bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2104: Evidence for E2F/EWS-FLI1 oncoprotein synergism using systems biology
Authors: Schwentner Raphaela , Papamarkou Theodore , Kauer Maximilian , Stathopoulos Vassilios , Yang Fan , Bilke Sven , Meltzer Paul S. , Girolami Mark , Kovar Heinrich
Published: August 2015

Cell cycle deregulation and enhanced proliferation of cells is one of the hallmarks of oncogenesis. In Ewing Sarcoma, a highly aggressive pediatric cancer, the chimeric transcription factor EWS-FLI1 deregulates cell cycle by targeting several cell cycle regulators including the E2F family of transcription factors. ChIP-seq studies showed a significant overlap of EWS-FLI1 and E2F3/4 binding in Ewing sarcoma cells. We show that EWS-FLI1 is able to directly activate E2F3, followed by the combinatorial binding of EWS-FLI1 and E2F3 on their target genes synergistically activating their transcription. Furthermore we propose a model where EWS-FLI1 directly exchanges repressive E2F4/p130 by E2F3/pRB thereby driving cells into enhanced cell proliferation. However, so far we were unable to experimentally demonstrate a physical interaction of EWS-FLI1 with any E2F or pocket protein.

As an alternative approach to the study of functional synergy between EWS-FLI1 and E2F3, we used time resolved RNA and protein expression data as a basis for mathematical modeling of EWS-FLI1 dependent E2F target gene regulation. By Bayesian model selection, we were able to postulate the formation of a complex between EWS-FLI1 and E2F3 as the most likely explanation for the observed ETS/E2F synergy. This study therefore provides an example of how computational systems approaches can complement experimental data in the discovery of disease mechanisms.

This study was supported in part by the Austrian Science Fund (FWF), [grant 22328-B09], and by the 7th framework program of the European Commission, [grant 259348] (‘ASSET’).

Citation Format: Raphaela Schwentner, Theodore Papamarkou, Maximilian Kauer, Vassilios Stathopoulos, Fan Yang, Sven Bilke, Paul S. Meltzer, Mark Girolami, Heinrich Kovar. Evidence for E2F/EWS-FLI1 oncoprotein synergism using systems biology. [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 2104. doi:10.1158/1538-7445.AM2015-2104
Abstract 2116: HDAC9 and 11 contribute to UV resistance in melanoma cells
Authors: Chang Elizabeth T. , Parekh Palak R. , Yang Qingyuan , Carrier France
Published: August 2015

We have previously shown that the Histone Deacetylase Inhibitor (HDACi) trichostatin A (TSA) can sensitize cancer cells to UV radiation by facilitating the formation of Cyclobutane pyrimidine dimers (CPD) without increasing the levels of HDAC1. Here we show that HDAC9 and HDAC11 are upregulated in response to UV radiation in a number of human melanoma cell lines. Both HDAC9 and HDAC11 have an hnRNP A18 signature motif in their 3′UTR. hnRNP A18 is a new regulator of protein translation that can confer growth advantages to tumor cells by specifically increasing the stability and translation of mRNA transcripts harboring its signature motif. Our data indicate that HDAC9 and 11 levels correspond to hnRNP A18 level in response to UV radiation in several melanoma cell lines while hnRNP A18 and HDAC 9 and 11 are not upregulated in response to UV radiation in the normal human fibroblasts Malme cells or the normal melanocytes HEMa-LP. Moreover, clonogenic colony survival assays indicate that melanoma cells expressing higher hnRNP A18 endogenous levels are more resistant to UV radiation than cells expressing lower hnRNP A18 levels. In addition, down regulation of hnRNP A18 prevents HDAC 9 and 11 upregulation in response to UV radiation. Furthermore, overexpression of hnRNP A18 increases further the levels of HDAC 9 and 11 in response to UV radiation. These data suggest that upregulation of HDAC9 and 11 in response to hnRNP A18 activation contribute to UV resistance and may increase sensitization to UV-induced cell killing by HDACi.

Citation Format: Elizabeth T. Chang, Palak R. Parekh, Qingyuan Yang, France Carrier. HDAC9 and 11 contribute to UV resistance in melanoma cells. [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 2116. doi:10.1158/1538-7445.AM2015-2116
Abstract 2117: Epigenetic mechanisms control switching between metastatic and proliferative subtypes in the SW13 adrenal adenocarcinoma cell line
Authors: Davis McKale R. , Hull Elizabeth E.
Published: August 2015

