DNA damage in obesity: Initiator, promoter and predictor of cancer

Epidemiological evidence linking obesity with increased risk of cancer is steadily growing, although the causative aspects underpinning this association are only partially understood. Obesity leads to a physiological imbalance in the regulation of adipose tissue and its normal functioning, resulting in hyperglycaemia, dyslipidaemia and inflammation. These states promote the generation of oxidative stress, which is exacerbated in obesity by a decline in anti-oxidant defence systems. Oxidative stress can have a marked impact on DNA, producing mutagenic lesions that could prove carcinogenic. Here we review the current evidence for genomic instability, sustained DNA damage and accelerated genome ageing in obesity. We explore the notion of genotoxicity, ensuing from systemic oxidative stress, as a key oncogenic factor in obesity. Finally, we advocate for early, pre-malignant assessment of genome integrity and stability to inform surveillance strategies and interventions

Susceptibility of microRNAs 145, 143 and 133b to epigenetic regulation in colorectal cancer cell lines; prediction and functional analysis of putative targets to associated microRNAs

A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Master of Science in Medicine Department of Internal Medicine, University of Witwatersrand, South Africa Johannesburg, 2016Colorectal cancer (CRC) is a significant health burden maintaining its position as the third most diagnosed cancer in men and women worldwide. Despite improvements in treatments for CRC, mortality rates still remain high. Genetic instability and epigenetic deregulation of gene expression are instigators of CRC development, resulting in genotype differences which herald treatment response variability and unpredictability. Over the past decade and a half, microRNAs (miRNA) have emerged as key contributors to the perturbed proteome in cancer cells, including CRC. MiRNAs are small non-coding RNA molecules (consisting of approximately 22 nucleotides) targeted to specific mRNAs through various target recognition mechanisms to repress protein translation or to induce mRNA degradation. Three miRNAs, miR-143, -145 and -133b, are most commonly downregulated in CRC and have been proposed as potential tumour suppressors. Although downregulation of these miRNAs in CRC is to a large extent unexplained, epigenetic silencing has been postulated as a causative regulatory mechanism. Potential epigenetic modulation of miRNA expression, by means of histone acetylation and DNA methylation, was assessed in this study by treating early (SW1116) and late stage (DLD1) CRC cells with the DNA demethylating agent, 5-aza-2’-deoxycytidine (5-Aza-2’C) and the histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), respectively. Subsequently quantifying miRNA expression, using miRNA TaqMan® PCR assays for each of miR-143, -145 and -133b, revealed that while all of these miRNAs are susceptible to DNA demethylation in early and late stage CRC cells, the susceptibility to DNA demethylation is significantly pronounced in the late stage DLD1 cells. Conversely, histone acetylation moderately affected miRNA expression in early stage CRC, but with a marginal effect on the expression of miRNAs in late stage CRC cells. These associations have been argued to correlate with genotypic differences between the microsatellite stable (MSS) SW1116 cell line and the microsatellite instability (MSI) of the DLD1 cells. To further evaluate the role that these miRNAs play in CRC development, this study utilised in silico miRNA target prediction tools to identify potential miRNA gene target lists. Once generated, these were strategically curated and filtered to allow for the election of suitable candidates for functional analysis. This approach yielded three candidates, KRAS, FZD7 and FBXW11/ß-TrCP as the most probable targets for miR-143, -145 and -133b, respectively, further supported by their inverse correlations to the associated miRNA expression in CRC. Proteomic expression of the predicted targets assessed pre- and post- transfection of HET-1A cells with anti-miR™ sequences of the associated miRNA revealed elevated protein expression with differential subcellular protein localization upon miRNA inhibition. Overall this study has provided further understanding of the contribution of epigenetics in regulation of putative tumour suppressor miRNAs in CRC. Additionally, KRAS targeting by miR-143 has been reaffirmed, while FZD7 and FBXW11/ß-TrCP expression analysis after anti-miR-145 and anti-miR-133b transfection, respectively, provides substantial evidence for their role as potential direct miRNA targets.MT201

DNA damage in paediatric obesity: a promoter and predictor of cancer in adulthood

