bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract IA04: ATRX mutations in human carcinogenesis
Authors: Papadopoulos Nickolas E.
Published: August 2014

The recent years, genome-wide analyses identified the genetic landscapes of many different tumor types. One of the most frequent class of genes found to be mutated were genes whose products regulate epigenetic mechanisms, like chromatin modification and remodeling, providing a link between genetic alterations and epigenetic changes in cancer. One of these genes is ATRX. This gene has been studied in the context of alpha thalassemia-mental retardation X-linked syndrome. In human sporadic solid tumors, it was first shown to be mutated in pancreatic neuroendocrine tumors (PanNETs). Approximately, 50% of PanNETs had mutations in either ATRX or DAXX. The proteins of these two genes form a complex that deposits H3.3 at heterochromatin and telomeres. ATRX, but not DAXX, has also been found to be mutated in certain types of central nervous system tumors. The prevalence of mutations in ATRX is different for the different types of gliomas. It is mutated more frequently in astrocytomas and oligoastrocytomas, but rarely in primary glioblastomas. Recently, we identified mutations in the promoter of TERT in the same set of glioma samples. TERT mutations were more prevalent in primary glioblastomas and oligodendrogliomas. Overall, mutations in ATRX and TERT were mutually exclusive, suggesting that these two genetic mechanisms result in equivalent growth advantages. Mutations in ATRX have almost perfect correlation with Alternative Lengthening of Telomeres (ALT) phenotype in PanNETs, CNS tumors and other cases with ATRX mutations, thus, TERT activation and ALT phenotype are mutually exclusive. This has implications for understanding the relationship between telomeres and tumorigenesis. These studies, in addition to providing clues about the underlying genetic changes that drive tumorigenesis, they also have clinical implications. Mutations in ATRX or DAXX in PanNETs associate with better prognosis. Mutations of ATRX and TERT aid in the classification and prognostication of brain tumors. Further understanding of the role of ATRX in tumorigenesis should help in uncovering new ways for therapeutic targeting of tumors with mutations in this gene.

Citation Format: Nickolas E. Papadopoulos. ATRX mutations in human carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA04.
Abstract IA06: Epigenetic regulation and heterogeneity in cancer
Authors: Bernstein Bradley E.
Published: August 2014

We are using emerging technologies to characterize chromatin state and regulation in human cells. Chromatin maps for diverse normal tissues and cells reveal the locations and cell type-specificities of distal regulatory elements and facilitate the interpretation of genetic variants associated with complex human diseases. Analogous studies of malignant tumors reveal regulatory programs that drive proliferation, while also emphasizing the importance of epigenetic heterogeneity in cancer biology and therapeutic resistance. I will highlight representative studies in these areas and touch on new technological approaches for the study of tumor heterogeneity.

Citation Format: Bradley E. Bernstein. Epigenetic regulation and heterogeneity in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA06.
Abstract IA25: Linking genetic features of human cancers and histone-modifying enzymes for future cancer therapies
Authors: Schreiber Stuart L.
Published: August 2014

Small-molecular therapeutics targeting chromatin-modifying enzymes offer promise for future treatment of cancers, but the most suitable ways to use such agents is still largely unknown. This lecture will explore several avenues of research aimed at advancing chromatin-based therapeutics, including the identification of cancer genetic dependencies involving chromatin, the creation of new cancer dependencies by altering cell states through chromatin modifications, and the discovery of novel small molecules that alter chromatin states.

Citation Format: Stuart L. Schreiber. Linking genetic features of human cancers and histone-modifying enzymes for future cancer therapies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA25.
Abstract PR04: Genomic and epigenomic interactions of an Ewing sarcoma-specific long noncoding RNA
Authors: Mitra Sheetal A. , Mitra Anirban P. , Buckley Jonathan D. , May William A. , Kapranov Philipp , Arceci Robert A. , Triche Timothy J.
Published: August 2014

Ewing sarcoma is a highly invasive pediatric bone malignancy with a 30% 5-year survival rate for patients with metastatic disease. The EWS-FLI1 fusion gene is detected in 90% of these cases, and functions as a transcription factor that regulates oncogenesis-related gene expression. The functions of EWS-FLI1 and other oncogenes have been explored in the past to detect potential therapeutic targets.

Here we report the functions and interactions of a long intergenic non-coding RNA (lincRNA) that is unique to Ewing sarcoma. Gene expression profiling studies identified a significant association of lincRNA, AK057037, with Ewing sarcoma. Chromatin immunoprecipitation (ChIP) and functional studies with EWS-FLI1 detected direct regulation of this multi-exonic, multivariant lincRNA by EWS-FLI1. Comparative RNA-seq analysis on primary and metastatic tumor cell lines showed increased transcriptional activity in the metastatic cell line. In vitro and in vivo functional studies with the lincRNA confirmed its role as an oncogene in promoting anchorage independence and metastasis of the Ewing sarcoma cell lines and xenograft models, respectively. Further, ChIP studies showed that AK057037 was bound to Ezh2, a methyltransferase in the polycomb repressor complex 2 (PRC2). ChIP-seq using histone antibodies showed changes in methylation pattern in promoter regions of genes affected by the lincRNA knockdown.

