, and their dimerization partners, can significantly influence clinical decisions. Herein we report a comprehensive analysis of key receptor tyrosine kinases (HER1, HER2, p95HER2, HER3, cMET, IGF1R, FGFRs, and others) and their downstream signal proteins (PI3K, Shc, AKT, MEK, ERK, PRAS40, RPS6, P70S6K, RSK), and dimerization partners (HER1:HER2, HER2:HER3, HER1:HER3, and others), and secreted growth factors (AREG, EREG, NRG1, HB-EGF, HGF, BTC, EGF, TGF, VEGF, and IGF-1) based on a multiplexed immunoarray CEER (Collaborative Enzyme Enhanced Reactive-immunoassay) platform. CEER platform utilizes the formation of an unique “triple-antibody-enzyme-channeling” immuno-complex. This complex requires co-localization of two detector-antibodies conjugated with corresponding channeling-enzymes once target proteins are bound by the capture antibodies. The immuno-collaboration between two detector enzymes, glucose oxidase (GO, conjugated to anti-target protein antibodies) and horseradish peroxidase (HRP, conjugated to anti-phosphorylated sites within the target) via target-specific, tyramide-mediated signal amplification enables target protein expression /phosphorylation profiling with validated analytical sensitivity between 1 to 10 cells. Using the CEER platform, quantitative modulations of target protein(s) in response to targeted agent(s) and treatment effect on associated downstream and upstream pathway proteins were measured. Due to the extreme sensitivity and specificity, CEER can be utilized in clinical samples with limited availability (e.g., FNA or CTCs). A comprehensive signaling pathway analysis can be a critical guide for selecting effective clinical strategies for targeted drug combinations or sequential therapeutic regimens. In addition, continued signaling pathway profiling after drug administration can monitor drug response and provide valuable information on potential drug resistance mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2562. doi:1538-7445.AM2012-2562
Abstract 2563: Characterization of a p53 knockout Sprague Dawley rat
Authors: McCoy Aaron M. , Cui Xiaoxia , Weinstein Edward
Published: August 2012

The tumor suppressor protein TP53 plays a crucial role in cancer biology, and the Tp53 gene is the most mutated gene in human cancer. The Tp53 knockout mouse models have been widely used in cancer etiology studies and in search for a cure of cancer with some limitations that other model organisms might help overcome. Via pronuclear microinjection of Zinc Finger Nucleases (ZFNs), we created a Tp53 knockout rat that contains an 11 bp deletion in exon 3. This deletion produces a frameshift and premature stop codons in the open reading frame. In cohorts of 30 homozygous, 35 heterozygous and 25 wild type rats, the homozygotes have an average life span of 126 days, and half of heterozygotes died by a year of age. Both heterozygous and homozygous knockouts developed a wide spectrum of tumors, most commonly, sarcomas. However, there is a strikingly high incidence of brain lesions, especially in homozygous animals. In addition, unlike the Tp53 knockout mice, both male and female homozygous Tp53 knockout rats are fertile. We believe this knockout rat line will be useful in studying cancer types rarely observed in mice and in carcinogenicity assays for drug development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2563. doi:1538-7445.AM2012-2563
Abstract 2567: DIM regulates anoikis through sonic hedgehog in ovarian cancer cells
Authors: Kandala Prabodh K. , Srivastava Sanjay K.
Published: August 2012

Ovarian cancer is the second leading gynecological cancer with poor prognosis. By the time of detection, it usually metastasizes to peritoneum or organs such as stomach and liver. Detachment of a cell from extracellular matrix (ECM) allows the cells to travel and lodge at metastatic site. Anoikis is an emerging form of cell death that is associated with the apoptosis of cells detached from ECM. Normal or immortal epithelial cells are sensitive to anoikis. However, carcinomas are generally associated with resistance to anoikis. Hence, induction of anoikis is an important step in preventing metastasis. We have recently shown that diindolylmethane (DIM), a compound present in cruciferous vegetables reduces the survival of ovarian cancer cells by inducing apoptosis. Here, we evaluated the effect of DIM in inducing anoikis in ovarian cancer cells. A2780, SKOV-3 and OVCAR-429 cells were grown on low attachment plates and treated with various concentrations of DIM for 24 hours and analyzed. Our results show that DIM treatment resulted in the concentration-dependent anoikis in all the cell lines. For example, anoikis induction was approximately 50-70% in all the cell lines treated with 75µM DIM. Furthermore, Integrin Linked Kinase 1 (ILK-1), an important regulator of anoikis was significantly down-regulated by DIM treatment. Western blotting revealed cleavage of caspase-3 and PARP by DIM treatment, confirming anoikis induction in our model. Few recent studies showed that Sonic Hedgehog (Shh) pathway is activated in ovarian tumors. Shh pathway is also associated with metastasis of various cancers. We therefore next determined whether Shh pathway was involved in anoikis induction by DIM. Our results demonstrated that DIM treatment reduced the expression of Shh and Gli1 in a concentration-dependent manner. Moreover, Shh secretion as well was reduced by DIM treatment in ovarian cancer cells. Importantly, Shh pretreatment significantly inhibited DIM-induced anoikis in A2780 ovarian cancer cells. Treatment of cells with Shh also reduced the cleavage of caspase-3 or PARP induced by DIM treatment indicating the involvement of Shh pathway in anoikis. Taken together, these results suggest that DIM induces anoikis in ovarian cancer cells by regulating sonic hedgehog signaling. Further in-depth mechanistic studies are in progress to establish anoikis induction by DIM and to validate it in an in vivo model. [Supported in part by R01 grants CA 1065953 and CA 129038 awarded to S.K.S by National Cancer Institute, NIH]

