bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1420: RRx-001 inhibits glucose erythrocyte and tumor glucose 6-phosphate dehydrogenase
Authors: Cabrales Pedro , Oronsky Bryan , Scicinski Jan
Published: May 2015

Cancer cells are characterized by an excess of reactive oxygen species (ROS) levels, which are balanced by high levels of glutathione (GSH) to protect against ROS-induced cytotoxicity. Glucose 6-phosphate dehydrogenase (G6PD), the first key enzyme of the pentose phosphate pathway (PPP), ensures NADPH supply for reduced glutathione regeneration. RRx-001, a novel redox active anticancer agent, derived from the aerospace industry, which has successfully completed a Phase 1 clinical trial and is scheduled to begin multiple Phase 2 studies, induces cell cycle-specific apoptosis through increase in intracellular ROS leading to free thiol depletion.

The purpose of the present study was to evaluate whether the mechanism by which RRx-001 targets the antioxidant capacity of tumor cells is related to G6PD inhibition. Since red blood cells (RBC) are particularly dependent on G6PD synthesis, we initially explored the effects of RRx-001 on the erythrocytic PPP.

Glucose consumption was higher for human RBC (hRBC) treated with RRx-001 (10 μM) compared to untreated cells. PPP was inhibited in RRx-001 treated hRBC. Methylene blue, which normally stimulates PPP flux, did not increase glucose utilization of treated cells. Analysis of PPP flux revealed that RRx-001 inhibits G6PD preventing conversion of glucose-6-phosphate to 6-phosphogluconolactone and reduction of NADP(+). Compared to G6PD inhibitors dehydroepiandrosterone and aminonicotinamide, RRx-001 was 10 fold more potent.

Given the critical importance of G6PD and NADPH to fuel both the proliferation of tumor cells and to detoxify ROS, we secondarily investigated whether RRx-001 has anti-proliferative and viability effects on hepatocellular carcinoma (Hep G2) and colorectal adenocarcinoma (Caco-2 and HT-29) cell lines. Through tetrazolium and bromodeoxyuridine incorporation assays, performed as indicators of cellular proliferation, RRx-001 inhibited cell growth in a dose dependent (1 - 20 μM) manner. The suppression of growth of the RRx-001 incubated cells was correlated with inhibition of G6PD (r=0.81, n=9, P<0.05). RRx-001 also reduced viability of all carcinoma cell lines. Given the key roles of the PPP in GSH metabolism, and the observation that cell lines overexpressing G6PD are associated with the formation of tumors in mice, inhibition of G6PD by RRx-001 is an attractive target, particularly in combination with standard of care radiotherapy or cytotoxic drugs that also generate ROS or free radical intermediates either directly or indirectly.

Citation Format: Pedro Cabrales, Bryan Oronsky, Jan Scicinski. RRx-001 inhibits glucose erythrocyte and tumor glucose 6-phosphate dehydrogenase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1420. doi:10.1158/1538-7445.AM2014-1420
Abstract 158: Sustained adrenergic signaling activates pro-inflammatory networks in ovarian carcinoma
Authors: Nagaraja Archana S. , Armaiz-Pena Guillermo , Allen Julie , Sadaoui Nouara C. , Zand Behrouz , Yang Peiying , Tan Lin , Cole Steve , Lutgendorf Susan , Sood Anil K.
Published: May 2015

Purpose: To determine the role of adrenergic activation in driving tumor inflammation through COX-2/Prostaglandin E2 (PGE2) axis as PGE2 is known to play an important role in the induction of a pro-inflammatory signature in the tumor microenvironment that promotes tumor growth and metastasis.

Methods We first analyzed global metabolic changes in tumors isolated from patients with known Center for Epidemiologic Studies Depression Scale (CES-D; depressive) scores and tumoral norepinephrine (NE) levels. Beta-adrenergic receptor (ADRB) positive cells (Skov3 and HeyA8) were used to study mechanism at gene and protein levels of PTGS2 (cyclooxygenase2), PTGES (prostaglandin E synthase) and metabolite levels (all prostaglandins, PGs) in vitro and in vivo.

Results Our results revealed that levels of PGs were significantly increased in patients with high CESD scores (>16). PGE2 was upregulated by 2.38 fold when compared to the low CES-D scores. A similar trend was also observed with other pro-inflammatory eicosanoids, such as 6-keto prostaglandin F1 Alpha (2.03), prostaglandin A2 (1.39) and prostaglandin E1 (1.39). Exposure to NE resulted in increased PTGS2 and PTGES (prostaglandin E2 synthase) gene expression in adrenergic receptor positive cell lines. Mass-spectroscopy analyses confirmed that upon treatment with NE and arachidonic acid (substrate), PGE2 levels were increased in conditioned medium from Skov3 cells. Treatment with a broad ADRB agonist (isoproterenol) or ADRB2 specific agonist (terbutaline) led to increases in expression of PTGS2 and PTGES. Conversely, treatment with a broad antagonist (propranolol) or an ADRB2 specific antagonist (butoxamine) in the presence of NE abrogated gene expression changes of PTGS2 and PTGES. Silencing PTGS2 resulted in significantly decreased migration (40%) and invasion (25%) of Skov3 and HeyA8 cells in the presence of NE. Importantly, silencing PTGS2 in a restraint stress orthotropic model decreased tumor burden by 70% and metastasis (tumor confined to the ovary) in Skov3-ip1 orthotopic mouse model as compared to control siRNA with restraint stress.