In conclusion, the lincRNA AK057037 behaves as an oncogene in Ewing sarcoma. Its association with the PRC2 complex allows for chromatin remodeling such that it promotes metastasis by perturbing transcription of genes involved in migration in this malignancy.

This abstract is also presented as Poster A33.

Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, William A. May, Philipp Kapranov, Robert A. Arceci, Timothy J. Triche. Genomic and epigenomic interactions of an Ewing sarcoma-specific long noncoding RNA. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR04.
Abstract PR06: CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression
Authors: Wang Lu , Zhao Zibo , Meyer Mark , Pike Wesley J. , Xu Wei
Published: August 2014

CARM1 belongs to the type I protein arginine (R) methyltransferase (PRMT) family and asymmetrically di-methylates proteins on arginines. Because CARM1 is highly expressed in high grade breast tumors and was shown to transactivate cancer-relevant transcription factors including ERα, NF-κB, p53 and E2F1, it has been strongly implicated in tumorigenesis. To fully characterize functions of CARM1 in breast cancer, we knocked-out CARM1 from several breast cancer cell lines using Zinc-Finger Nuclease technology. Western blot using an asymmetric di-methylated R antibody revealed striking differences between CARM1 KO and parental MCF7 cell lysates, indicating that CARM1 methylates a significant number of substrates in breast cancer cells. BAF155, a core subunit of SWI/SNF chromatin remodeling complex, was identified as a CARM1 substrate by mass spectrometry. Further biochemical analyses validated that BAF155 was methylated by CARM1 in vivo and in vitro and methylation was mapped to a single site, R1064. We next generated BAF155R1064K knock-in MCF7 and MDA-mb-231 cells and showed that methylation of BAF155 is essential for breast cancer cell growth, colony formation and migration. Further, we developed an antibody that selectively recognized methylated form of BAF155 (me-BAF155) and observed strong nuclear me-BAF155 staining in invasive ductal carcinoma but not in the normal breast tissue. Importantly, me-BAF155 immunostaining positively correlates with tumor stage and metastasis. To understand the molecular mechanism by which BAF155 methylation affects tumorigenesis, we performed ChIP-seq using ChIP-validated BAF155 antibody in the BAF155WT or BAF155R1064K knock-in MCF7 cells. Interestingly, C-Myc pathway genes were only identified in WT but not mutant BAF155 regulated categories. Individual ChIP assays validated differential binding of BAF155WT or BAF155R1064K to specific target genes and importantly, the mRNA levels of these genes were affected correspondingly. This result suggests that BAF155 methylation affects gene expression by altering genomic association of BAF155, and perhaps the SWI/SNF complex, to chromatin regions. Collectively, our study revealed novel mechanisms by which CARM1 perturbs the chromatin remodeling machinery to enhance tumor progression.

This work is supported by DOD ERA of HOPE Scholar Award and Shaw Scientist Award to W.X.

This abstract is also presented as Poster A37.

Citation Format: Lu Wang, Zibo Zhao, Mark Meyer, Wesley J. Pike, Wei Xu. CARM1 methylates BAF155 and perturbs chromatin remodeling machinery to enhance tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR06.
Abstract PR10: Modulation of chromatin structure and transcription by the SWI/SNF complex in acute lymphoblastic leukemia
Authors: Wu Jennifer , Pinello Luca , Yissachar Elinor , Yuan Guo-Cheng , Roberts Charles
Published: August 2014

SWI/SNF chromatin remodelers act as tumor suppressors in a wide variety of tissue types and cancers. Recent reports describe that as many as 20% of human cancers harbor mutations in one or more subunits of this multi-protein complex. Intriguingly, mutations in the SWI/SNF complex are rarely found in leukemias. However, it has long been understood that SWI/SNF is a key component of glucocorticoid-mediated transcription. Diminished expression of SWI/SNF subunits has also been linked to altered sensitivity to glucocorticoid-induced cell death in acute lymphoblastic leukemias (ALL). Glucocorticoid-induced cell death is a critical component of successful ALL therapy. We therefore use the glucocorticoid-mediated transcriptional program in a human ALL cell line to interrogate the genome-wide chromatin changes and contributions to transcription mediated by an intact SWI/SNF complex. We begin by elucidating the dynamic relationships between glucocorticoid receptor binding to chromatin and recruitment of SWI/SNF using ChIP-Seq. We further relate these glucocorticoid-inducible binding events to changes in position of modified histones, changes in chromatin accessibility by DNase-seq, and changes in gene expression. By better understanding the mechanisms by which SWI/SNF modifies the chromatin landscape within a specific transcriptional program, we will facilitate additional studies of how disruption of the SWI/SNF complex affects ALL responses to steroid therapy. Furthermore, we will contribute to future studies of how mutations in the SWI/SNF complex functionally affect chromatin structure and contribute to oncogenesis.

