bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1038: Cell-based screening identifies gene expression signature correlated with sensitivity to PI3K-mTOR dual inhibitor BEZ235
Authors: Zhang Jing , Guo Sheng , Bao Zhengzheng , Shang Limei , Wen Xiao , Wery Jean-Pierre , Ning Jinying
Published: August 2015

The PI3K-mTOR pathway is one of the major signaling cascades that promote tumor growth and cell survival. Multiple inhibitors targeting this important axis are currently under clinical investigation for cancer treatment and some are under preclinical development. Here we examined the anti-proliferation activity of BEZ235, a PI3K and mTOR dual inhibitor, in 243 human cancer cell lines of different caner types. We identified 35 lines that are sensitive to the inhibitor with IC50 values less than 0,04uM and 57 insensitive lines with IC50 values more than 0.4uM. We compared gene expression of the two groups by Affymetrix U219 arrays. With a p-value cutoff set at 0.00001, we identified 14 genes that are differentially expressed. These genes include epithelial membrane glycoprotein EPCAM and serine threonine kinase 31 (STK31). Furthermore, the mutation status of tubulin tyrosine ligase TTL is significantly correlated with sensitivity to BEZ235. These results suggested a potential genomic signature that could be predictive of response to BEZ235 and provided information that may help selecting patients that are likely to receive clinical benefit.

Citation Format: Jing Zhang, Sheng Guo, Zhengzheng Bao, Limei Shang, Xiao Wen, Jean-Pierre Wery, Jinying Ning. Cell-based screening identifies gene expression signature correlated with sensitivity to PI3K-mTOR dual inhibitor BEZ235. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1038. doi:10.1158/1538-7445.AM2015-1038
Abstract 1043: Blocking constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen receptor activity
Authors: Ghotbaddini Maryam , Hemphill Aaron , Powell Joann B.
Published: August 2015

PURPOSE: The aryl hydrocarbon receptor (AhR) is a member of the basic-helix-loop-helix family of transcription factors. AhR is widely known for regulating the transcription of drug metabolizing enzymes involved in the xenobiotic metabolism of carcinogens and therapeutic agents, such as cytochrome P450-1B1 (CYP1B1). Additionally, AhR has also been reported to interact with multiple signaling pathways during prostate development. Here we investigate the effect of sustained AhR signaling on androgen receptor function in prostate cancer cells. METHODS/RESULTS: Immunoblot analysis shows that AhR expression is increased in androgen independent (C4-2) prostate cancer cells when compared to androgen sensitive (LNCaP) cells. qRT-PCR studies revealed constitutive AhR signaling in C4-2 cells without the ligand induced activation required in LNCaP cells. A reduction of AhR activity by short RNA mediated silencing in C4-2 cells reduced expression of both AhR and androgen responsive genes. The decrease in androgen responsive genes correlates to a decrease in phosphorylated androgen receptor and androgen receptor expression in the nucleus. CONCLUSION: These data indicates that AhR is required to maintain hormone independent signaling by the androgen receptor in C4-2 cells. Collectively, these data provide evidence of a direct role for AhR in androgen independent signaling and provides insight into the molecular mechanisms responsible for sustained androgen receptor signaling in hormone refractory prostate cancer.

These studies were supported by the RCMI grant 2G12RR003062-22.

Citation Format: Maryam Ghotbaddini, Aaron Hemphill, Joann B. Powell. Blocking constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen receptor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2015-1043
Abstract 107: Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair
Authors: Hamamoto Ryuji , Sone Kenbun , Nakamura Yusuka
Published: August 2015

γ-H2AX, the serine 139-phosphorylated form of the histone protein H2AX, is a marker of activation of the DNA damage machinery and is often overexpressed in many malignancies. Although γ-H2AX deregulation in cancer has previously been reported, the relevant molecular functions during carcinogenesis as well as its relationship with other histone modifications are still largely unknown. In the present study, we found that the histone methyltransferase Suppressor of Variegation 3-9 Homolog 2 (SUV39H2) methylated lysine 134 on histone H2AX, which is located in a unique portion of H2AX among the histone H2A family. When lysine 134 of histone H2AX was substituted to alanine, the amount of γ-H2AX production was significantly abolished. We also found lower γ-H2AX activity after introduction of the double-strand breakage in Suv39h2-double null cells or when SUV39H2 was knocked down. Concordantly, tissue microarray analysis of clinical lung tissues demonstrated a significantly positive correlation between K134-methylated H2AX and γ-H2AX levels. Furthermore, introduction of K134-substituted histone H2AX enhanced radio- and chemo-sensitivity of cancer cells, implying a critical role of methylation on the regulation of γ-H2AX production and the DNA repair pathways in cancer cells. This is the first report describing the functions of methylation on histone H2AX and its important role in the regulation of γ-H2AX production in cancer.

