bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3226: GALNT6 stabilizes GRP78 protein by O-type glycosylation
Authors: Park Jae-Hyun , Chung Suyoun , Lin Jiaying , Ueda Koji , Katagiri Toyomasa , Nakamura Yusuke
Published: May 2015

Aberrant O-type glycosylation is predominantly detected in breast cancer and plays diverse roles in cancer cell proliferation, metastasis, and drug resistance. GALNT (polypeptide N-acetylgalactosaminyl transferase) family initiates the O-type glycosylation and its high activity accounts for aberrant O-type glycosylation in breast cancer. We previously suggested that GALNT6 is a promising molecular target in breast cancer and involved in O-type glycosylation of Mucin 1 and fibronectin. To further investigate the molecular function of GALNT6, we performed pull down assay using VVA (Vicia Villosa agglutinin) lectin and identified several O-glycosylated proteins specifically in GALNT6-positive stable cells. After in vitro and in vivo O-glycosylation assays, we confirmed GRP78 (glucose related protein 78) as a physiological substrate of GALNT6 and identified 5 O-glycosylation sites in GRP78 by mass spectrometry. GRP78 is a molecular chaperone that facilitates proper protein folding under cellular stress. Interestingly, accumulating evidences have indicated that GRP78 plays anti-apoptotic roles in cancer cells against chemotherapy. Exogenous induction of GALNT6 increased the amount of GRP78 protein without any changes in its transcriptional level. Concordantly, knockdown of GALNT6 significantly reduced the GRP78 protein. Collectively, our findings imply that GALNT6 stabilizes GRP78 via O-glycosylation and GALNT6 inhibitor will be able to sensitize breast cancer cells to chemotherapy.

Citation Format: Jae-Hyun Park, Suyoun Chung, Jiaying Lin, Koji Ueda, Toyomasa Katagiri, Yusuke Nakamura. GALNT6 stabilizes GRP78 protein by O-type glycosylation. [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 3226. doi:10.1158/1538-7445.AM2014-3226
Abstract 3244: Supplementation with selenium-enriched yeast but not selenomethionine reduces oxidative stress in healthy men in a randomized clinical trial
Authors: Kaag Matt G. , Richie John P. , Das Arun , Calcagnotto Ana , Sinha Raghu , Neidig Wanda , Liao Jiangang , Lengreich Eugene J. , Berg Arthur , Hartman Terryl J. , Ciccarella Amy , Baker Aaron , Boyiri Telih , Goodin Susan , DiPaola Robert S. , El-Bayoumy Karam
Published: May 2015

Epidemiological and laboratory studies indicate that dietary selenium is protective against prostate cancer. Results from clinical trials suggest that selenium-enriched yeast (SY) but not selenomethionine (SeMet) may be effective at reducing prostate cancer risk. Our objectives were to directly compare for the first time the effects of SeMet and SY on prostate cancer relevant biomarkers in men. We performed a randomized double blind, placebo-controlled trial of SY (200 or 285 μg/day) and SeMet (200 μg/day) administered for 9 months in 69 healthy adult males. Biomarkers including blood selenium, glucose, PSA and glutathione (GSH) and urine 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin-F2α (8-iso-PGF2α) were measured at baseline and various times thereafter. Compliance was high in all groups (>95%). Plasma selenium levels were increased 93%, 54%, and 86% after 9 months in the SeMet and low and high dose SY groups, respectively, and returned to baseline levels after a 3 month washout (P<0.05). Urinary biomarkers of oxidative stress, 8-OHdG and 8-iso-PGF2α, were decreased 34% and 28%, respectively, after 9 months in the high dose SY group (P<0.05). Decreases in oxidative stress biomarkers were greatest in individuals with low baseline plasma levels of selenium (<127 ng/ml). No changes in serum PSA or blood glucose and GSH were observed. We showed for the first time reductions in biomarkers of oxidative stress following supplementation with SY but not SeMet in healthy men. These findings suggest that selenium-containing compounds other than SeMet may be responsible for the decrease in oxidative stress and possibly cancer risk. This work was supported a National Cancer Institute grant (R01CA127729).

Citation Format: Matt G. Kaag, John P. Richie, Arun Das, Ana Calcagnotto, Raghu Sinha, Wanda Neidig, Jiangang Liao, Eugene J. Lengreich, Arthur Berg, Terryl J. Hartman, Amy Ciccarella, Aaron Baker, Telih Boyiri, Susan Goodin, Robert S. DiPaola, Karam El-Bayoumy. Supplementation with selenium-enriched yeast but not selenomethionine reduces oxidative stress in healthy men in a randomized clinical trial. [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 3244. doi:10.1158/1538-7445.AM2014-3244
Abstract 329: Rottlerin induced autophagy by targeting multiple sites that leads to the apoptosis in cancer stem cells
Authors: Kumar Dhruv
Published: May 2015