The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses the tumor-suppressor gene Brahma (BRM), SW13+, and one that does not, SW13-. Unlike many tumor supressor genes, which are often mutated, loss of BRM expression occurs post-transcriptionally and BRM expression can be restored via histone deacetylase (HDAC) inhibition. However, many of the previously characterized BRM inducing HDAC inhibitors are toxic, broad-spectrum inhibitors which have limited downstream use, and provide little insight into the specificity of BRM epigenetic suppression. In this work we sought to further characterize the oncogenic potential of the two SW13 subtypes, and to investigate the potential epigenetic regulators that contribute to the subtype switching. SW13 subtype morphology and growth rates were assessed by immunofluorescence and standard growth assays. Metastatic potential was characterized by measuring anchorage-independent growth with soft agar assays, collagenase activity with fluorescein-conjugated gelatin, and invasiveness into a 3D extracellular matrix. Differences in gene expression were measured by standard qRT-PCR and pathway-specific qRT-PCR profiling arrays. The capacity of specific HDAC inhibitors for their ability to induce switching between the SW13 subtypes was assessed by immunofluorescence and gene expression analyses. BRM deficient SW13- cells have both a faster doubling time and an increased rate of anchorage-independent growth, while SW13+ cells exhibit higher collagenase activity and invasive capacity. Significant differences in the expression of multiple genes involved in epigenetic chromatin remodeling as well as epigenetic chromatin modification were observed, indicating that these two subtypes also have unique gene expression profiles that could contribute to BRM regulation. Of those tested, inhibition of HDAC1 most efficiently increased the rate of conversion between SW13- and SW13+ cells, and did so in a dose dependent manner. After conversion to SW13+ subtype there is also increased acetylation on histone H3 but not histone H4 when compared to levels in SW13-, suggesting that the role of acetylation on the H3 tail may play an essential role in the subtype switch. These studies provide new insight into the functional consequences of BRM loss in the SW13 cell type and reveal potential targets for the reversal of BRM epigenetic silencing.

Citation Format: McKale R. Davis, Elizabeth E. Hull. Epigenetic mechanisms control switching between metastatic and proliferative subtypes in the SW13 adrenal adenocarcinoma cell line. [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 2117. doi:10.1158/1538-7445.AM2015-2117
Abstract 212: Infrared spectral diagnosis for predictive cancer medicine: application to the early diagnosis and prognosis of preinvasive bronchial intraepithelial lesions
Authors: Gaydou Vincent D. , Polette Myriame , Gobinet Cyril , Kileztky Claire , Manfait Michel , Birembaut Philippe , Piot Olivier
Published: August 2015

This study concerns the development of a new imaging diagnostic tool for bronchial cancer. This malignancy is a wide-spread cancer in the world, with an increasing incidence. Our objective is to assess the potential of infrared (IR) spectroscopy in the early diagnosis of the bronchial cancer. Our approach is divided into two steps: the first issue aims at highlighting specific spectroscopic markers of the bronchial cancer permitting to detect unambiguously the presence of cancer cells. The second step relies on establishing a chemometric model able to estimate the tumor invasivity of these malignant cells

For our approach, we used 4 different bronchial cellular lines displaying different in vitro invasive properties assessed by a modified Boyden chamber assay : the non invasive cell line 16HBE used as a control, the moderate invasive cell line Beas2B and the two highly invasive cell lines BZR and BZRT33. Four samples of each cell line were cultured and embedded in paraffin wax. Slices of 10 μm were prepared on CaF2 windows suitable for IR measurements. The infrared acquisitions were performed with an imager (Bruker, Germany) equipped with a Focal Plane Array detector allowing to image large tissue areas with a spatial resolution of a few micrometers.

The IR spectra were processed using chemometric and statistical multivariate treatments. A first stage permitted to correct spectral interferences originated from the paraffin and optical effects. Then, supervised models were established by means of PLS-DA (Partial Least Square Discriminant Analysis) method. A first model, based on sparse-PLS method, was optimized to highlight spectroscopic markers specific of the normal and cancerous states; this model has proved effective in terms of sensibility (96%) and specificity (90%). Based on these promising results, the study will be pursued by the analysis of tissue samples from biopsies and tumor samples. Subsequently, the next investigation will rely on the construction of a second model to model and quantify the aggressiveness level of the cell lines.

Citation Format: Vincent D. Gaydou, Myriame Polette, Cyril Gobinet, Claire Kileztky, Michel Manfait, Philippe Birembaut, Olivier Piot. Infrared spectral diagnosis for predictive cancer medicine: application to the early diagnosis and prognosis of preinvasive bronchial intraepithelial lesions. [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 212. doi:10.1158/1538-7445.AM2015-212
Abstract 2140: Dual Wnt and EGFR-MAPK dependency of BRAFV600E-mutant colorectal cancer
Authors: Wang Youzhen , Palmer Michael , Jaeger Savina , Bagdasarian Linda , Qiu Shumei , Woolfenden Steve , Meyer Ronald , Yang Guizhi , Green John , Pan Shifeng , Liu Jun , Gao Hui , Cao Z. Alexander , Myers Andrea , McLaughlin Margaret E.
Published: August 2015

