Nathan Ellis - US grants

proteomics; helicase; DNA; biomedical resource;

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) represents a serious public health problem that affects African Americans (AA) disproportionately. Both incidence rates and morbidity and mortality is higher in AAs than in other Americans. Family history, that is, genetic risk, remains one of the most important factors in recommendations for screening. Recent genome-wide association studies have identified 10 independent regions harboring genetic risk factors in CRC in populations of European ancestry. Using single-nucleotide polymorphisms (SNPs) from these studies, we have obtained evidence of association in four of these regions in AA CRC. Our goal is to identify the functional genetic risk factors, quantify their effects, and determine their functions. To pursue this goal, we propose four Aims. (1) We will validate previously identified candidate CRC-associated regions in AA CRC cases and controls. We will use Sequenom genotyping and tag SNPs from these regions to identify SNPs associated with CRC in AAs. We will use 100 ancestry informative markers to perform a structured logistic regression analysis that takes into account possible population stratification based on ancestry. Because AAs are more diverse than European Americans, these studies will very likely lead to better localization of the genetic risk factors. (2) We will use Next Generation Sequencing (NGS) technology to conduct a resequencing analysis of each candidate genetic region in 96 (48 CRC and 48 controls) persons with CRC. The region that we resequence will be guided by the genes and linkage disequilibrium observed in each CRC-associated region. (3) We will better quantify the effects of the genetic risk factors in each region. We will perform a bioinformatics analysis to identify putative functional variants. These functional candidates along with our novel genetic variants in CRC-associated regions will be genotyped in up to 2000 AA cases and 2000 AA control. We will perform structured logistic regression analysis to obtain the strongest genotype relative risks in each CRC-associated regions. (4) The foregoing analyses will provide a short-list of candidate genetic risk factors for each CRC-associated region. We will begin to characterize the molecular basis of CRC risk for each region by functional assays designed based on the putative mechanism caused by the genetic variants (regulatory or enzymatic). Molecular assays (for example, luciferase reporter assays) will be designed to determine the functional impact of candidate genetic risk factors. PUBLIC HEALTH RELEVANCE: Colorectal cancer affects African Americans disproportionately relative to other Americans. The discovery of genetic risk factors associated with colorectal cancer predisposition is tantamount to a fundamental understanding of the etiology of this significant health problem and to recommendations for cancer screening. We have found several candidate regions that contain genetic risk factors in African Americans. These and other regions have been implicated in colorectal cancer risk by validated genetic associations. We will determine the functional genetic variants that increase risk by genetic and functional studies. These studies will have important implications for early detection and possibly therapeutic intervention among African Americans.

The Genomic and Molecular Engineering (GME) Core of the DDRCC provides cutting-edge technologies for the analysis and manipulation of the genetic material. These new technologies empower DDRCC members to pinpoint and characterize genetic factors that influence the development and course of digestive diseases and to dissect gene functions In important pathways relevant to digestive diseases. The GME is a new Core, restructured from the former Molecular Biology and Biochemistry Core. The GME Core divided into two components. The Genotype Analysis component offers sen/ices relating to the genetic analysis of patient samples. The services of the Genotype Analysis component Include (1) customized single nucleotide polymorphism (SNP) genotyping based on the Sequenom Massanray genotyping platfonn, (2) standard SNP genotyping panels for high-Interest genes, (3) ultra-high throughput DNA sequence analysis for genotyping, (4) other genotype analysis methods (such as TaqMan), (5) DNA preparation, and (6) statistical genetics support. The Genetic Engineering component offers services relating to the manipulation of genes in cellular and organismal model systems. The services ofthe Genetic Engineering component include (1) somatic cell genetic manipulation of genes using homologous recombination to knock in or knock out mutations, (2) recombineering technologies to manipulate large DNA segments in bacterial artificial chromosomes, (3) the construction of gene expression constructs using lentiviral vectors for ectopic expression or silencing of genes, (4) support for realtime PCR, and (5) support for the Odyssey image analysis system. The GME Core supports members for genotype analysis and genetic engineering experiments by supporting labor cost, providing discounts for reagents, and training members in new technologies. The Administrative Directors of the GME Core, Drs. Nathan Ellis and David Boone, oversee the operations of the respective components. Directors are responsible for ensuring proper scientific direction and efficient use of services and facilities of the component resources. Usage of the GME Core rapidly increased from start-up because of substantial cost savings, relevance, and high quality of services and resources, and it is anticipated usage will grow substantially during the next cycle of this Grant. Each of the Components offers training of new and established investigators unfamiliar with the supported experimental approaches. The GME Core has helped to foster multidisciplinary collaborations and promote productive exchanges brought about by sharing of resources