This abstract is also presented as Poster B07.

Citation Format: Jennifer Wu, Luca Pinello, Elinor Yissachar, Guo-Cheng Yuan, Charles Roberts. Modulation of chromatin structure and transcription by the SWI/SNF complex in acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR10.
Abstract A27: Mapping of chemical-genetic interactions in breast cancer reveals a mechanism of resistance to PI3K inhibitors
Authors: Muellner Markus K. , Uras Iris Z. , Gapp Bianca , Nijman Sebastian M.
Published: August 2014

Linking the molecular aberrations of cancer to drug responses allows directing the choice of treatment and to explore new therapeutic applications. We set out to systematically map drug-gene interactions in breast cancer and established a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cells in response to a collection of drugs. This approach so far revealed several synthetic-lethal interactions and drug-resistance mechanisms, including previously discovered interactions from other model systems. Among the novel interactions NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

Citation Format: Markus K. Muellner, Iris Z. Uras, Bianca Gapp, Sebastian M. Nijman. Mapping of chemical-genetic interactions in breast cancer reveals a mechanism of resistance to PI3K inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A27.
Abstract IA19: Small-molecule modulators of the Hedgehog signaling pathway
Authors: Chen James K.
Published: August 2014

The Hedgehog (Hh) signaling pathway plays critical roles in embryonic patterning and tissue homeostasis, and its inappropriate activation can lead to basal cell carcinoma, medulloblastoma, and other cancers. Small molecules that modulate Hh signaling proteins can therefore be valuable probes of Hh pathway-dependent physiology and targeted therapies for Hh pathway-related diseases. While high-throughput screening campaigns in academia and industry have yielded several Hh pathway modulators that potently and selectively target the transmembrane protein Smoothened (Smo), there is a growing need for compounds that inhibit Hh target gene expression through other mechanisms. Such small molecules could provide new insights into the cellular processes that regulate Gli transcription factor function and possibly prevent or overcome the emergence of tumors that are resistant to Smo antagonists. Toward this goal, we have completed large-scale screens for Hh pathway inhibitors that act downstream of Smo, revealing several compounds that inhibit Gli-dependent transcription through distinct mechanisms of action. We have successfully determined the cellular target of one inhibitor from our screens, and mechanistic studies of other Gli antagonists are currently underway.

Citation Format: James K. Chen. Small-molecule modulators of the Hedgehog signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr IA19.
Abstract IA5: Designing synthetic regulatory RNAs: New tools for temporal and spatial control in biological systems
Authors: Smolke Christina D.
Published: August 2014

Advances in synthetic biology are transforming our ability to design and build synthetic biological systems. While progress has been made in the design of complex genetic circuits, capabilities for constructing large genetic systems currently surpass our ability to design such systems. This growing “design gap” has highlighted the need to develop methods that support the generation of new functional biological components and scalable design strategies for complex genetic circuits that will lay the foundation for integrated biological devices and systems.

As examples of functional RNA molecules playing key roles in the behavior of natural biological systems have grown over the past decade, there has been growing interest in the design and implementation of synthetic counterparts. Researchers are taking advantage of the relative ease with which RNA molecules can be modeled and designed to engineer functional RNA molecules that act as diverse components including sensors, regulators, controllers (ligand-responsive RNA regulators), and scaffolds. These synthetic regulatory RNAs are providing new tools for temporal and spatial control in biological systems. I will describe recent work in the design of RNA controllers and advances in addressing challenges faced in their broad implementation as user-programmed control systems in living cells. In particular, I will describe the development of high-throughput cell-based screens for rapidly generating synthetic regulatory RNAs with specified quantitative properties. I will also discuss how the application of synthetic regulatory RNAs as controllers in different biological pathways are leading to the elucidation of integrated systems design strategies and new capabilities for programming genetic systems.

Citation Format: Christina D. Smolke. Designing synthetic regulatory RNAs: New tools for temporal and spatial control in biological systems [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr IA5.
Abstract A08: Analysis of MRGBP and PPIL1 genes in colon cancer
Authors: Akisik Ebru , Dalay Nejat , Yamaner Sumer , Bugra Dursun
Published: August 2014

Colorectal cancer is one of the most common causes of cancer death throughout the world. Colorectal carcinogenesis is a multi-step process and in addition to genetic changes significant changes in DNA methylation and epigenetic events such as histone modifications can also lead to deregulation of gene expression. The histone proteins play a dynamic role in the chromatin structure and transcription. Aberrant protein acetylation, particularly on histones, has been related to cancer while abnormal expression of histone deacetlytransferases (HDACs) are observed in a broad range of cancer types. Therefore, HDACs have emerged as promising targets in cancer therapeutics.