Conclusion Increased adrenergic stimulation results in a pro-inflammatory milieu that drives tumor progression and metastasis.

Citation Format: Archana S. Nagaraja, Guillermo Armaiz-Pena, Julie Allen, Nouara C. Sadaoui, Behrouz Zand, Peiying Yang, Lin Tan, Steve Cole, Susan Lutgendorf, Anil K. Sood. Sustained adrenergic signaling activates pro-inflammatory networks in ovarian carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2014-158
Abstract 1968: Breast cancer cells escape from chemotherapy and hypoxia by distinct mechanisms
Authors: O'Brien Siobhan , Chen Liang , Zhong Wenyan , Armellino Douglas , Follettie Maximillian , Damelin Marc
Published: May 2015

Cancer relapse following treatment remains a significant barrier to achieving cures for many patients. An emerging framework for addressing this problem focuses on cancer stem cells (CSCs). Clinical and preclinical data suggest that CSCs survive chemotherapy and antiangiogenic therapy. In breast cancer, CSCs are marked by the cell surface expression of CD44, the major receptor for hyaluronic acid. The SKBR3 cell line was previously shown to be a clinically relevant model of breast CSCs in vivo, and we have further developed this model to enable mechanistic studies of tumor relapse in vitro. We found that chemotherapy and hypoxia both enriched for CD44hi populations in SKBR3, but surprisingly, the populations were phenotypically distinct. CD44hi cells from chemotherapy but not hypoxia exhibited increased tumor cell growth and increased sensitivity to the CSC-specific inhibitor salinomycin, compared to CD44lo cells. To examine these CD44hi and CD44lo populations further, we performed transcriptional profiling with sorted cells. We found that the growth factor TGF beta-2 was upregulated in chemotherapy-treated CD44hi cells and also enhanced the growth of these cells, which suggests that TGF beta-2 autocrine/paracrine signaling can promote the growth of surviving cells. We also observed an increase in xCT (SLC7A11) expression upon chemotherapy treatment and identified the CD44v8-10 variant expressed in the CD44hi SKBR3. CD44v could therefore stabilize xCT and promote survival by lowering intracellular reactive oxygen species (ROS). We have also established a functional role for CD44 in SKBR3 cell growth: CD44 knockdown prevented colony formation, and conversely the CD44 ligand hyaluronic acid enhanced colony formation. These findings indicate that CD44 not only marks CSC populations that arise in response to chemotherapy but also functions in their survival. Our work suggests that mechanisms of tumor relapse vary based on the particular therapy, and defining these mechanisms will allow for the development of novel therapeutic strategies to enable long-term responses in the clinic.

Citation Format: Siobhan O'Brien, Liang Chen, Wenyan Zhong, Douglas Armellino, Maximillian Follettie, Marc Damelin. Breast cancer cells escape from chemotherapy and hypoxia by distinct mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1968. doi:10.1158/1538-7445.AM2014-1968
Abstract 2008: CUL7 promotes non-small cell lung cancer cells migration and invasion
Authors: Song Qiong , Wang Lihui , Lu Yi , Zhang Jian , Fu Jiejun
Published: May 2015

Cullin7 (CUL7) is a scaffold protein of the CUL7 E3 ubiquitin ligase containing Fbxw8, Skp1 and ROC1. Germ line mutations of the CUL7 are linked to 3-M syndrome and Yakuts short stature syndrome, both of which are characterized by growth retardation and few bone abnormalities. We have previously shown that CUL7 promotes trophoblast cells migration and invasion. However, the role of CUL7 on cancer invasion and metastasis remains unknown. It has been reported that CUL7 mRNA is overexpressed in non-small cell lung cancer (NSCLC). In this study, we investigated the effects of CUL7 on NSCLC cells migration and invasion. Firstly, we evaluated CUL7 expression on tissue microarray slides by immunohistochemical staining. We found that CUL7 significantly overexpressed in NSCLC and its expression was associated with advanced TNM stage, nodal or bone metastasis and poor overall survival. Next, we determined CUL7, CUL1 and Fbxw8 expression in different NSCLC cell lines by real-time PCR and western blot. Higher level of CUL7 was observed in H1299, H358, H1437, and H1792 cells which derived from metastatic sites compared to H460 and human bronchial epithelial cells (BEAS2B). Knockdown CUL7 expression in NSCLC cells decreased cell motility and invasive ability using wound healing and matrigel invasion assay. Real-time PCR revealed that knockdown of CUL7 significantly decreased expression of CCL2, IL-6 and CXCL16. However, the expression of uPA, MMP-2 and MMP-9 were not changed. Furthermore, Knockdown of CUL7 in NSCLC cells decreased secretion of CCL2, CXCL16 and IL-6. This study indicates that CUL7 may be a potential biomarker as well as a novel therapeutic target in NSCLC progression. Supported by National Natural Sciences Foundation (NSFC) projects (81201669, 81171993 and 81272415); NSFC key project (81130046).

