bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3342: The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status
Authors: Bouley Stephanie J. , Dillon Lloye M. , Miller Todd W.
Published: May 2015

The class 1a phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, survival, and metabolism. These PI3Ks are heterodimeric lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (p110α, p110β, or p110δ). p110α and p110β play distinct roles in PI3K signaling in carcinoma cells. p110α is activated by growth factor receptor kinase signaling. In contrast, p110β was shown to play a role in insulin metabolic action, G protein-coupled receptor (GPCR) signaling, and oncogenic transformation. Cancer cells deficient in PTEN, the tumor suppressor phosphatase that antagonizes PI3K signaling, have been shown to be sensitized to pharmacological inhibitors of p110β. As a result, early clinical studies with p110β inhibitors are restricted to patients with PTEN-deficient cancers. However, analysis of data from the Genomics of Drug Sensitivity in Cancer database revealed that genetic lesions in PTEN or PIK3CA (encodes p110α) were significantly and independently associated with increased sensitivity to the p110β inhibitor AZD6284. Among 668 cancer cell lines, 96, 57, and 5 lines harbored alterations in PTEN, PIK3CA, or both, respectively. Among 61 cell lines with IC50 values ≤2 μM, only 25 harbored an alteration in PTEN and/or PIK3CA. Thus, a significant fraction of cancer cell lines (and tumors) without PTEN alterations are likely to be sensitive to p110β inhibition. To explore the role of p110β in PI3K signaling in ER+ breast cancer, we treated cells with the p110β inhibitor GSK2636771 and monitored effects on growth factor-induced activation of AKT and MEK/ERK. We found that p110β inhibition reduced basal pAKT and pERK levels in PTEN-deficient cells. Furthermore, p110β inhibition reduced insulin-like growth factor 1 (IGF-1)-induced pAKT levels and delayed the onset of AKT phosphorylation in both PTEN-deficient and PTEN-wild-type cells. In contrast, p110β inhibition sensitized both PTEN-wildtype and PTEN-deficient cells to heregulin stimulation. These results indicate that p110β desensitizes cells to IGF-1 stimulation and hyper-sensitizes them to heregulin stimulation and suggests that p110β regulates downstream AKT and MEK/ERK activation in response to growth factor receptor activation. Our findings advocate for the testing of p110β inhibitors in cell lines (and tumors) selected based on growth factor dependence and PTEN status.

Citation Format: Stephanie J. Bouley, Lloye M. Dillon, Todd W. Miller. The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status. [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 3342. doi:10.1158/1538-7445.AM2014-3342
Abstract 3344: Akt1 and Akt3 exert opposing roles in the regulation of vascular tumor growth
Authors: Phung Thuy L. , Xue Qi , Nhek Sokha , Gerald Damien , Perruzzi Carole , Ayyaswamy Sriram , Du Wa , Geeganage Sandaruwan , Benjamin Laura
Published: May 2015

Vascular tumors are endothelial cell neoplasms with a wide spectrum of clinical presentations, ranging from benign infantile hemangiomas in children to low-grade malignant hemangioendotheliomas and highly aggressive angiosarcomas in adults. To date, the cellular and molecular mechanisms leading to vascular tumor formation are poorly understood, and current therapies, particularly those for malignant vascular tumors, have not significantly improved the clinical outcome.

We examined the growth regulatory pathways in vascular tumors, and showed that human vascular tumors have increased Akt activation, and constitutive activation of Akt1 in endothelial cells is sufficient to drive de novo vascular tumor formation in a transgenic mouse model. Importantly, we have uncovered the opposing functions of Akt isoforms in this regulation, in which Akt1 promotes, whereas Akt3 inhibits tumor endothelial cell migration and growth in in vitro and in vivo tumor models. Consistent with these findings, we observed reduced levels of Akt3 expression in human vascular tumors. Mechanistically, Akt3 blocks tumor cell migration and growth by inhibiting p70 S6-Kinase (S6K) activation, and Akt3 does so in part by modulating the levels of Rictor, a component of the mammalian target of rapamycin complex-2. S6K in turn exerts negative feedback regulation on Akt3 expression. Moreover, vascular tumor cells with Akt3 knockdown have increased S6K signaling, and are sensitized to a novel S6K inhibitor. Our findings provide a new perspective on Akt signaling, in which there are “check-and-balance” functions by different Akt isoforms to modulate the overall Akt signaling output. These studies also highlight the potential clinical utility of treating malignant vascular tumors, such as angiosarcomas, with agents that effectively block the S6K pathway.