Autophagy plays an important role in maintaining cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via degradation of aggregated proteins and damaged organelles. Autophagy is associated with the several catabolic and pathological processes, including cancer. Cancer is a multifaceted disorder involving the perturbation of several different pathways that regulate cell proliferation, cell differentiation, and cell survival. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to apoptosis in cancer stem cells. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, rottlerin, from a medicinal plant (Mallotus philippinensis) that induces autophagy in various types of cancer stem cells through the formation of autophagosomes as measured by electron microscopy and GFP-LC3 puncta formation. By computational virtual docking analysis, rottlerin was shown to interact directly with the protein kinase-C delta, beclin-1, and autophagy related proteins. By biochemical assays, rottlerin was shown to increase autophagy induction through the activation of the kinase-AMP-activated protein kinase-mammalian target of rapamycin pathway. In addition, rottlerin treatment causes the induction of apoptosis, which was associated with the suppression of phosphorylated Akt and mTOR, upregulation of phosphorylated kinase-AMP, downregulation of Bcl-2, Bcl-XL, XIAP, and cIAP-1. Rottlerin also proved to be a potent autophagy inducer in cancer stem cells, by knocking down beclin-1, protein kinase-C delta, or Agt7. These results provide a detailed understanding of the mechanism of action of rottlerin, as a novel autophagic inducer by targeting multiple sites to induce autophagy, which leads to the apoptosis in cancer stem cells. Rottlerin has the potential of being developed into an anti-cancer agent by targeting cancer stem cells.

Citation Format: Dhruv Kumar. Rottlerin induced autophagy by targeting multiple sites that leads to the apoptosis in cancer stem 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 329. doi:10.1158/1538-7445.AM2014-329
Abstract 3302: CD146, a suppressor of breast cancer, is a novel target of CD44-signaling
Authors: Abdraboh Mohamed E. , Al-Riyami Hamad , Al-Farsi Yahya , Ouhtit Allal , Hollenbach Andrew D. , Gupta Ishita , Shanmuganathan Somya , Raj Madhwa H.
Published: May 2015

More than 80% of cancer related deaths are caused by cancer cell metastasis, a crucial step in the onset of tumor development. Among the molecules involved in promoting cancer metastasis is the cell adhesion molecule CD44, whose role in promoting cancer cell motility and metastasis is well known. Despite this knowledge, the molecular mechanism through which CD44 promotes tumor development and cell metastasis is still unclear. CD146 (MUC 18) is another member of the cell adhesion molecule family, first identified in highly metastatic melanomas. The absence of CD146 in normal melanocytes and its high expression in melanomas suggests its tumor promoting actions. Despite the association between CD146 expression and development of melanoma, the expression patterns and the role of CD146 in normal and metastatic breast tissues are still controversial. In this paper we provided evidence clarifying some of these discrepancies by presenting CD146 as a negative downstream target for CD44. Finally, this study demonstrates a new role of CD44 in regulating neovascularization and in promoting cancer cell transmigration of blood vessels via regulation of its downstream target CD146.

Citation Format: Mohamed E. Abdraboh, Hamad Al-Riyami, Yahya Al-Farsi, Allal Ouhtit, Andrew D. Hollenbach, Ishita Gupta, Somya Shanmuganathan, Madhwa Hg Raj. CD146, a suppressor of breast cancer, is a novel target of CD44-signaling. [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 3302. doi:10.1158/1538-7445.AM2014-3302
Abstract 3308: A-kinase anchoring protein 2 is required for calcitonin receptor-stimulated invasion of prostate cancer cells
Authors: Aljameeli Ahmed , Thakkar Arvind , Thomas Shibu , Shah Girish V.
Published: May 2015

Expression of neuropeptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. CTR-stimulated invasion of prostate cancer cells, which is abolished by specific, cell-permeable inhibitors of protein kinase A. Since PKA is ubiquitously distributed in cells, present study examined the mechanism(s) by which CTR-stimulated PKA activity is regulated in time and space.

CT reduced cell adhesion but increased invasion of prostate cancer cells, both actions were abolished by AKAP St-Ht31 inhibitor peptide suggesting the involvement of an AKAP in CT action. Next, we identified the AKAP associated with CT action by subtraction of AKAP candidates using siRNAs. Knock-down of AKAP2, but not other AKAPs, abolished CT stimulated invasion. AKAP2 was localized to the plasma membrane. Stable knock-down of AKAP2 in PC3-CTR cells remarkably decreased cell proliferation, invasion, clonogenicity and their ability to form orthotopic tumors and distant metastases in nude mice. Reexpression of AKAP2-wt restored these characteristics. Primary prostate cancer specimens displayed remarkable up-regulation of CTR/AKAP2 expression as compared to benign prostates. Metastatic cancers displayed significantly higher CTR/AKAP2 expression than localized cancers.

These results for the first time demonstrate that AKAP2 is expressed in human prostates, its expression is elevated in metastatic prostate cancer, and the knock-down of its expression remarkably decreased tumorigenicity and metastatic ability of prostate cancer cells. AKAP2 may serve as a critical component of CTR-mediated oncogenic actions.