The MRG-binding protein (MRGBP / C20orf20) is a component of the NuA4 histone acetyltransferase (HAT) complex and is involved in the transcriptional activation of specific genes. The MRGBP gene is frequently upregulated in tumors and is thought to play a role in cancer development. On the other hand removal of introns from pre-mRNA is carried out by a large macromolecular spliceosome complex. PPIL1 (Peptidyl-prolylisomerase-like 1) is a component of the human spliceosome and plays a primary role in pre-mRNA splicing for catalyzing isomerization of the peptide bonds. Overexpression of the PPIL1 protein in cancer suggest a potential role of PPIL1 in the in the development and promotion of colon cancer. In the present study we analyzed MRGBP and PPIL1 gene expression levels in matched tumor and normal tissue samples from patients with colorectal cancer.

Tissue samples were collected from 51 patients with colorectal cancer who underwent surgery for tumor resection. The normal tissue specimens used in the study were histologically confirmed to be free of cancer. The HPRT (hypoxanthine-Guanine phosphoribosyltransferase) gene was used as reference. The target and reference genes were amplified in the same multiplex PCR using the LightCycler 480 System. 8-9 nucleotide long UPL probes were labeled with fluorescein (FAM) and TAMRA at the 5′ and 3′ ends, respectively. Expression levels were determined using the Basic Relative Quantification analysis software and the results were evaluated by the Mann-Whitney U test.

Although the median MRGBP expression levels in the tumor tissue were higher than normal tissue (1,72 vs.1,41) the difference was not significant. The median expression levels of PPIL1 in the tumors and normal tissue samples (1,97 vs 1,86) were also similar. Our results indicate that neither MRGBP nor PPIL1 gene expression levels are upregulated in colon cancer cells indicating that these are not directly associated with colorectal cancer.

Citation Format: Ebru Akisik, Nejat Dalay, Sumer Yamaner, Dursun Bugra. Analysis of MRGBP and PPIL1 genes in colon cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A08.
Abstract A16: Role of Hsp90alpha-1, a splice variant of Hsp90alpha, in cancer
Authors: Ghosh Suman , Peterson Laura B. , Vielhauer George , Blagg Brian S. J
Published: August 2014

Molecular chaperone complexes, such as heat shock proteins 90 (Hsp90) and its co-chaperones, are essential for folding nascent polypeptides into their biologically active structures and for the refolding of aggregated and denatured proteins that occur upon cellular stress including cancer. Hsp90 exists as four isoforms in humans. Among them, Hsp90alpha is induced under stressful conditions, detected in the cytoplasm, nucleus and is secreted into extracellular space under metastatic conditions. Hsp90alpha is phosphorylated by DNA-PK at the nucleus under apoptotic conditions and localizes at the edge of the nucleus. Intrigued by the fact that Hsp90alpha might play a role in metastasis as well as in stress resistance, the mechanism of action of Hsp90alpha was investigated. Here we report that Hsp90alpha-1, a splice variant of Hsp90alpha, is up-regulated in cancer cells, predominantly associates with co-chaperone Aha1 in-vivo at the filopodia like extrusion of cell. The Hsp90alpha-1/Aha1 complex was partially disrupted by C- terminal inhibitors in-vivo conditions; and Hsp90alpha-1 and Aha1 redistributed to the cytoplasm from the filopodia following the treatment with the C- terminal inhibitors. Gene knock down analysis reveals that several P-Akt pathway and MAP Kinase pathway proteins were Hsp90alpha-1 dependent. P-Akt also co-localized with the Hsp90alpha-1/Aha1 complex at the filopodia region and suggests that Hsp90alpha-1 plays critical role in maturing/folding proteins that are important for establishing and maintaining cell polarity which is perturbed by the C- terminal inhibitors. Under apoptotic conditions the Hsp90alpha-1/Aha1 complex is present in the cytoplasm and nucleus and Hsp90alpha-1 is phosphorylated. Survivin, being a Hsp90alpha-1 dependent client protein, binds with the Hsp90alpha-1/Aha1 complex at normal and apoptotic conditions confirms that Survivin play dual role in mediating metastases as well as an Inhibitor of Apoptotic Protein (IAP).

