bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1984: Activated BMP signalling modulates multicellular spheroid formation and reattachment of ascites-derived human epithelial ovarian cancer cells in an in vitro model of metastasis
Authors: Peart Teresa M. , Bertrand Monique , Prefontaine Michel , Sugimoto Akira , DiMattia Gabriel , Shepherd Trevor
Published: August 2014

Epithelial ovarian cancer (EOC) comprises close to 90% of ovarian tumours, and over 75% of cases are not discovered until advanced metastatic stages. Therefore, there remains a critical need to identify the mechanisms controlling late-stage spread of disease and develop more effective treatment regimes. EOC cells have the ability to form multicellular aggregates in malignant ascites which dramatically alters cell signalling, survival, and metastatic potential. Primary human EOC cells derived from patient ascites secrete and are responsive to bone morphogenetic proteins (e.g. BMP2 and BMP4). BMPs can function in an autocrine fashion to enhance the malignant characteristics displayed by EOC cells in adherent monolayer culture. Herein, we demonstrate that EOC cells with enhanced BMP signalling form smaller, more loosely-aggregated spheroids than control cells when grown in suspension culture. Upon reattachment to standard tissue culture plastic, EOC spheroids readily attach and cells disperse from the attached spheroid; EOC spheroids with enhanced BMP signalling have an increased cell dispersion rate when compared with controls. Activated BMP signalling in EOC spheroids induces Smad1/5/8 phosphorylation, increases ID1 and ID3 proto-oncogene expression, and promotes epithelial-mesenchymal transition as evidenced by increased Snail and Slug and reduced E-cadherin markers. To identify additional target genes mediating the phenotypic changes in EOC spheroids due to activated BMP signalling, we performed genome-wide expression analyses using Affymetrix arrays. From this study, 102 genes were significantly upregulated and 52 downregulated in primary human EOC cells and spheroids with enhanced BMP signalling. A number of these genes have been previously identified as BMP targets or members of the BMP signalling pathway, several of which are involved in negative feedback regulation (e.g. noggin, Smad6 and Smad7), as well as several genes encoding proteins associated with cellular adhesion. In addition, using the online Connectivity Map resource (www.broadinstitute.org/cmap), we revealed that the PI3K-Akt-mTOR pathway may be impacted by an activated BMP pathway in EOC cells. Indeed, BMP signalling induces phosphorylation of Akt in both adherent primary human EOC cells and multicellular spheroids. Thus, we are currently investigating whether the PI3K-Akt pathway and the targets identified by microarray analysis are required for the BMP signalling effects on EOC spheroid formation and reattachment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1984. doi:10.1158/1538-7445.AM2011-1984
Abstract 1988: A bi-directional feedback loop between Wnt and ERK is important in maintaining the proliferative phenotype of normal human urothelial cells
Authors: Kirkwood Lisa A. , Georgopoulos Nikolaos T. , Southgate Jennifer
Published: August 2014

Introduction: The urothelial lining of the bladder is ordinarily mitotically quiescent, but can rapidly regenerate in response to injury. This regenerative capacity is thought to be important in restoring urinary barrier function and offers a primary target for deregulation in cancer. Previous work in our laboratory has implicated autocrine EGFR activation in this process, but other regulatory pathways may be involved.

Wnt signalling has been shown to be important in the renewal of a number of adult epithelial tissues, but to date, no conclusive evidence for a role for Wnt in urothelial regeneration has been demonstrated. In the absence of Wnt ligand, β-catenin is sequestered via the destruction complex. Activation of the canonical Wnt pathway by ligand, inactivates the destruction complex, allowing β-catenin to translocate to the nucleus and drive proliferation.

The aim of this study was to assess whether Wnt signaling was functional in normal human urothelial (NHU) cells, and to analyze the effects of Wnt and downstream EGFR signaling on proliferation.

Materials and Methods: NHU cells were grown as finite lifespan cell lines in proliferating cultures. Destruction complex antagonists were used to activate the Wnt pathway. Proliferation assays, immunofluorescence labeling and TOPFLASH luciferase reporter assays were used to assess NHU cell responses. Pathway crosstalk was analyzed using shRNA targeted to β-catenin, as well as antagonists of the EGFR signaling pathways.

Results: Pharmacological inhibition of the destruction complex or activation of the Wnt pathway with exogenous Wnt ligand led to nuclear accumulation of β-catenin and an increase in TCF/LEF transcription factor activity. These effects were only apparent when autocrine EGFR signaling was blocked. Knockdown of β-catenin led to significant changes in p-ERK and p-AKT downstream signaling.