Citation Format: Ahmed Aljameeli, Arvind Thakkar, Shibu Thomas, Girish V. Shah. A-kinase anchoring protein 2 is required for calcitonin receptor-stimulated invasion of prostate cancer 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 3308. doi:10.1158/1538-7445.AM2014-3308
Abstract 3314: Co-regulation of srGAP1 by Wnt and androgen receptor signaling in castration resistant prostate cancer
Authors: Yokoyama Noriko N. , Sakai Toshinori , Sun Zheng , Shao Shujuan , Huang Jiaoti , Hoang Bang H. , Zi Xiaolin
Published: May 2015

Androgen receptor (AR) and Wnt signaling both play a critical role during prostate cancer progression to castration-resistance prostate cancer (CRPC). Over expression of AR can activate transcriptional activities of Wnt signaling pathway and promote CRPC. Recent data in our laboratory showed a potential co-regulation of srGAP1 (Slit-Robo-GTPase activating protein1) and active Wnt and AR signaling in CRPC. srGAP1 is a downstream component of Slit-Robo signaling. Our data showed that srGAP1 is overexpressed in CRPC cell lines (androgen-insensitive prostate cancer) while absent in both androgen-sensitive cells and in normal prostate epithelial cells. We also detected increased srGAP1 expression in human CRPC tissues compared with normal epithelial prostate and androgen sensitive prostate cancer tissues by immunohistochemistry analysis. When androgen-sensitive LNCaP cells were grown under androgen deprived condition, we observed induced expression of srGAP1. Interestingly, srGAP1 expression was also increased when LNCaP cells were grown under Wnt stimulated conditions. And, srGAP1 expression was decreased when Wnt signaling was inhibited by a Wnt antagonist Wnt-inhibitory factor-1 (WIF-1) in CRPC cell lines. Functional studies using chromatin immmunoprecipitation and luciferase assays were performed to examine whether Wnt transcription factor LEF-1 or TCF-4 binds and activates the srGAP1 promoter. Overexpression of WIF-1 inhibited the promoter activity of srGAP1. Taken together, our results indicated that srGAP1 is co-regulated by both Wnt and AR signaling and srGAP1 may be a new potential Wnt target gene in castration-resistance prostate cancer.

Citation Format: Noriko N. Yokoyama, Toshinori Sakai, Zheng Sun, Shujuan Shao, Jiaoti Huang, Bang H. Hoang, Xiaolin Zi. Co-regulation of srGAP1 by Wnt and androgen receptor signaling in castration resistant prostate cancer. [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 3314. doi:10.1158/1538-7445.AM2014-3314
Abstract 3318: Using synthetic lethal screening to identify therapeutic targets for innately platinum resistant lung cancer
Authors: Marini Kieren D. , Rossello Fernando J. , Martelotto Luciano G. , Watkins D. Neil
Published: May 2015

Although platinum-based chemotherapy is the standard of care for most cases of advanced lung adenocarcinoma, its effectiveness is limited by the frequent incidence of innate chemoresistance. As a result, response rates rarely exceed 20%, even though cis-platinum and carboplatin are highly effective in other settings such as small cell lung, ovarian and testicular cancers. We hypothesized that innate chemoresistance in lung adenocarcinoma is mediated by one or more signalling pathways dependent on the expression of a single gene, and that these pathways could ultimately be targeted therapeutically. To address this question, we developed a synthetic-lethal high throughput siRNA screen using the innately resistant A549 lung adenocarcinoma cell line. Optimisation of the screen was performed using a siRNA death control (PLK1), which induced cell death in the absence of platinum, and a sensitization control (MTOR), which enhanced cell death only in combination with a sublethal concentration of carboplatin. These independent controls revealed that the screening protocol performed within acceptable limits of variability, quality and reproducibility as determined by Z’ factor analysis. Screening was then performed using a pool of four siRNAs targeting a single gene in conjunction with vehicle treatment, or with carboplatin. After screening siRNAs targeting the 720 kinases, 256 phosphatases and 4794 “druggable” targets of the human genome, we identified 50 candidate targets based on fold change difference between platinum and vehicle treatments, and statistical significance determined by multiple t-test corrected for false discovery rate. Preliminary integrated pathway analysis of RNA expression array and screening datasets revealed a highly significant enrichment of sensitizing genes in previously identified pathways as well as novel pathways. These data demonstrate that a synthetic-lethal approach can be used to identify therapeutic targets that could potentially sensitize lung adenocarcinoma to platinum-based chemotherapy.

Citation Format: Kieren D. Marini, Fernando J. Rossello, Luciano G. Martelotto, D. Neil Watkins. Using synthetic lethal screening to identify therapeutic targets for innately platinum resistant lung cancer. [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 3318. doi:10.1158/1538-7445.AM2014-3318
Abstract 3353: Docosahexaenoic acid enhances 2-deoxyglucose treatment in breast and lung cancer cells in vitro
Authors: Mouradian Michael , Ma Irvin V. , Vicente Erika D. , Chattin Amy M. , Pardini Ronald S.
Published: May 2015

Increased aerobic glycolysis is a common phenotype of cancer cells and is known as the Warburg effect. Recent studies have focused on targeting cellular metabolism to impede cancer cell growth. Previous findings in our laboratory have demonstrated that docosahexaenoic acid (DHA; C22:6 n-3), a long-chain omega-3 polyunsaturated fatty acid (PUFA), can inhibit cancer cell metabolism and reduce intracellular ATP levels. This study investigated the effects of concurrent treatment with DHA with 2-deoxyglucose (2DG), an inhibitor of glucose metabolism, in multiple cancer cell models in vitro, BT-474 human breast ductal carcinoma, MDA-MB-231 human breast adenocarcinoma, and A549 human lung adenocarcinoma. Dose-dependent decreases in intracellular ATP levels were observed with DHA and 2DG, both alone and in combination. In addition to the significant decreases in ATP levels, large decreases in extracellular lactate concentrations were observed in all three cell lines as well, suggesting that the combination of 2DG and DHA can augment treatment, mimic glucose deprivation and play a role in reducing the Warburg effect. Moreover, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were significantly reduced with the combination of DHA and 2DG, which suggests that the combination of DHA with other metabolic inhibitors, like 2DG, may sensitize cells to current treatment. Our findings show that combining DHA with 2DG enhances the efficacy of 2DG treatment alone, which may help to reduce the cytotoxic effects by 2DG on normal cells. Other metabolic inhibitors are being employed in combination with DHA to determine the mechanisms behind the DHA-induced sensitizing effects, which will provide additional treatment strategies for oncologists and cancer patients.