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2567. doi:1538-7445.AM2012-2567
Abstract 2571: The effect of 15-deoxy-α12,14-prostaglandin J2 on osteolytic bone metastasis by breast cancer cells
Authors: Kim Ki-Rim , Chung Won-Yoon , Park Kwang-Kyun
Published: August 2012

The majority of patients dying from cancer of the breast or prostate have metastases to bone. Bone is a dynamic tissue and metastatic breast cancer cells indirectly induce osteoclast-mediated bone resorption by secreting osteolytic factors, resulting in the increase of bone destruction. 15-deoxy-α12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for peroxisome proliferator-activated receptor-α, is the electrophilic cyclopentenone prostaglandin as one of the terminal products of the cyclooxygenase-2-catalyzed reactions. Through the post-translational modification of redox and inflammatory signaling molecules, this prostaglandin exerts various biological actions, such as potent anti-inflammatory, anti-proliferative, anti-fibrotic, and apoptotic activities. In particular, 15d-PGJ2 has been known to exhibit anti-tumor effects in a variety of cancers. This study explores the in vitro and in vivo effect of 15d-PGJ2 on osteolytic bone metastasis by breast cancer cells. 15d-PGJ2 inhibited the migration and invasion of MDA-MB-231 metastatic human breast cancer cells, in a dose-dependent manner. Treatment with 15d-PGJ2 inhibited osteoclast formation and reduced pit formation by osteoclast activity in receptor activator of the NF-κB ligand (RANKL)-treated mouse bone marrow macrophages (BMMs). In differentiated osteoclasts, the activities of matrix metalloproteinases (MMPs) and cathepsin K markedly decreased by 15d-PGJ2 treatment. Furthermore, we inoculated MDA-MB-231 cells into the left cardiac ventricle of nude mice and then subcutaneously injected 15d-PGJ2 for 6 weeks. The histomorphometrical analysis, including bone volume and microstructure, revealed that 15d-PGJ2 inhibited bone destruction by metastatic breast cancer cells. These results demonstrate that 15d-PGJ2 may inhibit migration/invasion of breast cancer cells, prevent RANKL-mediated osteoclast differentiation/ activity, and suppress breast cancer cells-mediated osteolytic lesion in mice. Thus, 15d-PGJ2 can be a therapeutic agent for cancer metastasis to bone.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2571. doi:1538-7445.AM2012-2571
Abstract 2573: Carnosol and carnosic acid, from rosemary, promote endoplasmic reticulum stress and androgen receptor degradation in prostate cancer cells
Authors: Petiwala Sakina M. , Li Gongbo , Johnson Jeremy J.
Published: August 2012