Note: This abstract was not presented at the meeting.

Citation Format: Qiong Song, Lihui Wang, Yi Lu, Jian Zhang, Jiejun Fu. CUL7 promotes non-small cell lung cancer cells migration and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2008. doi:10.1158/1538-7445.AM2014-2008
Abstract 2171: Weight change in relation to early breast cancer events in breast cancer patients
Authors: Kenéz Xuyian , Markkula Andrea , Simonsson Maria , Ingvar Christian , Rose Carsten , Jernström Helena C.
Published: May 2015

Background: Weight change may influence breast cancer prognosis. The aim was to investigate whether weight change between the preoperative and 1-year postoperative visit was associated with risk of early breast cancer events in a population-based cohort of breast cancer patients in Sweden.

Methods: Weight was measured by research nurses at the preoperative and the 1-year follow-up visits from 849 primary breast cancer patients operated between 2002 and 2011 in Lund, Sweden. Tumor characteristics, clinical data, and date of death were obtained from pathology reports, patient charts, and population registries.

Results: The median follow-up was three years and 79 early breast cancer events occurred during the follow-up period. In all patients, there was a non-significant tendency towards decreased risk of early events with weight loss (Ptrend = 0.11). Weight loss was significantly associated with lower risk of early events compared to stable weight or weight gain in patients with axillary nodal involvement (Ptrend = 0.012), but not in patients without axillary nodal involvement. Further analysis showed that weight loss was only significantly associated with lower risk of early events among patients younger than 70 years with axillary nodal involvement, while patients who had gained more than 5% of the preoperative weight had significantly earlier events compared to those with stable weight. The results remained significant after adjustment for age at diagnosis, natural logarithm of the preoperative weight, invasive tumor size, axillary nodal involvement, and ER status. Further adjustments for smoking and alcohol abstinence, ever use of hormone therapy, or for treatments did not materially change the result.

Conclusion: Weight loss was associated with significantly lower risk for early breast cancer events in patients younger than 70 years with axillary nodal involvement compared to stable weight or weight gain. If these results are confirmed, weight management may be incorporated in post-diagnosis management of breast cancer patients with axillary nodal involvement and have a significant beneficial impact on short-term prognosis.

Citation Format: Xuyian Kenéz, Andrea Markkula, Maria Simonsson, Christian Ingvar, Carsten Rose, Helena C. Jernström. Weight change in relation to early breast cancer events in breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2014-2171
Abstract 2211: Viral subtypes in hepatocellular carcinoma are associated with different mechanisms of WNT/CTNNB1 alteration
Authors: Covington Kyle R. , Donehower Lawrence A. , Creighton Chad , Slagle Betty L. , Goss John A. , Cotton Ronald T. , Gingras Marie-Claude , Shinbrot Eve , Guiteau Jacfranz J. , Nguyen Thao N. , Harring Theresa R. , Muzny Donna M. , Walker Kimberly , Doddapaneni HarshaVardhan , Gibbs Richard A. , Aburatani Hiroyuki , Shibata Tatsuhiro , Wheeler David A. ,   Japan ICGC HCC Project
Published: May 2015

Hepatocellular carcinoma (HCC) is one of the most prevalent human cancers, especially in Asia, and regions of endemic hepatitis infection. The primary risk factors for HCC are chronic hepatitis infection by hepatitis C (HCV) or hepatitis B (HBV). We performed whole exome sequencing on 549 matched tumor and normal samples from three centers (BCM HGSC, RCAST, NCC) to identify somatic changes in protein coding regions and changes in copy number. Here we describe variants associated with specific viral etiologies in HCC.

We found TP53, CTNNB1, ARID1A, ARID2, and AXIN1 to be among the most frequently mutated genes in the entire dataset, implicating DNA damage, WNT signaling, and chromatin remodeling as recurrently altered pathways in HCC. Our validation rate for mutations in significantly mutated genes was over 95%. We identified 18 genes which differed significantly (p < 0.05) in mutation frequency between viral subtypes. Of these, AXIN1 was mutated in over 15% of HBV-associated HCC but less than 5% of HCV and non-viral-associated HCC. Additionally, we performed GISTIC analysis based on read depth from our whole exome sequencing data. We found that WNT3A and MYC were significantly amplified and AXIN1 and TP53 were significantly deleted in HCV-associated HCC but not HBV or non-virus-associated HCC.

Our findings indicate that WNT/beta-catenin signaling is a frequently altered pathway in HCC. While aberrations in this pathway were present in all viral etiologies, HBV-associated HCC tended to mutate AXIN1 while HCV-associated HCC tended to have AXIN1 deletion. These differences may result from virus-specific oncogenic processes.