Aberrant Wnt pathway activation due to inactivating mutations in the gene encoding RNF43 (an E3 ubiquitin ligase that promotes degradation of the Wnt receptors Frizzled and LRP6) may contribute to the unresponsiveness of BRAFV600E-mutant colorectal cancer (CRC) to BRAF inhibitors. Analysis of The Cancer Genome Atlas (TCGA) CRC data set reveals a striking co-occurrence of the BRAFV600E mutation and truncating mutations in RNF43. RNF43 mutations are likely to be functionally significant, as RNF43 mutations and mutations in the β-catenin destruction complex component APC are almost completely mutually exclusive. The vast majority of BRAFV600E;RNF43-mutant CRCs are hypermutable [microsatellite instability (MSI)-high phenotype]. The mismatch repair deficiency in these tumors may directly contribute to RNF43 mutagenesis, as RNF43 mutations tend to be small insertions/deletions in homopolymeric tracts. To determine if RNF43 mutations confer Wnt dependency in BRAFV600E-mutant CRC, we treated three BRAFV600E;RNF43-mutant CRC patient-derived xenograft (PDX) models with the porcupine inhibitor WNT974 (formerly LGK974), which blocks the palmitoylation and secretion of Wnt ligands. Single agent WNT974 anti-tumor activity was observed in 2/3 PDX models, and correlated with decreased tumor cell proliferation and mucinous differentiation. Single agent anti-tumor activity with the BRAF inhibitor LGX818 was also observed in 2/3 PDX models. No single agent anti-tumor activity was observed with the EGFR inhibitor cetuximab. The double combinations of WNT974+LGX818 and LGX818+cetuximab, and the triple combination of WNT974+LGX818+cetuximab were efficacious in all three BRAFV600E;RNF43-mutant CRC PDX models. In summary, the Wnt pathway and the EGFR-MAPK pathway may jointly promote tumorigenesis of BRAFV600E-mutant CRC, providing a strong rationale to treat patients with BRAFV600E-mutant CRCs harboring upstream Wnt pathway mutations with combinations of WNT974, LGX818 and/or cetuximab.

Citation Format: Youzhen Wang, Michael Palmer, Savina Jaeger, Linda Bagdasarian, Shumei Qiu, Steve Woolfenden, Ronald Meyer, Guizhi Yang, John Green, Shifeng Pan, Jun Liu, Hui Gao, Z. Alexander Cao, Andrea Myers, Margaret E. McLaughlin. Dual Wnt and EGFR-MAPK dependency of BRAFV600E-mutant 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 2140. doi:10.1158/1538-7445.AM2015-2140
Abstract 2141: Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells
Authors: Herman Allison B. , Wurtzel Jeremy G.T. , Goldfinger Lawrence E.
Published: August 2015

In this study we investigated cellular and molecular mechanisms for breast cancer signaling by EGFR and Ras proteins, independent of activating mutations. Epidermal Growth Factor (EGF) ligation to its receptor, EGFR, at the plasma membrane (PM) activates Ras small GTPases, propagating Ras mitogenic signaling. However, activating Ras mutations are not associated with breast cancers. EGFR and palmitoylated (Pa-) Ras localize to lipid rafts at the PM. Pa-Ras isotypes (H- and N-Ras, but not K-Ras) cycle through vesicle trafficking pathways between the Golgi and PM. We have found that anterograde H-Ras vesicles are sorted in the endocytic recycling compartment (ERC) into vesicles which contain the epithelial-specific transport mediator Rab25 (Rab11c), and Rab11/FIP1C (Rab-coupling protein, RCP). RCP couples Rab25 to vesicles to mediate vesicle anterograde transport. Rab25 and RCP expression are highly correlated with breast cancer progression, aggressiveness, and invasion and metastasis. Rab25, unique among the large family of human Rab genes, has been identified as a major marker and potential stimulator of breast cancers. RCP was recently shown to be a breast cancer driver oncogene, supporting the suggestion that RCP and Rab25 status may be significant clinical markers. Moreover, RCP stimulates H-Ras activation. Meta-analysis of human breast cancer cell line panels shows that while most do not harbor H- or N-Ras mutations, both RCP and Rab25 are highly over-expressed and levels correlate with resistance to EGF and Ras signaling inhibitors. We demonstrate that Rab25, RCP, H-Ras and EGFR co-localized in the ERC and in transport vesicles in MCF10A mammary epithelial cells, and that dominant negative (DN) Rab25 sequestered H-Ras and EGFR from the plasma membrane. DN Rab25 or Rab25 knockdown by shRNA inhibited EGF-induced EGFR phosphorylation, H-Ras activation, and ERK phosphorylation (ppERK), demonstrating a functional link between Rab25 activity and EGFR and H-Ras signaling. Both ER/PR-positive and -negative human breast invasive ductal carcinoma tissues showed elevated ppERK correlated with elevated Rab25 expression over normal breast tissue, whereas HER2+ tumor tissues had elevated ppERK even with low Rab25 expression. Thus, Rab25 and RCP regulate EGFR and Ras trafficking to the plasma membrane, required for mitogenic signaling and associated with tumorigenic effects, even in Ras (WT) cells.

Citation Format: Allison B. Herman, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells. [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 2141. doi:10.1158/1538-7445.AM2015-2141
Abstract 2169: Genomic analysis of 207 rectal cancer samples
Authors: Hu Yue
Published: August 2015

As the 3rd most commonly diagnosed cancer, colorectal cancer makes up of about 10% of all cancer cases and afflicts 1.3million patient each year. It is also the 3rd leading cause of cancer death. Every year more than 50,000 patients die of this disease. To analyze the molecular mechanism of rectal cancer, we explore the gene expression of cancer samples of 207 patients; most of them have paired control. We also study SNPs as well as prognosis data of these patients. Rectal cancer samples show a significant downregulation of genes in metabolic pathways. The general expression pattern separates cancer samples into “control like” subgroup and “cancer like” subgroup. There is no significant prognosis difference between these two groups. We observe a strong correlation between the average gene expression and the copy number change of each chromosome of cancer samples. Several SNPs show differential survival among patients with different alleles. Clinical data shows that these SNPs affect prognosis independent of lymph node status.