Citation Format: Suman Ghosh, Laura B. Peterson, George Vielhauer, Brian S. J Blagg. Role of Hsp90alpha-1, a splice variant of Hsp90alpha, in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A16.
Abstract A29: Mdm2 E3 ubiquitin ligase function is dispensable for the degradation of Mdm2 itself
Authors: Kim Tae-Hyung , Zhang Yanping
Published: August 2014

Mdm2 has long been considered the major negative regulator of both p53 stability and activity. The RING finger domain of Mdm2 confers its E3 ubiquitin ligase activity, and through this function Mdm2 controls p53 stability, additionally, auto-ubiquitination of Mdm2 is believed to be the primary mechanism of Mdm2 degradation. In vitro, mutation or truncation of the RING domain in Mdm2 resulted in loss of ubiquitin ligase activity towards both Mdm2 and its substrate p53. In contrast, recent knock-in mouse models indicate that under physiological conditions the Mdm2 RING-mediated E3 ubiquitin ligase function is dispensable for Mdm2 degradation. To investigate the in vivo significance of Mdm2 auto-ubiquitination, we compared two Mdm2 knock-in mouse strains each harboring a single amino acid substitution in RING domain (C462A) or C-terminus (Y487A). Both knock-in mutants disrupt Mdm2 E3 ligase activity for p53. We observed that (1) mutant Mdm2 protein (C462A or Y487A) fails to degrade p53 protein. (2) Wild type and mutant Mdm2 are degraded similarly. Despite efficient disruption of p53 degradation, half-life assays demonstrated that neither of the Mdm2 mutants affected the rate of degradation of Mdm2 itself indicating not only that Mdm2 auto-ubiquitination is not the primary mechanism for Mdm2 degradation in vivo, but also that an additional mechanism is in action to regulate Mdm2. (3) Inhibition of proteasomal degradation by MG-132 resulted in similar levels of accumulation of both wild type and mutant Mdm2s suggesting Mdm2 degradation is mediated via the proteasome. In vivo ubiquitination assay reconfirmed that Mdm2 RING domain mutation does not affect degradation and accumulation of Mdm2 itself, further implicating the possibility of an outside E3 ligase activity. In conclusion, auto-ubiquitination of Mdm2 is not necessary for Mdm2 degradation in vivo. The identification of E3 ubiquitin ligases with specificity for Mdm2 is an important next step to gain mechanistic insight into the regulation of Mdm2 regulation, and with that, p53.

Citation Format: Tae-Hyung Kim, Yanping Zhang. Mdm2 E3 ubiquitin ligase function is dispensable for the degradation of Mdm2 itself. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A29.
Abstract A32: In vivo imaging of ERK activity of mouse breast cancer cells reveals heterogeneity in response to anti-cancer drugs and tumor forming capacity
Authors: Kumagai Yuka , Matsuda Michiyuki
Published: August 2014

The Ras-Raf-MEK-ERK pathway is critical for normal physiological development and for carcinogenesis. However, the activity of this signaling cascade has never been examined in living cancer tissues due to technical difficulty. We have recently developed transgenic mice expressing a Förster resonance energy transfer (FRET) biosensor for ERK, which allows us to monitor cellular ERK activity in living tissues under two-photon (2P) microscopes.

To study the role of ERK in mammary tumors, we crossed MMTV-Neu mice with the mice expressing the ERK FRET biosensor and observed in vivo under 2P. In the primary mammary tumors, the ERK activity exhibited significant divergence among the tumor cells. Such heterogeneity was significantly diminished in the secondary tumor cells. A MEK inhibitor PD0325901 and an EGFR/HER2 inhibitor Lapatinib suppressed ERK in both the primary mammary tumor cells and the secondary mammary tumor cells. Interestingly, we found that a small number of cells with low ERK activity in primary tumors that did not respond to Lapatinib. We sorted cells depending on ERK activity and obtained ERKlow and ERKhigh population.The ERKlow population exhibited higher tumorsphere formation in nonadherent culture condition and tumor formation in syngeneic mice. Notably, the tumors developed from ERKlow cells exhibited heterogeneous ERK activity similar to primary tumors. Taken together, our results suggest that the heterogeneous ERK activity might be related to cancer stem cell in tumors.

Citation Format: Yuka Kumagai, Michiyuki Matsuda. In vivo imaging of ERK activity of mouse breast cancer cells reveals heterogeneity in response to anti-cancer drugs and tumor forming capacity. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A32.
Abstract A44: The role of ultraconserved noncoding RNAs in oncogenic transformation
Authors: Nguyen Hung Thanh , Chan Lifang , Voorhoeve Mathijs
Published: August 2014

Ultra conserved regions are stretches of DNA in the genome of >200bp that are 100% conserved between human, mouse and rat. Their overall conservation in a wide variety of vertebrates is often >95%. There are ~ 500 such regions in the human genome. Their function and the reason for their extreme sequence conservation are unknown. A majority of these ultraconserved genes (UCG) is expressed as non-coding RNAs, and many of the UCGs are differentially expressed in tumors and locate to cancer-associated regions in the genome. Some UCGs show small sequence variants between cancer patients and control cohorts, further suggesting that they may be causally involved in tumorigenesis. We have employed a genetically defined model of carcinogenesis to screen a limited set of cancer-associated UCGs and found that expression of two of these UCGs can directly affect the p16-pRb tumor suppressor pathway and contribute to the transformation of primary human cells.