Citation Format: Kyle R. Covington, Lawrence A. Donehower, Chad Creighton, Betty L. Slagle, John A. Goss, Ronald T. Cotton, Marie-Claude Gingras, Eve Shinbrot, Jacfranz J. Guiteau, Thao N. Nguyen, Theresa R. Harring, Donna M. Muzny, Kimberly Walker, HarshaVardhan Doddapaneni, Richard A. Gibbs, Hiroyuki Aburatani, Tatsuhiro Shibata, David A. Wheeler, Japan ICGC HCC Project. Viral subtypes in hepatocellular carcinoma are associated with different mechanisms of WNT/CTNNB1 alteration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2014-2211
Abstract 2284: Cytotoxic and genotoxic effects of PEG-coated gold nanoparticles in HK-2 cells
Authors: Patlolla Anita K. , Epting Tammy , Thrasher Lisabeth , Tchounwou Paul B.
Published: May 2015

Biomolecular functionalization of gold nanoparticles (GNP's) have led to a dramatic expansion of their potential biomedical applications, including biosensorics, bioimaging, photothermal therapy, and targeted drug delivery. As the range of gold nanoparticle types and their applications continues to increase, human safety concerns are gaining attention, which makes it necessary to better understand the potential toxicity hazards of these novel materials. The main objective of this study was to determine the cytotoxic and genotoxic potential of poly-ethylene-glycol-coated gold nanoparticles (PEG-GNPs) in HK-2 cells. These cells were grown under sterile conditions in 96-well plate until 80% confluency. Four exposure concentrations (125, 250, 500 and 1000 µg/ml) of PEG-coated GNP's and a control were used. Following exposure to PEG-coated GNP's, cytotoxicity assay (MTS) was performed for 24 and 48 hrs using standard protocol. Genotoxic effects of GNP's were determined measuring DNA damage using comet assay. The results demonstrated a dose and time dependent decrease in cell viability with PEG-coated GNPs and an increase in percent DNA damage in exposed cells compared to control. The difference in the cytotoxic effect between 24 and 48 hrs was not statistically significant however, the genotoxic effect was statistically significant both in time and dose. The cellular findings reported here do suggest that GNPs has the potential to induce cell death and cause damage to DNA in HK-2 cells. However, more studies to clarify the role of encapsulation in the in vitro toxicity of GNP's, are required and parallel comparison is preferred.

Note: This abstract was not presented at the meeting.

Citation Format: Anita K. Patlolla, Tammy Epting, Lisabeth Thrasher, Paul B. Tchounwou. Cytotoxic and genotoxic effects of PEG-coated gold nanoparticles in HK-2 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2284. doi:10.1158/1538-7445.AM2014-2284
Abstract 2298: Methylated gene markers discovered in nasopharyngeal carcinoma by differential methylation hybridization (DMH) in high density CpG island DNA chips
Authors: Cheng Wai Wai , Ngan Roger K.C. , Kwong Dora L.W. , Huang Tim H.M. , Ma Victor W.S. , Law Stephen C.K. , Tse Loretta , Busson Pierre , Tsao George S.W. , Lung Maria L. , Yip Timothy T.C.
Published: May 2015

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in southern China and south east Asian countries. A variety of tumor suppressor (TS) genes including p16, RASSF1A, EDNRB, DAP-Kinase, RIZ1, E-Cadherin were found to be hypermethylated in NPC. This, however, could only be the tip of an iceberg. We made use of the high throughput differential methylation hybridization (DMH) technique in screening for methylated DNA markers in 244K CpG island DNA chip in three NPC xenografts (C15, C17, 99-186), a NPC tumor cell line (C666) versus an immortalized NP non-cancer cell line (NP69). This DMH screening discovered 100, 213, 372 and 977 genes respectively in 99-186, C17, C15 and C666 with ≥ 5 CpG sites in their gene promotors being methylated. Among these genes, 28 of them were found to have DNA methylation in all the 4 NPC xenografts/ tumor cell line. Bisulphite sequencing was performed in 17 pairs of tumor and normal biopsy samples from 17 NPC patients. This confirmed tumor specific methylation in the promotor regions of 8 gene markers, namely, NRP3, PCDH10, PENK, ADHFE1, IRX4, GSC, PFN2 & GFI1 with positive methylation ratios (+ve MR) in tumor biopsies of 86.7%, 85.7%, 80%, 72.7%, 54.5%, 50%, 41.7% &23.1% respectively and +ve MR of 18.2%, 0%, 0%, 0%, 0%, 37.5%, 12.5% & 0% respectively in normal biopsies. Methylation of CpG sites appears to occur either all or none with 15 to 45 consecutive CpG sites in these genes either all methylated or non-methylated in tumor biopsies probably indicating the need of methylation of a full cluster of CpG sites in order to affect the gene transcription of the discovered genes. Functions of the DMH identified genes includes cell adhesion (PCDH10), signal transduction (PENK), chloride ion binding (NPR3), regulation of DNA dependent transcription (IRX4) and xenobiotic metabolism (ADHFE1). In fact, PCDH10, PENK and GFI1 genes have been reported to be closely associated with NPC/esophageal carcinoma, pancreatic adenocarinoma and T-cell lymphoma respectively. Further analyses in these methylated markers could be useful in the diagnosis and study of etiology of NPC.