Note: This abstract was not presented at the meeting.

Citation Format: Yue Hu. Genomic analysis of 207 rectal 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 2169. doi:10.1158/1538-7445.AM2015-2169
Abstract 2176: Joint structural variant analysis of colorectal cancer whole genome sequencing data
Authors: Pitkänen Esa , Cajuso Tatiana , Katainen Riku , Lundgren Sofie , Tuupanen Sari , Kilpivaara Outi , Aaltonen Lauri A.
Published: August 2015

Colorectal cancer (CRC) is one of the most common cancers, with over million cases and over 600,000 deaths each year in the world. It is estimated that a significant part of CRCs may have a hereditary compo-

nent, but majority of this heritability remains unexplained. Structural variants (SVs) comprise an important type of variation that, until the availability of deep-coverage whole-genome sequencing (WGS), has been hard to study in genome-wide scale. It has been suggested that a significant portion of unexplained, or “missing”, heritability could be explained by structural variants. Somatic structural aberrations are known to play a role in tumorigenesis and tumor progression. However, structural variation discovery is often complicated by low sequencing coverage, sequencing artifacts, repeat regions and errors of the reference genome.

To study the role of structural variation in CRC, we analyzed deep-coverage WGS data of matched tumor-normal sample pairs of 213 Finnish CRC patients and additional in-house WGS data. In these data, we identified both germline and somatic structural variants using a recent structural variant caller DELLY.

A novel algorithm was used to combine DELLY paired-end structural variant calls across normal and tumor samples to improve both sensitivity and specificity of variant detection. A total of 4,854,576 unfiltered structural events were identified, consisting of 2,912,552 deletions, 1,304,636 tandem duplications, 71,724 inversions and 565,664 interchromosomal translocations. To call high-confidence somatic events, we identified 475,902 structural events in tumors that were not present in any normal sample. Calling accuracy was further improved by annotating events by mappability and proximity to repeat regions. Our results demonstrate the applicability of integrating structural variant data across a large number of samples to obtain high-quality structural variant calls.

Citation Format: Esa Pitkänen, Tatiana Cajuso, Riku Katainen, Sofie Lundgren, Sari Tuupanen, Outi Kilpivaara, Lauri A. Aaltonen. Joint structural variant analysis of colorectal cancer whole genome sequencing data. [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 2176. doi:10.1158/1538-7445.AM2015-2176
Abstract 2194: p21Waf1/Cip1 dependent chromatin modifications in colorectal tumor cells
Authors: Lennert Pablo , Erlenbach-Wuensch Katharina , Kroeger Carsten , Steinmann Sara , Huebner Kerstin , Paul Sushmita , Rausch Felix , Hartmann Arndt , Schneider-Stock Regine
Published: August 2015

Objectives: The cell cycle regulator p21Waf1/Cip1 belongs to the family of cyclin-dependent kinase inhibitors (CDKIs). Depending on the cellular condition, p21Waf1/Cip1 can either, inhibit the cell cycle or promote proliferation, prohibit or induce apoptosis and increase or decrease cell differentiation. The contradictory nature of p21Waf1/Cip1 encouraged us to investigate the effects of this CDKI on the expression of chromatin modifying enzymes in colorectal tumor cells.

Methods: Three different HCT116 cell lines were used for our studies. HCT116 WT, HCT116 p21-/- and HCT116 p53-/-. The isolated RNA was analyzed via Epigenetic Chromatin Modification Enzymes RT2 Profiler PCR Arrays from Qiagen. Western Blot analysis was performed to verify the results of the PCR arrays. In addition, immunohistochemical staining of different chromatin modifiers was performed on tissue microarrays of colon cancer patients. The protein structure of p21Waf1/Cip1 was generated by computational modelling techniques to investigate possible interactions between p21Waf1/Cip1 and chromatin modifying enzymes. Ex vivo crypt organoids from WT and p21 -/- mice were studied for chromatin signature.

Results: The PCR arrays provided us with astonishing results. Compared to the wild type HCT116 cells, we could see a clear deregulation of a high number of different chromatin modifying enzymes in the two knock out cell lines. SETD1B was up-regulated 1,8 times in p21-/- and HDAC1, which is a known interaction partner of p21Waf1/Cip1, was down regulated about 2 times compared to the WT cells. This could also be proven by Western Blot and immunohistochemical analysis. The data was further processed and analyzed using an R package termed as HTqPCR and it was observed that ATF2 was up-regulated, while AURKB, different HDACs, and DNMT3B genes were down-regulated in p21-/- cells compared to the WT cells. The interaction between p21Waf1/Cip1 and HDAC1 could be analyzed on an atomic scale by molecular dynamics simulations.

Conclusion: Our data strongly suggests that p21Waf1/Cip1 is very important for chromatin regulation in colon cancer cells and that its loss induces an active histone code. Interestingly most p21-dependent effects were independent of p53. These exciting and new findings have to be further investigated.

