bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1858: Development of a multiplex panel of biomarkers to assess energy metabolism in cancer.
Authors: Balakrishnan Meenakshi P. , Parchment Ralph E. , Hollingshead Melinda G. , Tomaszewski Joseph E. , Kinders Robert J. , Doroshow James H. , Srivastava Apurva K.
Published: August 2014

Background: Cancer cells have an altered glucose metabolism, which not only permits them to survive under hypoxic conditions but confers a distinct growth advantage by sustaining higher proliferation rates, invasiveness, and subsequent distant metastasis. This unique difference in glucose metabolism from healthy cells opens up a selective mechanism by which to kill cancer cells, bypassing normal healthy cells. Several enzymes in glucose metabolism are attractive targets for development of first-in-class therapeutic agents. We have undertaken development of a multiplex panel involving biomarkers involved in glucose metabolism to support drug development and to interrogate glycolytic pathway in cancer patients. Methods: The panel includes quantitative determinations of protein levels of hexokinase-2 (HK2), pyruvate kinase isoform-2 (PKM2), lactate dehydrogenase alpha (LDHa), and phosphorylated pyruvate dehydrogenase E1-alpha (phospho-PDHE1a). The sandwich immunoassays were built on the Luminex® xMAP technology platform using MagPlex magnetic beads and cloned proteins are employed as calibrators. The panel is designed to measure protein concentrations in fractionated cell extracts from flash frozen tumor biopsies. Results: We have completed development of three sandwich immunoassays for HK2, PK-M2, and LDHa on Luminex platform. Suitable capture antibodies as well as phycoerythrin labeled detection antibodies have been identified and tested for sandwich formation and multiplexing using recombinant calibrators and cell lysates. The multiplex is currently standardized for optimal assay performance. Known pharmacological modulators (3-bromopyruvate, bortezumib, oxamate, dichloroacetate) of HK2, PKM2, LDHa, and PDHE1a were used in vitro to demonstrate expected directional changes in biomarkers in the panel. Conclusions: We have developed a multiplex panel to measure protein levels of three key molecules involved in energy metabolism in cancer cells. The multiplex panel provides a critical tool, in the clinical setting, to support ongoing therapeutic strategies aimed at targeting the energy provision pathway in cancer patients. Importantly, the panel can be used to survey metabolic phenotype of different tumors to identify type and stage of cancer most susceptible to inhibition of glycolysis. Funded by NCI Contract No HHSN261200800001E.

Citation Format: Meenakshi P. Balakrishnan, Ralph E. Parchment, Melinda G. Hollingshead, Joseph E. Tomaszewski, Robert J. Kinders, James H. Doroshow, Apurva K. Srivastava. Development of a multiplex panel of biomarkers to assess energy metabolism in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1858. doi:10.1158/1538-7445.AM2013-1858
Abstract 1859: CYLD downregulation induces aerobic glycolysis in human oral squamous cell carcinoma cells.
Authors: Nakamura Takuya , Shinriki Satoru , Hayashi Mitsuhiro
Published: August 2014

Purpose: The hypermetabolic nature of cancer cells, especially their increased reliance on aerobic glycolysis which has been associated with more aggressive phenotype, is considered metabolic hallmarks of cancer cells including oral squamous cell carcinoma (OSCC) cells. However, its precise mechanisms remain unknown. Cylindromatosis (CYLD) is recognized as a tumor suppressor gene whereas little is available about its impact on cancer progression. Our unpublished data showed that lower CYLD expression was associated with poor prognosis in OSCC patients. The objective of our study was to address specific contributions of CYLD to the signature metabolic features of OSCC cells.

Materials and methods: We determined the level of glucose consumption as well as lactate production to evaluate the effects of CYLD knockdown by siRNA on aerobic glycolysis in human OSCC cell lines. In addition, we measured extracellular acidification and oxygen consumption rates in OSCC cells by using XF Extracellular Flux Analyzers.

Results: Downregulation of CYLD significantly suppressed cell proliferation through cell cycle arrest while it inhibited apoptosis induced by serum withdrawal in vitro. In addition, loss of CYLD led to the increase in both of glucose consumption and lactate production in several OSCC cell lines. Consistently, culture medium became more acidic by CYLD nockdown,compared with the control. Furthermore, glycolysis was promoted by CYLD downregulation whereas no significant difference was found in mitochondrial respiration.

Conclusions: Dysfunction of CYLD may contribute to aerobic glycolysis without apparent influence on mitochondrial respiration. Further investigation on the advantage of such increased glycolysis and the change in metabolome profiling may lead to development of effective therapeutic strategies for OSCC and other malignancies.