Citation Format: Wai Wai Cheng, Roger K.C. Ngan, Dora L.W. Kwong, Tim H.M. Huang, Victor W.S. Ma, Stephen C.K. Law, Loretta Tse, Pierre Busson, George S.W. Tsao, Maria Li Lung, Timothy T.C. Yip. Methylated gene markers discovered in nasopharyngeal carcinoma by differential methylation hybridization (DMH) in high density CpG island DNA chips. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2298. doi:10.1158/1538-7445.AM2014-2298
Abstract 23: bFGF induced VEGF expression and angiogenesis in human chondrosarcoma
Authors: Lin Kai-Wei , Tang Chih-Hsin
Published: May 2015

Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. Solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that express VEGF are able to grow and metastasize. In normal tissue, basic fibroblast growth factor (bFGF) is present in basement membranes and in the subendothelial extracellular matrix of blood vessels. It has been found that bFGF increased the formation of new blood vessels and angiogenesis during tumor growth and metastasis. However, the effect of bFGF in VEGF expression in human chondrosarcoma is largely unknown. In this study, we examined the effect of bFGF in VEGF expression in human chondrosarcoma. We found that the expression of bFGF and VEGF were correlated with tumor stage and significantly higher than that in normal cartilage. bFGF increased the VEGF expression and subsequently promoted migration and tub formation in endothelial progenitor cells (EPCs). Pretreatment of cells with p38, c-Src, or NF-κB inhibitor antagonized bFGF-induced VEGF expression and EPC migration as well as tube formation. Incubation with bFGF induced p38, c-Src and NF-κB phosphorylation. In addition knockdown bFGF decreased VEGF expression and also abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and matrigel-plug nude mice model in vivo. Furthermore, using xenograft tumor angiogenesis model, knockdown bFGF significantly reduced tumor growth and tumor-associated angiogenesis. Our results indicated that bFGF enhances the VEGF expression of chondrosarcoma cells. One of the mechanisms underlying bFGF-directed VEGF expression was through the p38/c-Src/NF-κB signal transduction pathway.

Citation Format: Kai-Wei Lin, Chih-Hsin Tang. bFGF induced VEGF expression and angiogenesis in human chondrosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2014-23
Abstract 2379: 96-Well CometChip validation for simultaneous treatment and measurement of DNA damage in a single platform
Authors: Woodgate Sandra R. , Whittaker Clare , George Jay , Sobol Robert W. , Schamus-Haynes Sandy , Engelward Bevin P. , Ge Jing
Published: May 2015

The 96-Well CometChip System is a high-throughput platform to simultaneously treat and measure DNA damage induced by different treatments, or among different cell types on a single slide using the comet assay. The CometChip is a consumable consisting of specifically sized micron pores patterned into agarose layered on a treated microscope slide. Ninety-six (96) separate wells are created by inserting the CometChip into a reusable 96-Well CometChip System, a magnetically-sealed cassette suitable for tissue culture incubators. Cells added to each well are captured by gravity into micropores and excess cells aspirated leaving an array of non-overlapping cells. Multiple experimental conditions are performed in parallel by the addition of different chemicals to respective wells. Once treatment is complete the CometChip is removed from the cassette and processed using standard alkaline comet conditions and imaging systems.

A distributable CometChip requires precision manufacturing to ensure robust and reproducible performance. CometChip tolerances were investigated and compared to normal comet using cryopreserved comet control cells with known levels of DNA damage. After setting specifications, 96-Well CometChips with 30 micron pores were distributed to evaluate both intra and inter-chip variations between laboratories. To assure experimental consistency between labs, magnetically sealable cassettes, cryopreserved control cells and identical electrophoresis systems were provided to each lab. Data is presented to demonstrate the feasibility of manufacturing the CometChip for reproducible results based on the coefficients of variance (CVs) obtained between different wells and laboratories. In addition, data will be presented demonstrating the absence of cross talk between CometChip wells using the 96-Well CometChip System. (Work was supported by R44ES021116).

Citation Format: Sandra R. Woodgate, Clare Whittaker, Jay George, Robert W. Sobol, Sandy Schamus-Haynes, Bevin P. Engelward, Jing Ge. 96-Well CometChip validation for simultaneous treatment and measurement of DNA damage in a single platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2014-2379
Abstract 2392: The role of ATM in lytic infection of EBV in nasopharyngeal epithelial cells
Authors: Tsao George Sai-Wah , Hau Pok Man
Published: May 2015

Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that reactivation of EBV in infected cells induces ATM-dependent DNA damage response (DDR). The functional role and involvement of ATM activation during lytic induction remains obscure. In this study, the essential role of ATM for viral DNA replication was examined in EBV-infected nasopharyngeal epithelial cells. Induction of lytic EBV infection triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using siRNA approach or specific chemical inhibitor diminished EBV DNA replication and production of infectious virion in EBV-infected cells induced to undergo lytic infection. Phosphorylation of Sp1 is a downstream event of ATM activation. The phosphorylation of Sp1 at serine 101 residue is crucial in promoting the accretion of EBV replication proteins at the replication compartment to facilitate viral DNA replication. Depletion of Sp1 suppresses viral DNA replication and localization of EBV replication proteins to the replication compartments. Our studies support an important role of ATM activation in lytic infection of EBV. Phosphorylation of Sp1 is an essential process mediating the recruitment of EBV replication protein to replication compartments. Our study highlights the importance of DDR pathway in lytic infection of EBV and suggests the potential application of specific DDR inhibitors to control the EBV replication in infected cells.