Citation Format: Ryuji Hamamoto, Kenbun Sone, Yusuka Nakamura. Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 107. doi:10.1158/1538-7445.AM2015-107
Abstract 1080: Computational and biochemical discovery of RSK2 as a novel target for epigallocatechin gallate (EGCG)
Authors: Chen Hanyong , Yao Ke , Chang Xiaoyu , Shim Jung-Hyun , Kim Hong-Gyum , Malakhova Margarita , Kim Dong-Joon , Bode Ann M. , Dong Zigang
Published: August 2015

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step mediating the effects of EGCG on the regulation of various key proteins involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and its activation requires phosphorylation of both the NTD and CTD. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting histone H3 Ser10 phosphorylation. Overall, these results indicate that EGCG is a novel natural compound for suppressing RSK2 kinase activity.

Citation Format: Hanyong Chen, Ke Yao, Xiaoyu Chang, Jung-Hyun Shim, Hong-Gyum Kim, Margarita Malakhova, Dong-Joon Kim, Ann M. Bode, Zigang Dong. Computational and biochemical discovery of RSK2 as a novel target for epigallocatechin gallate (EGCG). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1080. doi:10.1158/1538-7445.AM2015-1080
Abstract 1083: Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase, ACOX2, in breast cancer
Authors: Bjørklund Sunniva , Kristensen Vessela N. , Seiler Michael , Grenaker Alnaes Grethe I. , Ming Yao , Kerrigan John , Naume Bjørn , Sachidanandam Ravi , Bhanot Gyan , Børresen-Dale Anne-Lise , Ganesan Shridar
Published: August 2015

Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. In this study we show that an intronic start variant of ACOX2, peroxisomal Acyl-CoA oxidase 2 (ACOX2-i9), is expressed and translated into a 25kDa protein in HepG2 cells as well as in several breast cancer cell lines, where it is required for optimal cell proliferation. shRNA knock down of the ACOX2-i9 variant results in decreased cell viability as assessed by colony formation assay of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 is estrogen regulated, being induced by estradiol and inhibited by tamoxifen in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript is present predominantly in breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA Breast Cancer dataset. It is specifically enriched in ER+ breast cancers where it's expression is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast cancer.

Citation Format: Sunniva Bjørklund, Vessela N. Kristensen, Michael Seiler, Grethe I. Grenaker Alnaes, Yao Ming, John Kerrigan, Bjørn Naume, Ravi Sachidanandam, Gyan Bhanot, Anne-Lise Børresen-Dale, Shridar Ganesan. Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase, ACOX2, in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2015-1083
Abstract 1089: Dissecting the potential distinct role/s of the four RNA binding domains in a phosphoprotein nucleolin using bioinformatics tools
Authors: Steinberg Ruchama Chaya , Singh Shaneen , Saxena Anjana D.
Published: August 2015

Nucleolin is an abundant RNA binding protein within the nucleolus containing four copies of a consensus RNA-binding domain (RBD1-4). Two of these contiguous RNA-binding domains (RBD12) are sufficient to bind to a variety of RNAs (e.g. rRNA, mRNA, ncRNA) through their highly conserved RNP-1/2 motifs. Nucleolin controls gene expression either post-transcriptionally through mRNA stability or by post-translational regulation, targeting mRNAs with ARE (AU-rich elements), NRE (nucleolin recognition elements) and/or G-rich regions. Nucleolin's RNA binding functions are directly implicated in various pathological conditions e.g. Alzheimer's disease, cancer and viral infection. Interestingly, nucleolin RNA binding properties and phosphorylation pattern change during the cellular response to stress suggesting possible collaboration in regulating gene expression. However, most RNA binding properties of nucleolin studied to date exclude its highly acidic N-terminus phosphorylation domain. Importantly, NMR structures are only available for RBD12 although RBD1-4 of nucleolin is required to interact with the evolutionary conserved motif (ECM) on pre-RNAs, which have important role/s in RNA processing. Our work in accordance with others has shown that the N-terminus domain plays a vital role in cell proliferation and therefore has potential to influence RNA-binding properties of nucleolin to regulate gene expression. Here we use bioinformatics tools to elucidate the RNA binding functions of all four RBDs of nucleolin individually and in various combinations. Using I-TASSER, a tertiary structure prediction algorithm, and the available structural data for RBD12, we first predicted models for RBD3 and 4.These models displayed nearly perfect superimposition of RBD34 with RBD12 where RBD3/2 and RBD4/1 align. Our central hypothesis is that nucleolin RBD3/4 domains can replace RBD1/2 domains in binding to RNAs. To test our hypothesis we have generated a variety of theoretical constructs of RBD1-4 with mutations in the RNP1/2 conserved sites of individual RBDs. We present the computational analyses of these constructs to predict the role/s of RBD3/4. We further predict any role/s imparted by N-terminus phosphorylation and/or the C-terminus GAR (Gly-Arg rich) domains in regulating RNA binding by nucleolin. This bioinformatics-based knowledge will provide useful insights about nucleolin-RNA interactions that can be framed into rationally designed experiments.