Citation Format: Michael Mouradian, Irvin V. Ma, Erika D. Vicente, Amy M. Chattin, Ronald S. Pardini. Docosahexaenoic acid enhances 2-deoxyglucose treatment in breast and lung cancer cells in vitro. [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 3353. doi:10.1158/1538-7445.AM2014-3353
Abstract 3359: JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α-mediated glucose metabolism
Authors: Wang Hung-Jung , Hsieh Ya-Ju , Cheng Wen-Chi , Lin Chun-Pu , Lin Yu-Shan , Yang So-Fang , Chen Chung-Ching , Izumiya Yoshihiro , Yu Jau-Song , Kung Hsing-Jien , Wang Wen-Ching
Published: May 2015

JMJD5, a JmjC-domain containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with PKM2, pyruvate kinase M2, to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes HIF-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α are all recruited to the HRE (hypoxia response element) site in the LDHA and PKM2 loci, and mediates the recruitment of the latter two proteins. Our data uncovers a new mechanism whereby PKM2 can be regulated by factor-binding induced homo/hetero-oligomeric restructuring, paving the way to cell metabolic reprogram.

Citation Format: Hung-Jung Wang, Ya-Ju Hsieh, Wen-Chi Cheng, Chun-Pu Lin, Yu-Shan Lin, So-Fang Yang, Chung-Ching Chen, Yoshihiro Izumiya, Jau-Song Yu, Hsing-Jien Kung, Wen-Ching Wang. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α-mediated glucose metabolism. [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 3359. doi:10.1158/1538-7445.AM2014-3359
Abstract 3365: Aldolase A induces invasion/metastasis of lung cancer through modulating HIF1-α and is a marker for poor clinical outcome
Authors: Chang Yu-Chan
Published: May 2015

The dysregulated metabolism is an emerging hallmark of cancer. However, little is known about the metabolic requirements during cancer progression. Therefore, identifying metabolic genes that are critical for supporting cancer invasion/metastasis may provide novel targets for cancer diagnosis and therapy. By analyzing gene expression and proteomic profile of invasive lung cancer cells CL1-5 and its parental counterpart CL1-0, we identified Aldolase A (ALDOA), an enzyme of glycolysis pathway, is overexpressed in CL1-5 and is significantly associated with disease recurrence in lung cancer patient microarray datasets. Moreover, HIF-1α was identified as the most significant transcription factor in ALDOA-recurrence gene signature by IPA upstream regulator analysis. Furthermore, Immunohistochemistry analysis confirmed the expression of ALDOA was markedly higher in lung cancer tumors than in normal lung. High ALDOA expression was found significantly associated with lymph node involvement and disease recurrence. Moreover, overexpression of ALDOA was significantly associated with poor outcome in both overall and disease-free survival. Knockdown of ALDOA expression was found to suppress invasion potentials in highly invasive lung cancer cell lines while complementary overexpression of ALDOA in poorly invasive cells promoted it in vitro and in vivo. ALDOA overexpression also accelerated glucose consumption, ATP utilization and lactate production. Knockdown of ALDOA expression in CL1-5 abolished HIF-1α expression with concurrent Mesenchymal-to-Epithelial Transition (MET). Moreover, IHC analysis shows an inverse correlation between ALDOA and E-cadherin and combination of high ALDOA and low E-Cadherin showed an unfavorable outcome, in a sharp contrast, compared to patients with low ALDOA and high E-cadherin. Here, we identified ALDOA as a critical glycolytic enzyme involved in lung cancer progression and may be a marker for poor clinical outcome.

Citation Format: Yu-Chan Chang. Aldolase A induces invasion/metastasis of lung cancer through modulating HIF1-α and is a marker for poor clinical outcome. [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 3365. doi:10.1158/1538-7445.AM2014-3365
Abstract 3375: Targeting glucose metabolism by 2-deoxyglucose in gastrointestinal stromal tumors
Authors: Mühlenberg Thomas , Grunewald Susanne , Schuler Martin , Bauer Sebastian
Published: May 2015

Introduction

Most patients with gastrointestinal stromal tumors (GIST) respond to imatinib (IM) but eventually progress with then limited therapeutic options. Fluorodeoxyglucose (FDG) is used for visualizing tumors by positron emission tomography (PET) and has been particularly helpful for predicting tumor response in highly FDG-avid GIST. 2-deoxy-glucose (2DG) inhibits glucose metabolism in cancer cells, which depend exclusively on aerobe glycolysis for ATP production (Warburg Effect). 2DG is currently investigated in several clinical trials. Here we evaluated the therapeutic potential of 2DG in GIST in vitro and in vivo.