Citation Format: Takuya Nakamura, Satoru Shinriki, Mitsuhiro Hayashi. CYLD downregulation induces aerobic glycolysis in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1859. doi:10.1158/1538-7445.AM2013-1859
Abstract 1869: DCA and metformin synergistically induce apoptosis in breast cancer cells.
Authors: Haugrud Allison B. , Zhuang Yongxian , Miskimins Wilson K.
Published: August 2014

The unusual metabolism of aerobic glycolysis in cancer cells suggests that this phenomenon can be exploited for improved therapies. Metformin, a drug used to treat type 2 diabetics, has clinical evidence of cancer prevention, and some studies show that cancer patients have a better response when it is used with neoadjuvant therapy. One of the cellular targets of metformin is the inhibition of Complex I in the mitochondrial electron transport chain. This leads to an accumulation superoxide species that can cause oxidative damage to cells and eventually lead to cell death. Based on this, we hypothesized that drugs which promote mitochondrial metabolism will synergize with metformin to increase superoxide production and promote cell death of cancer cells. Cancer cells using aerobic glycolysis display a high level of glucose uptake that is processed through glycolysis to pyruvate, which is then converted to lactate and secreted from the cells. Diverting pyruvate to mitochondrial metabolism rather than its conversion to lactate was expected to enhance metformin cytotoxicity. Dichloroacetate (DCA) is well established drug that has been tested in humans for the treatment of certain metabolic diseases. DCA works through inhibition of pyruvate dehydrogenase kinase (PDK), thus reducing entry of pyruvate into mitochondrial oxidative phosphorylation pathways. Consequently DCA is expected to promote mitochondrial metabolism and reduce lactate secretion by cancer cells. We demonstrate that DCA and metformin synergistically kill cultured breast cancer cells although the drug combination has no effect on non-transformed MCF10A breast epithelial cells. The enhanced cell death of the breast cancer cells displayed is through apoptosis, as shown by caspase-dependent increases in cleaved PARP. DCA treatment was associated with reduced lactate secretion by the cells and increased pH of the growth medium which suggested it promoted oxidative phosphorylation. The combination of two drugs also promoted increased mitochondrial superoxide production compared to metformin alone which was associated with increased oxidative damage. These findings indicate that, as expected, DCA promotes metformin-mediated cell death by enhancing mitochondrial metabolism of pyruvate. Therefore, it is possible that the combination of metformin with DCA, or similar compounds, could enhance cancer therapy.

Citation Format: Allison B. Haugrud, Yongxian Zhuang, Wilson K. Miskimins. DCA and metformin synergistically induce apoptosis in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1869. doi:10.1158/1538-7445.AM2013-1869
Abstract 187: Clinically relevant efficacious doses of resveratrol induce changes in the AMPK/mTOR pathway, accompanied with senescence in preclinical models of colorectal cancer .
Authors: Kholghi Abeer O.
Published: August 2014

Resveratrol, a polyphenol present in red wine and peanuts exerts an array of beneficial effects, including anticancer activity and calorie restriction (CR) mimicry. Recent work from our laboratory has shown that low doses of resveratrol, comparable to amounts contained in a glass of red wine significantly reduced adenoma development in ApcMin mice when given with a high fat diet (Scott et al, unpublished). The protective effect was more pronounced than that caused by higher amounts, equivalent to doses used in clinical trials. The CR activity of resveratrol is mediated via the energy regulator AMP-activated kinase (AMPK). This study aims to investigate whether AMPK signalling contributes to the chemopreventive effect of resveratrol.

Intestinal mucosa from female ApcMin mice maintained on a high fat diet containing resveratrol (0.00007 or 0.014% in their diet) for 10 weeks was analysed by Western blotting to assess AMPK signalling, and the induction of autophagy and senescence. For more detailed mechanistic interrogation APC10.1 cells originally derived from adenomas of ApcMin mice were incubated with resveratrol (0.001-10μM) for 6 days and protein expression examined.

The extent of AMPK activation in vivo correlated with the degree of adenoma inhibition; AMPK expression and phosphorylation was evident in mucosa of resveratrol treated mice, but not in control animals, with highest levels in those that ingested the low dose. This chronic resveratrol treatment was also associated with increased expression of p21, a biomarker of senescence, particularly at the low dose. In APC10.1 cells resveratrol caused significant increases in AMPK phosphorylation at 0.01, 0.1 and 1μM, but not at 10μM, consistent with a bell-shaped dose-response. Activity was accompanied by significantly reduced phosphorylation of the mammalian target of rapamycin (mTOR), which is regulated by AMPK signalling, plus its downstream targets p70S6K and 4E-BP1. Furthermore, these concentrations also increased autophagy and senescence, as measured by LC3-II and p21 expression, respectively, and greater numbers of senescence-associated β-galactosidase positive cells.

The results suggest that activation of AMPK signalling and the induction of senescence may contribute to the cancer chemopreventive effects of resveratrol at dietary relevant intakes.