Citation Format: Ruchama Chaya Steinberg, Shaneen Singh, Anjana D. Saxena. Dissecting the potential distinct role/s of the four RNA binding domains in a phosphoprotein nucleolin using bioinformatics tools. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2015-1089
Abstract 1097: Knockdown of splicing factor ESRP1 affects multiple splicing factors
Authors: Gökmen-Polar Yesim , Neelamraju Yaseswini , Gu Xiaoping , Nallamothu Gouthami , Janga Sarath C. , Badve Sunil
Published: August 2015

Introduction: Tissue-specific alternative splicing (AS) is an important mechanism for regulating gene expression. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2), two RNA-binding proteins (RBPs) that promote splicing, are potential candidates that contribute to breast cancer recurrence and resistance to therapies. In our prior studies, we have shown that ESRPs are associated with endocrine resistance. In this study, we seek to investigate the impact of the ESRP1 knockdown in endocrine resistant breast cancer cells. Methods: Expression of ESRP1 was analyzed in endocrine resistant-LCC2 and LCC9 cells. Knockdown of ESRP1 expression in LCC2 and LCC9 cells was achieved using lenti-viral based shRNA approach (Mission TRC human shRNA constructs, Sigma). We next performed paired-end RNA-sequencing of LCC2 and LCC9 cells transfected with control and two clones representative of shRNA knockdown (Illumina HiSeq platform, SeqWright Genomic Services) according to the manufacturer's instructions. After processing and quality check of raw FASTQ data (Illumina CASAVA, FASTQ and FASTX toolkits), reads were aligned, and quantified using bioinformatics software tools (Tophat/Cufflinks and Sailfish). Results: Expression of ESRP1 is significantly elevated in ERα-positive endocrine -resistant cells (LCC2 and LCC9 cells). ESRP1 knockdown was confirmed using qRT-PCR analysis in LCC2 cell lines (clone1 - 80% reduction and clone 3- 95% reduction) and in LCC9 cells (clone 2- 90% reduction and clone 3- 50% reduction). RNA-seq analysis identified 5153 differentially regulated genes between ESRP1 knockdown and control LCC2 cell lines. Of the 5153 genes, 1117 and 2109 genes were unique to clone 1 and clone 3, respectively, while 1927 genes were common in both clones. Targeted RNA-seq analysis also revealed that ESRP1 regulates the expression of several genes including other splicing factors. Furthermore, a functional enrichment analysis of these genes revealed a possible role of altering important pathways such as cell-cell adhesion and cell signaling. Additionally, it was observed that neither ESR1 nor any of its downstream genes were affected by the knockdown of ESRP1. This data was similar to that obtained in knockdown of LCC9 cells. Conclusions: ESRP1 regulates splicing of multiple genes in ER+ breast cancer. The process is independent of ER pathway. Targeting splicing could be a novel modality for treating endocrine therapy resistant breast cancer.

Citation Format: Yesim Gökmen-Polar, Yaseswini Neelamraju, Xiaoping Gu, Gouthami Nallamothu, Sarath C. Janga, Sunil Badve. Knockdown of splicing factor ESRP1 affects multiple splicing factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1097. doi:10.1158/1538-7445.AM2015-1097
Abstract 110: The role of the p53 target Wig-1 in senescence and cancer
Authors: Jerhammar Fredrik , Bersani Cinzia , Djureinovic Dijana , Micke Patrick , Wiman Klas G.
Published: August 2015

The wild type p53-induced gene 1, Wig-1 (also known as Zmat3 or PAG608) binds to AU-rich elements in mRNAs and thereby regulates important tumor associated factors including p53, FAS, and N-Myc.

Focusing on normal human diploid fibroblasts (HDF), we are now exploring the role of Wig-1 in senescence. SiRNA-mediated Wig-1 depletion increases senescence markers such as Beta-galactosidase staining, H3K9me3, H4K20me3, and p21. Also, Wig-1 is spontaneously downregulated in cells undergoing replicative senescence.

The trimethylation of lysine 9 on histone 3 (H3K9me3) is an established marker of cellular senescence and increases upon siRNA-mediated Wig-1 knockdown in HDF cells. It is also increased in late passage (senescent) fibroblasts that express low Wig-1 levels.

Our RNA-immunoprecipitation sequencing (RIP-seq) data of tumor cells shows that Wig-1 binds histone-modifying enzymes SUV39H2 (which trimethylates H3K9), and subunits of PP1 (protein phosphatase 1, which dephosphorylates H3T10). These mRNAs have AU-rich elements in the 3´UTR. Importantly, we have observed that both SUV39H2 and its corresponding methylation (H3K9me3) is increased upon Wig-1 silencing.