Funding support: Research Grant Council of Hong Kong (GRF #779312 & 779713; AoE/M-06/08).

Note: This abstract was not presented at the meeting.

Citation Format: George Sai-Wah Tsao, Pok Man Hau. The role of ATM in lytic infection of EBV in nasopharyngeal epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2392. doi:10.1158/1538-7445.AM2014-2392
Abstract 2411: Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib
Authors: Osoegawa Atsushi , Gills Joell J. , Kawabata Shigeru , Sugio Kenji , Dennis Phillip A.
Published: May 2015

Poly (ADP-ribose) polymerase inhibitors (PARPi) have been developed and tested in a context of combining them with drugs that inhibit double-stranded (ds) DNA repair, because PARPi impair single-stranded (ss) DNA break repair, resulting in activation of dsDNA break repair machinery. Rapamycin, an mTOR inhibitor, has been widely prescribed for more than a decade, and recent studies revealed its ability to inhibit dsDNA break repair. The combination of the PARPi, olaparib, and rapamycin synergistically inhibited cell proliferation in non-small cell lung cancer (NSCLC) cells, as well as triple negative breast cancer (TNBC) cells that have BRCA1 mutations. Rad51, which forms a polymer on ssDNA upon dsDNA breaks, plays an essential role in homologous recombination. Olaparib induced Rad51 focus formation and rapamycin successfully inhibited it both in vivo and in vitro, suggesting this combination worked through blocking both ssDNA break repair and dsDNA break repair, resulting in cells that cannot go through G2/M checkpoint. The protein level of PARP was predictive for both PAR activity and Rad51 focus formation with this combination. Collectively, these data suggest that this combination could have a therapeutic potential to inhibit cancer with high PARP expression, or in combination with cytotoxic chemotherapy or radiation.

Citation Format: Atsushi Osoegawa, Joell J. Gills, Shigeru Kawabata, Kenji Sugio, Phillip A. Dennis. Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2411. doi:10.1158/1538-7445.AM2014-2411
Abstract 2413: Unstable genomes and the average cancer cell
Authors: Abdallah Batoul , Stevens Joshua B. , Horne Steven , Liu Guo , Heng Henry HQ
Published: May 2015

The high degree of multi-level heterogeneity represents a key challenge for cancer research. Despite its importance, both the theoretical concepts and appropriate tools for proper quantitative analysis are underdeveloped. Specifically, the characterization of heterogeneity in cancer cell populations of different genomic stabilities, and its effect on systems dynamics are poorly understood. We use a single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth, and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. We conclude: 1) the karyotype cannot be cloned when genome instability is high, and caution is needed for single cell analysis for cell populations that are not genetically identical; 2) the “average” cancer cell does not exist, as the statistical mean does not accurately describe systems behavior, especially genome instability is high; and 3) single cell analysis is needed when cells are not genomically identical. Our results not only provide an explanation for population heterogeneity but also explain how genome-level heterogeneity contributes to drug resistance and cancer evolution.

Note: This abstract was not presented at the meeting.

Citation Format: Batoul Abdallah, Joshua B. Stevens, Steven Horne, Guo Liu, Henry HQ Heng. Unstable genomes and the average cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2413. doi:10.1158/1538-7445.AM2014-2413
Abstract 2423: Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker
Authors: George Jay , Xu Hai
Published: May 2015

Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that becomes rapidly phosphorylated at Serine 139 by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis. γ-H2AX is an ideal Pharmacodynamic (PD) surrogate marker to measure molecular responses to a large number of drugs; however, methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, and not suitable for high throughput screening applications. To address this need, we have developed a novel high throughput ELISA assay to measure γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies.p

Background

Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that contains an evolutionarily conserved SQ motif at the C-terminus in eukaryotes. Serine 139 within this motif becomes rapidly phosphorylated by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis (1). During the past year investigators have confirmed the value of γ-H2AX as an important Pharmacodynamic (PD) marker (2) and genotoxicity endpoint (3). There are over 21 anticancer drugs that are known to result in γ-H2AX formation. As a result, γ-H2AX is an ideal Pharmacodynamic PD surrogate marker to measure molecular responses to a large number of drugs (4,5,6). While many of these drugs have already garnered regulatory approval, and are currently being used to manage various types of cancers, they are the subject of ongoing clinical studies to evaluate their efficacy when used alone or in combination with molecularly targeted drugs.

While methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, the ELISA method is the most quantifiable and easiest to validate, and not suitable for high throughput screening applications. To address this need Trevigen's quantitative pharmacodynamic HT γ-H2AX ELISA assay measures γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies.

