bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3036: The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing
Authors: Ellis Nathan A. , Yang Wei-Chih , Yagle Mary , Zhu Jianmei , Huang Jing , Seidman Michael , Matunis Michael J. 
Published: August 2015

Abstract
Regulation of dormant origin firing under conditions of replication stress is poorly understood. The Bloom syndrome DNA helicase BLM functions in maintaining replication fork stability, with sumoylation regulating this function, and BLM deficiency is associated with increased dormant origin firing. We found here that BLM sumoylation levels increase in response to replication stalling by hydroxyurea (HU) and to proteasome inhibition; depletion of the SUMO-targeted ubiquitin ligase RNF4 also causes increased levels of BLM sumoylation. RNF4 directly interacts with BLM and can ubiquitylate sumoylated BLM in vitro. These data indicate that sumoylated BLM is an RNF4 substrate. RNF4 is recruited to DNA repair foci in response to replication stalling. RNF4 depletion causes the accumulation of increased numbers of HU-induced BLM foci, whereas the numbers of RPA, RAD51, and gamma-H2AX foci are unaffected by fork stalling in RNF4-depleted cells. Sister chromatid exchanges are normal in RNF4-depleted cells. These data indicate that RNF4 can regulate BLM retention at stalled forks without affecting repair by homologous recombination. RNF4 depletion reduces cell proliferation and survival of untreated cells, and it confers hypersensitivity to replication stalling by HU. Studies of cell-cycle progression using bromodeoxyuridine incorporation and flow cytometry show that RNF4 depletion causes a delay of re-entry into S phase in HU-treated cells and the replication delay is partially rescued by BLM mutation. The delay is not caused by modification of checkpoint kinase activities. Analysis of replication dynamics using the DNA fiber assay show that RNF4 depletion causes an increase of permanently stalled replication forks and a decrease in activation of dormant origins following recovery from HU-induced replication stress. Co-depletion of RNF4 and BLM partially rescues these defects. Replication dynamics is unaffected by RNF4 depletion in untreated cells. These data indicate that RNF4 regulates BLM's functions in replication fork stability and dormant origin firing, without affecting BLM's roles in homologous recombination. We propose that sumoylation of BLM at stalled replication forks leads to RNF4-mediated ubiquitylation and subsequent proteasome-dependent degradation of BLM, which relieves BLM's active inhibition of dormant origin firing.
Citation Format: Nathan A. Ellis, Wei-Chih Yang, Mary Yagle, Jianmei Zhu, Jing Huang, Michael Seidman, Michael J. Matunis. The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2015-3036
Abstract 304: Formation of uniform and reproducible 3D cancer spheroids in a high throughput plate format
Authors: Neeley Cindy , Revankar Chetana M. 
Published: August 2015

Abstract
Cells grown on flat two dimensional (2D) tissue culture substrates can differ considerably from the three dimensional (3D) physiological environment. There is growing evidence that tumor cell aggregates or cancer spheroids are more representative of tumors in vivo and they exhibit several physiological traits including similar morphology, the formation of cell-cell contacts, decreased proliferation, increased survival rates and a hypoxic core. However, variability in cancer spheroid culture has been a persistent problem for researchers. This variability is linked to medium composition and volume, cell density, duration in culture, and most importantly, the cellular interactions with the culture dish itself. Here, we demonstrate that a hydrophilic polymer coated surface, Thermo Scientific Nunclon Sphera, inhibits cell attachment to the culture dish by blocking binding of the extracellular matrix (ECM). The U bottom wells in the Nunclon Sphera plate provide the ideal size and shape for single spheroid formation in each well, offering consistency and reproducibility to the 3D tumor model system in vitro. The growth of cancer spheroids in the Nunclon Sphera 96U-well plate is superior to that of the non-treated plate with less “satellite colonies” and faster cell aggregation at low seeding density. Cancer cell viability and spheroid health are also evaluated by fluorescence- and colorimetric- based assays in the Nunclon Sphera plates. The Nunclon Sphera 96U-well plate provides an excellent solution for reproducibly generating uniform 3D cancer spheroids that are suitable for many cell-based drug discovery procedures, co-culture studies, and high-throughput drug screening.
Citation Format: Cindy Neeley, Chetana M. Revankar. Formation of uniform and reproducible 3D cancer spheroids in a high throughput plate format. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2015-304
Abstract 305: An innovative 3D porous scaffold-based perfusion bioreactor system for the in vitro maintenance and expansion of primary breast cancer tissue
Authors: Muraro Manuele G. , Muenst Simone , Spagnoli Giulio C. , Oertli Daniel , Weber Walter P. , Soysal Savas D. 
Published: August 2015