In a cohort of 350 lung cancer patients, we found Wig-1 gene amplification in a large portion of squamous cell carcinomas, whereas in lung adenocarcinomas the Wig-1 gene was unchanged. Interestingly, strong Wig-1 staining in this cohort correlates to worse prognosis.

We think Wig-1 attenuates senescence via downregulating important senescence markers in both tumor and normal cells. Evidence point to Wig-1 playing an inhibitory role on the mRNAs of histone modifying enzymes, thereby acting on well established senescence markers of gene expression and chromatin regulation.

Citation Format: Fredrik Jerhammar, Cinzia Bersani, Dijana Djureinovic, Patrick Micke, Klas G. Wiman. The role of the p53 target Wig-1 in senescence and cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 110. doi:10.1158/1538-7445.AM2015-110
Abstract 1115: Targeted RNA sequencing improves transcript analysis in cancer samples
Authors: Lin Ling , Abo Ryan , Dolcen Deniz , Paquette Rachel , Laing Angelica , de Waal Luc , Thorner Aaron , Ducar Matthew , Ziaugra Liuda , Wollison Bruce , Breneiser Marc , Hahn William , Meyerson Matthew , Van Hummelen Paul , MacConaill Laura
Published: August 2015

RNA sequencing (RNA-seq) is a transcriptome profiling technology that provides multiple levels of insight into the genome. In addition to expression levels (transcript abundance), it generates endpoints such as alternative splicing, somatic mutations and rearrangements, which may have functional consequences in cancer. Although somatic mutations are generally identified by DNA sequencing, RNA-seq has the advantage of detecting allele-specific expression affecting a variant allele, as well as functional chimeric transcripts that result from structural rearrangements. Compared to microarray technologies, RNA-seq can provide additional information about novel transcripts. Due to the complexity of the human transcriptome and the variability of gene abundance, the cost of whole transcriptome sequencing to achieve sufficient coverage to detect these types of alterations remains high. To explore the feasibility of a more cost-effective method, we compared the performance of three different RNA-seq methods: whole-transcriptome-, exome-, and targeted RNA-seq, using RNA derived from cancer cell lines and Formaldehyde Fixed-Paraffin Embedded (FFPE) samples. For whole-transcriptome preparation, we used the Illumina TruSeq Stranded mRNA and total RNA kits for cell line and FFPE samples, respectively. Exome-RNAseq was performed using the Illumina Access kit. The libraries from whole-transcriptome RNAseq were subjected to hybridization capture using OncoPanel-an Agilent SureSelect baitset of 500 cancer-related genes. Compared to whole-transcriptome, exome- and targeted-RNA-seq demonstrated (1) higher coding exon coverage and multiplexing capability; (2) reduced rRNA composition to 1%; (3) comparable gene abundance information and (4) over 90% of reads aligned to coding exon regions in FFPE samples, compare to ∼30% when using whole transcriptome method. In conclusion, we demonstrated that exome- and targeted RNA-seq provide a cost-effective way to analyze a subset of the transcriptome. Furthermore, targeted RNA-seq can be highly multiplexed and is therefore amenable for large-scale tumor profiling in clinical or research settings.

Citation Format: Ling Lin, Ryan Abo, Deniz Dolcen, Rachel Paquette, Angelica Laing, Luc de Waal, Aaron Thorner, Matthew Ducar, Liuda Ziaugra, Bruce Wollison, Marc Breneiser, William Hahn, Matthew Meyerson, Paul Van Hummelen, Laura MacConaill. Targeted RNA sequencing improves transcript analysis in cancer samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1115. doi:10.1158/1538-7445.AM2015-1115
Abstract 1140: PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit
Authors: Cerniglia George John , Day Souvik , Gallagher-Colombo Shannon M. , Daurio Natalie , Tuttle Stephen , Busch Theresa M. , Busch Theresa M. , Lin Alexander , Esipova Tatiana V. , Vinogradov Sergei A. , Koumenis Constantinos , Maity Amit
Published: August 2015

The PI3K/mTOR pathway plays a central role in coupling metabolic processes to the cellular proliferative state. Pharmacologic inhibitors of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in transformed cell lines in vitro by 30-40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, an inhibitory modification of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a glioblastoma cell line with mutant PTEN led to an increase in PDH E1α phosphorylation and a decrease in OCR. The decrease in OCR with PI3K/Akt/mTOR inhibition was recapitulated by knocking down PDH E1α expression using siRNA. Pre-treatment with dichloroacetate (DCA), a known inhibitor of the pyruvate dehydrogenase kinases (PDKs) that phosphorylate PDH E1α, prevented the upregulation in PDH E1α phosphorylation, induced by inhibition of the pathway and also blunted the decrease in OCR. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway.