Citation Format: Abeer O. Kholghi. Clinically relevant efficacious doses of resveratrol induce changes in the AMPK/mTOR pathway, accompanied with senescence in preclinical models of colorectal cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 187. doi:10.1158/1538-7445.AM2013-187
Abstract 1873: Effect of intermittent calorie restriction on cancer stem cells.
Authors: Luo Yi , Kitadai Yasuhiko , Chihara Yoshitomo , Fujiwara Rina , Fujimoto Kiyohide , Kuniyasu Hiroki
Published: August 2014

Effect of intermittent calorie restriction (ICR) on cancer is controversial. We then examined the effect of ICR and food contents on cancer using mouse model. CT26 mouse colon cancer cells were inoculated into the back of syngeneic BALB/c mouse. A 24 hours-fasting was performed once a week for 4 weeks. A control diet and a high calorie diet were used for feeding. The body weight at week 4 of mice was not different among control diet (Group C), control diet+ICR (Group CI) and high fat diet+ICR (Group FI). The tumor weight, nodal metastasis and CD133-positive cancer stem cells (CSC) in the tumor were more pronounced in Group CI and FI than those in Group C and F. Blood CD133 mRNA levels after fasting breakage were increased in Group CI and FI. We have reported the prometastatic effect of trans fatty acids in colorectal cancer. Here effect of oral intake of trans fatty acids on tumor stem cells with ICR was examined using the same model. Mice fed with 10% elaidic acid diet with ICR- (Group E) or ICR+ (Group EI) were compared with Group C and CI. The tumor weight and tumor MIB1 index was Group EI, Group E, Group CI and Group C in high order. In Group EI, nucleostemin (NS)-positive tumor cells were transiently increased on the next day of ICR. During 12 ICR cycle, NS-positive CSCs were gradually increased in Group EI. These findings suggest that ICR might enhance cancer stem cell-activating factors.

Citation Format: Yi Luo, Yasuhiko Kitadai, Yoshitomo Chihara, Rina Fujiwara, Kiyohide Fujimoto, Hiroki Kuniyasu. Effect of intermittent calorie restriction on cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1873. doi:10.1158/1538-7445.AM2013-1873
Abstract 1879: Myc induces lipogenesis and suppresses fatty acid oxidation in human P493-6 B-lymphoma cells.
Authors: Gouw Arvin Matthew , Mancuso Anthony , Hsieh Annie Lee , Altman Brian , Wolfgang Michael , Van Dang Chi
Published: August 2014

Myc has been known to control cell growth and proliferation through regulating thousands of genes, of which a substantial amount are involved in metabolism. We have previously shown that Myc increases ribosomal biogenesis, nucleotide metabolism, mitochondrial biogenesis, glycolysis and glutaminolysis in cancer cells. However, Myc's role in fatty acid metabolism is not well-documented, notwithstanding the expectation that Myc induction of cell growth must be accompanied by membrane biogenesis. Other studies have shown that fatty acids can be utilized as energy fuel by cancer cells or as building blocks for proliferation. The P493 human B cell line contains a tetracycline (Tet)-repressible human MYC transgene, permitting robust control of cell growth and proliferation through MYC. Having previously documented that Myc stimulates glucose and glutamine metabolism, we sought to determine whether Myc affects P493 use of fatty acids for oxidation and fatty acid synthesis. We found that resting P493 cells, in which ectopic MYC was off, can use labeled palmitate for respiration, while Myc activated P493 cells demonstrated an inability to oxidize palmitate. We also observe through microarray analysis and RT-PCR that Myc increases the levels of mRNAs for lipogenesis genes. Chromatin immunoprecipitation (ChIP) with an anti-Myc antibody documents that Myc binds to the promoters of lipogenesis genes. Moreover, immunoblot analyses with antibodies available for selected enzymes also indicate that Myc increases the protein levels of ACACA and FASN. Uniformly labeled 13C-glucose and 13C,15N-glutamine were found to be incorporated into lipids when Myc was induced in P493 cells. Cerulenin, an inhibitor of FASN, dramatically inhibits Myc-induced P493 cell proliferation. Similarly, TOFA-inhibition of ACACA suppresses P493 cell proliferation as well. Our data suggest that in P493 Burkitt lymphoma model cells, Myc inhibits fatty acid oxidation and upregulates fatty acid synthesis through transcription of target genes.

Citation Format: Arvin Matthew Gouw, Anthony Mancuso, Annie Lee Hsieh, Brian Altman, Michael Wolfgang, Chi Van Dang. Myc induces lipogenesis and suppresses fatty acid oxidation in human P493-6 B-lymphoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1879. doi:10.1158/1538-7445.AM2013-1879
Abstract 188: 4-methylumbelliferone: Dietary supplement turned chemo-preventive and anti-metastatic agent for prostate cancer.
Authors: Yates Travis , Lopez Luis , Hupe Marie , Ortiz Nicolas , Lokeshwar Vinata B.
Published: August 2014

INTRODUCTION: Hyaluronic acid (HA), a glycosaminoglycan, is associated with prostate cancer (PCa) progression and metastasis. 4-methylumbelliferone (4-MU) is a HA-synthesis inhibitor and we have shown antitumor and anti-invasive effects of 4-MU on PCa cells in vitro and in xenografts. In this study we evaluated the efficacy of 4-MU to prevent PCa growth and metastasis in the TRAMP model. We also evaluated the effects of 4-MU on epithelial-mesenchymal transition (EMT).