DESCRIPTION (provided by applicant): Homologous recombination (HR) is an essential mechanism that maintains genomic integrity in cells by repairing double-strand breaks and repairing damaged replication forks. The RAD51 recombinase catalyzes the central step in the HR pathway, forming joint molecules through homology-dependent strand invasion. The BLM helicase, which is the gene mutated in Bloom's syndrome (BS), regulates RAD51. BLM can promote RAD51 function, for example by generating substrate for RAD51 binding, or it can inhibit RAD51 function by unwinding the products of RAD51 catalysis, which prevents the accumulation of toxic recombination intermediates. Thus, through its regulation of RAD51, BLM has both pro- and anti-recombinogenic functions in HR. We have shown that BLM is modified by small ubiquitin-related modifiers (SUMOs) and that BLM SUMOylation regulates BLM's functions at stalled replication forks, stimulating HR repair. In addition, RAD51 is SUMO-binding, and BLM SUMOylation stimulates BLM's interaction with RAD51 in vitro. Our findings support a model in which BLM SUMOylation controls a switch between BLM's anti- recombinogenic and pro-recombinogenic functions. To test this hypothesis, we have four specific aims: (1) characterization of the role of BLM SUMOylation in stabilization of replication forks; (2) characterization of the role of RAD51 SUMO binding in replication fork stability and HR repair; (3) analysis of the effects of BLM SUMOylation on BLM's biochemical activities and RAD51 function; and (4) analysis of the effects of BLM SUMOylation and RAD51 SUMO binding in genomic integrity. Our studies will elucidate the molecular mechanisms that regulate HR at damaged replication forks, providing insights into the dynamic functions of BLM and RAD51 in HR repair and leading to a deeper understanding of how these functions are regulated by the SUMO pathway. Because HR repair mechanisms are often dysregulated in cancer cells, our work will lead to a better understanding of genomic instability in cancer and will facilitate the exploitation of this instability in cancer treatments.

The Genomic and Molecular Engineering (GME) Core of the DDRCC provides cutting-edge technologies for the analysis and manipulation of the genetic material. These new technologies empower DDRCC members to pinpoint and characterize genetic factors that influence the development and course of digestive diseases and to dissect gene functions In important pathways relevant to digestive diseases. The GME is a new Core, restructured from the former Molecular Biology and Biochemistry Core. The GME Core divided into two components. The Genotype Analysis component offers sen/ices relating to the genetic analysis of patient samples. The services of the Genotype Analysis component Include (1) customized single nucleotide polymorphism (SNP) genotyping based on the Sequenom Massanray genotyping platfonn, (2) standard SNP genotyping panels for high-Interest genes, (3) ultra-high throughput DNA sequence analysis for genotyping, (4) other genotype analysis methods (such as TaqMan), (5) DNA preparation, and (6) statistical genetics support. The Genetic Engineering component offers services relating to the manipulation of genes in cellular and organismal model systems. The services ofthe Genetic Engineering component include (1) somatic cell genetic manipulation of genes using homologous recombination to knock in or knock out mutations, (2) recombineering technologies to manipulate large DNA segments in bacterial artificial chromosomes, (3) the construction of gene expression constructs using lentiviral vectors for ectopic expression or silencing of genes, (4) support for realtime PCR, and (5) support for the Odyssey image analysis system. The GME Core supports members for genotype analysis and genetic engineering experiments by supporting labor cost, providing discounts for reagents, and training members in new technologies. The Administrative Directors of the GME Core, Drs. Nathan Ellis and David Boone, oversee the operations of the respective components. Directors are responsible for ensuring proper scientific direction and efficient use of services and facilities of the component resources. Usage of the GME Core rapidly increased from start-up because of substantial cost savings, relevance, and high quality of services and resources, and it is anticipated usage will grow substantially during the next cycle of this Grant. Each of the Components offers training of new and established investigators unfamiliar with the supported experimental approaches. The GME Core has helped to foster multidisciplinary collaborations and promote productive exchanges brought about by sharing of resources.