Abstract
Introduction: Development of cancer treatment protocols has traditionally been based on studies in experimental animal models or taking advantage of human established tumor cell lines for in vitro and in vivo assays. Here we present an innovative method to culture breast cancer tissues in porous 3D scaffolds by using a perfusion-based bioreactor system.
Experimental procedures: Freshly excised estrogen-receptor positive breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between porous scaffolds under perfusion flow, with “on-line” monitoring of environmental condition. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium in the presence or absence of estrogens. The ability of tumor cells to survive and expand into the scaffold in perfusion culture as well as their ability to recapitulate features of the original breast cancer specimens was histologically assessed.
Results: With this innovative method we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of estrogen to the culture medium was associated with increased expansion of cancer cells into the scaffold starting at 14 days of culture.
Conclusions: Culture of breast cancer tissue in a 3D scaffold-based bioreactor preserves malignant, interstitial and immunocompetent cells present in surgically excised breast cancer samples, thus allowing direct testing of basic cancer research hypotheses in intact, complex human tumor microenvironments. Furthermore, it might allow a direct evaluation of the effects of various treatments on malignant and non-transformed cells. This engineered in vitro model could be extended beyond the context of our primary scientific interest, namely breast cancer, as a broader platform allowing animal-free testing of innovative approaches for the treatment of human malignancies, possibly in the direction of personalized medicine.
Citation Format: Manuele G. Muraro, Simone Muenst, Giulio C. Spagnoli, Daniel Oertli, Walter P. Weber, Savas D. Soysal. An innovative 3D porous scaffold-based perfusion bioreactor system for the in vitro maintenance and expansion of primary breast cancer tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2015-305
Abstract 3052: NAMPT inhibition induces mitochondrial dysfunction leading to apoptosis in chronic lymphocytic leukemia cells
Authors: Bouchard Eric DJ , Mejia Edgard M. , Gehrke Iris , Poeppl Armando G. , Hewitt Donna , Johnston James B. , Gibson Spencer B. , Hatch Grant M. , Banerji Versha 
Published: August 2015

Abstract
We have previously shown that the NAD depletion following inhibition of NAMPT by the NA-mimetic FK866 leads to loss of mitochondrial membrane potential, depletion of cellular ATP, cytochrome C release and caspase activation in primary Chronic Lymphocytic Leukemia (CLL) cells in vitro, and that these events are coupled with an increased production of reactive oxygen species uncharacteristic of metabolic inhibition. In the present study we characterize the effect of NAD depletion on CLL mitochondria and the downstream mechanism of cell death.
We profiled the mitochondrial respiration of CLL cells and control B-lymphocytes, by extracellular flux analysis, over the 48 hours following FK866 treatment and found time-dependant inhibition of respiratory capacity consistent with disruption of the electron transport chain (ETC) in CLL cells. This led to suppression of basal respiration concomitant with the rise in ROS and approximately one day before loss of cellular viability. Interestingly, Zap70-positive CLL cells exhibited increased mitochondrial respiration and respiratory capacity over Zap70-negative CLL cells, but responded similarly to NAMPT inhibition. Additionally, mitochondrial respiration was not effected by NAMPT inhibition in control B-lymphocytes.
Increase autophagic flux, caspase-dependant apoptosis and mitochondrio-nuclear translocation of Apoptosis Inducing Factor (AIF) were detected by western blot. However, neither pan-caspase inhibition with Z-VAD-fmk nor inhibition of autophagy with 3-methyladenine were sufficient to prevent FK866-induced CLL cell death. We conclude that NAD depletion following inhibition of NAMPT leads to disruption of the mitochondrial ETC in CLL cells, leading to mitochondrial initiation of the intrinsic apoptosis pathway. As healthy B-lymphocytes are resistant to this NAD depletion, these downstream effects are also selective for malignant cells.
We are currently working to characterize the roles of NAD and Zap70 in the regulation and function of the CLL ETC. This will not only lead to an improved understanding of CLL metabolism, but will also inform new therapeutic strategies that will effectively employ NAMPT inhibition to treat this yet-incurable disease.
Citation Format: Eric DJ Bouchard, Edgard M. Mejia, Iris Gehrke, Armando G. Poeppl, Donna Hewitt, James B. Johnston, Spencer B. Gibson, Grant M. Hatch, Versha Banerji. NAMPT inhibition induces mitochondrial dysfunction leading to apoptosis in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3052. doi:10.1158/1538-7445.AM2015-3052
Abstract 3056: Mitochondrial DNA copy number and risk of second primary tumors in long-term breast cancer survivors
Authors: Santibanez Miguel , Rodriguez Alma , Hildebrandt Michelle A. , Chang David W. , Wu Xifeng 
Published: August 2015