Citation Format: Thuy L. Phung, Qi Xue, Sokha Nhek, Damien Gerald, Carole Perruzzi, Sriram Ayyaswamy, Wa Du, Sandaruwan Geeganage, Laura Benjamin. Akt1 and Akt3 exert opposing roles in the regulation of vascular tumor growth. [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 3344. doi:10.1158/1538-7445.AM2014-3344
Abstract 335: Aspirin, di-aspirin and their toxicity to colorectal cancer
Authors: Kankipati Chandra S. , Perry Christopher J. , Nicholl Iain D.
Published: May 2015

Introduction

The lifetime risk for developing colorectal cancer (CRC) is approximately 7%. In the USA over 140,000 people a year develop CRC, with approximately 51,000 deaths/year. Epidemiological studies indicate that long-term aspirin usage reduces the incidence of CRC, and may be protective against other non-CRC related adenocarcinoma, including esophageal cancer. We have investigated the mechanism by which aspirin exhibits toxicity to CRC by synthesising novel analogues of aspirin in an effort to identify functionally significant moieties. We previously suggested that “di-aspirin” (bis-carboxyphenol succinate, BCS) and analogues thereof retain relatively specific toxicity to colorectal cancer cells cultured in vitro.

The cellular functions of the Inhibitors of Apoptosis (IAPs) correlate with suppression of apoptotic cell death, tumor progression and metastases. Aspirin triggered apoptosis correlates with its capacity to decrease expression of Survivin, a member of the IAP family. Survivin exhibits dual functions; as an inhibitor of apoptosis and regulator of mitosis. We hypothesised that studying the alterations in the expression of IAPs would give an insight into the cytotoxic nature of the compounds.

Experimental procedure

A cell viability assay (MTT) was employed to investigate the cytotoxicity of the analogues in various CRC (SW480, HCT116 and LoVo) and non-CRC cell lines (A549, MCF7 and EA.hy926). Expression levels of IAPs (c-IAP1, cIAP2, XIAP, Livin and Survivin) were examined in the SW480 cell line by immunoblotting. Survivin expression was further examined by immunofluorescence analysis. Changes in Survivin expression in the CRC and non-CRC cell lines was also determined by immunoblotting.

Summary of data

Herein, we show that the “di-aspirin” analogues are relatively more toxic to colorectal cancer cells compared to non-colorectal cancer cell lines. In addition we suggest that these analogues can potently reduce the expression of Survivin in comparison to other IAPs.

Conclusion

We conclude that the analogues of aspirin used in the study promote cell death in CRC and non-CRC cell lines. We propose that the compounds induce apoptosis by decreasing the expression of Survivin. We also propose that the anti-proliferative activity of “di-aspirins” and their analogues have potential benefit in tackling human cancer.

Citation Format: Chandra S. Kankipati, Christopher J. Perry, Iain D. Nicholl. Aspirin, di-aspirin and their toxicity to colorectal 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 335. doi:10.1158/1538-7445.AM2014-335
Abstract 3350: CIP2A regulates cell proliferation via akt signaling pathway in human lung cancer
Authors: Lei Ningjing , Peng Bo , Zhang Jiangying
Published: May 2015

CIP2A is an intracellular endogenous protein phosphatase 2A (PP2A) inhibitor with oncogenic activities. CIP2A was initially identified as a tumor-associated antigen (TAA) in gastric and liver cancer patients, and it was overexpressed in a variety of cancer types. Recently, we found that CIP2A was overexpressed in human lung cancer specimens as compared to normal lung tissues. The elevated expression of CIP2A in lung cancer cells results in an increased cell proliferation. The observation on the association between CIP2A and AKT phosphorylation at S473 in our lab and other labs suggested the role of CIP2A in regulating AKT phosphorylation. Our preliminary data indicated that CIP2A might promote cell proliferation through AKT signaling pathway under growth factor stimulation. Interestingly, the depletion of CIP2A in lung cancer cells did not induce a global change of AKT phosphatase activity, which implied that CIP2A might recognize specific AKT targets and play certain roles in the signaling pathway. In this study, we identified several AKT substrates specifically targeted by CIP2A, such as Hsp70 and mTOR, by proteomic-based systematic screening. The candidates obtained from our study will expand the understanding on protein-substrate interaction and therefore direct the cancer drug design. Therefore, our study addressed a novel role of CIP2A in mediating cancer progression through interacting with AKT pathway in a substrate-dependent manner.

Citation Format: Ningjing Lei, Bo Peng, Jiangying Zhang. CIP2A regulates cell proliferation via akt signaling pathway in human 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 3350. doi:10.1158/1538-7445.AM2014-3350
Abstract 3356: Temozolomide resistance in human glioblastoma is associated with marked increase in PDK1 and MGMT expression
Authors: Lee William , Velpula Kiran Kumar , Tsung Andrew J.
Published: May 2015

Temozolomide has become the standard of care in glioblastoma (GBM) treatment, but a significant number of patients despite optimal treatment are met with recurrence. It is important to understand the molecular mechanisms underlying TMZ resistance for the development of better treatment strategies. In the present study, we evaluated the dynamic relationship between MGMT and resistance to TMZ. We generated a TMZ-resistant 5310R cell line by continuous treatment with 100 µM concentrations of TMZ (3 months) to parent 5310 cells. We tested the efficacy of TMZ on 5310R cells by FACS analysis. 5310R cells showed increased expression of MGMT when compared to parental controls. Also, we observed that the 5310R cells showed alterations in cell morphology, enhanced cell adhesion, increased migration and invasion capacities. Moreover, we observed PDK1, MGMT and EGFR expression to be up-regulated in 5310R cells compared to parental controls. Our previous data suggested that inhibition of PDK1 reverses the Warburg effect, and here we demonstrate that the acquired TMZ resistance in glioma may be associated with promotion of the malignant phenotype. Furthermore, we observed a significant correlation between PDK1, MGMT and EGFR expression in human GBM specimens using immunohistochemistry and Western blot analysis. As treatment with TMZ becomes more ubiquitous in GBM treatment, the significance of the resistant 5310R cell line and an understanding of the metabolic and epigenetic changes will become critical in the development of targeted and effective therapies.