Methods

Two IM-sensitive (IM-S; GIST-T1, GIST882) and 3 IM-resistant (IM-R; GIST430, GIST48, GIST48B) cell lines were studied. Cell viability was evaluated by Sulforhodamin B (SRB) assay after 6 days of 2DG treatment and combinations with IM and ABT-263, a BH3-mimetic. Immunoblotting was done for KIT- and KIT-downstream signaling and markers of apoptosis. For in vivo experiments nude mice bearing GIST-T1 xenografts were treated for 21 days with 500mg/kg/d 2DG or vehicle control.

Results

Cell viability assays in KIT-expressing cell lines displayed IC50 values for 2DG between 0.5mM (GIST882) and 2.5mM (GIST48), while KIT-negative GIST48B was less sensitive (6mM). Notably, treating GIST-T1 in low-glucose media (1g/L, LG) reduced IC50 (1.1mM to 0.33mM) in a glucose dependent manner. Immunoblot studies revealed a dose- and glucose dependent inhibition of KIT glycosylation by 2DG with complete inhibition at 1mM in LG, which coincided with inhibition of KIT-phosphorylation and KIT-downstream signaling. The combination of 2DG with IM had additive antiproliferative effects in IM-S cell lines. In IM-R GIST430 a synergistic induction of apoptosis could be observed when combining KIT-inhibiting concentrations of 2DG with ABT-263 1µM. Treatment of mice with 2DG alone did not affect tumor growth. At this dose 2DG dependent inhibition of KIT-glycosylation or -phosphorylation was not observed.

Conclusions

2DG shows promising GIST-specific antiproliferative effects by inhibiting KIT-glycosylation and -phosphorylation in vitro. These effects were additive when combined with imatinib. Further studies will investigate alternate treatment schedules as well as drug combinations with the aim of translating 2DG into a clinical trial in GIST.

Citation Format: Thomas Mühlenberg, Susanne Grunewald, Martin Schuler, Sebastian Bauer. Targeting glucose metabolism by 2-deoxyglucose in gastrointestinal stromal tumors. [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 3375. doi:10.1158/1538-7445.AM2014-3375
Abstract 3376: Targeting metabolic flux of tumor cells to prevent pancreatic cancer associated cachexia
Authors: Shukla Surendra K. , Singh Pankaj K.
Published: May 2015

Cancer associated cachexia is a complex metabolic syndrome characterized by excessive loss of skeletal muscle and adipose tissue. Cancer-induced cachexia accounts for nearly 20% of all cancer-related deaths and is most prevalent in patients with gastric, pancreatic, colon and lung cancer. More than 80% pancreatic cancer patients exhibit cachectic phenotype. Although association of cachexia with cancer is known for a long time, the molecular mechanism of cancer-induced cachexia is still not well understood. Better understanding of the cancer-related cachexia syndrome may provide novel therapeutic targets for enhancing the quality of life of cancer patients. Several studies have demonstrated that multiple cytokines and cachectic factors secreted by tumor cells contribute to cancer associated cachexia. Because most of the cancer cells present metabolic alterations, which may contribute to secretion of cachectic cytokines and metabolites involved in cancer-induced cachexia. By culturing differentiated C2C12 and 3T3-L1 cells in the presence of pancreatic adenocarcinoma cell or immortalized epithelial cell supernatants, we established cell culture-based cachexia model systems to investigate the depletion of muscle mass and fatty acid content. To study the role of metabolic pathways in tumor cells on cachectic factor secretion, we pre-treated pancreatic cancer cells with different metabolic inhibitors like 2-Deoxy-D-glucose (2-DG), 3-Bromopyruvic acid (3-BrPA) , bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulphide (BPTES) and collected conditioned medium from pre-treated cancer cells at different time points. Further, we accessed the cachectic potential of these conditioned media along with control conditioned medium. Physiological and biochemical alterations during muscle fiber breakdown and adipocyte fat depletion were assayed by performing real-time PCR and cell imaging. We observed that inhibition of glycolysis with 3-BrPA & 2-DG and inhibition of glutaminolysis with BPTES in tumor cells causes decreased muscle fiber breakdown and adipocytes fat depletion. Our results demonstrates crucial role of tumor cell metabolic alterations in cancer-induced cachexia.

Citation Format: Surendra K. Shukla, Pankaj K. Singh. Targeting metabolic flux of tumor cells to prevent pancreatic cancer associated cachexia. [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 3376. doi:10.1158/1538-7445.AM2014-3376
Abstract 3378: Glutaminase activity determines cytotoxicity of L-asparaginases on leukemia cell lines
Authors: Parmentier Jean-Hugues , Maggi Maristella , Tarasco Erika , Scotti Claudia , Avramis Vassilios , Mittelman Steven D.
Published: May 2015

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine (Asn) and glutamine (Gln). We and others have shown that its cytotoxicity depends more on Gln than Asn depletion. Novel ASNases with different activities have been developed in an attempt to decrease side effects and allergic reaction. To evaluate the importance of glutaminase activity, we developed a novel form of Helicobacter pylori ASNase (dm HpA) using site-directed mutagenesis, with amino acid substitutions M121C/T169M. This mutant form was designed to have the same asparaginase activity as wild type (wt), but lack glutaminase activity. WT and dm HpA were compared with the clinically used ASNases from E. coli (EcA-II) and Erwinia chrysanthemi (ErA).