METHODS: TRAMP mice (n = 8-11/group) were treated with vehicle or 4-MU (450 mg/kg) by starting stage-specific treatment at 8 (PIN stage), 12 (adenocarcinoma) and 22 (invasive carcinoma) wks of age. At 28 wks, 50% of animals in 4-MU treated groups were sacrificed and the other 50% were left untreated up to 52 wks. At necropsy prostate (P) and seminal vesicles (SV) were weighed, tissues were analyzed by histology, immunohistochemistry and quantitative PCR for EMT markers and serum chemistry was evaluated. PC3-ML cells treated with 4-MU were analyzed for EMT markers.

RESULTS: At 28 wks, 100% of vehicle treated mice developed prostate tumors, but tumor growth and progression was inhibited in all 4-MU treated groups; P+SV weight (g): vehicle: 3.2±2.9; 8 wk: 0.43±0.14; 12 wk: 0.35±0.04; 22 wk: 1.0±0.35 g. The P+SV weight in TRAMP mice at 22 wks of age was 0.9±0.04, showing that late stage administration of 4-MU halted further tumor progression. Animals in 8 and 12 wk groups remained tumor free until 52 wk and 44 wk respectively. While 55% of vehicle treated animals developed metastasis, none of the treated groups had metastasis. No serum/organ toxicity or weight loss was observed in the treatment groups. HA, HA receptors (CD44, RHAMM) and EMT markers (β-catenin, Zeb2) were downregulated (>2-fold), but E-cadherin was upregulated (4-fold) in the treatment groups. 4-MU treatment (0-60 μg/ml) similarly modulated the expression of EMT markers and HA receptors in PC3-ML cells.

CONCLUSION: 4-MU is a promising non-toxic, oral chemopreventive agent, which inhibits PCa growth and metastasis by plausibly abrogating HA-signaling and reversing EMT.

Support: R01 CA 123063-04 (VBL); R01 CA 72821-11 (VBL)

Citation Format: Travis Yates, Luis Lopez, Marie Hupe, Nicolas Ortiz, Vinata B. Lokeshwar. 4-methylumbelliferone: Dietary supplement turned chemo-preventive and anti-metastatic agent for prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 188. doi:10.1158/1538-7445.AM2013-188
Abstract 1888: Metabolomic analysis provides insights into uncoupling-induced cancer resistance in the K5-UCP3 mouse model.
Authors: Solmonson Ashley , Keaton Mignon , Nowinski Sara M. , Morin-Kensicki Elizabeth , Mills Edward M.
Published: August 2014

Metabolic reprogramming is a hallmark of tumorigenesis and malignancy across a variety of cancer types. Reduced oxidative phosphorylation and increased ATP synthesis by glycolytic upregulation is thought to provide both the energy and metabolic precursors necessary to meet the increased biosynthetic and energetic demands associated with malignancy. We hypothesized that since uncoupling protein 3 (UCP3) dissipates the mitochondrial electrochemical proton gradient, it may lead to the consumption or depletion of biosynthetic substrates necessary for carcinogenesis. In support, previous studies have shown that mice expressing a keratin 5-UCP3 transgene are completely resistant to chemically- and Ras-induced skin carcinogenesis, however the metabolic consequences of UCP3 overexpression in relation to cancer resistance are unclear. To examine the metabolic changes associated with UCP3 expression and this profound skin cancer resistance, we performed metabolomic analysis of more than 350 metabolites in epidermis from K5-UCP3 and wild type FVB/N littermate mice. Results of this analysis show significant UCP3-dependent changes in metabolites indicative of increased energy harvesting via glycolysis, beta-oxidation and branched chain amino acid metabolism. Consistent with decreased energy reserve, K5-UCP3 epidermis exhibits increased uncoupled respiration, decreased ATP and increased AMP levels, which correspond to increased AMP Kinase (AMPK) activation (Thr172 phosphorylation). Taken together, these data suggest that uncoupling proteins might function to oppose the metabolic demands of tumorigenesis and suggest that targeting mitochondrial uncoupling may be a novel and effective strategy in cancer prevention and treatment.

Citation Format: Ashley Solmonson, Mignon Keaton, Sara M. Nowinski, Elizabeth Morin-Kensicki, Edward M. Mills. Metabolomic analysis provides insights into uncoupling-induced cancer resistance in the K5-UCP3 mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1888. doi:10.1158/1538-7445.AM2013-1888
Abstract 1903: Up-regulation of vitamin B1 homeostasis genes in breast cancer.
Authors: Zastre Jason , Hanberry Brad , Sweet Rebecca , McGinnis Cary , Venuti Kristen , Bartlett Michael , Govindarajan Raj
Published: August 2014

Malignant cells exhibit alterations in metabolic enzymes requiring an adequate supply of cofactors to support enzymatic activity. In particular, Vitamin B1 (thiamine) is an important component of cellular growth and metabolism within all cells, functioning as an essential enzyme cofactor in key metabolic pathways within in the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and the conversion of thiamine to the active coenzyme, thiamine pyrophosphate (TPP) by thiamine pyrophosphokinase (TPK1). Therefore, the objective was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. This work will aid in linking the metabolic phenotype of cancer cells with the adaptive regulation of thiamine homeostasis. This may provide a molecular basis for dietary interventions and new therapeutic targets directed towards cancer cell metabolism.