Citation Format: George John Cerniglia, Souvik Day, Shannon M. Gallagher-Colombo, Natalie Daurio, Stephen Tuttle, Theresa M. Busch, Theresa M. Busch, Alexander Lin, Tatiana V. Esipova, Sergei A. Vinogradov, Constantinos Koumenis, Amit Maity. PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2015-1140
Abstract 1141: LPA stimulates glycolytic shift of ovarian cancer cells via gip2 oncogenes
Authors: Ha Ji Hee , Radhakrishnan Rangasudhagar , Dhanasekaran Danny
Published: August 2015

Ovarian cancer patients show increased levels of lysophosphatidic acid (LPA) in their serum and ascitic fluid. Our previous studies have established a critical role for LPA in inducing proliferation as well as migration of ovarian cancer cells. In the present study, we focused on identifying novel therapeutic targets in the oncogenic signaling pathway regulated by LPA. Using a commercial pathway reporter array of 45 transcription factors, we found that hypoxia-induced factor-1 alpha (HIF-1 alpha) was the most activated transcription factor induced by LPA. Both stable as well as transient silencing of Galphai2, a downstream signaling mediator for LPA, abrogates LPA induced increase in HIF-1 alpha expression. In addition, our results indicate that LPA stimulated an increase in the expression of Hexokinase-2 (HK2) and Glucose transporter 1 (GLUT1), which are targets of HIF-1 alpha, are also mediated via Galphai2 signaling pathway. To determine the outcome LPA induced increase in HK2 and GLUT1 on glycolytic rates, we measured the extracellular acidification rate (ECAR) in ovarian cancer cells following treatment with LPA, using an XFe analyzer. In both SKOV3-ip and OVCA429 cells, LPA increased ECAR in a dose-dependent manner. Silencing Galphai2 as well as HIF-1 alpha in ovarian cancer cells reduced ECAR indicative of reduced glycolytic rate. The current study, points to the identification of LPA(R)-Galphai2-HIF-1 alpha-GLUT1 signaling axis as potential metabolism-targets in ovarian cancer treatment.

Citation Format: Ji Hee Ha, Rangasudhagar Radhakrishnan, Danny Dhanasekaran. LPA stimulates glycolytic shift of ovarian cancer cells via gip2 oncogenes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1141. doi:10.1158/1538-7445.AM2015-1141
Abstract 1144: Antioxidant enzyme-mediated survival of ECM-detached breast cancer cells requires AMPK activation
Authors: Durbin Sienna M. , Versagli Calli , Pape Daniel , Schafer Zachary
Published: August 2015

Normal epithelial cells require attachment to the extracellular matrix (ECM) for proper maintenance of metabolic function and survival. However, it has recently become clear that cancer cells must alter their metabolism during times of ECM-detachment in a fashion that promotes their survival. We have discovered that breast cancer cells utilize the activity of antioxidant enzymes to eliminate the detachment-induced elevation in reactive oxygen species (ROS). This decrease in ROS relieves the inhibitory effects that ROS have on fatty acid oxidation (FAO) which in turn facilitates ATP production during times of energy deprivation. Interestingly, this critical role of antioxidant enzymes is not evident in ECM-attached cells suggesting that targeting antioxidant enzymes may be an effective strategy to eliminate ECM-detached cells. Furthermore, our studies have revealed that the molecular mechanisms controlling the regulation of FAO by antioxidant enzymes involve modulation of AMPK protein levels. Thus, our data reveal that antioxidant enzymes and AMPK signaling may be attractive targets for the design of novel therapeutics aimed at eliminating ECM-detached cancer cells.

Citation Format: Sienna M. Durbin, Calli Versagli, Daniel Pape, Zachary Schafer. Antioxidant enzyme-mediated survival of ECM-detached breast cancer cells requires AMPK activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1144. doi:10.1158/1538-7445.AM2015-1144
Abstract 115: SMYD2-mediated methylation regulates PTEN activity
Authors: Nakakido Makoto , Nakamura Yusuke , Hamamoto Ryuji
Published: August 2015

PTEN is a well characterized tumor suppressor protein. PTEN counteract PI3K-AKT pathway, which is one of the key signaling pathway for human tumorigenesis, through its lipid phosphatase activity. Whereas PTEN is known to be modified with various kinds of post-translational modifications such as phosphorylation, acetylation, and ubiquitination, methylation has not been reported to date. In this study, we elucidated SMYD2 (SET and MYND domain containing 2), a protein methyltransferase related to human tumorigenesis, methylated PTEN at lysine 313 in C2 domain. SMYD2 knockdown caused downregulation of PI3K-AKT pathway and led to cell growth suppression, suggesting that SMYD2-mediated PTEN methylation would negatively regulate PTEN activity. Given the critical role of PTEN as a tumor suppressor, our finding would not only contribute to further understanding of the mechanism of SMYD2 depending human tumorigenesis, but also may lead to a novel anti-cancer therapeutic strategy.