Abstract
Background. Mitochondria is constantly exposed to reactive oxygen species (ROS), an important factor in carcinogenesis. Altered mitochondrial DNA (mtDNA) copy number in peripheral blood has been associated with increased risks of several cancers, including breast cancer. However, the association of mtDNA copy number with clinical outcomes of breast cancer has not been reported. This study aimed to assess the association of mtDNA copy number with second primary tumors (SPT) in long-term breast cancer survivors.
Methods. We measured mtDNA by quantitative PCR in patients in 68 long-term breast cancer survivors with SPT, 100 long-term breast cancer survivors without SPT and 97 newly diagnosed breast cancer cases. Cases with and without SPT were matched by years since diagnosis, age at time of sample collection and race. Differences between mtDNA copy number among groups were analyzed using the Wilcoxon rank sum test.
Results. MtDNA copy number was significantly higher in long-term breast cancer survivors (either with SPT or not SPT) compared with newly diagnosed breast cancer patients (Mean (SD), 1.03 ± 0.25 vs 0.95 ± 0.27; p = 0.005). We did not observe significant differences of mtDNA copy number between long-term survivors with SPT and without SPT. However, in stratified analyses by epidemiological and clinical variables, we observed lower mtDNA copy number in HER2-positive long-term survivors with SPT than those without SPT (0.84 ± 0.13 vs 1.27 ± 0.44; p = 0.010) and in pre-menopause long-term survivors with SPT than those without SPT (0.95 ± 0.20 vs 1.06 ± 0.22; p = 0.034). A borderline significance of mtDNA copy number between SPT and non-SPT ER-negative long-term survivors was also noted (0.90 ± 0.17 vs 1.18 ± 0.41, p = 0.056).
Conclusion. Higher mtDNA copy number was found in long-term breast cancer survivors compared to newly diagnosed cases. There were significant associations between lower mtDNA copy number and SPT in long-term breast cancer survivors who are HER2+ or are pre-menopause. These results indicate a role of mtDNA copy number in long-term breast cancer survivorship.
Citation Format: Miguel Santibanez, Alma Rodriguez, Michelle A. Hildebrandt, David W. Chang, Xifeng Wu. Mitochondrial DNA copy number and risk of second primary tumors in long-term breast cancer survivors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3056. doi:10.1158/1538-7445.AM2015-3056
Abstract 3057: Potentiation of LPS-induced toxicity in human hepatoma HepG2 cells by aspirin and its protection by N-acetyl cysteine
Authors: Raza Haider , John Annie , shafarin Jasmin 
Published: August 2015