Citation Format: William Lee, Kiran Kumar Velpula, Andrew J. Tsung. Temozolomide resistance in human glioblastoma is associated with marked increase in PDK1 and MGMT expression. [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 3356. doi:10.1158/1538-7445.AM2014-3356
Abstract 3358: Significant increase in survival of triple negative breast cancer animal model in response to BPM31510 alone or in combination with standard of care: BPM31510 mediated dynamic metabolic (Warburg) shift in breast cancer as potential mechanism
Authors: Narain Niven R. , Diers Anne R. , Walshe Tony E. , Lee Arleide , Ouro-Djobo Rakib , Vishnudas Vivek K. , Benaim Ely , Sarangarajan Rangaprasad
Published: May 2015

This study demonstrates that BPM 31510, a Coenzyme Q10 containing proprietary formulation to influences cellular metabolism that is deregulated in cancers and is now considered to be a “hallmark of cancer”. These studies demonstrate that BPM 31510 alone or in combination with standard of care significantly improved survival of triple negative breast cancer (TNBC) animal model. To further tease out beneficial effects of BPM31510, human breast cancer cells of varying receptor status (SKBR3, MDA-MB231) were subjected to either (a) pretreatment with BPM 31510 (6 h) followed co-incubation with chemotherapeutic agents for 48-72 h or (b) co-treatment with BPM 31510 and chemotherapeutic agents, and cancer cell responses were compared to non-tumorigenic mammary cells (MCF12A). Cellular bioenergetics profiling revealed that BPM 31510 shifted cellular metabolism from glycolysis to mitochondrial metabolism, and this metabolic shift was associated with significant increases in reactive oxygen species (ROS). Both BPM 31510 alone or pretreatment and cotreatment strategies with BPM 31510 plus standard of care resulted in significant decreases in viable breast cancer cells when compared to chemotherapeutic agents; however, minimal effects were observed in MCF12A cells. In contrast, BPM 31510 in combination with chemotherapeutic agents amplified caspase 3 activation and apoptotic cell death, indicating BPM 31510 enhances apoptotic signaling. Taken together, these data demonstrate that BPM 31510 is a novel agent that reengages the cellular metabolic and apoptotic machinery of cancer cells independent of genetic make-up underlying malignancy. In addition, BPM 31510 enhances the cytotoxicity of standard-of-care chemotherapeutic agents in breast cancer cells through regulation of mitochondrial metabolism and oxidative stress. These findings confirm that BPM 31510 is a novel, paradigm shifting agent with multiple utility (as a single agent or in combination) in breast cancer including TNBCs that otherwise have poor prognosis and limited therapeutic options.

Citation Format: Niven R. Narain, Anne R. Diers, Tony E. Walshe, Arleide Lee, Rakib Ouro-Djobo, Vivek K. Vishnudas, Ely Benaim, Rangaprasad Sarangarajan. Significant increase in survival of triple negative breast cancer animal model in response to BPM31510 alone or in combination with standard of care: BPM31510 mediated dynamic metabolic (Warburg) shift in breast cancer as potential mechanism. [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 3358. doi:10.1158/1538-7445.AM2014-3358
Abstract 3361: EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma
Authors: Makinoshima Hideki , Takita Masahiro , Matsumoto Shingo , Yagishita Atsushi , Owada Satoshi , Esumi Hiroyasu , Tsuchihara Katsuya
Published: May 2015

Recent accumulating evidences suggest that genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancerous cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer metabolism has not yet been clearly elucidated. In this study, we measured extracellular lactate production, glucose consumption and the glucose-induced extracellular acidification rate (ECAR) after the inhibition of EGFR signaling in LAD cell culture. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomic techniques, the major intracellular metabolites in EGFR-mutated LAD cells were quantified in the absence or presence of the tyrosine kinase inhibitor (TKIs) erlotinib. The molecular profile regulating the metabolic enzymes at the transcriptional and post-translational levels was characterized by RT-PCR, Western blotting and flow cytometry. We demonstrated that EGFR signaling was required for lactate production, glucose consumption and glucose-induced ECAR, indicating that EGFR signaling promoted the Warburg effect in LAD cells. Moreover, comprehensive metabolomic analysis revealed that the levels of key intermediate metabolites in glycolysis and the pentose phosphate pathway (PPP) were decreased after treatment with EGFR inhibitors. The glucose transport carried out by GLUT1 and GLUT3 was the most rapid and critically regulated function of glucose metabolism linked to EGFR signaling, although the expression of MYC-dependent glycolytic genes and the phosphorylation of pyruvate kinase muscle (PKM2) were down-regulated by EGFR inhibition at a later time point. A modest down-regulation of both total and membrane-bound GLUT3 was also observed after EGFR inhibition. Despite an equivalent total amount of GLUT1 in LAD cells, the levels of membrane-bound GLUT1 was decreased by EGFR-TKI treatment, suggesting that EGFR signaling controlled GLUT1 function through post-translational modification. We concluded that EGFR signaling regulated aerobic glycolysis in EGFR-mutated LAD cells. Our data suggests that the glycolytic pathway plays a central role in the pathogenesis of EGFR-mutated lung cancer. This regulatory pathway could be an attractive target for the development of targeted therapies to better treat patients with EGFR-mutated LAD.