Citation Format: Jay George, Hai Xu. Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2014-2423
Abstract 2430: Targeting estrogen-related receptor alpha using pyrrole-imidazole polyamide
Authors: Li Yang , Gallagher John , Olenyuk Bogdan , Stiles Bangyan , Kassa Ankeste
Published: May 2015

Estrogen-related receptor α (ERRα), an orphan nuclear receptor, is a prominent regulator of mitochondrial biogenesis and function. We previously showed that liver-specific PTEN deletion and the subsequent activation of PI3K/AKT pathway lead to tumorigenesis. Here we show that Pten-null hepatocytes established from Pten-null mice exhibit dramatically increased ERRα expression, accompanied by elevated mitochondrial mass, cellular oxygen consumption and reactive oxygen species production. Silencing ERRα by siRNA in Pten-null hepatocytes not only suppresses mitochondrial biogenesis and respiration but also attenuates hepatocytes' proliferation and colony-forming potential. Cohort analyses of clinical datasets from liver tissues revealed a negative correlation between expressions of PTEN and ERRα in patients with liver cancer. Therefore, targeting ERRα might provide therapeutic implication for treating tumors bearing abnormal PTEN-ERRα signaling. ERRα exerts transcriptional activity towards its target genes via binding to consensus DNA sequences named estrogen-related receptor α response elements (ERRE). The DNA-binding activity of ERRα could be modulated by polyamides, a class of synthetic amino acid oligomers capable of binding to DNA minor groove with sequence specificity. By designing and employing polyamide that specifically targets ERRE in Pten-null hepatocytes, we found the mRNA levels of two ERRα target genes, cytochrome c (cyt c) and ATP synthase β (ATP5b), are reduced. Biochemical gel-shift assay showed the binding between ERRα and ERRE is significantly diminished by polyamides in vitro. Moreover, polyamide treatment in hepatocytes reduced mitochondrial respiration and colony-forming ability. In conclusion, our findings show that ERRα is significantly induced via PTEN-AKT signaling pathway and in turn activates mitochondrial bioenergetics, which likely provides growth and survival advantages. The crucial role in mitochondrial metabolism and the association with PTEN signaling make ERRα a useful target for cancer therapy. Synthetic polyamide that disrupts transcription factor-DNA interaction can be utilized to modulate ERRα's activity, highlighting its potential as a future therapeutic target.

Citation Format: Yang Li, John Gallagher, Bogdan Olenyuk, Bangyan Stiles, Ankeste Kassa. Targeting estrogen-related receptor alpha using pyrrole-imidazole polyamide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2430. doi:10.1158/1538-7445.AM2014-2430
Abstract 2432: Phosphorylation of Raptor by pemetrexed-activated AMPK is sufficient to suppress mTORC1 activity promoted by loss of p53 and TSC2
Authors: Agarwal Stuti , Bell Catherine M. , Moran Richard G.
Published: May 2015

The key sensor of energy status, AMP-dependent kinase (AMPK), can be activated by intracellular ZMP generated directly from the aminoimidazolecarboxamide ribonucleoside (AICAR) or from inhibition of purine synthesis by the antifolate pemetrexed (PTX). In spite of this common mechanism, signaling from AMPK activated by PTX or AICAR differed: PTX-activated AMPK phosphorylated the mTORC1 component Raptor but not tuberin (TSC2), whereas AICAR-activated AMPK phosphorylated both targets. This dichotomy proved to be due to p53 function: although p53 accumulated after either treatment, AICAR robustly activated the transcriptional profile of p53, including expression of p53 target genes TSC2 and sestrin2 whereas, in PTX-treated cells, p53 was virtually inactive as a transcriptional activator. Accordingly, activation of AMPK by PTX initiated a cell growth inhibition that was p53 and TSC2 independent, whereas p53 and TSC2 wild-type HCT116 colon carcinoma cells were more sensitive to AICAR than isogenic cells null for p53 or both p53 and TSC2. Furthermore, in cells null for p53, mTORC1 was hyperactive, apparently caused by lower TSC2 function, higher Rheb bound to mTORC1, and a concomitantly lower inhibition of mTORC1 by PRAS40. Although TSC2 was deficient and mTORC1 activity enhanced in the absence of p53 function, Raptor phosphorylation by AMPK following PTX was robust and independent of both p53 and TSC2. This provides insight into the therapeutic activity of PTX against human lung cancers lacking p53 function and with hyperactive mTORC1, a feature atypical of other antifolates. We concluded that phosphorylation of Raptor by AMPK after PTX treatment was both necessary and sufficient to suppress the kinase activity of mTORC1, even under conditions of limited TSC2 and enhanced mTORC1 function, suggesting the use of pemetrexed for treatment of Tuberous Sclerosis.