Citation Format: Hung Thanh Nguyen, Lifang Chan, Mathijs Voorhoeve. The role of ultraconserved noncoding RNAs in oncogenic transformation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A44.
Abstract A51: p53 and p21 mRNA and protein expression in treated synthetic estrogen in mouse transgenic animal model
Authors: Salleh Mohd Nazil , Cheng Tan Mei , Hashim Thuaibah , Lai Henkie Isahwan Ahamd Mulyadi , Ramli Wan Khairuzzaman Wan
Published: August 2014

Studies with Diethylstilbestrol (DES) in humans and rodents have resulted in a spectrum of toxic and carcinogenic effects. Previous findings on gene expression profiles following DES treatment showed that p53, p21 and bax was transcriptionally regulated in this model. In the present studies,Reverse Transcriptase in situ Polymerase Chain Reaction and immunohistochemistry techniques for localization and expression of p53, p21 and bax at cellular levels was applied. Animal were housed individually and treated with 500μmole/kg b.w of DES, (ip) once daily up to 4 days. Our results have shown, the expression of p53, p21 and bax mRNA were greater in wild-type compared to p53+/- knockout mice. In addition, p53, p21 and bax mRNA were significantly higher in DES-treated compared to control-vehicle animals. Collectively, similar patterns of expression also were seen in p53 and p21 proteins and scored according to the percentage of positive nuclear staining. Therefore, the combination of p53 and p21 were concluded to be a valuable markers and prognostic for carcinogenesis and progression of endometrial cancer.

Citation Format: Mohd Nazil Salleh, Tan Mei Cheng, Thuaibah Hashim, Henkie Isahwan Ahamd Mulyadi Lai, Wan Khairuzzaman Wan Ramli. p53 and p21 mRNA and protein expression in treated synthetic estrogen in mouse transgenic animal model. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A51.
Abstract A56: Spontaneous aggregation and novel signalosome organization of BENTA-associated gain-of-function CARD11 mutants in lymphocytes
Authors: Stinson Jeffrey Richard , Snow Andrew L.
Published: August 2014

Antigen receptor (AgR)-induced NF-κB activation is a critical determinant of lymphocyte growth and survival. However, constitutive NF-κB activity can drive excessive lymphoproliferation and oncogenesis. We recently described a novel human lymphoproliferative disorder designated as BENTA (B cell Expansion with NF-κB and T cell Anergy), caused by germline gain-of-function mutations in the lymphocyte-specific scaffolding protein CARD11. Similar, somatic CARD11 mutations are found in ~10% of activated B cell-type diffuse large B cell lymphomas (ABC-DLBCL). Normally, AgR ligation converts CARD11 into an active conformation that nucleates a large multi-protein signalosome incorporating BCL10, MALT1, and other components that ultimately facilitate activation of IκB kinase (IKK) and nuclear translocation of NF-κB. We have now identified four different heterozygous CARD11 missense mutations (E134G, G123S, G123D, C49Y) in affected patients. Similar to BENTA patient cells and DLBCL tumors, ectopic expression of each mutant triggers constitutive NF-κB activation in B and T cell lines without AgR stimulation. Using confocal microscopy, we now demonstrate that these CARD11 mutants form large, spontaneous perinuclear aggregates with a novel, shell-like substructure. While BCL10 is largely excluded, other signaling components such as MALT1 and phospho-IκB kinase (IKK) are found within or closely colocalized with CARD11 shells, suggesting these aggregates are sites of active signaling. Our results provide the first evidence that gain-of-function CARD11 mutants assemble into a compartmentalized, active signalosome with a distinct structural organization. Further imaging and biochemical analysis of these aggregates will provide new insights into aberrant CARD11 signaling in BENTA patient lymphocytes and DLBCL tumors harboring active CARD11 mutations.

Citation Format: Jeffrey Richard Stinson, Andrew L. Snow. Spontaneous aggregation and novel signalosome organization of BENTA-associated gain-of-function CARD11 mutants in lymphocytes. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A56.
Abstract B23: Cysteine (C)-X-C receptor 4 induces oxidative stress through NADPH oxidase-2 in prostate cancer cells
Authors: Jones Kia J. , Chetram Mahandranauth A. , Bethea Danaya A. , Hinton Cimona V.
Published: August 2014