Abstract
Bacterial lipopolysaccharides (LPS) induce cytotoxicity in vitro and inflammation-induced toxic responses in vivo. Use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, has been reported to be beneficial in inflammation-associated diseases like cancer and diabetes. Their precise molecular mechanisms, however, are not clearly understood. Our previous studies on aspirin treated HepG2 cells strongly suggest cell cycle arrest and induction of apoptosis associated with mitochondrial dysfunction. In the present study, we have further demonstrated that HepG2 cells treated with LPS alone or in combination with aspirin induces subcellular toxic responses which are accompanied by increase in reactive oxygen species (ROS) production, oxidative stress, apoptosis and mitochondrial respiratory dysfunction. The LPS/Aspirin induced toxicity was attenuated by pretreatment of cells with N-acetyl cysteine (NAC). Oxidative stress and glutathione-dependent redox-homeostasis in the mitochondria were altered more severely compared to the extramitochondrial compartment of cells. Pre-treatment of HepG2 cells with NAC has demonstrated selective protection in redox homeostasis and mitochondrial dysfunction. Our results suggest that altered glutathione (GSH)-redox metabolism, oxidative stress and mitochondrial function in HepG2 cells play a critical role in LPS/aspirin-induced cytotoxicity. These results may help in better understanding the pharmacological and toxicological properties of NSAIDs in cancer cells exposed to bacterial endotoxins. (Supported by Sheikh Hamdan Medical Research Grant and Terry Fox Cancer Research Funds and a fund from CMHS Research Committee).
Citation Format: Haider Raza, Annie John, Jasmin shafarin. Potentiation of LPS-induced toxicity in human hepatoma HepG2 cells by aspirin and its protection by N-acetyl cysteine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3057. doi:10.1158/1538-7445.AM2015-3057
Abstract 3065: MicroRNA-137 confers chemoresistance by suppressing CASP3 in lung cancer cells
Authors: Shu Te-Jen , Yu Sung-Liang , Chen Jeremy J.W. 
Published: August 2015

Abstract
Lung cancer is the leading cause of cancer-related death. Although molecular targeting therapy (MTT) could benefit patients with EGFR activating mutations, chemotherapy still is the first choice of EGFR wild type patients, MTT failed ones and patients of developing countries without regarding their EGFR status. However most of these chemotherapy-treated patients will develop drug resistance eventually. Here, we identified miR-137 might contribute to chemo-resistance in lung cancer in part. Our previous study identified 5 miRNAs (let-7a, miR-182*, miR-372, miR-137 and miR-221) whose expression can be an independent marker to predict the outcome of non-small cell lung cancer (NSCLC) patients. Among them, miR-137 acts as a risky miRNA. Caspase-3 (CASP3) was predicted as one of miR-137 potential targets. CASP3 is a critical effector caspase that promotes programmed cell-death when activated in the apoptotic cells both by extrinsic and intrinsic pathways that are highly conserved and related to drug resistance. We found miR-137 could regulate CASP3 through binding to its 3′UTR. Endogenous CASP3 can be up-regulated by over-expressing miR 137 in CL1-5, H1437 and A549 cells and down-regulated by silencing miR-137 in H1299, CL1-0 and A549 cells. Suppression of CASP3 by miR-137 providing lung cancer cells with ability of anti-apoptosis under UV-induced cell death. MiR-137 also increase IC50 of CL1-5, H1437, A549 and H1975 cells under cisplatin treatment to at least 2 folds. Restoration of CASP3 could reverse the effect. Our IHC data also shows the CASP3 protein expression is negative correlated with miR-137 level. In summary, manipulation of miR-137 might provide a potential treatment for EGFR wild-type and MTT failed NSCLC.
patients in the future.
Citation Format: Te-Jen Shu, Sung-Liang Yu, Jeremy J.W. Chen. MicroRNA-137 confers chemoresistance by suppressing CASP3 in lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3065. doi:10.1158/1538-7445.AM2015-3065
Abstract 3066: Regulation of oncogenic Zeb1 by microRNA-23b in bladder cancer
Authors: Majid Shahana , Dar Altaf A. , Saini Sharanjot , Yamamura Soichiro , Shahryari Varahram , Mitsui Yozo , Tanaka Yuichiro , Dahiya Rajvir , Deng Guoren 
Published: August 2015