Conclusion: Wnt signaling can be activated in NHU cells, verifying the presence of a functional pathway. Activation of the pathway is however masked by EGFR signaling, indicating pathway crosstalk. The effect of β-catenin knockdown on p-ERK and p-AKT indicates a bi-directional positive feedback loop between the Wnt and EGFR signaling cascades in NHU cells. This study identifies the Wnt signaling pathway as an important signaling cascade in regulating the regenerative phenotype of NHU cells and indicates at least two pathways that may be arrogated by urothelial cells during neoplastic transformation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2011-1988
Abstract 1996: Chronic arsenic exposure and changes in global levels of histone modifications in a human population in Bangladesh
Authors: Chervona Yana , Arita Adriana , Wu Fen , Sun Hong , Kiok Kathrine , Kluz Thomas , Tseng Hsiang-Chi , Zavadil Jiri , Leroy Gary , Gamble Mary , Costa Max
Published: August 2014

Arsenic and arsenic compounds have been classified as group 1 human carcinogens by IARC. Naturally occurring inorganic arsenic, in particular, has been implicated in the development of human skin, lung, and bladder cancers and has also been associated with the development of liver and prostate cancers. However, despite being a formidable carcinogen, arsenic is a poor mutagen and it has been suggested that arsenic-induced carcinogenesis maybe governed by epigenetic mechanisms. We have detected differences in the levels of several histone epigenetic marks, such as H3K9acetylation, H3K9dimethylation and H3K27trimethylation in human peripheral mononuclear cell collected from individuals exposed to varying levels of inorganic arsenic in their drinking water. The changes in the global levels of these epigenetic marks were primarily measured using a novel ELISA method, while changes of arsenic-induced histone modifications in peripheral blood lymphocytes treated in vitro have also been examined via Mass Spectrometry and Western Blotting. Furthermore, we have also found a profound increase in the expression of histone genes after in vitro treatment of peripheral blood lymphocytes with sodium arsenite and will be examining this finding further. To the best of our knowledge, this study is the first in its class to measure arsenic-induced epigenetic modifications in humans and how they relate to changes in gene expression in a human population.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2011-1996
Abstract 1998: Aberrant methylation of BRCA/HR pathway genes and response to platinum-taxane therapy in ovarian cancer
Authors: Maradeo Marie E. , Morgan Mark A. , Burger Robert A. , Cairns Paul
Published: August 2014

Women diagnosed with ovarian cancer generally receive debulking surgery in combination with platinum-taxane based chemotherapy. While this treatment is currently the most effective, women diagnosed with Stage III and IV ovarian cancer have a less than a 20% five-year survival rate. Ovarian tumorigenesis is a multi-step process that results from the sequential accumulation, and interplay, of genetic and epigenetic mutations in critical genes. It is likely that such mutations underlie the different responses to therapy.

There are several reports that demonstrate that women with germline mutations in BRCA genes and inherited ovarian cancer have a favorable survival as compared to women with sporadic ovarian cancer that is matched for pathological stage. The BRCA1 and BRCA2 genes function to repair DNA damage and maintain genetic stability through the homologous recombination (HR) repair pathway. Much of the superior survival is likely due to hypersensitivity to platinum-containing chemotherapy that in turn is due to incompetent DNA repair of platinum DNA adducts due to faulty HR repair of double stranded DNA breaks (DSBs).

It is hypothesized that sporadic ovarian tumors, which constitute the vast majority of ovarian cancer, with epigenetic suppression of BRCA or BRCA-associated proteins, e.g. aberrant promoter hypermethylation of BRCA1, will be relatively hypersensitive to platinum as compared to ovarian tumors with an intact BRCA/HR pathway. To answer this important question, we have used bisulfite sequencing and quantitative methylation specific PCR to examine the incidence of epigenetic impairment of BRCA/HR pathway genes, including BRCA1, PALB2 and other genes, in primary human epithelial ovarian cancer and, using these results, then examined the relative frequency of BRCA/HR pathway impairment in sporadic ovarian tumors separated into cohorts by time to progression after platinum-taxane therapy and other measures of outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1998. doi:10.1158/1538-7445.AM2011-1998
Abstract 200: Identification of proteins involved in c-Myc induced mammary epithelia apoptosis in 3D culture system
Authors: Huang Ling , Tai Elizabeth , Muthuswamy Senthil K.
Published: August 2014

