• NF-kB2 mutation targets survival, proliferation and differentiation pathways in the pathogenesis of plasma cell tumors

      McCarthy, Brian A.; Yang, Liqun; Ding, Jane; Ren, MingQiang; King, William; ElSalanty, Mohammed; Zakhary, Ibrahim; Sharawy, Mohamed; Cui, Hongjuan; Ding, Han-Fei; et al. (2012-05-29)
      Background: Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines.
    • Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease.

      Mojica, Wilfrido; Hawthorn, Lesleyann; GHSU Cancer Center (2010-02-18)
      BACKGROUND: Studies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing. RESULTS: We present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells. CONCLUSIONS: The findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.
    • Novel Somatic Mutations to PI3K Pathway Genes in Metastatic Melanoma

      Shull, Austin Y.; Latham-Schwark, Alicia; Ramasamy, Poornema; Leskoske, Kristin; Oroian, Dora; Birtwistle, Marc R.; Buckhaults, Phillip J.; GHSU Cancer Center (2012-08-17)
      Background: BRAFV600 inhibitors have offered a new gateway for better treatment of metastatic melanoma. However, the overall efficacy of BRAFV600 inhibitors has been lower than expected in clinical trials, and many patients have shown resistance to the drugâ s effect. We hypothesized that somatic mutations in the Phosphoinositide 3-Kinase (PI3K) pathway, which promotes proliferation and survival, may coincide with BRAFV600 mutations and contribute to chemotherapeutic resistance.
    • De novo transcriptome sequencing in a songbird, the dark-eyed junco (Junco hyemalis): genomic tools for an ecological model system

      Peterson, Mark P; Whittaker, Danielle J; Ambreth, Shruthi; Sureshchandra, Suhas; Buechlein, Aaron; Podicheti, Ram; Choi, Jeong-Hyeon; Lai, Zhao; Mockatis, Keithanne; Colbourne, John K.; et al. (2012-07-9)
      Background: Though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. In contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. It is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. We used GS-FLX Titanium Sequencing (Roche/454-Sequencing) of two normalized libraries of pooled RNA samples to characterize a transcriptome of the dark-eyed junco (Junco hyemalis), a North American sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior.
    • Ploidy status and copy number aberrations in primary glioblastomas defined by integrated analysis of allelic ratios, signal ratios and loss of heterozygosity using 500K SNP Mapping Arrays.

      Gardina, Paul J; Lo, Ken C; Lee, Walter; Cowell, John K.; Turpaz, Yaron; GHSU Cancer Center (2008-10-30)
      BACKGROUND: Genomic hybridization platforms, including BAC-CGH and genotyping arrays, have been used to estimate chromosome copy number (CN) in tumor samples by detecting the relative strength of genomic signal. The methods rely on the assumption that the predominant chromosomal background of the samples is diploid, an assumption that is frequently incorrect for tumor samples. In addition to generally greater resolution, an advantage of genotyping arrays over CGH arrays is the ability to detect signals from individual alleles, allowing estimation of loss-of-heterozygosity (LOH) and allelic ratios to enhance the interpretation of copy number alterations. Copy number events associated with LOH potentially have the same genetic consequences as deletions. RESULTS: We have utilized allelic ratios to detect patterns that are indicative of higher ploidy levels. An integrated analysis using allelic ratios, total signal and LOH indicates that many or most of the chromosomes from 24 glioblastoma tumors are in fact aneuploid. Some putative whole-chromosome losses actually represent trisomy, and many apparent sub-chromosomal losses are in fact relative losses against a triploid or tetraploid background. CONCLUSION: These results suggest a re-interpretation of previous findings based only on total signal ratios. One interesting observation is that many single or multiple-copy deletions occur at common putative tumor suppressor sites subsequent to chromosomal duplication; these losses do not necessarily result in LOH, but nonetheless occur in conspicuous patterns. The 500 K Mapping array was also capable of detecting many sub-mega base losses and gains that were overlooked by CGH-BAC arrays, and was superior to CGH-BAC arrays in resolving regions of complex CN variation.
    • Protection Against Colonic Inflammation and Colon Cancer by Commensal Bacterial Metabolites: An Obligatory Role for the Short- Chain Fatty Acid Transporter Slc5a8