Citation Format: Pablo Lennert, Katharina Erlenbach-Wuensch, Carsten Kroeger, Sara Steinmann, Kerstin Huebner, Sushmita Paul, Felix Rausch, Arndt Hartmann, Regine Schneider-Stock. p21Waf1/Cip1 dependent chromatin modifications in colorectal tumor cells. [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 2194. doi:10.1158/1538-7445.AM2015-2194
Abstract 2199: Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity
Authors: Morris Benjamin L. , Damle Priyadarshan , Nawaz Zaid , Grossman Steven R.
Published: August 2015

C-terminal binding proteins (CtBP) 1 and 2 are transcriptional coregulators that are upregulated in several cancers, including a majority of studied breast, colorectal, and ovarian tumor samples. CtBPs drive many cellular oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes, such as E-cadherin, PTEN, BRCA1, and p16. CtBPs encode an intrinsic dehydrogenase activity regulated by both intracellular NADH concentration and the putative substrate 4-methylthio-2-oxobutyric acid (MTOB). NADH binding induces CtBP dimerization, which regulates the recruitment of transcriptional regulatory complexes. High levels of MTOB appear to inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. However, the function of the substrate-binding domain has yet to be examined with regard to CtBP's oncogenic activity. To this end, we have created several point mutations in the substrate-binding pocket of CtBP and have determined key residues for CtBP's enzymatic activity using an in vitro dehydrogenase assay. We have found that a conserved tryptophan in the catalytic domain, and specifically its aromatic moiety, is imperative for enzyme activity. This tryptophan is unique to CtBP family proteins and distinguishes the CtBP substrate-binding domain from that of all other families of dehydrogenases. Additionally, we found arginine and histidine residues lining the substrate pocket that are necessary for CtBP dehydrogenase function. Knowledge of these residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Moreover, to investigate the importance of the catalytic domain on CtBP's oncogenic potential, we have transfected human cancer cell lines with several CtBP substrate-binding domain mutants and observed the effects on cellular processes, including growth, survival, migration, and recruitment of key components of the transcriptional repressor complex. From this work we have investigated the utility of CtBP, and specifically the CtBP substrate-binding domain, as a target for cancer therapeutics. We also provide preliminary insights into the function of this domain in cellular models of cancer.

Citation Format: Benjamin L. Morris, Priyadarshan Damle, Zaid Nawaz, Steven R. Grossman. Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic 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 2199. doi:10.1158/1538-7445.AM2015-2199
Abstract 22: Genetic re-programming of the acute myeloid leukemia cell line HL-60: Regulation of cell proliferation, differentiation, and programmed cell death
Authors: Roberts Michael , Holbert Cassandra , Ghoto Mansoor , Schneider Juliana , Marriott Abigail , Forrester Jeffrey
Published: August 2015

The human acute myeloid leukemia cell line, HL-60, in response to phorbol esters, undergoes genetic re-programming leading to cell cycle arrest, macrophage differentiation, and apoptosis. The induced program overrides the oncogenic mutations driving the leukemia program, illustrating that changes in gene expression can reverse the cancer phenotype. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. We define a subset of 186 genes functionally assigned to the biological processes of regulation of cell proliferation, differentiation, and/or programmed cell death showing specific temporal expression patterns, and suggest that a subset of these genes, which are down-regulated during the reversal of the leukemia phenotype while showing overexpression in AML patient samples, may represent new therapeutic targets in AML. Among the genes showing immediate early up-regulation (2-fold or greater by one hour post-PMA treatment), promoter analysis reveals highly significant overrepresentation of binding sites for the transcription factors STAT5A, HIF1A and NF-kB. Delayed early up-regulated genes (2-fold or greater by three hours post-PMA treatment), have promoters highly enriched in binding sites for EGR1, SP1:SP3, ZFP206 and E2F4. A phorbol ester response “signature” related to reversal of the leukemic phenotype and initiation of apoptosis has been defined and examined in two additional AML cell lines (THP-1 and Kasumi-1). Among the significantly up-regulated genes are MCL1, CDKN1A, NR4A1, and BCL6. Down-regulated genes include MYC, KIT, LGALS12, and ZBTB16. HL-60 cell lines with CRISPR-mediated knockouts of several of these genes are currently being evaluated for their effects on the leukemic phenotype and phorbol ester-induced apoptosis.

Citation Format: Michael Roberts, Cassandra Holbert, Mansoor Ghoto, Juliana Schneider, Abigail Marriott, Jeffrey Forrester. Genetic re-programming of the acute myeloid leukemia cell line HL-60: Regulation of cell proliferation, differentiation, and programmed cell death. [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 22. doi:10.1158/1538-7445.AM2015-22
Abstract 2200: A rational approach for discovery of inhibitors of YAP-TEAD interaction
Authors: Fromond Claudia , Chene Laurent , Soude Anne , Barth Martine , Montalbetti Christian , Broqua Pierre
Published: August 2015