Identification of proteins involved in c-Myc induced mammary epithelia apoptosis in 3D culture system We have previously discovered that depletion of a cell polarity gene Scribble in mammary epithelial cells disrupted cell polarity, apoptosis and morphogenesis in three dimensional cell culture as well as inducing dysplasia that progressed into tumor in mice. Loss of Scribble also inhibited c-Myc induced apoptosis to cause cell transformation in vitro and tumor formation in vivo. Scribble mutant with deficiency in membrane localization resulted in similar phenotypes. These results suggest that Scribble is a tumor suppressor in mammary gland epithelial cells and its functions depends on correct cellular localization. Furthermore, Scribble is required to inhibit Myc-induced tumorigenesis by blocking Myc-induced apoptosis. I am interested in identifying proteins that are involved in Scribble dependent apoptosis as well as discovering other polarity proteins that cooperate with c-Myc for mammary tumor progression. To address the two questions, I use MCF10A three dimensional culture system and RNAi technology to screen proteins participating in apoptosis during c-Myc induced mammary epithelial cell transformation. We will report a screen to identify proteins that are involved in c-Myc induced mammary epithelial cell apoptosis in 3D culture, using a lentiviral shRNA library.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2011-200
Abstract 2000: Investigation of the molecular events associated with gene resilencing following withdrawal of epigenetic therapies
Authors: Hesson Luke B. , Patil Vibha , Sloane Mathew , Ward Robyn L.
Published: August 2014

Epigenetic therapies are a potential new class of anticancer medication for the treatment of solid tumors as well as haematological malignancies. These therapies reverse changes to DNA methylation and histone modifications allowing re-expression of epigenetically inactivated genes. However, upon drug withdrawal reactivated genes undergo re-silencing. This study investigates the mechanism of gene re-silencing following withdrawal of epigenetic therapies, in an effort to identify strategies which deliver a permanent re-expression of epigenetically inactivated genes. Using the mismatch repair gene MLH1 as a model we investigated the molecular events associated with the re-silencing process in the colorectal cancer cell line RKO. These cells lack MLH1 expression due to biallelic promoter DNA hypermethylation. We treated RKO cells for 72hrs with a combination of 2.56µM 5-aza-2’deoxycytidine (a DNA methyltransferase (DNMT) inhibitor) and 19nM Trichostatin A (a histone deacetylase (HDAC) inhibitor). We observed MLH1 re-expression by quantitative RT-PCR, concomitant with de-methylation at the MLH1 promoter as determined by bisulphite sequencing. Methylation at the MLH1 promoter decreased from 90.9% in mock-treated cells to 74.7, 61.7, 62.4 and 65.9% at 0, 3, 5 and 7 days after drug withdrawal, respectively. We observed a reproducible re-silencing of MLH1 gene expression, which began three days after drug withdrawal and was complete by 120 days. We further demonstrate the recruitment of a nucleosome, located immediately upstream of the transcription start site, prior to the re-silencing of MLH1 expression that remained high whilst MLH1 transcript levels decreased. We propose that the recruitment of promoter nucleosomes precedes a reduction in transcriptional output and the re-methylation of promoter DNA. Ongoing work aims to dissect the re-silencing process further by investigating the dynamics of RNApolII occupancy and histone modifications at the MLH1 promoter. This study provides important information on the molecular events associated with gene re-silencing by addressing the temporal relationship between changes to gene expression, nucleosomal occupancy and DNA methylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2000. doi:10.1158/1538-7445.AM2011-2000
Abstract 393: The chemokine receptor, CXCR2/IL8RB, contributes to the survival of pancreatic adenocarcinoma, and may play a role in stroma-tumor communication
Authors: Ustach Carolyn V. , Watanabe Aprill , Aziz Meraj , Diep Caroline , Hostetter Galen , Michael Demeure , Han Haiyong , Von Hoff Daniel D.
Published: August 2014

The failure of most chemotherapeutic agents to treat pancreatic cancer could be related to poor delivery of drug due to the physical barrier of dense fibrosis surrounding the tumor. Targeting and attenuating the fibrotic stroma may result in better access of drug to tumor. Our current studies show that CXCR2/IL8RB is over-expressed in the surrounding stroma of pancreatic ductal adenocarcinoma tumors. CXCR2/IL8RB and its ligands are well known to be involved in fibrosis, and recently published papers suggest a role for CXCR2 in tumor proliferation, as well. Therefore, CXCR2 is a good target for pancreatic cancer therapy, because it has the potential to target both the stroma surrounding the tumor, as well as the tumor itself. When comparing matched samples of patient tumor and adjacent normal tissue, the mean IHC score of stroma surrounding normal pancreatic ducts is 0.61 (95% CI 0.38-0.84), while the mean IHC score of stroma surrounding ductal adenocarcinoma is 1.6 (95% CI: 1.3-1.9; Mann-Whitney test p<.0001; n=31). We previously reported that a CXCR2 ligand, CXCL5, was highly expressed in patient tumor samples, but not in the stroma. Interestingly, our present data shows CXCR2 is also expressed in tumor cells, therefore, CXCR2 may be involved in both autocrine and paracrine signaling inducing tumor proliferation, as well as induction of fibrosis in the stroma. Supporting CXCR2's role in pancreatic tumor proliferation, specific knockdown of CXCR2 expression by siRNA reduces cell growth by approximately 50%. Additionally, drug dose response curve experiments show pancreatic cancer cell lines which express CXCR2 are more sensitive to the CXCR2 antagonist, SB225002, than cells which do not express CXCR2. For example, the CXCR2 expressing cell line PANC-1 is approximately 7 times more sensitive to SB225002 than the CXCR2 negative cell line BxPC-3 (0.12uM vs. 0.88uM in IC50). We are currently assessing the mechanism of death induced by CXCR2 inhibition in the cancer cells. CXCR2's role in tumor cell proliferation, as well as its over-expression in the stroma surrounding tumors, make it a good target for both weakening the protective stroma surrounding the tumor, and reducing tumor cell survival.