Project Summary / Abstract: Cancer Biology Program (CBP) The goal of the Cancer Biology Program (CBP) is to identify etiologic mechanisms underlying cancer development and progression. As the main basic science of cancer platform for the University of Arizona Cancer Center, the Cancer Biology Program seeks to advance fundamental knowledge of the complex biological networks that are deranged in cancer and to characterize interactions between these complex biological networks and the environment that promote carcinogenesis and tumor progression. Translation of current findings and development of novel approaches to cancer prevention and treatment is facilitated through inter-programmatic collaborations and Cancer Center support. The program is organized into three major themes, including Genomic Instability and Epigenetic Control of Gene Expression, Signaling Networks in Carcinogenesis and Tumor Progression, and Invasion and Metastasis, with four aims: (i) to investigate mechanisms of cancer initiation and progression and to characterize cellular mechanisms that control cancer metastasis, (ii) to identify networks and regulatory pathways as potential markers or targets in prevention and treatment, (iii) to promote intra- and inter-programmatic collaborations to enhance translational research along the continuum from pre-clinical mouse models and human tissue correlates to clinical trials, and (iv) to foster research directions of particular relevance to individuals in Arizona and the Southwest. The Cancer Biology Program achieves these aims through establishment of working groups, conferences and seminars, and development of inter-disciplinary scientific teams; faculty recruitment, membership development and mentoring, and the use of developmental funds to spur targeted research; and, guidance and support of the shared resources. The CBP has 49 Members representing 21 different departments at the University of Arizona. CBP Members have published 390 cancer-relevant manuscripts, of which 30% were intra- programmatic and 36% were inter-programmatic. As of September 1, 2015, the CBP Program secured $11.8M total annual grant dollars with $2.5M from the NCI and $8.3M in other peer-reviewed funding. The CBP has regional impact through its members' basic research into the carcinogenic mechanisms of arsenicals that are pervasive environmental carcinogens in the Southwest. Finally, the Program is invested in using modern, high throughput genomic platforms to develop, enhance, and implement precision medicine. The expanding representation of Hispanic Americans in the State's population impels increased research into cancer health disparities in this population.

The incidence of colorectal cancer (CRC) is higher in African Americans (AAs) compared with US whites (NHWs) overall, but the disparity is more extreme in persons with earlier age of onset. The median age of onset is 65 in AAs compared to 72 in NHWs, and early-onset CRC is more than twice as frequent in AAs than in NHWs. In addition, epidemiologic evidence suggests that younger age CRCs may have a more rapid progression through the steps of carcinogenesis. Yet, younger age CRC is not well characterized in any population, and we have no explanation for its higher incidence in AAs. In a recent genomic analysis of a series of Chicago African American CRC cases, we found an excess of CRCs lacking mutation in the tumor suppressor gene APC (APC mutation-negative CRCs). Microsatellite stable APC mutation-negative CRCs were associated with younger age of diagnosis, fewer numbers of somatic mutations, and microsatellite and chromosome stability. Importantly, we discovered that APC mutation-negative CRCs exhibited a novel methylation profile characterized by increased levels of methylation in key cancer driver genes including those in stem-cell maintenance and the WNT signaling pathway. Based on our preliminary data, we hypothesize that epigenetic dysregulation in APC mutation-negative CRCs drives specific DNA methylation changes and gene regulatory networks that maintain a stem-like cancer phenotype. The overall goal of the project is to characterize the molecular mechanisms that drive this novel subtype of CRCs. We have three aims. Aim 1. Identify and characterize significant differentially methylated regions in AA CRC. Hypothesis: Tumor- specific differentially methylated regions are associated with carcinogenesis in APC mutation-negative CRCs. APC mutation-negative CRCs will be associated with earlier age of onset and with distinct molecular and clinicopathological features. Aim 2. Identify and characterize differentially expressed genes and regulatory networks in AA CRCs. Hypothesis: Specific regulatory networks that maintain a stem-like cancer phenotype are associated with APC mutation-negative CRCs. Aim 3. Determine driver gene dependencies of AA CRCs in organoid cancer models. Hypothesis: Suppression of specific WNT signaling factors and epigenetic modulators will induce increased epithelial differentiation in APC mutation-negative organoids in comparison to APC mutation-positive organoids. The proposed studies will provide essential knowledge of the DNA methylation and gene expression changes underlying AA CRCs and will characterize cancer cell responses to chemical challenge. The new knowledge will provide translatable information, including diagnostic and predictive biomarkers and precision-medicine approaches, that could be used to treat a novel subtype of CRC that occurs in excess in AAs and is associated with earlier age of onset.