Methods: cDNA arrays of breast cancer and normal breast tissues were employed to determine expression of SLC19A2, SLC19A3, SLC25A19 and TPK-1. Western blotting was used to determine whole cell protein expression. To test the extent of cell surface localization of the transporters, cell surface proteins were biotinylated and run on Western Blot. Transport assays were used to quantify thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells (HMEC).

Results: SLC19A2, SLC25A19 and TPK-1were found to be significantly up-regulated in clinical tissues and breast cancer cell lines 6.48 (P=0.0013), 5.97 (P=0.0003), and 2.33 (P=0.0063) folds, respectively. Additionally, thiamine transporters THTR1 and THTR2 exhibited different cellular localization and expression depending on the ER status of the cancer cell. The increase in thiamine homeostasis genes correlated with an increase in intracellular free thiamine levels but not with the coenzyme TPP.

Conclusions: Overall, these findings demonstrate an adaptive response by breast cancer cells to bolster an increase in the cellular availability of thiamine.

Citation Format: Jason Zastre, Brad Hanberry, Rebecca Sweet, Cary McGinnis, Kristen Venuti, Michael Bartlett, Raj Govindarajan. Up-regulation of vitamin B1 homeostasis genes in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1903. doi:10.1158/1538-7445.AM2013-1903
Abstract 1908: 19q13.2 amplification occurs in a subset of SMAD4-deficient pancreatic cancers.
Authors: Min Bosun , Lai Albert , Bardeesy Nabeel , Deshpande Vikram
Published: August 2014

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. The PDAC genomes show evidence of high genomic instability leading to complex genomic rearrangements and copy number alterations. Major genetic events involved in PDAC progression have been characterized, but the subsequent alterations that contribute to different tumor behavior or patient outcomes are not well understood. Smad4 tumor suppressor gene mutation or deletion in pancreatic cancer correlates with metastatic disease and poorer patient outcome. We found that in a subset of PDACs with Smad4 expression loss, the chromosomal region 19q13.2 is focally amplified. Genomic amplifications found in cancers have been shown to contain driver genes that contribute to tumorigenesis. We are currently investigating the contribution of 19q13.2 amplicon resident genes in pancreatic cancer cell growth. Our characterization of a cancer amplicon in relation to known major genetic events in pancreatic cancer revealed that the 19q13.2 amplicon is closely associated with a subset of SMAD4-deficient tumors. Through additional in-depth tumor analyses we may better understand the heterogeneity and complexity of PDAC biology.

Citation Format: Bosun Min, Albert Lai, Nabeel Bardeesy, Vikram Deshpande. 19q13.2 amplification occurs in a subset of SMAD4-deficient pancreatic cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1908. doi:10.1158/1538-7445.AM2013-1908
Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.
Authors: Gomes Evan , Goodison Steven , Miyake Makito , Ross Shanti , Zhang Ge , Rosser Charles
Published: August 2014

Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression.

Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones.

Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death.

Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes.

Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG).

Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922
Abstract 1923: Constitutional mismatch repair-deficiency syndrome is a rare cause of cancer even in a highly consanguineous population.
Authors: Bu Rong , Siraj Abdul K. , Bavi Prashant , Belgaumi Asim , Al-Haqawi Wael , Al-Dayel Fouad , Uddin Shahab , Alkuraya Fowzan S. , Al-Kuraya Khawla S.
Published: August 2014

Background:

Biallelic germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2 cause a recessive childhood cancer syndrome characterized by early onset malignancies and signs reminiscent of neurofibromatosis type 1 (NF1). This syndrome is known as constitutional mismatch repair-deficiency (CMMR-D) syndrome. Because consanguinity unmasks autosomal recessive disorders, we hypothesized that the frequency of CMMR-D is inflated in the highly consanguineous population of Saudi Arabia.

Methods:

372 patient samples from Saudi Arabia that cover the tumor spectrum of CMMR-D syndrome were analyzed for biallelic germline mutations in the MLH1, MSH2, MSH6 and PMS2 with the use of direct genomic sequencing.

Results:

None of the 372 patients involved in the study was found to have biallelic pathological mutations of MLH1, MSH2, MSH6 or PMS2.

Conclusion:

CMMR-D is exceptionally rare among pediatric cancer patients and adult early onset cancer patients, even in the highly consanguineous Saudi population. Our findings suggest that larger cohorts will be needed, particularly in outbred populations, to determine the frequency of CMMR-D and that routine screening for this syndrome among cancer patients is not warranted.