Reactive oxygen species (ROS), and the coupled oxidative stress, have long been implicated in the onset and progression of many cancers, including prostate cancer (PCa). One of the mechanisms by which ROS contributes to cancer is by acting as a second messenger, relaying signals from the cell surface to important signaling proteins, thereby regulating a variety of cellular processes. We have demonstrated that ROS increased the expression and activity of the chemokine (CXC) receptor, type 4 (CXCR4), which resulted in proliferation, invasion and trans-endothelial migration of PCa cells. Similarly, a study in human hepatoma cells revealed that CXCR4, and its ligand, stromal cell derived factor 1 alpha (SDF-1α), promoted ROS accumulation; however, the source (s) of ROS following growth factor stimulation have not been investigated. Identifying the sources of ROS that leads to oxidative stress in the tumor microenvironment remain areas of intense research. A recent study demonstrated that the increased ROS generation observed in PCa cell lines of varying aggressiveness was largely contributed by the NADPH oxidase family of enzymes. Herein, we observed that SDF-1α promoted ROS accumulation in C4-2 human PCa cells. We also established that NOX, an extramitochondrial ROS-generating system, was the primary source of ROS instead of the mitochondria. Moreover, expression of the isoform, NOX2, was preferentially regulated by CXCR4 at transcriptional and post-transcriptional levels. Finally, NOX2 expression correlated with PCa aggressiveness in microarray datasets. Therefore, we hypothesize that the SDF-1α /CXCR4 signaling axis mediates ROS production in PCa through NADPH-oxidases, resulting in oxidative stress that encourages tumor progression. Results generated from this study will further elucidate the mechanism (s) by which growth factor signaling promotes oxidative stress to enhance tumor development. Understanding the relationship between known cancer promoting elements, such as CXCR4, ROS and NOX, will facilitate the development of more comprehensive therapeutics to enhance current clinical strategies to combat cancer metastasis.

Citation Format: Kia J. Jones, Mahandranauth A. Chetram, Danaya A. Bethea, Cimona V. Hinton. Cysteine (C)-X-C receptor 4 induces oxidative stress through NADPH oxidase-2 in prostate cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B23.
Abstract B35: Amentoflavone inhibits angiogenesis and stimulates apoptosis in cells
Authors: Liu Zhihe , Zhang Jinli , Cao Wenjuan
Published: August 2014

Amentoflavone (8-[5-(5,7-dihydroxy-4-oxo-chromen-2-yl)-2-hydroxy-phenyl]-5,7-dihydroxy -2-(4-hydroxyphenyl) chromen-4-one, AF) is a biflavonoid derived from the extracts of Selaginella tamariscina. It has been shown that AF has diverse biological effects including anti-inflammatory, anti-viral, inhibition of fatty acid synthesis, and anti-tumor. It is well known that high cell proliferation and angiogenesis are two key factors in tumorigenesis. In this study, we report that AF inhibited proliferation, and stimulated apoptosis in fibroblasts. Incubation of fibroblast with AF showed inhibitory effect on cell viability and exhibited a series of cellular changes that were consistent with apoptosis. By western blot analysis, our date indicated significant increases in the amounts of cleaved-caspase 3, 8, 9 and Bax, several apoptotic promoters, and a significant decrease in TCTP, an apoptotic inhibitor, in fibroblast treated with AF. Furthermore, AF showed significant inhibitions on the proliferation, migration, and tube formation of endothelial cells, which are associated with angiogenesis. In conclusion, this study suggests that AF stimulates apoptosis in fibroblasts and inhibits angiogenesis of endothelial cells. Therefore, AF is a promising molecule which can be used in cancer treatment.

Note: This abstract was not presented at the conference.

Citation Format: Zhihe Liu, Jinli Zhang, Wenjuan Cao. Amentoflavone inhibits angiogenesis and stimulates apoptosis in cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B35.
Abstract B40: Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties
Authors: Mohamed Mona Mostafa , El-Ghonaimy Eslam A. , Nouh Mohamed A. , Schneider Robert J. , Sloane Bonnie F. , El-Shinawi Mohamed
Published: August 2014

Introduction: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and is characterized by rapid progression and dismal outcome. Although there is a growing literature describing the role of macrophages in non-IBC, the role of macrophages in IBC is unclear. The aims of the present study were to: 1) assess macrophages infiltration within the tumor tissues of non-IBC and IBC patients; 2) isolate and profile cytokines of macrophages drained from breast tumor axillary tributaries of non-IBC and IBC patients and 3) test whether major cytokines secreted by tumor draining macrophages induce motility of the IBC cell line SUM149.

Methods: Tumor-infiltrating macrophages were measured by immunohistochemical (IHC) techniques using CD14 and CD68 monoclonal antibodies. Blood drained from axillary vein tributaries was collected from non-IBC and IBC patients during breast cancer surgery and total mononuclear cells isolated by density gradient centrifugation using Histopaque. The percentage of monocytes/macrophages (CD14+) in the total isolated leukocytes was calculated by flow cytometry using fluorochrome-labeled monoclonal antibodies (APC-CD14 and PerCP-CD3). CD14+ population was separated from total mononuclear cells using an immuno-magnetic bead separation technique. CD14+ cells were seeded overnight in appropriate culture media at 37°C in a humidified CO2 incubator and conditioned media were collected and cytokines profiled. To test whether cytokines secreted by CD14+ enhances motility and invasion of SUM149 cells, we used wound healing and invasion assays.