Citation Format: Hideki Makinoshima, Masahiro Takita, Shingo Matsumoto, Atsushi Yagishita, Satoshi Owada, Hiroyasu Esumi, Katsuya Tsuchihara. EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma. [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 3361. doi:10.1158/1538-7445.AM2014-3361
Abstract 3362: Tyr-phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate pyruvate dehydrogenase complex and promote the Warburg effect
Authors: Fan Jun , Gu Ting-Lei , Boggon Titus , Kang Sumin , Chen Jing
Published: May 2015

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homoeostasis in mammalian cells. The current understanding of PDC regulation involves serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDHK), which inhibits PDH and subsequently PDC, whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here we report that lysine acetylation of PDHA1 and PDP1 is induced by EGF-stimulation in human cells, and is common in diverse human cancer cells. We found that K321 acetylation inhibits PDHA1 by recruiting PDHK1 and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important for the metabolic switch to glycolysis in cancer cells and promote tumor growth. Moreover, we identified mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) as the upstream acetyltransferase of PDHA1 and PDP1 using a novel shRNA library that targets the majority of acetyltransferases in the human genome, and SIRT3 as the deacetylase. Knockdown of ACAT1 results in decreased cancer cell proliferation and tumor growth. Furthermore, we found that EGFR and other tyrosine kinases including FGFR1 that are frequently dysregulated in human cancers phosphorylate PDP1 at Y381, which dissociates SIRT3 and recruits ACAT1 to PDC.

Our findings suggest a novel mechanism where lysine acetylation of PDHA1 and PDP occurs in both normal and cancer cells, and contributes to inhibitory regulation of PDC, providing complementary insight into the current understanding of PDHA1 regulation. We also demonstrate that hierarchical, distinct post-translational modifications crosstalk to regulate PDC by controlling its molecular composition. Moreover, our findings for the first time link a mitochondrial acetyltransferase, ACAT1, to the Warburg effect and tumor growth, suggesting that the ACAT1-PDP1-PDHA axis represents a promising anti-cancer target.

Citation Format: Jun Fan, Ting-Lei Gu, Titus Boggon, Sumin Kang, Jing Chen. Tyr-phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate pyruvate dehydrogenase complex and promote the Warburg effect. [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 3362. doi:10.1158/1538-7445.AM2014-3362
Abstract 3363: Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3)
Authors: Lea Michael A. , Geherty Raymond , David Rachelle , desBordes Charles
Published: May 2015

In human colon cancer cells, glycolytic activity shows a positive correlation with proliferation. PFKFB3 is an important enzyme regulating glycolysis in many tumor cells. We have studied the action of an established inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation in four colon cancer cell lines (Caco-2, HCT116, HT29 and SW1116). Glucose uptake and cell proliferation were inhibited by 3PO and butyrate and additive effects were seen with 10 µM 3PO and 1 mM butyrate. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The large induction of alkaline phosphatase activity by butyrate seen in most colon cancer cell lines was not observed with 3PO. Similar contrasting effects on induction of alkaline phosphatase activity were seen with rat Morris hepatoma 7777 cells where 2-3 fold induction was seen with 1 µM vorinostat or 1 mM butyrate but no significant effect was seen with 3PO in the range of 5-30 µM. The biguanides, metformin and phenformin, increase glycolysis but inhibit proliferation of colon cancer cells. After combination treatment with 3PO, glycolysis was inhibited, as judged by glucose uptake and acidification of the medium, and additive inhibitory effects on cell proliferation could be observed. For example, after a 72 hour incubation of Caco-2 cells with 10 µM 3PO, 25 µM phenformin or 1 mM metformin, as single agents, proliferation relative to controls was decreased 20%, 17% and 34%, respectively. For combinations of 3PO and phenformin or 3PO and metformin the decreases relative to controls were 48% and 58%, respectively. In conclusion, 3PO was an inhibitor of proliferation of colon cancer cells, especially the rapidly growing HCT116 and HT29 cell lines. 3PO may be a useful agent in combination with other drugs that inhibit colon cancer cell proliferation.