Citation Format: Makoto Nakakido, Yusuke Nakamura, Ryuji Hamamoto. SMYD2-mediated methylation regulates PTEN activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 115. doi:10.1158/1538-7445.AM2015-115
Abstract 1152: The regulatory effect of fructose-bisphosphate aldolase C in oral squamous cell carcinoma progression
Authors: Li Yueju , Huang Tse-Hung , Lin Been-Ren , Chang Cheng-Chi
Published: August 2015

In previous studies, glycolysis machinery has been identified as a regulating pathway in cancer cell. However, the role of glycolysis in oral squamous cell carcinoma (OSCC) progression is still largely unknown. Fructose-bisphosphate aldolase C (ALDOC), one of the key enzymes in glycolysis, expresses in OSCC patients and cell lines and detects by real-time quantitative RT-PCR. The functions of ALDOC were determined by gain and loss function approaches and the orthotopic animal model was performed. ALDOC mRNA and protein expression was significantly decreased in advanced migratory cell and negatively correlated with lymph node metastasis and advanced tumor TNM stage in OSCC patients. In conclusion, ALDOC plays as an OSCC prognosis marker clinically, and functions to suppress OSCC cell migration and invasion. ALDOC could be a potential therapeutic target to block OSCC progression.

Citation Format: Yueju Li, Tse-Hung Huang, Been-Ren Lin, Cheng-Chi Chang. The regulatory effect of fructose-bisphosphate aldolase C in oral squamous cell carcinoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1152. doi:10.1158/1538-7445.AM2015-1152
Abstract 1157: Potential mechanism for mitochondrial uncoupling protein 2 promotes skin carcinogenesis
Authors: Zhang Chunjing , Zhao Yunfeng
Published: August 2015

Mitochondrial uncoupling has recently been proposed as a novel survival mechanism for cancer cells, However, there is no direct evidence supporting that uncoupling proteins promote carcinogenesis. Herein, we examined whether mitochondrial uncoupling affects mouse skin carcinogenesis using uncoupling protein 2 (UCP2) homozygous knockout mice, and explored the mechanisms of tumor promotion by mitochondrial uncoupling by overexpressing UCP2 in JB6 P+ cells, which are the only characterized skin cell model to study tumor promotion. The vivo results indicate that the rates of oxygen consumption were only decreased in the carcinogen-treated UCP2 knockout mice whereas glycolysis was only increased in the carcinogen-treated wild-type mice. The levels of metabolites pyruvate, malate and succinate showed different trends after carcinogen treatments between the wild-type and UCP2 knockout mice. In addition, UCP2 knockout decreases 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2, especially on the regulation of the key enzyme in glycolysis), CyclinB1 and the oncoprotein Fra-1 expression during skin carcinogenesis. The vitro results indicate that overexpression of UCP2 enhanced colony formation in response to tumor promoter treatment. In addition, overexpression of UCP2 induced the upregulation of Fra-1 and PFKFB2 expression. Although UCP2 overexpression could suppress apoptosis in vitro, But UCP2 Knockout did not cause increase in apoptosis. This study provides the first in vivo evidence using UCP2 mice supporting that UCP2 promotes carcinogenesis. Although the current data are unable to answer the question of how UCP2 knockout suppresses skin cancer formation, an interesting and important finding is the adverse changes of the three metabolites and especially on the regulation of the key enzyme in glycolysis pathway during carcinogenesis, which provides a clue for future studies. Together with early studies showing that UCP2 is overexpressed in a number of human cancers and knockout of UCP2 significantly reduced the formation of both benign (papilloma) and malignant (squamous cell carcinoma) tumors in mice, UCP2 could be a potential target for cancer prevention and/or therapy.

Citation Format: Chunjing Zhang, Yunfeng Zhao. Potential mechanism for mitochondrial uncoupling protein 2 promotes skin carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1157. doi:10.1158/1538-7445.AM2015-1157
Abstract 116: Targeting EZH2 catalytic independent functions in breast cancer
Authors: Lim Keng Gat
Published: August 2015

Advances in drug discovery have enabled the development of catalytic inhibitors or inhibitors that disrupt protein-protein interactions. One validated drug target is the polycomb repressive complex 2 (PRC2), which promotes cancer development and reduces patient survival in multiple cancers. The core of PRC2 consists of Enhancer of Zeste Homolog 2 (EZH2) and Embryonic Ectoderm Development (EED). Currently, inhibitors targeting the catalytic site of EZH2 were efficacious in inhibiting a subset of blood cancers. In addition, a recently developed peptidomimetic inhibitor that disrupts the EED-EZH2 interaction also inhibits PRC2 activity and decreases proliferation of solid tumors. In this study, we investigated the effects of EZH2 protein depletion versus EZH2 catalytic inhibition with two small molecule inhibitors, GSK126 (an EZH2 catalytic inhibitor) and DZNep (a histone methylation inhibitor that depletes EZH2). We found that catalytic inhibition of EZH2 was insufficient to block cancer cell proliferation whereas depletion of EZH2 was effective at killing cancer cells. This study suggests that depleting EZH2 to inhibit both catalytic dependent and independent functions could be useful in the treatment of solid tumors such as breast cancer.