Asparaginase and glutaminase activities of each enzyme were measured with Nessler's assay at pH 8.6. The ratio of glutaminase/asparaginase activity was 1.4% for dm HPase, 7.5% for wt HPase, 41% for EcA-II, and 99% for ErA (at 0.6 IU), respectively.

L-Asparaginases (0.01 to 3 IU/ml over 72 hrs) were tested on 8 human leukemia cell lines: pre B ALL (BV173, Nalm-6, RCH-ACV, RS4;11, SEM, SupB15), T cell ALL (Molt-4), and acute promyelocytic leukemia (HL-60). Viable cells were determined by trypan blue exclusion.

Two cell lines which we had previously shown to be Asn-dependent (RS4;11 and SupB15) were equally sensitive to the asparaginase isoforms (>90% kill at 0.03 IU/mL). The other 6 lines were more sensitive to isoforms with higher glutaminase activities (Table). ErA was the most effective on all three cell lines on which it was tested.

These data show that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full efficacy. These sensitivities to ASNases match previous data on Asn- and Gln-dependent cell proliferation (Ehsanipour et al., Cancer Research, 2013).

% cell growth inhibition at 3 IU/ml ASNase (n=1-2 for each cell line; nd: not done)Cell linedm HpAwt HpAEcA-IIErARS4;11919291ndSupB15919193ndRCH-ACV13362866SEM22484165BV17324326ndNalm-61621574HL-60204033ndMolt4325963nd

Citation Format: Jean-Hugues Parmentier, Maristella Maggi, Erika Tarasco, Claudia Scotti, Vassilios Avramis, Steven D. Mittelman. Glutaminase activity determines cytotoxicity of L-asparaginases on leukemia cell lines. [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 3378. doi:10.1158/1538-7445.AM2014-3378
Abstract 338: Mcl-1 mediates TWEAK/Fn14-induced non-small cell lung cancer survival and therapeutic response
Authors: Whitsett Timothy G. , Mathews Ian T. , Cardone Michael H. , Lena Ryan J. , Pierceall William E. , Bittner Michael , Sima Chao , LoBello Janine , Weiss Glen J. , Tran Nhan L.
Published: May 2015

Lung cancer is the leading cause of cancer-related mortality in the world killing more than one million people each year. Therapeutic resistance remains a significant clinical hurdle, contributing to a five-year survival rate of less than 5% in advanced lung cancer. The mechanisms of therapeutic resistance remain poorly understood and thus difficult to combat therapeutically. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling axis is known to promote cancer cell survival via NF-κB activation and up-regulation of pro-survival Bcl-2 family members. Mcl-1 is over-expressed across many tumor types and correlates with poor patient prognosis and therapeutic resistance. We hypothesize that activation of the TWEAK-Fn14 signaling axis can induce therapeutic resistance through Mcl-1, and that Mcl-1 suppression can sensitize lung cancer cells to therapeutic insult. Here we demonstrate that both Fn14 and Mcl-1 are over-expressed and correlated in primary patient lung tumors. Mcl-1 expression correlates with both advanced stage lung cancer and poor patient prognosis. TWEAK stimulation of non-small cell lung cancer (NSCLC) cell lines induces Mcl-1 protein expression in a NF-κB-dependent manner. TWEAK exposure completely abrogates the cell killing effects of cisplatin or radiation treatment in NSCLC cell lines, and this TWEAK-induced protection is dependent on Mcl-1 expression. The suppression of Mcl-1 through RNAi or a novel Mcl-1 inhibitor (EU-5148) enhances the cell killing effects of cisplatin or radiation and completely blocks TWEAK-induced cell survival, whereas Bcl-2 and Bcl-xL inhibition have lesser effect on TWEAK-induced survival. The specificity of EU5148 is demonstrated through abrogation of the protein-protein interaction of Mcl-1 and Bim, with no effect on the interaction of Bcl-xL and Bim. Moreover, EU-5148 treatment inhibits cell survival across a panel of NSCLC cell lines, representing a range of histological subtypes and driver mutations, such as EGFR and KRAS. Collectively, these results position TWEAK-Fn14 signaling through Mcl-1 as a significant mechanism for NSCLC tumor cell survival, and open new therapeutic avenues to abrogate the high mortality rate seen in advanced NSCLC.

Citation Format: Timothy G. Whitsett, Ian T. Mathews, Michael H. Cardone, Ryan J. Lena, William E. Pierceall, Michael Bittner, Chao Sima, Janine LoBello, Glen J. Weiss, Nhan L. Tran. Mcl-1 mediates TWEAK/Fn14-induced non-small cell lung cancer survival and therapeutic response. [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 338. doi:10.1158/1538-7445.AM2014-338
Abstract 339: Inhibition of NANOG/NANOGP8 downregulates MCL-1 in colorectal cancer (CRC) cells, enhances the efficacy of BH3 mimetics and inhibits clonogenicity
Authors: Mattoo Abid R. , Zhang Jingyu , Espinoza Luis A. , Thorgeirsson Snorri S. , Jessup J. M.
Published: May 2015