Citation Format: Michael A. Lea, Raymond Geherty, Rachelle David, Charles desBordes. Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3). [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 3363. doi:10.1158/1538-7445.AM2014-3363
Abstract 3368: Down-regulation of LDH-A reduced lactate production and changed the expression of glycolysis-associated transporters
Authors: Schroer Henrike , Fabian Christian G. , Kreutz Marina , Goetze Kristina , Mueller-Klieser Wolfgang
Published: May 2015

In the clinic, tumors showing a pronounced Warburg effect (high lactate tumors) are associated with a higher risk of metastasis and a decreased probability of survival of patients. Therefore, manipulations of the glycolytic pathway may alter tumor cell metabolism and might be a way to influence tumors’ aggressiveness, e.g. by down-regulating intracellular lactate levels.

To test for this hypothesis B16.SIY E12 murine melanoma cells were stably transfected with shRNA down-regulating LDH-A expression (Lac Low cells). As a control cell line B16.SIY E12 was transfected with a scrambled shRNA construct (Lac High cells). These cell lines were characterized regarding their metabolic properties in vitro, especially lactate production in medium supernatants, the extracellular acidification rates (ECAR) and oxygen consumption rates (OCR). Furthermore, the expression of glycolysis-associated transporters (GLUT1, GLUT3, MCT1, MCT4 and CD147) was determined at the protein level. In a first in vivo set-up ATP, lactate and glucose content of syngeneic experimental tumors were measured using induced metabolic bioluminescence imaging (imBI).

Changing LDH-A expression induced differences in lactate production in the two tumor cell lines in vitro with Lac Low cells producing significantly less lactate in 24 h than Lac High cells. Accordingly, Lac Low cells revealed a significantly lower ECAR and a higher OCR than Lac High cells. The protein expression of glycolysis-associated transporters was also influenced by changes in LDH-A expression with Lac High cells showing a significantly higher protein expression of MCT1 and MCT4. The general expression of GLUT1 was unchanged (Western Blot) but membrane localization was higher in Lac High cells (immunofluorescence staining). Measuring metabolite content in experimental tumors of the same cell lines showed that the cells maintained their glycolytic properties with Lac Low tumors showing lower lactate content than Lac High tumors.

In summary, down-regulation of LDH-A expression reduced lactate production and membrane localization of glycolysis-associated transporters. The fact, that these characteristic are maintained during the transition from in vitro to in vivo makes this model system useful for systematic studies on therapeutic manipulation of tumor glycolysis and associated pathways.

Supported by the Deutsche Forschungsgemeinschaft KFO262, DE 835/2-1 (WMK) + KR1418/8-1 (MK)

Citation Format: Henrike Schroer, Christian G. Fabian, Marina Kreutz, Kristina Goetze, Wolfgang Mueller-Klieser. Down-regulation of LDH-A reduced lactate production and changed the expression of glycolysis-associated transporters. [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 3368. doi:10.1158/1538-7445.AM2014-3368
Abstract 3369: Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines
Authors: Farah Ibrahim O. , Lewis Veshell L. , Ayensu Wellington K. , Cameron Joseph A.
Published: May 2015

Lung cancer is a one of the most prevalent and deadly cancers in United States. Experimental evidence support that cancer cells do exhibit higher glycolytic rates than normal cells (Warburg effect). To exploit this unique cancer-dependent ATP generation phenomenon, we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would negatively impact their survival and alter their growth and viability resulting from the vast decrease in their essential glycolytic ATP production; no negative consequences will be seen on normal lung cells. The human lung fibroblast cell line MRC-5 and the human alveolar epithelial cell line A549 were used as models for normal lung and lung cancer in vitro in this study. Using standard methods, both cell lines were maintained and exposed to honey and D-glucose reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging. Our results indicate that exposure of both cell lines to these natural nutraceutic organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Honey and D-glucose showed statistically significant (p<0.05) differential negative effects on the A549 line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line. Results show a promising role of honey and D-glucose as metabolites of interest for selective management of cancerous cells.

Citation Format: Ibrahim O. Farah, Veshell L. Lewis, Wellington K. Ayensu, Joseph A. Cameron. Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 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 3369. doi:10.1158/1538-7445.AM2014-3369
Abstract 3371: Iodoacetate and 3-bromopyruvate exert differential effects on human neuronal tumor SK-N-SH and U-87 cells
Authors: Bhushan Alok , Chatterji Tanushree , Wong Wendy , Rizvi Nisha , Balaraju Anurag K. , Neti Aishwarya , Lai James C.K.
Published: May 2015