This work was supported by NIH P01 Grant CA109552.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 393. doi:10.1158/1538-7445.AM2011-393
Abstract 3933: Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling
Authors: Wagle Nikhil , Emery Caroline , Berger Michael F. , Davis Matthew J. , Sawyer Allison , Pochanard Panisa , Kehoe Sarah M. , Johannessen Cory M. , Macconaill Laura E. , Hahn William C. , Meyerson Matthew , Garraway Levi A.
Published: August 2014

Although targeted anticancer agents often result in dramatic responses, tumors invariably become resistant to these agents. A detailed understanding of the mechanisms by which tumors acquire resistance to such inhibitors should speed the development of more durable treatment strategies. The promise of RAF inhibition in metastatic melanoma is exemplary in this regard. Mutations in the BRAF oncogene have been detected in more than 50% of metastatic melanomas, and inhibition of mutant BRAF has shown high response rates in early clinical trials of patients with melanoma. However, resistance to RAF inhibition invariably develops. Mechanisms of de novo and acquired resistance to RAF inhibition remain poorly understood. Moreover, the clinical application of genomic approaches that might be capable of diagnosing salient resistance mechanisms remains underdeveloped.

Here, we describe an approach to characterize genetic mechanisms of resistance through systematic tumor mutation profiling. We performed massively parallel sequencing of 138 cancer genes in a tumor specimen from a melanoma patient who developed resistance to PLX4032 after a dramatic initial response. The resulting profile identified a novel mutation in the downstream kinase MEK1 that was absent in the corresponding pre-treatment tumor. This MEK1 mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Our results provide an instructive framework both for assessing mechanisms of acquired resistance to kinase inhibition and deploying new technologies for elaboration of resistance mechanisms in a manner that may accelerate personalized cancer medicine.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3933. doi:10.1158/1538-7445.AM2011-3933
Abstract 3937: Global methylation patterns, genome copy number alterations and expression analysis distinguish two distinct subclasses of hepatocellular carcinoma
Authors: Revill Kate , Tim Wang , GInther Charles L. , Anderson Lee , Aleshin Alexey , Busuttil Ronald W. , Powers Scott , Finn Richard S.
Published: August 2014

Background: Hepatocellular carcinoma (HCC) is a devastating disease ranking as the third most frequent cause of cancer deaths worldwide. In the US, the incidence of HCC tripled between 1975 and 2005 and at present day survival rates are as low as 20% due to limited treatment options. Future therapeutic advances rely on a greater understanding of the full range of genetic and epigenetic defects displayed by this cancer. Recent advances in whole genome methylation profiling have allowed us to investigate these alterations in a cohort of HCC and normal liver samples.

Methods: Using the Illumina Infinium platform, we analyzed the methylation patterns in over 50 human HCC and normal liver samples obtained from surgical resection and transplant specimens. We also investigated the expression and gene copy number (CGH) status on the same cohort using the Agilent platform.

Findings: We have been able to identify two very distinct groups of HCC based on their methylation status. In each class of tumors, there are both hypo- and hypermethylated genes relative to normal liver. Several of these tumor-specific alterations have been validated by pyrosequencing. In one class of tumors, many genes that are normally repressed by polycomb group proteins in stem cells are hypermethylated, consistent with the tumor cell of origin being a progenitor stem cell. The other class of tumors does not show this association and are likely to originate from a different cell of origin. Intriguingly, these two classes also correspond to distinct groups based on both DNA copy number analysis and expression analysis.

Conclusion: Combining these three independent methods of global analysis; expression profiling, epigenome analysis and CGH data, we have identified a robust new classification scheme for HCC. This approach should provide insight for future therapeutic advances.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3937. doi:10.1158/1538-7445.AM2011-3937
Abstract 3945: Association of inflammation-related microRNA gene expression in inflammatory bowel disease and IBD-associated colon cancer
Authors: Schetter Aaron J. , Welsh Judith A. , Bowman Elise D. , Harris Curtis C.
Published: August 2014

Introduction: There is an undeniable link between chronic inflammation and cancer. Inflammatory bowel disease (i.e. ulcerative colitis [UC] and Crohn's disease [CD]) are conditions that lead to chronic inflammation of the colon and increased risk of colon cancer. Indentifying factors that contribute to inflammatory bowel disease (IBD) and IBD-associated cancers may allow for the development of therapeutic and preventive strategies for these diseases. Altered expression of microRNAs may contribute to these diseases.