      Gurav, Ashish; Georgia Cancer Center (2014-11)
      Dietary fiber consumption has long been known to protect against inflammatory bowel diseases and colorectal carcinogenesis. In mammals, large intestinal microorganisms ferment dietary fiber to generate energy, while releasing short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate. Interestingly, SCFAs are also known to protect against intestinal inflammation and colorectal carcinogenesis, although the molecular mechanisms behind these actions are still being investigated. For most of their biological effects, SCFAs must be transported from lumen into the intestinal tissue, where they activate multiple biological processes. We and others have reported Slc5a8 as a high affinity transport mechanism for SCFAs, which would remain fully functional, even when SCFA concentration drops to sub-millimolar range, whereas other transport mechanisms are rendered inefficient. The aim of the current study was to test protective role of Slc5a8 against intestinal inflammation and colorectal carcinogenesis during suboptimal intake of dietary fiber. We observed that Slc5a8 is obligatory for HDAC-inhibition in colonic epithelium and intestinal barrier function, only when the animals were fed a dietary fiber-free diet (FF diet), and not when the animals were fed diet containing optimal amounts of fibers (FC Diet). Compared to WT, Slc5a8-/- animals demonstrated higher susceptibility to AOMDSS- mediated intestinal inflammation and colorectal carcinogenesis under FF dietary conditions, but not under FC dietary conditions. At molecular level, we found that butyrate and propionate could induce potent immunosuppressive enzymes Indoleamine 2,3-dioxygenase and Aldehyde Dehydrogenase 1A2 in dendritic cells obtained from WT animals, but not from Slc5a8-/- animals. Butyrate, transported via Slc5a8 enabled DCs to suppress conversion of naïve T cells to interferon-γ secreting pro-inflammatory T cells and Slc5a8-/- animals harbored higher proportion of interferon-γ+ CD4+ T cells in vivo. Taken together, our data provide crucial evidence for critical role of Slc5a8 mediating protective effects of dietary fiber metabolites, SCFAs in protecting against intestinal inflammation and colorectal carcinogenesis.
    • Regulation and Function of the Major Stress-Induced HSP70 Molecular Chaperone in vivo: Analysis of Mice with Targeted Gene Disruption of the HSP70.1 or HSP70A1

      Huang, Lei; Georgia Cancer Center (6/3/2002)
      (First Paragraph) The cellular response to stress, including exposure to environmental (UV radiation, heat shock, heavy metals), pathological (infection, fever, inflammation, malignancy, ischemia) or physiological (growth factor, hormonal stimulation, tissue development) stimuli is represented at the molecular level by synthesis of groups of protein named heat shock proteins [hsp(s)] (Benjamin 1998; Feder and others 1992; Jolly and Morimoto 2000; Li and Mivechi 1986; Lindquist 1986; Smith 1998). The presence of hsp(s) protect host cells from the damage caused by thermal stress, and after induction of hsp expression, cells are protected well from higher temperatures than they can normally tolerate. This phenomenon is defined as themiotoleranee (Gemer 1975; Li and Mivechi 1986). The protective role of hsp(s) is attributed to several functional properties, including active participation in maintaining proteins in their native correctly folded states, promoting degradation and refolding of misfolded proteins, and minimizing aggregation and incorrect interactions between proteins (Agashe and Hartl 2000; Gething and Sambrook 1992). In addition, hsp(s) can function in cellular protection by modulating the engagement and progression of apoptosis induced by a variety of stress stimuli (Beere and Green 2001). Besides the recognition of the cytoprotective function of hsp(s) under stress conditions, widespread clinical interests exist in their chaperone function during a range of human pathologies, including neurodegenerative conditions, such as amyloidosis, prion disease, and Alzheimer's disease, and cardiovascular diseases, such as myocardial ischemia, cardiac hypertrophy, stroke, and blood vessel injury (Benjamin 1998; Planas and others 1997; Smith 1998).
    • T Cell Immune Response in Persistent Infection of Lymphocytic Choriomeningitis Virus (LCMV)

      Ou, Rong; Georgia Cancer Center (2004-07)
      The m urine LCMV system provides a ciassic model to study the mechanism of immunological tolerance, an efficient strategy used by virus to establish a persistent infection by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset o f infection drives responding cells into functional unresposiveness (anergy) that can, be followed by their physical elimination. In this study, the downregulation o f the virus-specific CD8^-T-ceil response was studied during a persistent infection o f adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense, or perforin-, Fas/FasL-, or TN FR l-m ediated cytolysis in regulating T-cell homeostasis. Since LCMV infects a broad range o f host tissues, the functional properties o f virus-specific CD8'^ T cells in different tissues during LCMV infection were also evaluated. Infection of mice deficient in receptor for type I (IFN-a/p), type II (IFN-y), or both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-celi exhaustion, revealed a critical role for IFN -a/p in restricting LCMV spread at the onset o f infection while IFN-y has impact on effector cells. The production o f IF N -a/p and/or IFN-y critically regulates the virus-host balance during the acute phase o f infection, such that a high viral burden drives responding cells into different programs o f exhaustion. Infection o f mice deficient in perferin, FasL or TNFRl with the Docile or Aggressive strains of LCMV revealed comparable kinetics of expansion and functional inactivation o f virusspecific C D ^ T cells in the early phase o f Infection in C57BL/6 controls. However, the data underscore a critical role for these molecules in the persistence o f the virus-specific CD8"‘-T-ceil population once it has become anergic. Study o f the functional properties of virus-specific CD8'^ T cells in different tissues during LCMV infections showed that a centra! role for the viral load in lymphoid tissue in the induction and maintenance of clonal exhaustion. The data strongly suggest that CD8^ T ceils may be differentially regulated in the environments o f lymphoid versus nonlymphoid tissues, and the pattern of T cell exhaustion observed with mice is likely a common feature o f the immune response during chronic infections in humans.
    • Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing

      Lee, Eun-Joon; Pei, Lirong; Srivastava, Gyan; Joshi, Trupti; Kushwaha, Garima; Choi, Jeong-Hyeon; Robertson, Keith D.; Wang, Xinguo; Colbourne, John K.; Zhang, Lu; et al. (2011-10-23)
      We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21â 408 CGIs and more than 15â 946 transcriptional regulatory regions. Of the CpGs analyzed, 77â 84% fell on or near capture probe sequences; 69â 75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5â ²-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.
    • Targeting HSP90 for cancer therapy

      Mahalingam, D; Swords, R; Carew, Jennifer S; Nawrocki, S T; Bhalla, Kapil N.; Giles, F J; GHSU Cancer Center (2009-04-28)
      Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis.
    • The temporal and spatial expression pattern of the LGI1 epilepsy predisposition gene during mouse embryonic cranial development

      Silva, Jeane; Wang, Guanghu; Cowell, John K.; GHSU Cancer Center; Department of Neurology; Institute of Molecular Medicine and Genetics (2011-05-13)
      Background: Mutations in the LGI1 gene predispose to a rare, hereditary form of temporal epilepsy. Currently, little is known about the temporal and spatial expression pattern of Lgi1 during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses GFP under the control of Lgi1 cis-regulatory elements.
    • Toward integrative cancer immunotherapy: targeting the tumor microenvironment

      Emens, Leisha A; Silverstein, Samuel C; Khleif, Samir; Marincola, Francesco M; Galon, Jérôme; GHSU Cancer Center (2012-04-10)
      The development of cancer has historically been attributed to genomic alterations of normal host cells. Accordingly, the aim of most traditional cancer therapies has been to destroy the transformed cells themselves. There is now widespread appreciation that the progressive growth and metastatic spread of cancer cells requires the cooperation of normal host cells (endothelial cells, fibroblasts, other mesenchymal cells, and immune cells), both local to, and at sites distant from, the site at which malignant transformation occurs. It is the balance of these cellular interactions that both determines the natural history of the cancer, and influences its response to therapy. This active tumor-host dynamic has stimulated interest in the tumor microenvironment as a key target for both cancer diagnosis and therapy. Recent data has demonstrated both that the presence of CD8+ T cells within a tumor is associated with a good prognosis, and that the eradication of all malignantly transformed cells within a tumor requires that the intra-tumoral concentration of cytolytically active CD8+ effector T cells remain above a critical concentration until every tumor cell has been killed. These findings have stimulated two initiatives in the field of cancer immunotherapy that focus on the tumor microenvironment. The first is the development of the immune score as part of the routine diagnostic and prognostic evaluation of human cancers, and the second is the development of combinatorial immune-based therapies that reduce tumor-associated immune suppression to unleash pre-existing or therapeutically-induced tumor immunity. In support of these efforts, the Society for the Immunotherapy of Cancer (SITC) is sponsoring a workshop entitled "Focus on the Target: The Tumor Microenvironment" to be held October 24-25, 2012 in Bethesda, Maryland. This meeting should support development of the immune score, and result in a position paper highlighting opportunities for the development of integrative cancer immunotherapies that sculpt the tumor microenvironment to promote definitive tumor rejection.
    • Towards Curative Cancer Immunotherapy: Overcoming Posttherapy Tumor Escape

      Zhou, Gang; Levitsky, Hyam; GHSU Cancer Center; Department of Medicine (2012-05-31)
      The past decade has witnessed the evolvement of cancer immunotherapy as an increasingly effective therapeutic modality, evidenced by the approval of two immune-based products by the FDA, that is, the cancer vaccine Provenge (sipuleucel-T) for prostate cancer and the antagonist antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) ipilimumab for advanced melanoma. In addition, the clinical evaluations of a variety of promising immunotherapy drugs are well under way. Benefiting from more efficacious immunotherapeutic agents and treatment strategies, a number of recent clinical studies have achieved unprecedented therapeutic outcomes in some patients with certain types of cancers. Despite these advances, however, the efficacy of most cancer immunotherapies currently under clinical development has been modest. A recurring scenario is that therapeutic maneuvers initially led to measurable antitumor immune responses in cancer patients but ultimately failed to improve patient outcomes. It is increasingly recognized that tumor cells can antagonize therapy-induced immune attacks through a variety of counterregulation mechanisms, which represent a fundamental barrier to the success of cancer immunotherapy. Herein we summarize the findings from some recent preclinical and clinical studies, focusing on how tumor cells advance their survival and expansion by hijacking therapy-induced immune effector mechanisms that would otherwise mediate their destruction.