Recent interests in the “Warburg effect” stimulated a variety of studies that have demonstrated many aggressive cancers have adapted to hypoxia and shifted to a glycolytic phenotype depending on their micro-environment. However, the mechanisms underlying their adaptive behavior have not been delineated. To further investigate our hypothesis that glycolytic enzymes are targets for new anti-cancer drug discovery, we have examined the effects of iodoacetate (IAA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, and 3-bromopyruvate (3-BP), an inhibitor of hexokinase II, on human neuroblastoma SK-N-SH and astrocytoma U-87 cells. Our results from survival assays showed that both IAA and 3-BP induced concentration- and time-dependent lowering of survival of both cell types, with the effects of IAA being more potent than those of 3-BP in both cell types while SK-N-SH cells being more susceptible than U-87 cells to 3-BP-induced cytotoxicity. Employing lactate dehydrogenase release from cells to medium as a marker for necrosis, we also found IAA was more potent than 3-BP in inducing necrosis in both cells types. Further investigation employing flow cytometric analysis revealed that at 1-10 µM, IAA induced more apoptosis in SK-N-SH than in U-87 cells while at the higher levels in this range, IAA induced more necrosis relative to apoptosis in both cell types. However, at 10-40 µM, 3-BP induced mainly apoptosis but little necrosis in both cell types. Our results are therefore consistent with the notion that IAA and 3-BP exert differential effects in inducing apoptosis and necrosis in SK-K-SH and U-87 cells. Studies are ongoing to further elucidate the signaling mechanisms mediating these effects induced by IAA and 3-BP. Thus, our findings may have pathophysiological and therapeutic implications in employing glycolysis as a target for discovering new drugs for treating aggressive brain tumors.

Citation Format: Alok Bhushan, Tanushree Chatterji, Wendy Wong, Nisha Rizvi, Anurag K. Balaraju, Aishwarya Neti, James C.K. Lai. Iodoacetate and 3-bromopyruvate exert differential effects on human neuronal tumor SK-N-SH and U-87 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 3371. doi:10.1158/1538-7445.AM2014-3371
Abstract 3373: Contribution of microRNA-122 to the homeostasis of liver energy metabolism
Authors: Tsou Ann-Ping , Wang Shuo-Ting , Tao Tse-Wen
Published: May 2015

MicroRNA-122 (miR-122) is a liver specific miRNA known to regulate lipid metabolism and to prevent liver tumorigenesis. Recent data suggest that miR-122 regulates the metabolism of mitochondria but its mechanistic role is unclear. We investigated how energy metabolism is regulated by miR-122 using the Mir122a-/- mouse model. We identified a distinctive expression profile of mitochondrially localized genes between WT and Mir122a-/- mice by both microarray and proteomics. KEGG and IPA database analysis further showed decreased mitochondrial respiration activity and Acetyl-CoA synthesis. Besides, the expression of genes involved in antioxidant mechanism is increased.

The primary hepatocytes were used to study the energy metabolism. The physiological activities measured by XF-24 extracellular flux analyzer revealed a higher mitochondrial respiration activity (OCR) accompanied with a proton leak in the mitochondrial inner membrane of the Mir122a-/- hepatocytes. In addition, a higher glycolytic activity (ECAR) was detected in Mir122a-/- hepatocytes. Poor ATP production was detected from Mir122a-/- mitochondria. Several lines of evidence demonstrated that Mir122a-/- hepatocytes have low mitochondria membrane potential: excess cytosolic reactive oxygen species, less mitochondrial superoxide anion and lower staining with Rhodamine 123 and Mitotracker red. We further treated cells with mitochondrial inhibitors and found an excellent antioxidant capacity and ATP homeostasis in Mir122a-/- primary hepatocytes. Interestingly, we detected lower protein expression of pyruvate dehydrogenase complex subunit E1, which may partly contribute to the defective mitochondrial activity. The liver homeostasis of Mir122a-/- mice, however, is stabilized by a vigorous antioxidant activity and higher glycolysis capacity. As a result, the livers of young Mir122a-/- mice appear to have switched to aerobic glycolysis (Warburg effect), which is the hallmark of cancer cells. Therefore, correction of mitochondrial dysfunction is likely to become one of new therapeutic means for hepatocellular carcinoma (HCC).

Citation Format: Ann-Ping Tsou, Shuo-Ting Wang, Tse-Wen Tao. Contribution of microRNA-122 to the homeostasis of liver energy 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 3373. doi:10.1158/1538-7445.AM2014-3373
Abstract 3377: Glutamine mediated aggressiveness and drug sensitivity in ovarian cancer cells
Authors: Yang Lifeng , Moss Tyler J. , Marini Juan C. , Mangala Selanere , Wahlig Stephen , Win Julia , Su Dan , Sood Anil K. , Ram Prahlad T. , Nagrath Deepak
Published: May 2015

Platinum-based drugs such as cisplatin, carboplatin and xoaliplatin hav been introduced into clinical trials and their outcomes has progressed the treatment on several tumors, including ovarian cancer. However, cancer cells acquire chemo-resistance in the long term incubation of drugs, which turns out to be the major limitation for cancer therapy. Recent studies have shown that cisplatin can accumulate in mitochondria and induce cytochrome C release, thereby resulting in apoptosis. We found that glutamine plays a vital role in maintaining mitochondrial integrity and function by regulating NAD+ and NADH. Using bioenergetics and isotopomer analysis we show that chemodrugs in presence of glutamine affect oxidative phosphorylation in drug sensitive parental ovarian cancer cells. Addition of NAD+ substrates increased NAD/NADH ratio abrogating cisplatin's effect on cell death and mitochondrial dysfunction. Furthermore, drug resistant cancer cells had relatively higher glutamine dependence when compared with parental cells. Importantly, we found that when glutamine's entry into TCA cycle was inhibited ovarian cancer cells became more sensitive to chemotherapy. Our findings are based on the quantification of metabolites, related to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acids and nucleotide metabolism, and comparison of these pathways fluxes in drug sensitive and drug resistant ovarian cancer. The insights obtained present a unique opportunity for overcoming the drug resistance limitation in clinical trials in ovarian cancers.