Methods: We examined the expression of 29 specific microRNAs by Taqman-based quantitative RTPCR using the Fluidigm 96.96 dynamic array system. The 29 microRNAs were selected based on a combination of our own preliminary microarray data on inflammatory bowel disease and published literature on microRNAs involved in inflammation or inflammatory bowel disease. RNA was extracted from colon tissues of 34 patients with CD, 33 patients with UC, 64 sporadic colon cancer patients, 14 patients with IBD-associated colon cancer and 18 patients with adenomas. We identified microRNAs associated with gross inflammation in UC and CD patients. We also compared microRNA expression profiles of sporadic tumors with IBD-associated tumors.

Results: The expression of 18 microRNAs were significantly altered in gross inflammation of CD and 19 microRNAs were altered in gross inflammation of UC. In general, the expression patterns were similar comparing UC and Crohn's tissues, but 3 microRNAs were significantly different between the two diseases. Fifteen microRNAs were altered in sporadic colon cancer. The microRNA expression patterns of IBD-associated tumors were quite different from the expression patterns of sporadic colon tumors.

Summary: We find evidence supporting that the chronic inflammation of both ulcerative colitis and Crohn's disease leads to a systematic change in microRNA expression patterns. Altered expression of microRNA may be in part responsible for the phenotypic affects of gross inflammation. The expression of miR-21, miR-135b and miR-192 were altered in similar ways in colon tumors as they were in chronic inflammation, suggesting that alteration of these specific microRNAs may in part be responsible for the elevated risk of cancer in these populations. We also find that sporadic tumors have different expression profiles that IBD-associated tumor, suggesting that the different etiology of these tumors results in different microRNA transcriptomic expression patterns.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2011-3945
Abstract 3951: The ERG 3′-UTR is a target of miR-145 and expression patterns of ERG isoforms are altered in prostate cancer
Authors: Wach Sven , Hart Martin , Nolte Elke , Szczyrba Jaroslaw , Wullich Bernd , Graesser Friedrich
Published: August 2014

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs.

Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries and microarray analyses. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. The abundance of ERG as one of the identified target genes was assessed by Western blotting using a self-generated polyclonal antiserum.

Results: One of the miRNAs consistently deregulated in primary prostate cancer samples was miR-145 which was down regulated more than 1.5-fold in deep sequencing and 1.44-fold in microarray experiments (p<0.001, ANOVA). Independent target prediction databases (including miRanda, Targetscan and PicTar) indicated that the 3’ untranslated region (3’-UTR) of the ERG gene is a regulative target for miR-145. This prediction could be confirmed using a luciferase reporter gene vector containing the ERG 3’-UTR. Co-expression of the reporter gene construct with miR-145 resulted in a reduction of reporter gene activity while mutation of the miRNA binding site caused a loss of responsiveness to the respective miRNA. We generated a polyclonal antiserum directed against the 100 C-terminal amino acids of the longest ERG isoform and discovered that transient expression of miR-145 reduced the amount of ERG protein by 26% in the HEK293 cell line. Western blots of 26 corresponding pairs of prostate cancer and nonmalignant tissue samples showed, that ERG protein is overexpressed in the majority of cancer samples and that the relative abundance of several identified isoforms of ERG is altered in cancer samples. As expected, miR-145 expression was reduced in 24 of the 26 tumor samples.

Conclusion: Our findings demonstrate that the 3’-UTR of ERG is one regulative target of miR-145, which is commonly deregulated in prostate cancer. The ERG gene is commonly overexpressed in prostate cancer and the relative abundance of ERG isoforms is altered in samples of primary prostate cancers. This is the first report to show that ERG is a target of miR-145.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3951. doi:10.1158/1538-7445.AM2011-3951
Abstract 4043: Characterization of the enzymatic activity, biochemical requirements and inhibition of deubiquitinating enzymes, a novel oncology target class
Authors: Wrigley Jonathan D. , Eckersley Kay , Plant Helen , Preston Marian , Millard Lindsey , Hardern Ian , Nowak Thorsten , Simpson Peter B. , Andrews David , Hudson Kevin
Published: August 2014

Deubiquitinating enzymes (DUBs) represent promising opportunities for therapeutic intervention in oncology, and this study reports key information about the catalytic activity, assaying and inhibition of this novel enzyme class. The DUBs are proteases responsible for the cleavage of Ubiquitin from substrate molecules, and the resulting effects on the levels or activity of the substrates has been shown to play key roles in a range of cellular processes.