Citation Format: Stuti Agarwal, Catherine M. Bell, Richard G. Moran. Phosphorylation of Raptor by pemetrexed-activated AMPK is sufficient to suppress mTORC1 activity promoted by loss of p53 and TSC2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2014-2432
Abstract 2463: Suppression of hepatocyte nuclear factor 4α by nuclear factor κB in hepatoma cells
Authors: Ning Bei-Fang , Zhang Xin , Xie Wei-Fen
Published: May 2015

BACKGROUND & AIMS: HCC predominantly arises from cirrhotic liver where there usually has been repeated wound-healing response upon chronic inflammation. However, the mechanistic link between hepatic inflammation and carcinogenesis remains largely unknown.

METHODS: Expression of HNF4α, RelA(p65) or microRNAs in human HCCs was determined by Real-time PCR, western blot or immunohistochemistry. 3′UTR reporter assay was conducted to determine the regulation of HNF4α by microRNAs. The recombinant adenoviruses carrying HNF4α gene were injected into diethylinitrosamine(DEN)-administrated Wistar rats through tail vein. Tissue microarray was utilized to assess the expression of HNF4α and RelA HCC tissues.

RESULTS: Overexpression or interference of RelA remarkably decreased or increased HNF4α expression in hepatoma cells respectively, suggesting a negative regulation of HNF4α by NF-κB. Interestingly, ecotopic RelA expression dramatically reduced the activation of HNF4α-3′UTR indicating microRNA could be involved in the feedback regulation. MiR-21 was predicted to be regulated by NF-κB using Targetscan analysis, and overexpression of RelA evidently enhanced the miR-21 expression. Delivery of miR-21 mimic notably suppressed HNF4α-3′UTR activity and HNF4α expression. Moreover, treatment of miR-21 inhibitor increased HNF4α levels and attenuated the down-regulation of HNF4α by RelA. Consistently, downregulation of HNF4α and upregulation of RelA was detected during DEN-induced rat hepatocarcingenesis, negative correlation of HNF4α and RelA was also observed in human HCCs. CONCLUSIONS: These findings not only broaden the knowledge on hepatic inflammation and cancer initiation, but also indicates novel strategy in HCC prevention.

Note: This abstract was not presented at the meeting.

Citation Format: Bei-Fang Ning, Xin Zhang, Wei-Fen Xie. Suppression of hepatocyte nuclear factor 4α by nuclear factor κB in hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2014-2463
Abstract 25: Targeting c-Myc in pediatric sarcoma xenografts with the BET bromodomain inhibitor, JQ1, disrupts angiogenesis
Authors: Bid Hemant Kumar , Phelps Doris A. , Xiao Linlin , Baker Laurence , Qi Jun , Houghton Peter J.
Published: May 2015

Histone acetylation regulates activation and repression of multiple angiogenesis and inflammatory genes known to play critical roles in the pathogenesis of various diseases. The current study was designed to elucidate the therapeutic potential of JQ1, an inhibitor of the BET class of human bromodomain proteins (“readers” of histone acetylation marks), in pediatric sarcomas. Surprisingly, our results demonstrated that in-vivo xenografts showed similar sensitivity to JQ1, despite their cell lines showing >20 fold differences in sensitivity in-vitro. cMYC expression significantly down regulated in most rhabdomyosarcoma cell lines after JQ1 treatment, however there was no significant down regulation of cMYC in Ewings sarcoma lines, despite similar sensitivities to JQ1. When administered to mice bearing human sarcoma xenografts, JQ1 (50mg/kg/d) significantly retarded tumor growth. Whereas Ki67 staining remained unchanged in treated tumors, CD34-positive staining decreased after 2 weeks of treatment. Microvessel density (CD34-positive cells) in tumors of JQ1-treated animals was reduced by at least 60% compared with controls (P < 0.01) indicating anti-angiogenic activity. Assay of angiogenic factors in JQ1 treated tumor xenografts showed that 7 factors were significantly downregulated compared to control tumors in all four sarcoma lines. To test the anti-angiogenic activity of JQ1 directly we exposed primary cultured human umbilical vascular endothelial cells (HUVEC) to JQ1 (500 nM). JQ1 significantly inhibited vascular endothelial growth factor (VEGF) induced proliferation, migration, invasion, adhesion and capillary-like structure formation by HUVECs in a concentration-dependent manner. Furthermore, JQ1 (50 mg/kg/d) abrogated VEGF-induced vascular formation and reduced hemoglobin levels in the mouse Matrigel plug assay in vivo. To understand the molecular mechanism of these activities, we next examined the signaling pathways in HUVECs treated with JQ1 using a proteome based angiogenesis array as well as transcription factor activation assay. JQ1 treatment considerably decreased various angiogenic factors as well as dramatically decreasing AP1 expression and it transcriptional activity. This study provides evidence that JQ1 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis and demonstrate that the role of BRD4 inhibitors as a therapeutic strategy to target vasculature. Supported by a grant from SARC.

Citation Format: Hemant Kumar Bid, Doris A. Phelps, Linlin Xiao, Laurence Baker, Jun Qi, Peter J. Houghton. Targeting c-Myc in pediatric sarcoma xenografts with the BET bromodomain inhibitor, JQ1, disrupts angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 25. doi:10.1158/1538-7445.AM2014-25