Background: Prostate cancer is one of the most prevalent forms of cancer among men, especially in the United States. Previously, we have shown that carnosol, a dietary diterpene from rosemary (Rosmarinus officinalis), has a unique function in that it is a dual disruptor of androgen and estrogen receptor alpha. Herein, we examined how carnosol and another dietary diterpene from rosemary, carnosic acid, promote endoplasmic reticulum (ER) stress and modulate the activity and expression of androgen receptor (AR). Materials and Methods: Experiments were performed using the prostate cancer cell lines 22Rv1 and LNCaP cells. Cell proliferation and cell viability were studied using BrdU and MTT assay respectively. Western blotting, ELISA and siRNA were used to evaluate the relationship between ER stress and androgen receptor degradation. Results: Both, carnosic acid and carnosol, inhibited growth and decreased the cell viability of 22Rv1 and LNCaP cells in a dose-dependent manner. The anti-proliferative effect was associated with apoptosis as evidenced by a significant increase in levels of cleaved caspase-3 protein. Interestingly, these diterpenes modulated proteins associated with ER stress including BiP, IRE1 and CHOP. Furthermore, we saw a dose-dependent decrease in expression of AR in 22Rv1 and LNCaP cells when treated with either carnosol or carnosic acid. The proteasome has been shown to be regulated by multiple proteins including proteins associated with ER stress. The AR expression increased in presence of proteasome inhibitor, MG-132, suggesting that decrease in AR expression was due to proteasomal degradation. Carnosic acid and carnosol, also showed a decrease in expression of prostate-specific antigen (PSA) levels in a dose-dependent manner. Conclusion: Both, carnosic acid and carnosol, induce ER stress-mediated apoptotic cell death in prostate cancer cell lines, 22Rv1and LNCaP cells, leading to disruption of AR expression. Our data suggests that these natural diterpenes possess strong anticancer activity and should be evaluated further as potential chemopreventive and/or chemotherapeutic agents for controlling prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2573. doi:1538-7445.AM2012-2573
Abstract 2575: DNA microarray and connectivity map analysis reveals estrogen-like activity of Chinese medicinal formula Si-Wu-Tang
Authors: Liu Mandy M. , Wen Zhining , Wang Zhijun , Zuo Zhong , Chow Moses S.S. , Shi Leming , Huang Ying
Published: August 2012

To pursue a systematic approach for discovery of potential mechanisms of action and new therapeutic use of traditional herbal medicines, we used DNA microarray, bioinformatics and the “Connectivity Map” (CMAP) analysis to investigate the gene expression profiling of Chinese herbal medicines. We demonstrated that this approach can be used to elucidate novel molecular mechanisms for the Chinese medicinal formula Si-Wu-Tang (SWT) which is widely used for women's health in Asian countries. The human breast cancer MCF-7 cells treated with beta-estradiol (E2, 0.1 µM) or SWT extract (0.0256, 0.256, and 2.56 mg/ml) for six hours showed significant gene expression changes. The differentially expressed genes related to SWT treatment were used to compare with the gene expression profiles of 1,309 compounds in the CMAP database. Such comparison revealed that the CMAP profile of E2-treated MCF-7 cells showed an excellent match with SWT treatment (permutation p<0.00001), consistent with SWT's widely claimed use for women's diseases and suggesting a phytoestrogen effect. Many genes strongly up-regulated by E2 were similarly upregulated by SWT, e.g., GREB1, a well-known estrogen regulated gene. Of interest with regard to the potential cancer preventive activity of SWT, the oncogenes MYBL1 and cyclin D1 were found to be strongly induced by E2 but not by SWT. In addition, SWT inhibited expression of estrogen receptor (ER)-alpha, but not ER-beta. Consistently, at low concentration (1.5 and 3.0 mg/ml), SWT stimulated MCF-7 (ER-positive) cell growth, while at high concentration (> 6.0 mg/ml), it inhibited the growth of MCF-7. The combination of SWT with tamoxifen, a selective estrogen receptor modulator, demonstrated enhanced growth inhibitory effects on MCF-7 cells. The results our study suggest that SWT may possess a non-toxic chemopreventive effect for human breast cancer. This study also demonstrated the feasibility of using microarray gene expression profiling in combination with the CMAP data mining to discover new molecular signature that are present in natural products.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2575. doi:1538-7445.AM2012-2575
Abstract 2577: Blocking Janus activated kinase-2 activation by DIM suppresses ovarian tumor growth in vitro and in vivo
Authors: Kandala Prabodh K. , Srivastava Sanjay K.
Published: August 2012