Here we have utilized a range of in vitro and cellular assays of DUB activity to characterize various DUB enzymes, generate high throughput screens and profile DUB inhibition by hit molecules, to further our understanding in this area.

The characterization of different DUBs has enabled us to highlight both differences and similarities in their behavior and requirements, particularly with respect to protein subunits, reducing conditions, and activity against different substrates. Profiling of small-molecule hits targeting these enzymes, under various in vitro conditions, and also in a cellular environment, has not only yielded information on the compounds, but has also demonstrated key considerations about the inhibition of these cysteine proteases.

Together these findings further our knowledge of this novel target class, as well as providing valuable information to aid the hunt for therapeutic agents modulating these enzymes in a range of oncology settings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2011-4043
Abstract 4050: Quantitation of sirtuin activity with a one-step luminescent assay
Authors: Kirkland Thomas A. , Evans Nathan , Niles Andrew L.
Published: August 2014

The Sirtuin family of histone/protein deacetylating enzymes has gained considerable interest both for their recognized importance in processes such as gene silencing and expression and in cellular metabolism. This has led to identification of these enzymes as important actors in diverse pathologies including diabetes, Alzheimer's disease and oncology. We have developed a convenient and sensitive screening methodology to detect and measure both inhibitors and activators of sirtuin activity. Here we describe a one-step, homogeneous luminescent assay for the detection of activity for multiple sirtuin isotypes. The reagent formulation contains a pro-luminescent sirtuin peptide substrate, NAD+, ATP, thermostable luciferase, and a developer enzyme that cleaves deacetylated lysine residues. The addition of an active sirtuin enzyme source leads to a robust luminescent signal within minutes with “glow-type” characteristics lasting for several hours. Data will be presented showing characterization of the assay using a diverse assortment of established sirtuin inhibitors, followed by suitability for screening in 384-well formats and the incorporation of a control substrate to identify false positives.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4050. doi:10.1158/1538-7445.AM2011-4050
Abstract 4066: Combination metformin and ODC siRNA inhibit human melanoma proliferation and migration
Authors: Hsueh Eddy C. , Zhang Yanping , Peng Guangyong
Published: August 2014

Metformin can exert an antiproliferative effect on human tumor cells through the AMP-activated kinase (AMPK) pathway. Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis pathway in cancer cell proliferation and tumor progression. We hypothesized that the combination of Metformin and small interfering RNA (siRNA) against ODC could inhibit human melanoma cell growth and migration in vitro.

Human melanoma cell line cells (SK-MEL23, MEL1861, MEL938, MEL3910, and MEL888) were evaluated. ODC siRNA and contol siRNA were transfected into the human melanoma cells using lipofectamine per manufacturer's instructions (Santa CruZ. CA). Transfected cell were then treated with 1uM, 2uM, 5uM Metformin and assess for cell proliferation and migration assay at various time points up to 72 hours. Cell proliferation assay was performed in triplicates using Cell Titer Blue Assay system according to manufacturer's protocol (Promega). Migration was assessed by creating an artificial homogenous wound onto a cell culture monolayer with a sterile plastic 10µl micropipette tip. Migration of cells into the wound was observed and photographed up to 48 hours. A total of three areas were selected randomly from each well and the cells in three wells of each group were quantified. All Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant.

Human melanoma cells were infected with ODC siRNA or control siRNA. On quantitative RT-PCR and Western blot analysis, >95% suppression of ODC expression was achieved. The siRNA-ODC significantly inhibited cells growth up to 50% at 72 hour (P<0.01). Inhibition of melanoma cell proliferation was also observed with Metformin in a dose-dependent fashion in all 5 melanoma cell line cells. Combination of ODC siRNA and Metformin significantly inhibited cell growth compared to ODC siRNA or MF alone (P<0.01) in all 5 melanoma cell line cells. The addition of Metformin to siRNA-ODC also significantly inhibited melanoma migration in the wounding assay model.

Metformin treatment and inhibition of ODC expression can have anti-proliferative effect on human melanoma cells. The combination also retard melanoma cell migration in a wounding model. The combination of Metformin and ODC inhibition may be a novel treatment strategy for human melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2011-4066
Abstract 4113: Kinase activity based biomarkers: Identification of prognostic and erlotinib response prediction markers in NSCLC patients
Authors: Ruijtenbeek Rob , Thijssen Victor , Schaake Eva , Houkes Liesbeth , de Wijn Rik , van de Heuvel Michel , van Suylen Robert-Jan , van Pel Rene , Nederlof Petra , Hilhorst Riet , Dingemans Anne-Marie , Klomp Houke
Published: August 2014

Background: Reliable diagnostic tests are needed to identify early stage non-small cell lung carcinoma (NSCLC) patients with poor prognosis. Concomitantly there is a clear need for tests that enable the selection of patients who will benefit from targeted therapy with kinase inhibitors. We evaluated kinase activity profiles in two groups of early stage NSCLC patients, either for prognosis of long- or short-term survival, or for predicting erlotinib drug response.