Citation Format: Keng Gat Lim. Targeting EZH2 catalytic independent functions in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 116. doi:10.1158/1538-7445.AM2015-116
Abstract 1160: CtBP1 is the molecular link that associates breast cancer and metabolic syndrome
Authors: De Luca Paola , Dalton Nicolás , Moiola Cristian Pablo , Flumian Carolina , Scalise Georgina , Porretti Juliana , Massillo Cintia , Kordon Edith , Todaro Laura , Vazquez Elba , Meiss Roberto , De Siervi Adriana
Published: August 2015

Breast cancer is still one of the most important public health problems in the entire world. Obesity and metabolic syndrome (MS) increases the incidence and aggressiveness of breast cancer. C-Terminal Binding Protein (CtBP1) is a transcriptional corepressor of tumor suppressor genes and is considered a molecular sensor of cell metabolic state due to is activated in high energy conditions (high NADH). In this work we studied the effects of the activation of CtBP1 pathway by metabolic syndrome on breast tumor development and progression. We generated a murine model of MS by chronic high fat diet (HFD) administration. By histological and whole mount methods, we found that breast tissue of animals receiving HFD presented higher levels of immature adipose tissue and an increased glandular area with more generation of lateral branches and terminal end buds of mammary ducts. Breast tissue of HFD animals also showed higher expression of the proliferation markers (cyclin D1) and epithelial markers (E-cadherin). Interestingly, HFD induced CtBP1 expression in the mammary ducts. Furthermore, the number and size of mamospheres generated with LM38-LP breast cancer cells were significantly increased when cells were incubated with serum from HFD fed mice compared to the serum of animals under control diet (CD). In addition, to investigate CtBP1 role in tumor progression we performed xenografts in nude mice fed with CD or HFD by subcutaneous injecton of breast tumor cells MDA MB 231 with depleted CtBP1 expression (shRNA CtBP1) or control cells (shRNA scramble). We found that CtBP1 depletion dramatically decreased tumor growth and KI67 expression relative to control tumors. Furthermore, xenografts developed in HFD fed mice were less differentiated compared to CD. Finally, CtBP1 diminished expression tumors showed lower mesenchymal markers expression, progenitor cells markers and markers involved in mammary development. Our studies demonstrated for the first time that gene transcription regulation by CtBP1 provides an important molecular link among MS, CtBP1 function and tumor growth. Hence, these results suggest an association to understand metabolism and breast cancer.

Citation Format: Paola De Luca, Nicolás Dalton, Cristian Pablo Moiola, Carolina Flumian, Georgina Scalise, Juliana Porretti, Cintia Massillo, Edith Kordon, Laura Todaro, Elba Vazquez, Roberto Meiss, Adriana De Siervi. CtBP1 is the molecular link that associates breast cancer and metabolic syndrome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1160. doi:10.1158/1538-7445.AM2015-1160
Abstract 1161: Metformin has an inhibitory effect on cell proliferation but does not induce death in colorectal cancer
Authors: Mogavero Angela , Maiorana Maria Valeria , Bertan Claudia , Bozzi Fabio , Pierotti Marco A. , Gariboldi Manuela
Published: August 2015

Metformin, a widely used and well-tolerated antidiabetic drug, may reduce cancer risk and improve prognosis of certain malignancies. In vitro and in vivo studies on several cancer models have evaluated the potential antitumorigenic effects of metformin and many clinical trials are looking to its use for suppressing tumor growth. However, the mechanism for its anti-cancer effect remains uncertain and needs to be clarified. Here we investigated the anti-cancer activity of metformin on colorectal cancer growth by treating three colorectal cancer cell lines, HT29, HCT116, HCT116p53-/-, with the drug. We found that, at a dose of 5mM, metformin reduced cell proliferation in all the colorectal cancer cells analyzed, as shown by BrdU incorporation assays. Metformin induced cell cycle arrest in G0/G1 phase that was accompanied by a strong decrease of cyclin D1, c-Myc expression, and inhibition of pRB phosphorylation. The antiproliferative activity of the drug resulted mediated by suppression of mTOR and IGF-1/AKT pathways. In particular, we observed inactivation of mTOR and of its downstream targets S6 and 4EBP1, both in an AMPK dependent and independent way, and also downregulation of the activity of IGF1R. We analysed the biological mechanisms by which metformin inhibited cell proliferation by evaluating its ability to induce apoptosis, autophagy or senescence, as observed in other cancer cell lines. Unexpectedly, we found that metformin did not stimulate any of these mechanisms in colorectal cancer cells. In support of these results, a colony formation assay showed that metformin did not arrest growth of cells, but only slowed down cell ability of forming colonies. Our findings highlight that metformin inhibits the proliferation of colorectal cancer cell lines as a consequence of promoting cell cycle arrest in the G0/G1 phase, but does not induce cell death. Further investigations are needed to better elucidate the mechanisms altered by the drug in colorectal cancer and caution should be used when treating cancer patients with metformin.