Citation Format: Rong Bu, Abdul K. Siraj, Prashant Bavi, Asim Belgaumi, Wael Al-Haqawi, Fouad Al-Dayel, Shahab Uddin, Fowzan S. Alkuraya, Khawla S. Al-Kuraya. Constitutional mismatch repair-deficiency syndrome is a rare cause of cancer even in a highly consanguineous population. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1923. doi:10.1158/1538-7445.AM2013-1923
Abstract 1927: Polyploidy across breast cancer subtypes.
Authors: Fothergill Amy , Burkard Mark
Published: August 2014

Introduction: Breast cancer remains the most common cancer in women. Although treatment advances have improved the prognosis for many women, many existing therapies have significant associated toxicities. A major goal of current research is to develop targeted therapies based on characteristics of each individual's cancer.

Polyploidy can provide a potential difference between cancer and normal cells that can be harnessed to selectively destroy cancer cells. We hypothesize that polyploidy is common in aggressive triple negative breast cancer. If this is the case, polyploidy could provide a target for therapy in triple negative cancers.

Methods:We conducted a retrospective review of data collected by the University of Wisconsin Tumor Registry and HER2 testing by the state lab of hygiene between 2003 and 2011. Patients from the two data sets were matched by name and/or medical record number. Tumors were considered polyploid if ≥3 CEP17 signals were detected. Chi square testing and student T-tests were used to identify statistical correlations.

Results: A total of 1211 were identified meeting eligibility criteria and 170 women had polyploid tumors. Of these, 72 had unknown or equivocal results for ER/PR or HER2 and were excluded from analyses. Polyploid tumors correlated with TNBC (p=0.028) and hormone receptor negative cancer (p=0.02). Subgroup analysis showed a positive correlation between HER2 positive tumors and polyploid tumors (p=0.0005). Polyploidy tumors were more likely to be either stage I or stage IV (p=0.0019). Vital status at last contact and age at diagnosis were not significantly different between the two groups (p=0.2875 and 0.7826, respectively).

Discussion:We conclude that polyploid tumors cross all known subtypes of breast cancer, but are enriched among triple negative cancers. This may represent a viable group of cancers that can be targeted with specialized therapy.

Table 1 Polyploid Euploid P HER2- 122 857 0.0005 Hormone- 38 164 0.02 Triple negative cancers 29 121 0.028 Stage I 80 612 0.002 Stage II 54 275 Stage III 29 99 Stage IV 4 49 Unknown stage 3 6 Mortality 33 168 0.29 Mean Age 59 58 0.78

Citation Format: Amy Fothergill, Mark Burkard. Polyploidy across breast cancer subtypes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1927. doi:10.1158/1538-7445.AM2013-1927
Abstract 1936: The increased frequency of ERG oncoprotein expression in high grade prostate cancers in Caucasian American in comparison to African American men.
Authors: Young Denise , Farrell James , Chen Yongmei , Cullen Jennifer , Petrovics Gyorgy , Rosner Inger , McLeod David G. , Srivastava Shiv , Sesterhenn Isabell A.
Published: August 2014

Introduction & Objectives: ERG is one of the most commonly over expressed oncogene (50-70%) in prostate cancer (CaP) due to TMPRSS2-ERG gene fusion. Prior studies suggested that the fusion is more common in Caucasian American (CA) than in African Americans (AA) men with CaP. Previous studies from our laboratory have also shown that in AA patients the higher grade index tumors were predominantly ERG negative. We sought to quantify the association between race and ERG expression in a cohort of high grade CaP patients. We then examined whether the prevalence of ERG has prognostic significance for CaP disease-specific outcomes.

Materials & Methods: The CPDR multi-center national database was queried to identify patients with high grade CaP who underwent radical prostatectomy between 1994 and 2011. Sixty-three AA patients were age-matched with 63 CA patients (n=126). Immunohistochemistry was performed to detect fusion status in representative whole mount prostate specimens using an ERG monoclonal antibody (9FY). Tumors were classified as ERG positive or negative and compared by race. Multivariate analysis and Kaplan-Meier curves were used to evaluate the clinical significance of ERG oncoprotein positivity.

Results: The frequency of ERG-positive index tumors in high grade CaP was significantly greater among CA patients compared to AA patients (49% vs.16%, P < .0001) by chi-squared analysis. This observation persisted when all ERG-positive tumor foci were assessed between CA and AA patients (59% vs. 33%, P = .0042). Neither ERG prevalence nor race was predictive of biochemical recurrence in this cohort.

Conclusions: This study examined the prevalence of ERG oncoprotein expression in high grade CaP patients stratified by race. ERG expression (predominantly due to TMPRSS2-ERG gene fusion) is significantly more common in high grade CaP of CA compared with AA patients. Genetic alterations in high grade CaP in the AA population need to be further determined. This study underscores that typing of prostate tumors for ERG may enhance our understanding of biological differences between ethnic groups.