The Hippo signalling pathway plays a major role in organ growth regulation and tumorigenesis. By sensing contact inhibition, the central kinase network of the hippo pathway controls nuclear translocation and activity of the YAP transcription factor. Once translocated in the nucleus, YAP interacts with the TEAD family of transcription factors and drives expression of gene involved in cell survival and proliferation. Deregulation of the hippo pathway frequently occurs in cancers and high nuclear expression of YAP has been documented in many tumors including lung, colorectal, ovarian and skin cancers. These data suggest that targeting the hippo pathway is a new therapeutic option for the treatment of a large number of cancers. Several approaches are currently proposed to target the hippo pathway such as inhibition of kinases and GPCRs that are upstream regulators of YAP nuclear translocation. However, because YAP and TEAD are the downstream effectors of the Hippo pathway, we believe that targeting YAP-TEAD interaction is the most promising approach to selectively counteract abnormal activity of the Hippo pathway. We have started a drug discovery program aimed at identifying potent inhibitors of YAP-TEAD interaction, based on a combined FBLD/HTS strategy. Druggability of the TEAD protein was assessed by a fragment screen using NMR and SPR technologies. This approach, conforted by assigned HSQC protein NMR information, provided fragment hits which are used as starting points for the identification of more potent binders. We have also designed a dedicated AlphaScreen assay to validate the ability of our compounds to disrupt the YAP-TEAD interaction. Fifty thousand compounds of the Inventiva's library were screened using the AlphaScreen. Several hits in the micromolar range were identified and further confirmed using SPR. Hits are currently tested for their ability to disrupt YAP-TEAD interaction in a cellular context with a transactivation assay and for their ability to block tumoral cell proliferation.

Citation Format: Claudia Fromond, Laurent Chene, Anne Soude, Martine Barth, Christian Montalbetti, Pierre Broqua. A rational approach for discovery of inhibitors of YAP-TEAD interaction. [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 2200. doi:10.1158/1538-7445.AM2015-2200
Abstract 2205: Identification of transcript abundance difference at lung cancer and COPD risk genes in normal bronchial epithelial cells
Authors: Yeo Jiyoun , Zhang Xiaolu , Crawford Erin , Willey James
Published: August 2015

Background: Lung cancer and chronic obstructive pulmonary disease (COPD) and are leading causes of morbidity and mortality both in the United States and worldwide. Inhalation of cigarette smoke is the primary known and preventable cause but only 10-15% of heavy smokers develop these diseases. This suggests that cigarette smoke exposure interacts with inherited susceptibility factors to determine risk. There is urgent need to identify heritable susceptibility factors that explain the majority of lung cancer and COPD risk. In prior studies, we and/or others have observed large inter-individual variation in normal bronchial epithelial cell (NBEC) expression of antioxidant, DNA repair, and cell regeneration control genes and altered regulation in NBEC of subjects with lung cancer or COPD. We collected normal bronchial epithelial cell (NBEC) samples from over 500 subjects with or demographically at risk for lung cancer. In this pilot study, we assessed total expression of multiple putative risk genes in NBEC samples from 78 subjects.

Methods: A targeted competitive multiplex next generation sequencing (NGS) method (Blomquist et al, PLOS one 2013) was used to quantify transcript abundance at 70 marker sites among 33 target genes in NBEC total RNA from 78 subjects (18 cancer cases and 60 non-cancer controls). Approximately half of the cancer group (N = 10) and non-cancer group (N = 30) had COPD (FEV1/FVC<0.7).

Results: TP73 was expressed higher and XRCC1 and MUC5B lower in cancer. GSTP1, MUC5B, OGG1, and TP73 were expressed at significantly higher level in COPD vs control by t-test in non-cancer group.

Conclusions: This study of 78 subjects provides evidence for transcript abundance difference in selected genes with high prior likelihood for contributing to lung cancer and COPD risk. Those genes will be the focus of additional studies in BEC samples from over 500 subjects with or at risk for lung cancer and/or COPD in effort to further optimize current tests for lung cancer and COPD risk.

Note: This abstract was not presented at the meeting.

Citation Format: Jiyoun Yeo, Xiaolu Zhang, Erin Crawford, James Willey. Identification of transcript abundance difference at lung cancer and COPD risk genes in normal bronchial epithelial cells. [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 2205. doi:10.1158/1538-7445.AM2015-2205
Abstract 2218: Qualitative and quantitative analysis of IDH1 mutation in progressive gliomas by allele-specific quantitative RT-PCR and Western blot analysis
Authors: Timmer Marco , Perrech Moritz , Röhn Gabriele , Dreher Lena , Goldbrunner Roland
Published: August 2015

Diagnostic of the IDH1 mutation (IDH1mut) is so far based on DNA sequencing and immunohistochemistry (IHC), methods limited in terms of sensitivity and ease of use. Recently, measuring the IDH1mut level by real time PCR was introduced as alternative method. Therefore, we aimed to (i) validate this new technique in a larger patient cohort, compare it to control tissue, and (ii) investigate if the expression of both, IDH1mut and IDH1 wildtype (IDH1wt) correlates with the course of disease and different treatment regimens.

A total of 113 tumor samples were obtained intraoperatively from 84 glioma patients with diagnosis of diffuse glioma (WHO°II), anaplastic glioma (WHO°III), secondary glioblastoma +/- chemotherapy (CTx), primary glioblastoma +/- CTx (WHO°IV). Tumor samples were snap frozen and processed for sectioning, RNA and protein isolation. The quantitative expression of IDH1 mRNA was assessed using real-time PCR with specific primers for IDH1mut and -wt; protein expression was verified by Western Blot analysis and IHC. Additionally, 19 samples of LGG and their consecutive HGG were analyzed at different time points of disease.