Combining lentiviral (LV) delivered shRNA against NANOG (shNG-1) or NANOGP8 (shNp8-1) with BH3 mimetics (ABT-737 and ABT-199) was undertaken to determine whether inhibition of NANOG/NANOGP8 enhances the cytotoxic effect of these BH3 mimetics in Colorectal Cancer (CRC) cells. NANOG is a key transcription factor important for both pluripotency in embryonic stem cells and malignant transformation and progression of colorectal carcinoma (CRC). Inhibition of NANOG or its paralog NANOGP8 reduces the proliferation, stemness and tumorigenicity of CRC cells. The cytotoxic potential of ABT-737 or ABT-199, as single agents, was tested in the CRC cell lines- Clone A, CX-1 and LS174T that have high levels of anti-apoptotic BCL-2 family proteins. Treatment with ABT-737 or ABT-199 showed variable cytotoxicity in the three CRC lines with LS174T being the most resistant. Inhibition of NANOG and NANOGP8 with LVshNG-1 or LVshNp8-1 in these cell lines decreased expression of MCL-1. The combination of LVshNG-1 and/or LVshNp8-1, with ABT-737 produced enhanced killing of the CRC cells by caspase-dependent apoptosis. These results were were confirmed when LV shNG-1 or LV shNp8-1 was used in combination with ABT-199. siRNA targeting MCL-1 reproduced the effects of NANOG/NANOGP8 inhibition. The CRC cells that survived the combination treatment showed lower regrowth potential and reduced clonogenicity when re-plated in fresh media. These results demonstrate that inhibition of NANOGP8 or NANOG enhances the cytotoxicity of BH3 mimetics that target BCL-2 family members through inhibition of MCL-1 expression.

Citation Format: Abid R. Mattoo, Jingyu Zhang, Luis A. Espinoza, Snorri S. Thorgeirsson, J. M. Jessup. Inhibition of NANOG/NANOGP8 downregulates MCL-1 in colorectal cancer (CRC) cells, enhances the efficacy of BH3 mimetics and inhibits clonogenicity. [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 339. doi:10.1158/1538-7445.AM2014-339
Abstract 3408: FOXM1 regulates doxorubicin resistance through the non homologous end joining pathway and is associated with recurrence of bladder cancer
Authors: Roh Yun-Gil , Lee Se-Ra , Kim Won-Tae , Kim Seon-Kyu , Chu In-Sun , Leem Sun-Hee
Published: May 2015

Non-muscle invasive bladder cancer (NMIBC) remains a very troublesome cancer to care because of high recurrence rates. In the previous study, we characterized a gene expression dataset from 102 NMIBC patients of the Korean cohort to identify a signature associated with disease recurrence. In recurrent bladder cancers, the expression of FOXM1 (Forkhead Box M1) was increased compared to primary cancers. Although there is a relationship between DNA repair pathway and FOXM1, it remains unclear whether FOXM1 is associated with DNA repair mediated cancer recurrence. To investigate this association, we used five different human bladder cancer cell lines to examine the expression level of FOXM1. Two cancer cell lines (5637 and EJ cells) showed the increased level of FOXM1 than other three cancer cell lines (UC5, LD611 and UC9 cells). We examined the chemoresistance with chemotherapy reagent (Doxorubicin) and checked the possible regulation of DNA repair pathway by FOXM1 in these cell lines. The cell lines which elevated FOXM1 expression showed high expression levels of Non-homologous end joining (NHEJ) pathway genes (Ku70, Ku80, LIG4 and XRCC4). To examine the direct interaction between the FOXM1 and NHEJ pathway genes, the binding activity was checked by a promoter assay and chromatin immunoprecipitation. We revealed that the expression levels of NHEJ pathway genes are elevated on activation of exogenous FOXM1. Therefore, we suggest a novel activity of FOXM1 has a role in cancer recurrence through the NHEJ pathway in bladder cancer cells.

Citation Format: Yun-Gil Roh, Se-Ra Lee, Won-Tae Kim, Seon-Kyu Kim, In-Sun Chu, Sun-Hee Leem. FOXM1 regulates doxorubicin resistance through the non homologous end joining pathway and is associated with recurrence of bladder cancer. [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 3408. doi:10.1158/1538-7445.AM2014-3408
Abstract 3417: The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation
Authors: Hendricks William P. , Zismann Victoria , Button Berry , Legendre Christophe , Tembe Waibhav , Yin Holly , Bittner Michael , Berry Donald , Huentelman Matthew , Khanna Chand , LoRusso Patricia , Breen Matthew , Sekulic Aleksandar , Trent Jeffrey
Published: May 2015