Ovarian cancer is one of the leading causes of gynecological deaths in the United States and several other countries. Genetic alterations including overexpression of EGFR and STAT-3 play a crucial role in the signal transduction pathways that regulate key cellular functions such as cell survival, proliferation, metastases etc. Several epidemiological studies suggested a decreased risk of ovarian cancer in the population consuming Brassica vegetables. 3,3′-diindolylmethane (DIM) is an indole compound present in Brassica vegetables. In our previous studies, we have demonstrated that DIM induces apoptosis in ovarian cancer cells by inhibiting the activation STAT-3. However, the exact mechanism of STAT-3 regulation by DIM was not clear. Accumulating evidences suggest that Janus Activated Kinases (JAK's) play a crucial role in regulating STAT-3. Our current results show that DIM inhibits the activation of JAK-2 by blocking its phosphorylation at Tyr-1007/1008 in a concentration-dependent manner in SKOV-3 and OVCAR-3 cells. Inhibition of JAK-2 phosphorylation by DIM was as early as one hour after DIM treatment. The expression of survivin was significantly reduced by DIM. IL-3 is a cytokine that specifically activates JAK-2. Treatment of SKOV-3 or OVCAR-3 cells with 10ng/ml IL-3 for 15min not only increased the activation of JAK-2 and STAT-3, but also induced the expression of STAT-3. DIM treatment almost completely blocked IL-3 induced phosphorylation of JAK-2 and STAT-3 in SKOV-3 and OVCAR-3 cells. Several studies have shown that JAK's are regulated by EGFR, hence to test this, cells were stimulated with EGF, a specific ligand of EGFR. As expected, EGF treatment increased the activation of JAK-2 and STAT-3 in SKOV-3 cells. Nonetheless, DIM treatment blocked the phosphorylation of JAK-2 and STAT-3 induced by EGF. Furthermore, our in vivo studies demonstrated that oral administration of 3 mg DIM for 40 days significantly suppressed the growth of ovarian tumors xenografts in athymic nude mice. The tumors of DIM treated mice exhibited reduced phosphorylation of JAK-2, EGFR and STAT-3 and increased apoptosis. These results indicate that DIM induces apoptosis in ovarian tumor cells by regulating JAK-2 activation in vitro and in vivo. [Supported in part by R01 grants CA 106953 and CA 129038 awarded to S.K.S by National Cancer Institute, NIH]

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2577. doi:1538-7445.AM2012-2577
Abstract 2578: Anticancer action of garcinol in vitro and in vivo is mediated through inhibition of STAT-3 signaling
Authors: Ahmad Aamir , Aboukameel Amro , Ali Shadan , Li Yiwei , Bao Bin , Kong Dejuan , Banerjee Sanjeev , Sarkar Fazlul H.
Published: August 2012

Garcinol, obtained from Garcinia indica, has exhibited some promising anticancer activity. In particular, our earlier work has demonstrated its ability to inhibit cell proliferation, and induce apoptosis in multiple cancer cell lines originated from breast, prostate as well as pancreatic cancer. However, its exact mechanism of action remains largely unclear. Here we show that garcinol targets signal transducer and activator of transcription-3 (STAT-3) signaling pathway. STAT-3 is frequently found to be activated in many cancer types and this is the first report on such action of garcinol leading to its anticancer effects. Garcinol inhibited total as well as phosphorylated STAT-3 in breast, prostate and pancreatic cancer cell lines, and was also found to inhibit cell migration and colony formation of all the cancer cell lines tested. STAT-3 phosphorylation was inhibited by garcinol in a dose-dependent manner. We also observed an inhibitory effect of garcinol on IL-6-induced STAT-3 phosphorylation and production of uPA and VEGF which might explain the reduced migration and aggressiveness in cells treated with garcinol. The results were further verified in vivo in a MDA-MB-231 breast cancer mouse xenograft model where administration of garcinol significantly inhibited tumor growth, and western blot analysis of tumor lysates indicated reduced STAT-3 expression and activation. These results indicate that garcinol may have translational potential as chemopreventive or therapeutic agent against multiple cancers through inhibition of STAT-3 signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2578. doi:1538-7445.AM2012-2578
Abstract 2579: Capsaicin inhibits β-catenin signaling in pancreatic cancer cells by disrupting nuclear β-catenin/TCF-1 complex: critical role of STAT-3
Authors: Pramanik Kartick C. , Boreddy Srinivas Reddy , Srivastava Sanjay K.
Published: August 2012