Citation Format: Lifeng Yang, Tyler J. Moss, Juan C. Marini, Selanere Mangala, Stephen Wahlig, Julia Win, Dan Su, Anil K. Sood, Prahlad T. Ram, Deepak Nagrath. Glutamine mediated aggressiveness and drug sensitivity in ovarian 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 3377. doi:10.1158/1538-7445.AM2014-3377
Abstract 3379: Metabolic influences of pancreatic tumor microenvironment on pancreatic cancer cell's metabolism
Authors: Baddour Joelle , Yang Lifeng , Marini Juan C. , Franco-Barraza Janusz , Cukierman Edna , Hu Chaoxin , Maitra Anirban , Nagrath Deepak
Published: May 2015

Pancreatic cancer, the most lethal of solid tumors, is associated with a five-year survival rate and high mortality. The lethality of this tumor stems from lack of early symptoms, inability for detection of cancerous pancreatic lesions, and a diagnosis window that is accompanied by tumor resistance and metastasis. The bulk of the tumor mass, the fibrotic stroma, has been deemed an active player in the initiation and progression of pancreatic ductal adinocarcinoma (PDAC). A number of studies have elucidated the interactions between stromal cells and pancreatic cancer cells (PCCs), and the extracellular matrix (ECM) and PCCs. However, the role of the tumor microenvironment on the metabolic machinery of PCCs remains an active field of investigation. In this study, we investigate the effect of pancreatic stellate cells (PSCs), cancer-associated fibroblasts (CAFs), with or without ECM components, in a two-dimensional or three-dimensional setting on the glycolytic and mitochondrial pathways of patient-derived PCCs. The modulation of the expression levels of metabolic enzymes by the tumor microenvironment was also investigated. The metabolic reprogramming induced in PCCs by normal and reactive pancreatic stroma was investigated by isotopomer flux analysis. Our results reveal that pancreatic reactive stroma differentially upregulates glutamine and arginine metabolism in PCCs. The insights obtained from our work will lead to the development of targeted therapies for stroma and pancreatic cancer cells.

Citation Format: Joelle Baddour, Lifeng Yang, Juan C. Marini, Janusz Franco-Barraza, Edna Cukierman, Chaoxin Hu, Anirban Maitra, Deepak Nagrath. Metabolic influences of pancreatic tumor microenvironment on pancreatic cancer cell's 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 3379. doi:10.1158/1538-7445.AM2014-3379
Abstract 3388: The CD44s splice isoform is essential for breast tumor initiating cell properties
Authors: Brown Rhonda , Liu Sali , Reinke Lauren , Cheng Chonghui
Published: May 2015

Tumor initiating cells (TICs) constitute a small fraction of tumor cells that display stem cell characteristics, including the ability to self-renew and generate heterogeneous tumors. A CD44+/CD24- profile has been widely used to identify TICs in a variety of tumors. However, it has remained unknown whether CD44 is functionally required for TICs and whether a specific CD44 isoform is enriched in TICs. Here, we report that the CD44s isoform is the major splice isoform expressed in a CD44+/CD24- breast stem cell population. Functionally, CD44s is required for expression of a breast stem cell (BSC) gene signature and is essential for mammosphere formation. Modulation of CD44 alternative splicing by the splicing regulator ESRP1 prevents CD44s production and, in turn, inhibits mammosphere formation and expression of the BSC signature. Furthermore, CD44s is required for limiting numbers of TICs to form tumors in an animal model of breast cancer progression. Together, these data illustrate that the CD44s splice isoform is enriched in CD44+/CD24- breast stem cells and serves as an important regulator of stem cell properties. Our results also suggest that CD44s-dependent TIC properties can be controlled at the level of alternative splicing by splicing factors that regulate production of specific CD44 isoforms.

Citation Format: Rhonda Brown, Sali Liu, Lauren Reinke, Chonghui Cheng. The CD44s splice isoform is essential for breast tumor initiating cell properties. [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 3388. doi:10.1158/1538-7445.AM2014-3388
Abstract 3391: Alteration of mammary tumor cell behavior by FLIZ1
Authors: Anderson Torri , Cali Christopher , Bell Katherine , Danielson Keith G. , Klein Ashley , Martin Christina , Radecki Sara , Santoro Adam , Schmidt John A. , Knepper Janice E.
Published: May 2015