Method: Retrospective studies were performed on fresh frozen resection material of two groups of early stage NSCLC patients. The first group consisted of 48 short- and long-term survivors who underwent a complete surgical resection (5+ years follow-up). The second group consisted of 14 NSCLC patients who received 3 weeks of neo-adjuvant treatment with erlotinib prior to complete surgical resection. Response evaluation to neo-adjuvant treatment was based on histopathological examination of the surgical specimens. For both studies, kinase activity profiles of lysed cryosections of tumour tissues were generated in the presence and absence of protein tyrosine kinase inhibitors on PamChip® peptide micro-arrays, comprising 144 tyrosine containing peptides, derived from known phosphorylation sites of human proteins. Partial least square discriminant analysis was used to construct prediction models. ClustalW alignment algorithms were used to investigate the most informative phosphorylation sites.

Results: Kinase activity profiles obtained in the absence of inhibitor did not distinguish between subgroups (long- versus short-term survival, responder or non-responder to TKI), whereas ratios of inhibited versus non-inhibited signals resulted in distinct classifiers predicting survival for the first group, and response for the second group. Multivariate unsupervised analysis with leave-one-out cross-validation resulted in an error rate for survival prediction of 29%. In the drug response prediction 13 out of 14 patients were correctly classified.

Conclusion: This is the first study to show that kinase activity profiles of tumour tissue exposed to a kinase inhibitor can be used to identify NSCLC patients likely to respond to erlotinib treatment. Furthermore, based on kinase activity profiles of early stage NSCLC tumours, a prognostic classifier, for a set of 48 patients, was obtained.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2011-4113
Abstract 4115: Prediction of drug sensitivity based on correlation of molecular and genetic signatures between lung tumor cell lines and tumor tissue samples from NSCLC cancer patients
Authors: Gong Hua C. , Beer David , Singh Sharat , Webster Edvelyn , Meyer Gary
Published: August 2014

To establish transition of preclinical response profiles to the subsequent clinical treatment, we profiled pathway activation and genomic signature in tumor cell lines and human tumor tissues. This profiling has allowed us to define molecular pathways and these models may serve as an excellent platform to evaluate the efficacy of targeted therapies. We profiled pathway activation, mutation status, and clinical histology of a panel of representative lung tumor cell lines and 50 human lung adenocarcinoma samples. We cluster patients samples with corresponding tumor cell lines based on such signature and predicted treatment options for the patients based on response to selected candidate treatment of the cell lines. Subsets of patients were identified as candidates to treatment with inhibitors of EGFR, cMET, IGFR, MEK, and PI3K/mTOR. EGF pathways in EGFR mutant cell lines NCI-H1975, HCC827 and NCI-H1650 are highly activated, and those cells were sensitive to inhibitors blocking EGFR signaling. A group of patients were identified to share the similar profiles, and we predict those are candidates for EGFRi therapies. While H1973 is a cMET amplified line with cMET and EGFR activated, patients sharing this genotype may respond to cMET inhibitor treatment. IGF1R expressing A549 and NCI-H358 cells were sensitive to IGF1R inhibition, the patients sharing the similar profile may be candidates to this class of therapies. PI3K/mTOR inhibitor, BEZ 235, effectively inhibited tumor cell growth in most of the cell lines except NCI-H1993 and NCI-H358 cells; while MEK inhibitor PD-325901 was less active in blocking growth of k-RAS mutant cell lines NCI-H460, NCI-H1734, NCI-H358 and A549. Patients with a similar signature may benefit from those treatments. By combining pathway biomarker, mutation status, and clinical features, we were able to cluster clinical patient samples to the lung tumor cell lines and propose treatment options for the patients based on their signature overlap.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4115. doi:10.1158/1538-7445.AM2011-4115
Abstract 4116: Predictive biomarkers for the PI3 kinase inhibitor GDC-0941 in NSCLC
Authors: Spoerke Jill , O'Brien Carol , Boyd Zach , Mohan Sankar , Pandita Ajay , Berry Leanne , Fridlyand Jane , Friedman Lori , Hampton Garret , Amler Lukas , Lackner Mark
Published: August 2014