Results: Our results revealed that there was an increase in the number of infiltrated macrophages (CD14+ cells) in IBC tumor tissues and axillary tributaries drained blood as compared to non-IBC. Cytokine profiling of IBC-CD14+ cells revealed a significant increase in tumor necrosis factor-alpha (TNF-α); monocyte chemoattractant protein-1 (MCP-1) also known as CC-chemokine ligand 2 (CCL-2); interleukin-10 (IL-10) and interleukin-8 (IL-8) as compared to non-IBC CD14+ cells. TNF-α, IL-8 and IL-10 cytokines significantly increased motility and invasion of SUM149 cells.

Conclusion: IBC cancer tissues are characterized by high infiltration of monocytes/macrophages that secrete cytokines that may contribute to dissemination of IBC cells.

Citation Format: Mona Mostafa Mohamed, Eslam A. El-Ghonaimy, Mohamed A. Nouh, Robert J. Schneider, Bonnie F. Sloane, Mohamed El-Shinawi. Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B40.
Abstract B55: Chemoprevention of obesity- and diabetes-related liver carcinogenesis by branched-chain amino acids and acyclic retinoid
Authors: Shimizu Masahito , Moriwaki Hisataka
Published: August 2014

Obesity and diabetes-related metabolic abnormalities, including insulin resistance, alterations in the IGF/IGF-1R axis, state of chronic inflammation, and induction of oxidative stress increase the risk of hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis, which is frequently associated with obesity and diabetes, can progress to cirrhosis and HCC. On the other hand, these findings indicate that the metabolic disorders caused by obesity and diabetes might be effective targets to prevent HCC development. For instance, branched-chain amino acids (BCAA), which improve protein energy malnutrition in patients with liver cirrhosis, improve insulin resistance and reduce the risk of HCC in obese cirrhotic patients. In an experimental study, BCAA supplementation inhibits the development of liver tumors in obese and diabetic mice by inhibiting the activation of IGF/IGF-1R axis, improving insulin resistance, reducing serum leptin levels, and attenuating hepatic steatosis and fibrosis. BCAA supplementation also inhibits the early phase of obesity-related liver tumorigenesis by attenuating chronic inflammation in both the liver and white adipose tissue. In addition, acyclic retinoid (ACR), which is a promising agent for the chemoprevention of HCC, prevents liver tumorigenesis in obese and diabetic mice by inhibiting the activation of Ras and phosphorylation of ERK and RXRα proteins. ACR administration also inhibits this tumorigenesis via improvement of insulin sensitivity and attenuation of the chronic inflammation induced by excessive fatty deposits. These findings suggest that pharmaceutical and nutraceutical approaches for targeting and restoring metabolic disorders, especially insulin resistance and chronic inflammation, may be an effective strategy for preventing the development of obesity- and diabetes-related HCC. We indicate that BCAA and ACR are considered some of the most promising agents for achieving this purpose.

Citation Format: Masahito Shimizu, Hisataka Moriwaki. Chemoprevention of obesity- and diabetes-related liver carcinogenesis by branched-chain amino acids and acyclic retinoid. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B55.
Abstract B59: Epithelial-mesenchymal interaction in cancer as potential target for anticancer therapy
Authors: Szabo Pavol , Smetana Karel , Dvorankova Barbora , Kodet Ondrej , Strnad Hynek , Kolar Michal
Published: August 2014

Many results demonstrate that cancer cells need for their growth and spread through organism a specific microenvironment'the tumor stroma. Carcinomas represent highly complex tissue composed from cancer cells and stroma including fibroblasts producing extracellular matrix and bioactive substances, inflammatory cells and blood vessels. We focused our research on most abundant cell component of cancer stroma on cancer-associated fibroblasts. We isolated stromal fibroblasts from tumours originated from the squamous epithelium such as basal and squamous cell carcinoma, melanoma and skin metastasis of breast cancer. We expanded them and in vitro evaluated their biological effect on normal keratinocytes and breast cancer keratinocytes (EMG-3). The results were compared with control experiments using normal human dermal fibroblasts, 3T3 mouse fibroblasts, and 3T3 fibroblasts influenced by the fibroblasts prepared from the basal cell carcinoma. Our results demonstrated that expression of luminal marker keratin 8 was influenced only by cancer associated fibroblasts prepared from any tested tumors. In contrast, all tested types of fibroblasts showed a strong stimulatory effect on the expression of basal/myoepithelial marker keratin 14. Since keratin 14 is a marker of basal myoepithelial cells and keratin 8 is a marker of luminal cells, these double-positive cells can be considered for precursor cells with properties close to stem cells. Their presence in clinical samples indeed signals very poor prognosis in cancer-suffering patiens. In conclusion, our data indicate that cancer associated fibroblasts are able to influence the phenotype of a breast cancer cell line and this effect is based on a tumor type-unspecific mechanism.

Citation Format: Pavol Szabo, Karel Smetana, Jr., Barbora Dvorankova, Ondrej Kodet, Hynek Strnad, Michal Kolar. Epithelial-mesenchymal interaction in cancer as potential target for anticancer therapy. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B59.