Citation Format: Angela Mogavero, Maria Valeria Maiorana, Claudia Bertan, Fabio Bozzi, Marco A. Pierotti, Manuela Gariboldi. Metformin has an inhibitory effect on cell proliferation but does not induce death in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1161. doi:10.1158/1538-7445.AM2015-1161
Abstract 1162: Investigating the NAD metabolome in Ewing Sarcoma
Authors: Mutz Cornelia N. , Schwentner Raphaela , Kauer Maximilian O. , Ban Jozef , Aryee Dave N. T. , Erhardt Sophie , Fuchs Dietmar , Heitger Andreas , Kovar Heinrich
Published: August 2015

Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in generation of the chimeric transcription factor EWS-FLI1.

Recently, ES has been reported to be exquisitely sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1). This enzyme uses NAD as substrate and was demonstrated to regulate EWS-FLI1 in a feed-back mechanism. NAD is a key metabolite essential for sustaining cellular energy metabolism. It plays a central role in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. The other major mammalian NAD consumer is the deacetylase SIRT1 which we observed to be specifically highly expressed in ES metastases. PARP1 and SIRT1 are crucial for coupling cellular metabolism to transcriptional gene regulation as well as to stress response. Usually, NAD is regenerated from nicotinamide in the NAMPT-dependent salvage pathway or from the reduction of pyruvate via LDHA (Warburg effect), but can also be synthesized de novo from tryptophan. Interestingly, the knockdown of EWS-FLI1 in ES cells comes along with alterations in the expression of multiple enzymes involved in NAD biosynthesis and regeneration, including TDO2, KMO, NMNAT1, NAPRT, NAMPT, and LDHA. We are interrogating the role of EWS-FLI1 mediated modulation of these enzymes and of specific small molecule inhibitors on cellular tryptophan consumption, kynurenine production and intracellular NAD levels of Ewing sarcoma cells. Additionally, we are investigating the functional consequences thereof on constitutive and stress-induced protein poly(ADP)-ribosylation and acetylation.

Preliminary results suggest that NAMPT inhibition diminishes PARP1 activity due to low NAD, implying a pivotal role for the regenerative salvage pathway in ES cells. TDO2-dependent tryptophan consumption increases in the absence of the oncogene.

These studies aim at a better understanding of factors influencing Ewing sarcoma sensitivity to therapies targeting PARP1 and the NAD metabolome.

Citation Format: Cornelia N. Mutz, Raphaela Schwentner, Maximilian O. Kauer, Jozef Ban, Dave N. T. Aryee, Sophie Erhardt, Dietmar Fuchs, Andreas Heitger, Heinrich Kovar. Investigating the NAD metabolome in Ewing Sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1162. doi:10.1158/1538-7445.AM2015-1162
Abstract 117: DOT1L is a human chromosome stability gene
Authors: Guppy Brent , McManus Kirk
Published: August 2015

The loss of genome stability is an early event that drives the development and progression of virtually all tumor types. Recent studies have revealed that certain histone post-translational modifications exhibit dynamic and global increases in abundance that coincide with mitosis and exhibit essential roles in maintaining genomic stability. Histone H2B ubiquitination at lysine 120 (H2Bub1) is regulated by RNF20, an E3 ubiquitin ligase that is mutated/altered in numerous tumor types. Through an evolutionarily conserved trans-histone pathway, H2Bub1 is an essential pre-requisite for subsequent downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Although the role that RNF20 plays in tumorigenesis has garnered much attention, the downstream components of the trans-histone pathway, H3K4me2 and H3K79me2, and their potential contributions to genome stability remain largely overlooked. In this study, we employ single-cell imaging, and biochemical approaches to investigate the spatial and temporal patterning of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. Using two independent human cell lines, we show that H2Bub1, H3K4me2 and H3K79me2 exhibit distinct temporal progression patterns throughout the cell cycle. Most notably, we demonstrate that H3K79me2 is a highly dynamic histone PTM that reaches maximal abundance during mitosis in an H2Bub1-independent manner. Using RNAi and chemical genetic approaches, we identify DOT1L as the histone methyltransferase responsible for mitotic-associated increases in H3K79me2. We also demonstrate that the loss of mitotic H3K79me2 levels correlates with increases in chromosome numbers and increases in mitotic defects. Collectively, these data suggest that H3K79me2 dynamics during mitosis are normally required to maintain genome stability and further implicate the loss of H3K79me2 during mitosis as a pathogenic event that contributes to the development and progression of tumors.

Citation Format: Brent Guppy, Kirk McManus. DOT1L is a human chromosome stability gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 117. doi:10.1158/1538-7445.AM2015-117