Citation Format: Denise Young, James Farrell, Yongmei Chen, Jennifer Cullen, Gyorgy Petrovics, Inger Rosner, David G. McLeod, Shiv Srivastava, Isabell A. Sesterhenn. The increased frequency of ERG oncoprotein expression in high grade prostate cancers in Caucasian American in comparison to African American men. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1936. doi:10.1158/1538-7445.AM2013-1936
Abstract 1941: Identification of microRNA(miRNA) signature genes for chemoresistance through high throughput profiling of cancer related miRNAs (OncomiRs) in ovarian cancer cell lines and formalin fixed, paraffin embedded (FFPE) ovarian tissues.
Authors: Guan Yinghui , Lu Shan , Ryner Lisa , Wang Yulei
Published: August 2014

The standard treatment for ovarian cancer after cytoreductive surgery is platinum combined with taxane based chemotherapy. However, most patients quickly develop resistance to these therapies. Many miRNA have been implicated to play critical roles in regulating genes involved in chemoresistance.

In order to understand mechanism of potential drug resistance to small molecule agents used in our pipeline antibody drug conjugates, we first optimized and developed a gene expression panel for detecting 96 OncomiRs that have previously been identified as either oncogenic or tumor suppressive genes in multiple cancer types using the Fluidigm 96.96 dynamic array platform. A synthetic miRNA pool and a 22-tissue total

RNA pool were first titrated and tested against these 96 OncomiRs to determine the feasibility of multiplexing of 96 genes in the reverse transcription and pre-PCR amplification steps and the dynamic range of the assay. The slopes of Ct vs. RNA input in most assays showed over 90% amplification efficiency and more than 1000 fold detection range. We then profiled 53 ovarian cancer cell lines using this 96-OncomiR panel.

Unsupervised hierarchical clustering showed that expression of 96 genes in these cell lines could separate them into two distinctive clusters with overexpression of either markers for epithelial or for mesenchymal subtypes, indicating the OncomiR panel could represent both molecular subtypes. By correlating miRNA expression levels of these cell lines with their responses to several chemotherapeutic compounds, we have identified distinctive miRNA signatures of sensitivity or resistance to each compound. The overexpression of some miRNAs is consistently correlated with resistance to multiple compounds implying their target mRNA genes might confer multidrug resistance. We have also profiled 130 FFPE samples from ovarian cancer patients who were either platinum treatment sensitive or resistant using the OncomiR panel. miRNA expression markers for predicting platinum sensitivity and resistance will be also presented. In conclusion, our OncomiR

Fluidigm panel can provide an important tool for predicting clinical outcome and drug response.

Citation Format: Yinghui Guan, Shan Lu, Lisa Ryner, Yulei Wang. Identification of microRNA(miRNA) signature genes for chemoresistance through high throughput profiling of cancer related miRNAs (OncomiRs) in ovarian cancer cell lines and formalin fixed, paraffin embedded (FFPE) ovarian tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1941. doi:10.1158/1538-7445.AM2013-1941
Abstract 1946: Downregulated microRNAs in salivary rinse from patients with head and neck squamous cells carcinoma.
Authors: Pelarin Maria Fernanda A. , Horst Maria Aderuza , Carvalho Ana Carolina , Kowalski Luiz Paulo , Vettore André Luiz , Karia Bruno
Published: August 2014

Head and neck Squamous cell carcinoma (HNSCC) is a disease with high incidence and mortality, and the sixth most common cancer worldwide. The diagnosis is usually made late, after the appearance of visible lesions. The discovery of altered epigenetic profiles in cancer awakened to new paradigms by which molecular mechanisms contribute significantly to carcinogenesis. To date, approximately 1000 miRNAs have been described and it is estimated that they may influence the expression of up to one third of human genes. The deregulation of the expression of miRNAs can directly influence cell proliferation and differentiation and therefore, they can act as oncogenes or tumor suppressors. The aim of this study is to compare the miRNAs expression profiles in saliva samples from patients with HNSCC and normal controls in order to identify potential diagnostic markers for this cancer. Comprehensive miRNA profile was done in 4 salivary rinse samples from HNSCC patients and 3 samples from healthy donors using a Megaplex TaqMan Low Density miRNA Arrays (TLDA) to assess the expression status of miRNAs. 101 miRNAs differentialy expressed were identified. From that, 21 were upregulated in the HNSCC samples while 80 were downregulated in these samples. After a deep revision of the information available from them, we selected candidates to be validated in an independent group of 100 salivary rinses collected from HNSCC patient at diagnoses. A preliminary result indicated that four selected miRNA were down regulated with 90-91% of specificity and 53-64% of sensibility. Our hypothesis is based on the fact that the down regulation confirmed in our results may be related to the potential of tumor suppressor activity of these miRNAs; Besides the validation obtained by our tests, important studies in the literature show agreement with this hypothesis and may add relevant data in developing this study.

Supported by Fapesp (Grant 2011/15122-9).