Despite remarkable therapeutic advances in malignant melanoma, an incomplete understanding of melanoma's extraordinary genetic diversity, complex disease course and ability to resist treatment continues to impede rational, curative clinical intervention. This is particularly true for the understudied “dark matter” of the melanoma genome, i.e. cases that lack mutations in the three most commonly-mutated melanoma genes (BRAF, NRAS, NF1) and that display extensive mutational heterogeneity. Clearly a pressing need exists for transformative approaches to melanoma medicine. We have therefore broadened our traditional focus on melanoma in human patients to include canine melanoma as a model of human disease. Given canine melanoma's clinicopathological resemblance to human non-cutaneous triple-negative subtypes, study of this disease in each species may inform the other. Further, the accelerated clinical course of canine melanoma and flexibility in veterinary clinical trial design may allow for evaluation of novel treatment designs that would not be possible in human cancer trials. Thus, we have undertaken a first-in-dog genomic characterization study of canine melanomas from a cohort of 37 dogs to further explore the landscape of this comparative model. This analysis combines comprehensive genomics (whole genome, whole genome long-insert, RNA sequencing, and array comparative genomic hybridization) in 7 matched canine melanoma/normal tissues with targeted analysis in an additional 27 tumors and 3 cell lines. These canine tumors display striking patterns of mutation and subtype heterogeneity resembling that of their non-cutaneous human counterparts with the majority being BRAF, NRAS, and NF1 wild type and lacking recurrent single nucleotide variants. Despite heterogeneity at the single nucleotide level, shared patterns of structural variation and gene expression exist and these patterns suggest core pathways that may define therapeutic vulnerabilities in these disease subtypes. These data provide the genomic framework for further clinical pursuit of melanoma in the canine model as a paradigm for drug development.

Citation Format: William P. Hendricks, Victoria Zismann, Berry Button, Christophe Legendre, Waibhav Tembe, Holly Yin, Michael Bittner, Donald Berry, Matthew Huentelman, Chand Khanna, Patricia LoRusso, Matthew Breen, Aleksandar Sekulic, Jeffrey Trent. The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation. [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 3417. doi:10.1158/1538-7445.AM2014-3417
Abstract 3420: TERT promoter mutations are rare in uveal melanoma
Authors: AMARO Adriana Agnese , Gualco Marina , Dono Maria , Pfeffer Ulrich , Maric Irena , Mosci Carlo
Published: May 2015

Uveal melanoma is the most frequent primary tumor of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight UV-exposure on the etiology of uveal melanoma is matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. We also analyzed the frequency of other recently discovered uveal melanoma specific mutations in the genes GNAQ, GNA11, BAP1, SF3B1, and EIFAX1, and we analyzed the association of these mutation with histopathological and clinical features including progression free survival. Finally, we analyzed TERT expression in these tumors.

The mutation frequencies observed confirmed the prevalence of GNAQ mutations in cases with disomy of chromosome 3 that have a better prognosis whereas GNA11 was more frequent in monosomic cases. Together these two mutations account for more than 85% of the cases. BAP1 mutations are associated with chromosome 3 monosomy but not with progression free survival. SF3B1 and EIF1AX mutations occur in 10.3 and 22.5% of uveal melanomas, in disomic cases only. We detected a TERT mutation in only one case of a 57-year old white male with clinical and histo-pathological features typical for uveal melanoma. The tumor showed mutations in GNA11 and EIF1AX. No mutations were detected in GNAQ, BAP1, and SF3B1. Both copies of chromosome 3 were retained. Several tumors among which the one carrying the TERT promoter mutation showed elevated TERT expression.

These data indicate that TERT mutations are rare in uveal melanoma, consistent with a reduced etiological influence of sunlight. No conclusion can be drawn on the potential influence of TERT mutations on tumor progression.

Citation Format: Adriana Agnese AMARO, Marina Gualco, Maria Dono, Ulrich Pfeffer, Irena Maric, Carlo Mosci. TERT promoter mutations are rare in uveal melanoma. [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 3420. doi:10.1158/1538-7445.AM2014-3420
Abstract 3428: Validation and utilization of next generation sequencing in the clinical assessment of gliomas
Authors: Nasrallah MacLean P. , Martinez-Lage Maria , Fox Alan , Sukhadia Shrey , Desai Arati , O'Rourke Donald M. , Brem Steven , Roth David , Morrissette Jennifer J.D. , Daber Robert D.
Published: May 2015

Background. Identifying prognostic and potentially therapeutic genetic alterations in gliomas is crucial to improve clinical outcome and guide future targeted therapies in this devastating disease. Here, we establish the feasibility of next generation sequencing (NGS) profiling of glioma samples in a clinical setting.

Methods. We optimized and validated a 47 gene panel for use on both fresh and Formalin Fixed Paraffin embedded (FFPE) tissue specimens in a CLIA-certified laboratory. Validation was performed in 27 samples by comparing mutation detection on two different NGS platforms, and we have since incorporated routine molecular tumor profiling into the standard workup of glioma patients seen at our institution.

Results. From analysis of 120 clinical samples, we found disease-associated changes in 90 patients (75%). Overall 184 changes including disease-associated point mutations and variants of unclear significance were identified across 98 samples, resulting in an average of 1.85 mutations per sample and a range of 1- 6 mutations. This included 49 amplifications across several genes (EGFR, PDGFRA and KIT).

Conclusions. The final validated assay allows for a cost effective and efficient analysis of a spectrum of clinically relevant and actionable biomarkers including point mutations, insertions, deletions and gene amplifications. Given the high fraction of tumors presenting with known disease associated changes, routine genomic profiling has the promise of improving patient outcomes and allow for access to targeted therapies.

Citation Format: MacLean P. Nasrallah, Maria Martinez-Lage, Alan Fox, Shrey Sukhadia, Arati Desai, Donald M. O'Rourke, Steven Brem, David Roth, Jennifer J.D. Morrissette, Robert D. Daber. Validation and utilization of next generation sequencing in the clinical assessment of gliomas. [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 3428. doi:10.1158/1538-7445.AM2014-3428