The class I phosphatidylinositol 3’ kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting the PI3K pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. GDC-0941 is a pan-inhibitor of all 4 isoforms of class 1 PI3K that is currently undergoing testing in the clinic. We were interested in the predictive value of candidate biomarkers in NSCLC to help inform clinical trial biomarker strategies in this indication. We assembled a focused panel of cell lines with key alterations in the PI3K pathway isolated from other genetic events to the extent possible. The approach involved pharmacogenomic and proteomic profiling. We found that models harboring key pathway alterations were exquisitely sensitive to GDC-0941. In particular, cell lines with EGFR mutations, PIK3CA mutations or amplification were the most sensitive. Inhibition of PI3K in mechanism of action studies showed effects consistent with induction of apoptosis, cell cycle arrest, and impairment of translation in models with these key alterations. In addition, gene expression changes of genes downstream of PI3K were seen upon treatment with GDC-0941 in sensitive cell lines but not those that were resistant. We extended these studies to NSCLC tumor samples and evaluated the prevalence of important pathway alterations, including PTEN loss, PIK3CA amplification, LKB1 loss in tumors representing distinct histopathological subtypes. We find that certain candidate biomarkers show subtype specific associations, and will discuss the implications for the development of PI3K-targeting agents in NSCLC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2011-4116
Abstract 4117: A non-invasive system for monitoring resistance to EGFR tyrosine kinase inhibitors with plasma DNA in lung cancer patients
Authors: Aragane Naoko , Nakamura Tomomi , Iwanaga Kentaro , Sato Akemi , Komiya Kazutoshi , Abe Tomonori , Ureshino Norio , Hayashi Shinichiro , Hosomi Toshiya , Hirai Mitsuharu , Sueoka Eisaburo , Kimura Shinya
Published: August 2014

Although EGFR tyrosine kinase inhibitors (EGFR-TKIs) have great response to lung adenocarinoma with EGFR activating mutations, acquired resistance eventually developed. The half of these patients has the gatekeeper T790M mutation of EGFR in lung cancer tissue. A non-invasive mutation detection system is desired considering the difficulty in obtaining tissue specimens during disease progression_the majority of lung cancer recurrences occur in distant sites. We report a novel non-invasive monitoring system, MBP-QP (mutation-biased PCR and quenching probe) method, to detect the mutation using plasma DNA. The MBP-QP method is combined with mutated biased PCR and genotyping based on the analysis of a melting curve of probe DNA which is bound to the target mutated site using a fluorescence quenching probe. The detection limit was 2 copies of control plasmid, and 0.2 ng of genomic DNA isolated from a lung caner cell line harboring T790M. 0.3 % mutant plasmid could be detected in the mixture of plasmids inserted with EGFR exon 20 with or without T790M. With this method, T790M mutations were detected in plasma DNA of 50% of patients who acquired resistance, but not in primary EGFR-TKI non-responders, patients responding to treatment, or patients not treated with EGFR-TKI, which is consistent with the clinical course. The non-invasive MBP-QP method enabled us to monitor T790M repeatedly and will be useful for determining appropriate lung cancer treatment strategies in practice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4117. doi:10.1158/1538-7445.AM2011-4117
Abstract 4119: Association of plasma hepatocyte growth factor (HGF) and insulin-like growth factor binding protein (IGFBP)-3 with gefitinib resistance in patients with advanced non-small cell lung cancer
Authors: Han Ji-Youn , Kim Jin Young , Lee Soo-Hyun , Lee Geon Kook , Lee Jin Soo
Published: August 2014

Purpose: It has been suggested that hepatocyte growth factor (HGF) and insulin-like growth factor binding protein (IGFBP)-3 are associated with gefitinib resistance in non-small cell lung cancer (NSCLC). We investigated the predictive and prognostic roles of these proteins in NSCLC patients treated with gefitinib.

Methods: Of 106 patients enrolled in the randomized phase II study of gefitinib, 97 had plasma samples available for ELISA testing. Of these samples, seven and eight, respectively, had HGF and IGFBP-3 values that could not be measured. Therefore, the correlations between clinical outcomes and plasma levels of HGF and IGFBP-3 were evaluated in 90 and 89 patients, respectively.

Results: Plasma HGF levels were significantly higher in older patients, male patients, patients with squamous cell carcinoma, current smokers, and patients with EGFR wild-type tumors. Low HGF levels were significantly associated with higher response rate (RR), and longer progression-free survival (PFS) and overall survival (OS) irrespective of EGFR mutation status. In a multivariate analysis, the presence of EGFR mutations (HR, 0.42; 95% CI, 0.24 to 0.73; P=0.002) and low HGF levels (HR, 0.58; 95% CI, 0.35 to 0.95; P=0.031) were independently predictive of longer PFS, and an ECOG PS of 0 (HR, 2.95; 95% CI, 1.57-5.54; P=0.001) and low HGF levels (HR, 0.40; 95% CI, 0.22-0.72; P=0.002) were independently predictive of longer OS. No statistically significant differences were found for IGFBP-3.

Conclusion: High HGF levels are significantly associated with resistance to gefitinib and can be used as a predictive marker for the differential outcome of gefitinib treatment in NSCLC irrespective of EGFR mutation status.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2011-4119