β-catenin/TCF signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers including pancreatic cancer. In our previous study, we have shown that capsaicin-induced apoptosis in pancreatic cancer cells was associated with mitochondrial depolarization and oxidative stress. In the present study, we evaluated the effect of capsaicin on β-catenin/TCF signaling. In a concentration and time-dependent study, we observed that capsaicin treatment inhibits the activation of disheveled (Dsh) and phosphorylation of STAT-3 at Tyr705 in L3.6PL, Panc-1 and MiaPaCa-2 pancreatic cancer cells. Capsaicin treatment activated GSK-3β by inhibiting its phosphorylation and further activated APC and Axin multicomplex leading to the ubiquitination and degradation of β-catenin. Pre-treatment of cells with MG-132 blocked capsaicin-mediated proteasomal degradation of β-catenin. The expression of TCF-1 and β-catenin-responsive proteins c-myc and cyclin D1, were also decreased in response to capsaicin treatment. Significant cleavage of caspase-9, caspase-3 and PARP was observed in all the cell lines. To establish the involvement of β-catenin in capsaicin-induced apoptosis, cells were treated with LiCl or SB415286, inhibitors of GSK-3β. Our results reveal that capsaicin treatment suppressed LiCl-mediated activation of β-catenin signaling. Our results further showed that capsaicin blocked the nuclear translocation of β-catenin and TCF-1. The immunoprecipitation results also indicated that capsaicin treatment reduced the interaction of β-catenin with TCF-1 in the nucleus. Interestingly, STAT-3 over expression or STAT-3 activation by IL-6, significantly increased the levels of β-catenin and attenuated the effects of capsaicin in inhibiting β-catenin signaling. These observations indicate the direct regulation of β-catenin by STAT-3 in our model. Taken together, our results suggest that capsaicin-induced apoptosis in pancreatic cancer cells was associated with the inhibition of β-catenin signaling due to the dissociation of β-catenin/TCF-1 complex leading to apoptosis, and this process was orchestrated by STAT-3. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2579. doi:1538-7445.AM2012-2579
Abstract 2580: Induction of estrogen receptor alpha by GW9662 sensitizes mammary glands to tamoxifen treatment in organ culture
Authors: Mehta Rajendra G. , Peng Xinjian , Roy Sarbani , Mehta Rajeshwari , Kopelovich Levy
Published: August 2012

Peroxisome proliferator-activated receptor gamma (PPARδ) are ligand activated nuclear receptor and binds to natural and synthetic ligands. Earlier we had reported that PPARδ agonist troglitazone inhibited the development of precancerous lesions induced by DMBA in mouse mammary gland organ culture (MMOC). In the present study we evaluated the effects of PPARδ agonist GW7845 and antagonist GW9662 on the development of 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary alveolar lesions (MAL) in ER negative mammary glands in MMOC. The results showed that both GW7845 and GW9662 inhibited development of ER- MAL in MMOC. Since PPARδ and ER cross talk has been reported in the literature, we determined the effects of GW9662 on the induction of ER and PR in this model. Real-time PCR studies and immunohistochemical analyses showed that GW9662 induced ERα in the absence of estradiol, whereas GW7845 did not induce ERα under these conditions. Both GW7845 and GW9662 failed to suppress mRNA expression of PPARδ in the mammary glands that are otherwise ER negative. However GW9662 suppressed the expression of PPARδ response genes adipsin and aP2. Conversely, GW7845 an agonist of PPARδ enhanced the expression of aP2 and adipsin. These results suggested that the effects of these agents are mediated by PPARδ in MMOC. Finally, Tamoxifen does not inhibit MAL in MMOC, however sequential treatments with GW9662 followed by Tamoxifen suppressed development of DMBA-induced MAL in these glands. Since the ER negative breast cancer patients are generally not treated with SERM, inducing ERα in the mammary cells by GW9662 possibly could make them responsive to Tamoxifen or other SERM. [This work was supported by N01 CN433303]

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2580. doi:1538-7445.AM2012-2580
Abstract 2582: 2-ME2-induced biological effects in prostate cancer: Role for miR127-3p/SET8
Authors: Xie Jianping , Batth Izhar Singh , Bedolla Roble , Li Guiming , Chen Hung-I Harry , Chen Yidong , Ghosh Rita , Kumar Addanki Pratap
Published: August 2012

Prostate cancer is the second most common cause of death related to cancer in Western societies. 2-Methoxyestradiol (2-ME2), an endogenous metabolite of 17-α estradiol inhibits tumor cell proliferation in various cancer cells, including the prostate. Previous studies from our laboratory showed that 2-ME2 (i) inhibits proliferation of both androgen responsive and independent cells through induction of apoptosis involving G2/M check point block; (ii) prevents the development of prostate tumors in transgenic adenocarcinoma of mouse prostate (TRAMP) model. Although various molecular targets have been proposed, the mechanism of action behind its antiproliferative activity is still uncertain. Here we investigated the possible role for 2-ME2 induced antiproliferative activity by examining the altered regulation of microRNA (miRNAs). MicroRNA expression profiling identified miR127-3p as most significantly up regulated in response to 2-ME2 treatment in androgen independent (PC-3) cells. We have also identified histone methylase SET8 as a potential target of miR 127-3p. We have validated these data using Q-PCR. Further 2-ME2 treatment reduced binding of multiple transcription factors including Sp1, Sp3 and NFκB to the FLIP promoter as evidenced by transient expression assays, ChIP and gel-shift assays. Our findings indicate that 2-ME2 suppresses proliferation and induces apoptosis in prostate cancer cells possibly through miR127-3p/SET8 mediated regulation of FLIP. Supported by NIH CA 135451 (APK).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2582. doi:1538-7445.AM2012-2582