Allele-specific quantitative RT-PCR does work in larger patient cohorts and seems to be an easy and ∼10 times more sensitive method for quantitative IDH1mut evaluation compared to the ones used so far. Our results with this new method confirmed previous data showing that most astrocytomas and secondary GBM bear the mutation (conc. 0.13 to 0.2) whereas most primary GBM do not (conc. ∼0.025). Quantitative analysis revealed that IDH1mut expression is upregulated in sGBM (mean±SEM: 3.52±0.55) compared to lower grade glioma (II° = 1.54±0.22; III° = 1.67±0.23). By contrast, IDH1wt expression is upregulated in all glioma grades (con. >0.1) compared to control brain tissue (0.007± 0.0016). Western Blot analysis showed a high concordance to both, sequencing and RT-PCR results in qualitative analysis of IDH1mut status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in sGBM.

Taken together, recurrent disease and radiochemotherapy do not alter the general IDH1 status. Moreover, we found an increase of IDH1mut gene expression in sec. GBM compared to lower grade glioma indicating a pivotal role of IDH1mut not only in gliomagenesis but also in glioma progression.

Citation Format: Marco Timmer, Moritz Perrech, Gabriele Röhn, Lena Dreher, Roland Goldbrunner. Qualitative and quantitative analysis of IDH1 mutation in progressive gliomas by allele-specific quantitative RT-PCR and Western blot analysis. [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 2218. doi:10.1158/1538-7445.AM2015-2218
Abstract 2219: Development of an assay to detect transcription misincorporation errors in Escherichia coli
Authors: Irizarry-Caro Jorge A. , Ernst Mary , Court Carolyn , Rattray Alison , Bubunenko Mikhail , Jin Ding , Court Donald , Strathern Jeffrey
Published: August 2015

In contrast to errors in DNA replication which can product heritable mutations, errors in transcription cause only transient alterations. The temporary nature of these transcription mistakes has made them difficult to detect and genetic screens to identify mutations that lower the fidelity of transcription have been problematic. We addressed this problem by developing methods to turn transient transcription errors into permanent genetic changes. The principle is based on suppression of a mutation in the active site of the cre recombinase. This assay is based on two parts: a source of Cre, which is actually the transcription misincorporation substrate, and a genetic reporter with a phenotypic change created by Cre-mediated recombination between lox sites. To develop a substrate for E. coli we chose the galactokinase gene (galK) as our reporter. From an alignment of the galK protein from E.coli, H. sapiens and L. lactis we identified sites that might be able to accept a 12aa insertion encoded by the LoxP sequence without disrupting its function. We selected four different sites that were away from the GalK active site and on the surface of the protein. We started our project using the strain MG1655, which is galK (+), able to grow in galactose as a carbon source and red on a MacConkey galactose plate (MAcGal). Using oligo recombineering, we first inserted a 7 base sequence, which caused a frame-shift mutation, the cells became galK (-) and, therefore white on MacGal. To put it back in frame, we next replaced the 7 bases with a 36 base loxP site, which, if it does not disrupt the function of galK will make the cells galK (+) again. To our surprise, the insertion of this LoxP site was completely successful, putting the gene back in frame and making the cells galK (+) and, therefore, red at each of the four sites we chose. A derivative of this reporter was made with the a portion of the galK gene inverted at one of the loxP sites. Active Cre recombinase can be detected by flipping the inversion to restore galK function. The active site mutant cre-Y324C exhibits a very low inversion rate. Mutations that reduce the accuracy of transcription, such as cells defective in greA, exhibit an elevated frequency of galK (+) inversions. This system will be used to identify additional mutations that reduce the fidelity of transcription in E. coli.

Citation Format: Jorge A. Irizarry-Caro, Mary Ernst, Carolyn Court, Alison Rattray, Mikhail Bubunenko, Ding Jin, Donald Court, Jeffrey Strathern. Development of an assay to detect transcription misincorporation errors in Escherichia coli. [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 2219. doi:10.1158/1538-7445.AM2015-2219
Abstract 2227: Effects of cathepsin S in differentiated and stem-like glioblastoma cells
Authors: Foster Robyn A. , Al Rashid Shahnaz T. , Burden Roberta E. , Scott Christopher J. , Prise Kevin M. , Flannery Thomas J.
Published: August 2015

Background:

Glioblastoma (GBM) is the most common and aggressive brain tumour subtype with a poor prognosis of ∼15 months. A GBM stem-like population within the tumour is hypothesised to promote resistance to current treatment and invasion through normal tissue. Cathepsin S (Cat S) is a lysosomal cysteine protease that remains catalytically active at neutral pH and has been shown to play a role in radiation resistance and invasion of cancer cells. It is also an independent predictor of GBM patient survival.

Materials and methods:

GBM cell lines (U87MG and LN229) with lentiviral shRNA knockdown of Cat S are grown in serum-free medium with growth factors that allow de-differentiation of the cells into a stem-like state. A transwell system is used for quantifying monolayer invasion. Under stem conditions, spheroid structures are formed and their invasion in 3D is analysed with GFP expression.

Protein expression determined via Western blot. Radiation response determined via clonogenic survival assay. Proliferation measured using MTT assay. Cat S-like activity measured using fluorogenic peptide substrate cleavage.