Citation Format: Maria Fernanda A. Pelarin, Maria Aderuza Horst, Ana Carolina Carvalho, Luiz Paulo Kowalski, André Luiz Vettore, Bruno Karia. Downregulated microRNAs in salivary rinse from patients with head and neck squamous cells carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2013-1946
Abstract 1954: Circulating microRNA-21 predicts the response to hypomethylating agents in patients with myelodysplastic syndromes.
Authors: Min Yoo Hong , Cho Jae Yong , Kim Yundeok , Kim Yeo-Kyeoung , Kim Soo Jeong , Kim Jin Seok , Cheong June-Won , Kim Hyeuong Joon
Published: August 2014

Background: Although hypomethylating agents therapy have considerably improved the outcomes of patients with myelodysplastic syndromes (MDS), the discovery of sensitive non-invasive biomarkers that can facilitate the prediction of response to hypomethylating agents (HMA) and therapeutic outcomes are highly desirable to determine optimized strategies of epigenetic therapy for MDS. Circulating microRNAs have been evaluated as potential biomarkers for cancer diagnosis and prognosis prediction.

Purpose: We evaluated the significance of miR-21 expression in serum as a biomarker for predicting response in MDS patients treated with HMA.

Patients and methods: Serum miR-21 level was measured by quantitative real-time PCR in 58 MDS patients at diagnosis and 14 healthy controls. We analyzed the correlation between serum miR-21 level and clinical characteristics and response to HMA and survival.

Results: We found that serum miR-21 level was significantly downregulated in patients with MDS than those in healthy controls (P<0.001). Moreover, miR-21 level was significantly lower in patients with response (CR, mCR, PR and SD with HI) to HMA compared to those without response (median, 0.9414±0.6349 vs. 1.1443±0.7707; P=0.041). Receiver-operator characteristic curve analysis indicated that serum miR-21 was useful in differentiating responder from non-responder with AUC of 0.648. At a cut-off value of 1.2613 for serum miR-21 level, the optimal sensitivity and specificity were 83.3% (95% CI: 68.6-93.3%) and 45.8% (95%CI: 25.5-67.1%), respectively. Patients, whose miR-21 level at diagnosis was higher than cutoff level, were defined as high miR-21 group (n=17) and the low miR-21 group representing the remaining 41 patients. We found that patients in high miR-21 grooup, compared with those in low miR-21 group, had a shorter median PFS (P=0.001). On multivariate analysis, Serum miR-21 level remained as a significant prognostic factor for response to HMA and PFS.

Conclusion: In this study, we evaluated the levels of serum miR-21 in patients with MDS, and found that serum miR-21 level predicts response to HMA and PFS in MDS. Although this prognostic significance should be validated in an independent set of patients receiving HMA, serum miR-21 level is a promising biochemical marker for response prediction to HMT.

Citation Format: Yoo Hong Min, Jae Yong Cho, Yundeok Kim, Yeo-Kyeoung Kim, Soo Jeong Kim, Jin Seok Kim, June-Won Cheong, Hyeuong Joon Kim. Circulating microRNA-21 predicts the response to hypomethylating agents in patients with myelodysplastic syndromes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1954. doi:10.1158/1538-7445.AM2013-1954
Abstract 1960: Implication of miR-34a in radioresistance of bladder cancer .
Authors: Mansure Jose J. , Nassim Roland , Kassouf Wassim
Published: August 2014

Purpose : Investigate resistance of radiation via miR-34a expression in human urothelial carcinoma

Methods : Human urothelial carcinoma cell lines derived from well-differentiated superficial bladder tumors as well as from high-grade invasive tumors were screened for their sensitivity to radiation. Growth inhibition was monitored by clonogenic assays. qRT-PCR analysis was used to evaluate expression of miR-34a among the human urothelial carcinoma cell lines. Methylation-specific polymerase chain reaction (MSP) was performed to determine CpG methylation status of miR-34a gene promoter among the cell lines. Association between miR-34a and sensitivity to radiation was also investigated by ectopic expression of miR-34a. Western blot was performed to evaluate protein expression.

Results: Remarkable differences in sensitivity to radiation have been shown among those cell lines in vitro. Of interest, miR-34a expression correlates with human bladder cancer cell response to radiation, with resistant cell lines showing lower expression of miR-34a and that is due to CpG methylation of its promoter. Additionally, an inverse correlation was observed between miR-34a and expression of sirtuin1 (SIRT1). Ectopic expression of miR-34a in cell lines resistant to radiation showed a positive effect of miR-34a to the response therapy. Growth inhibition observed after treatment could be explained, in part, by the increased expression of the cell cycle inhibitor p21.

Conclusion: Preliminary results suggest that detection of miR-34a expression may potentially be used as a predictor of therapeutic efficacy, and its regulation represent an attractive mechanism for the management of bladder cancer therapy.

Citation Format: Jose J. Mansure, Roland Nassim, Wassim Kassouf. Implication of miR-34a in radioresistance of bladder cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2013-1960