Fetal liver zinc finger protein (FLIZ1, ZC3H8) was previously described as a negative regulator of GATA3 expression in T cells. GATA3 also is an important determinant of mammary cell fate and breast tumor cell behavior. We hypothesized that FLIZ1 also regulates GATA3 in the mammary gland, thereby influencing normal and tumor cell processes controlled by GATA3. We identified tumor cell lines with high levels of FLIZ1 expression, and transfected them with vectors driving expression of FLIZ1-interfering RNAs. Stable transfectants (Flizout cells) were evaluated for ability to migrate in a wound healing assay, and were found to have decreased mobility. Flizout cells also grew more slowly and with more pronounced epithelial morphology in media containing charcoal-stripped serum, suggesting increased dependence on steroid hormones. Flizout cells were compared to control cells in proliferation assays, and were found to grow at equivalent rates in standard media, but Flizout cells grew at decreased rates in the presence of tamoxifen. We implanted Flizout cells and controls into the mammary glands of syngeneic female mice, which were then treated with tamoxifen or peanut oil vehicle alone. Flizout cells grew more slowly in tamoxifen-treated animals, and tissue weights were significantly less than controls. In order to determine if FLIZ1 plays a role in the normal mammary gland, we assessed its expression during stages of mouse mammary gland development by immunoblot, and found FLIZ1 expression increased during mammary involution. These data are consistent with a role for FLIZ1 in regulation of mammary cell growth and tumor cell behavior.

Citation Format: Torri Anderson, Christopher Cali, Katherine Bell, Keith G. Danielson, Ashley Klein, Christina Martin, Sara Radecki, Adam Santoro, John A. Schmidt, Janice E. Knepper. Alteration of mammary tumor cell behavior by FLIZ1. [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 3391. doi:10.1158/1538-7445.AM2014-3391
Abstract 3392: Thromboxane-A2 receptor β-isoform regulates the activity of the tumor suppressor FOXO3 via phosphorylation by ERK in urothelial cancer
Authors: Sobolesky Philip M. , Halushka Perry V. , Garrett-Mayer Elizabeth , Moussa Omar
Published: May 2015

The tumor suppressor FOXO3 is a transcription factor whose activity and stability are regulated by phosphorylation on S294 via extracellular regulated kinases (ERK1/2). Previous data by our group has demonstrated overexpressing the thromboxane A2 isoform-β receptor (TPβ) increased ERK activation and induced malignant transformation of immortalized bladder cells. These data describe a novel mechanism of TPβ mediated regulation of FOXO3 via phosphorylation by ERK. Immunohistochemical studies revealed increased expression of nuclear pFOXO3-S294 in late stage bladder tumors, whereas total FOXO3 nuclear expression was reduced in high grade tumors. Both cytoplasmic and nuclear pFOXO3-S294 staining scores positively correlated with TPβ staining scores. Overexpressing TPβ in non-transformed urothelial cell lines, UROsta and SV-HUC, induced phosphorylation of FOXO3 at -S294. Knockdown of FOXO3 expression by shRNA increased cell migration and invasion. Conversely, overexpression of mutated FOXO3 that is resistant to ERK phosphorylation reduced the overall and TP agonist (U46619) mediated increase in malignant UMUC3 cell migration and invasion. Stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression which could be attenuated with TP antagonist (PTXA2) pre-treatment. A dual luciferase assay demonstrated that U46619 stimulation decreased overall FOXO3 transcriptional activity. Additionally, U46619 stimulation initially caused nuclear accumulation of pFOXO3-S294, but prolonged stimulation increased FOXO3 cytoplasmic localization. In summary, the data describe a novel mechanism of TPβ signaling in bladder cancer that results in the ERK mediated regulation of FOXO3 activity, which could contribute to tumor progression.

Citation Format: Philip M. Sobolesky, Perry V. Halushka, Elizabeth Garrett-Mayer, Omar Moussa. Thromboxane-A2 receptor β-isoform regulates the activity of the tumor suppressor FOXO3 via phosphorylation by ERK in urothelial 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 3392. doi:10.1158/1538-7445.AM2014-3392
Abstract 3394: In-depth gene expression profiling of seminomatous testicular germ cell tumors
Authors: Patel Sunali , Chu Shirley , Degoricija Lovorka , Lee Kathy Y. , Gillis Ad J.M , Rijlaarsdam Martin , Dorssers Lambert C.J , Looijenga Leendert H.J
Published: May 2015

In depth Whole Transcriptome Analysis by RNA-Seq was used to compare the expression profile of testicular germ cell tumors with the histology of seminoma and normal testis. From this analysis we identified a large number of genes that showed differential expression with statistically high significance, (p<0.01, >2X FC). The list included genes related to stem cell pleuripotency (NANOG, POU5F1), proliferation (KRAS, CCND2), a number of non-coding RNAs (snoRNAs, miRNA precursors, XIST), and testicular cancer related genes (LDHB, AKAP4). From this list we identified a subset of genes for validation and further screening. We used the OpenArray® RT-qPCR platform to quantitatively screen up to 224 coding and non-coding genes using samples from different testicular germ cell tumors (seminoma and non-seminoma) and normal tissue. Results from these screening experiments will be presented.

For research use only. Not for diagnostic procedures.

Citation Format: Sunali Patel, Shirley Chu, Lovorka Degoricija, Kathy Y. Lee, Ad J.M Gillis, Martin Rijlaarsdam, Lambert C.J Dorssers, Leendert H.J Looijenga. In-depth gene expression profiling of seminomatous testicular germ cell 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 3394. doi:10.1158/1538-7445.AM2014-3394