Abstract
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation. In this study we show that miRNA-23b (miR-23b) acts as a tumor suppressor in bladder cancer. Quantitative real-time PCR analysis showed that miR-23b is significantly down-regulated in bladder cancer cell lines and tumor tissues compared to non-malignant cells and normal tissue samples. We also demonstrate that miR-23b expression has a potential to be diagnostic and prognostic biomarker in bladder cancer. High miR-23b expression is positively correlated with higher overall survival of bladder cancer patients as revealed by Kaplan-Meier analysis. ROC analysis showed that miR-23b expression can distinguish between normal and bladder cancer tissues. Further we elucidated the biological significance of miR-23b in bladder cancer. Over-expression of miR-23b in bladder cancer cells inhibited cell proliferation and impaired colony formation. Fluorescence activated cell sorting (FACS) analysis revealed that re-expression of miR-23b in bladder cancer cells induced G0/G1 cell cycle arrest and apoptosis while inhibiting cell migration and invasion. Luciferase reporter assays demonstrated that Zeb1, a crucial regulator of epithelial-to-mesenchymal transition (EMT), is a direct target of miR-23b in bladder cancer. These results show that loss of miR-23b confers a proliferative advantage and promotes bladder cancer cell migration and invasion. Furthermore, re-expression of miR-23b may be a beneficial therapeutic strategy for the treatment of human bladder cancer.
Citation Format: Shahana Majid, Altaf A. Dar, Sharanjot Saini, Soichiro Yamamura, Varahram Shahryari, Yozo Mitsui, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng. Regulation of oncogenic Zeb1 by microRNA-23b in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2015-3066
Abstract 3067: Expression of miR-301a regulates metastasis and act as a lethal marker for prostate cancer
Authors: Das Trinath P. , Suman Suman , PapuJohn Arokya Ms , Ankhem Akhila , Rao Sandhya R. , Messer Jamie C. , Alatassi Houda , Ankhem Murali M. , Damodaran Chendil 
Published: August 2015

Abstract
Prostate Specific Antigen (PSA) screening contributes as a biomarker for diagnosis of prostate cancer; however, PSA screening has been considered an over diagnosis in some cases. This has lead to the identification of novel invasive markers which could eventually differentiate patients with lethal prostate cancer from those with indolent disease. Emerging evidences suggest that a small set of microRNAs (miRNA) are linked to prostate cancer pathogenesis leading to castration resistant prostate cancer (CRPC).
In our studies of genome wide miRNA profiling, we have found that miR-301a is highly expressed in prostate cancer (CaP) cell lines as compared to normal prostate epithelial cells. While analyzing CaP samples, we found higher expression of miR-301a at specific stages. Interestingly, the miR-301a induction varies from 10 to 250 fold in aggressive tumors which clearly correlate with metastases. To confirm these findings, we utilized metastatic lesions from transgenic mice model of Trans Adenocarcinoma of Mouse Prostate (TRAMP) tissues, where higher levels of miR301a expression were observed in prostate tumors in comparison with normal prostate. Further, micro dissecting the tumors (metastasis) from lung showed ∼15 fold induction of miR301a expression compared to the normal lung components. Silencing specifically miR-301a resulted in the inhibition of proliferation, colony forming abilities, invasion, and migration and cell adhesion properties in CaP cell lines. Our molecular studies also suggest that miR-301 regulates Rho GTPases family proteins and transcriptionally activates Epithelial-Mesenchymal-Transition (EMT) markers like slug and β-catenin.
Our ongoing studies may not only reveal whether miR-301 regulates Rho-associated kinases (ROCK) but also the potential role of EMT in CaP. Furthermore, we intend to perform a case control study by screening an additional 100 CaP cases besides indolent controls. As mentioned earlier, besides revealing the functional role in CaP pathogenesis, results from these findings will also suggest whether miR-301 expression distinguishes lethal CaP from indolent disease.
Citation Format: Trinath P. Das, Suman Suman, Arokya Ms PapuJohn, Akhila Ankhem, Sandhya R. Rao, Jamie C. Messer, Houda Alatassi, Murali M. Ankhem, Chendil Damodaran. Expression of miR-301a regulates metastasis and act as a lethal marker for prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2015-3067