Results:

Radiation induces Cat S protein expression and protease activity in both GBM cell lines at 24 hours. Knockdown of Cat S induces radiation sensitivity in both cell lines when differentiated. In stem-like conditions, this is observed for U87MG but not for LN229 cells.

Similarly, Cat S knockdown has no effect on proliferation of cell lines, except LN229 stem-like cells where it is increased.

Finally, Cat S knockdown reduces the invasive capacity of both differentiated cell lines and U87MG stem-like cells 3D invasion through both collagen and matrigel, but has no effect on LN229 stem-like cells. Cat S-like activity is reduced in all but stem LN229, despite complete knockdown.

Further investigation into differential effects in stem-like cells will examine possible role of autophagy, which is linked to radiation resistance and Cat S inhibition.

Conclusion

Cat S is a potential drug target in GBM as it reduces invasion and radio-resistance of glioma cell lines. The differential effect seen in stem-like cells suggests that the genetic background of tumours may be an important factor in determining utility of Cat S therapy in targeting the stem-like GBM population thought to be responsible for recurrence.

Citation Format: Robyn A. Foster, Shahnaz T. Al Rashid, Roberta E. Burden, Christopher J. Scott, Kevin M. Prise, Thomas J. Flannery. Effects of cathepsin S in differentiated and stem-like glioblastoma cells. [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 2227. doi:10.1158/1538-7445.AM2015-2227
Abstract 2237: Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells
Authors: Berardi Damian E. , Flumian Carolina , Diaz Bessone Maria I. , Cirigliano Stefano M. , Bal de Kier Joffe Elisa D. , Urtreger Alejandro J. , Todaro Laura B.
Published: August 2015

Retinoids exert different effects on cell differentiation and malignant phenotype reversion through the modulation of Retinoid Acid Receptors (RAR).

In this work we have analyzed the involvement of retinoid system induction on proliferation, self-renewal and differentiation of cancer stem cells (CSC) using the triple negative mammary cell line LM38-LP.

In order to obtain a CSC enriched culture, cells where cultured under mammosphere conditions. We performed mammosphere cultures in order to enrich in CSC.

The treatment with All-trans retinoid acid (ATRA1μM for 96 h) reduced CSC growth rate evaluated as mammosphere diameter (Control: 176±8 μm vs. ATRA: 129±10 μm; p≤0.05). Surprisingly, ATRA pre-treatment for 96 h increased CSC self-renewal evaluated as the number of secondary mammospheres (Control: 313±19; ATRA: 495±21; p≤0.05) and as the clonogenic capacity of LM38-LP CSC (Control: 6±0.5; ATRA: 11±1.2; p≤0.05). Concomitant with these biological effects, the same treatment showed, by RT-PCR, an increase in RARβ and RARγ levels in LM38-LP- CSC.

Through the use of specific RAR antagonists, we found that RARγ inhibition impaired ATRA effects on self-renewal but RARβ inhibition increased the number of secondary mammospheres (Control: 382±27; ATRA: 735±11; ATRA/RARγ antagonist: 352±17; ATRA/RARβ Antagonist: 1260±177; p≤0.05). A similar result was observed when clonogenic capacity in RARγ blocked CSC was analyzed.

Finally, we performed a matrigel 3D culture using cells from LM38-LP mammospheres. Under these conditions, colonies presented large irregular structures while ATRA treatment led to the formation of differentiated colonies with evidence of lumen.

Our findings suggest that RARγ down modulation and RARβ induction could lead to CSC self-renewal inhibition and differentiation in triple negative mammary cancers.

Citation Format: Damian E. Berardi, Carolina Flumian, Maria I. Diaz Bessone, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells. [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 2237. doi:10.1158/1538-7445.AM2015-2237
Abstract 2241: MDM2 silencing promotes tumor-initiating cells in osteosarcoma and breast cancer
Authors: Olivos David J. , Mayo Lindsey D.
Published: August 2015

Chemotherapy has made remarkable strides in the past century to alleviate tumor burden across most cancer types, yet it remains ineffective at achieving complete tumor remission. To improve chemotherapy efficacy and patient survival, novel drugs will require a greater mechanistic understanding of tumor initiating cell (TIC) regulatory pathways. Intratumoral heterogeneity is a major obstacle for conventional targeted treatment modalities, which select for aggressive phenotypes with acquired drug resistance and TIC enrichment. Here we focus on elucidating the role of Mdm2 in mediating TIC levels in osteosarcoma (MG-63, Saos2) and breast cancer (MDA 231, MDA 468, and BT474) cell lines. We demonstrate Mdm2 silencing by ShRNA results in increased levels of Nanog, Oct4a, and Sox2, and diminished expression of ERα and ERβ by western blot. We observed an increase in the immunophenotypic expression of CD133 in ShMdm2 osteosarcoma and breast cancer cell lines by flow cytometry. Our data provides the first evidence that loss of Mdm2 results in increased TIC population. Further analysis will examine the affects of TIC using small molecular inhibitors to Mdm2. This insight will contribute to the molecular foundations defining the role of Mdm2 in TIC and will determine if targeting Mdm2 therapeutically is a prognostic indicator for recurrence.

Citation Format: David J. Olivos, Lindsey D. Mayo. MDM2 silencing promotes tumor-initiating cells in osteosarcoma and 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 2241. doi:10.1158/1538-7445.AM2015-2241