Obesity in children is one of the most serious, global, public health challenges of the 21st century. The accumulation of adipose tissue is associated with a range of metabolic complications including diabetes, cardiovascular disease and dyslipidaemia. Epidemiological evidence links obesity in childhood with developing certain types of cancer later in life. It is postulated that excess adipose tissue and consequent inflammation derived oxidative stress may inflict an accumulation of deleterious DNA mutations and promote genome instability and drive carcinogenesis. Furthermore, a deficiency in micronutrients that are essential for DNA repair may exacerbate this pathological state. This research combined the assessment of anthropometric, inflammatory, micro-nutritional and DNA damage biomarkers via non-invasive techniques. In total, 112 children were recruited from schools and NHS obesity clinics. Anthropometric markers assessed were waist to hip ratio, body fat percentage via bioelectrical impedance, and body mass index standard deviation scores (BMI-SDS). These markers were used to classify participants as obese or nonobese and used for correlational analysis. Inflammation and micronutrient status were determined via C-reactive protein and vitamin D Enzyme Immune Assay (EIA) in saliva. DNA damage assessments include a microscopic assessment of nuclear anomalies via the buccal cytome assay, salivary telomere length via quantitative Polymerase Chain Reaction (qPCR) and urinary 8- hydroxyguanosine (8-OHdG) via EIA. The results from this study indicate obesity to be concurrent with increased inflammation and vitamin D deficiency in this cohort of participants. In addition, obesity was associated with increased oxidative DNA damage (8-OHdG) in urine and DNA damage events in the buccal mucosa. Salivary telomere length was positively correlated with obesity and the total frequency of nuclear anomalies found in buccal epithelial cells. Furthermore, there was a negative correlation between vitamin D and the frequency of nuclear anomalies in the oral cavity. Importantly, odds ratio analysis indicates a high BMI Z-score, waist circumference, body fat percentage, salivary CRP and low salivary vitamin D to be independent risk factors for increased nuclear anomalies in the buccal mucosa. This research is the first to accrue evidence for acquired DNA damage in multiple tissues obtained non-invasively from children with obesity. Our findings instigate that biomonitoring of ‘genome health’ for pre-cancerous molecular and morphological markers in obese patients may inform prioritization and severity of clinical intervention measures to prevent malignancy

Analysis of alterations in the human cancer genome

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Aneuploidy, an abnormal complement of chromosomes, is present in approximately 90% of human malignancies. Despite over 100 years of research, many questions remain regarding the contribution of aneuploidy to the cancer phenotype. In this thesis, we develop computational methods to infer the presence and specific patterns of aneuploidy across thousands of primary cancer tissue specimens. We then combine these inferences with clinical and genomic features of the cancer samples to refine our understanding of both the clinical implications of aneuploidy, and how it evolves in various human cancers. We identified a signature of chromosomal instability from specific genes whose expression was consistently correlated with aneuploidy in several cancer types, and which was predictive of poor clinical outcome multiple cancer types. Current genomic characterization techniques measure somatic alterations in a cancer sample in units of genomes (DNA mass). The meaning of such measurements is highly dependent on the tumors purity and its overall ploidy; they are hence complicated to interpret and compare across samples. Ideally, copy-number should be measured in copies-per-cancer-cell. Such measurements are straightforward to interpret and, for alterations that are fixed in the cancer cell population, are simple integer values. We develop two computational methods to infer tumor purity and malignant cell ploidy directly from allelic analysis of DNA. First we describe HAPSEG, a probabilistic method to interpret bi-allelic marker data in cancer samples in order to produce genome-wide estimates of homologue specific copy-ratios. Second, we describe ABSOLUTE, a method that infers purity, ploidy, and absolute copy-numbers from the estimates produced by HAPSEG. In addition, ABSOLUTE can analyze point mutations to detect subclonal heterogeneity and somatic homozygosity. We used ABSOLUTE to analyze ovarian cancer data and discovered that 54% of somatic point mutations were, in fact, subclonal. In contrast, mutations occurring in key tumor suppressor genes, TP53 and NF1 were predominantly clonal and homozygous. Analysis of absolute allelic copy-number profiles from 3,155 cancer specimens revealed that genome-doubling events are common in human cancer, and likely occur in already aneuploid cells in many cancer types. By correlating genome-doubling status with mutation data, we found that homozygous mutations in NF1 occurred predominantly in non-doubled samples. This finding suggests that genome doubling influences the pathways of tumor progression, with recessive inactivation being less common after genome doubling.by Scott L. Carter.Ph.D

GENOME-WIDE ANALYSIS OF LOSS HETEROZYGOSITY AND DISCOVERY OF NOVEL TUMOR SUPPRESSOR GENES IN GASTRIC CANCER

Ph.DDOCTOR OF PHILOSOPH

Investigating the relationship between miRNA expression and epithelial mesenchymal transition in colorectal cancer

Introduction: Epithelial-mesenchymal transition (EMT) is characterized by the loss of an epithelial phenotype and gain of a mesenchymal phenotype, i.e., migratory and metastatic properties. The EMT process is therefore characterized by a low expression of E-cadherin and high expression of mesenchymal markers (e.g., N-cadherin, snail and vimentin). It is stated that cells which have undergone EMT also gain stem cell features. Therefore, both EMT and stem cell phenotypes have been implicated in carcinogenesis and metastasis of tumour cells. Furthermore, EMT is regulated by small non-coding molecules (miRNAs) that either function as tumour suppressors or oncogenes (oncomirs). Tumour suppressor miRNAs reverse EMT while oncomirs activate it. Therefore, investigating the relationship between miRNAs and EMT is important in addressing metastasis of colorectal cancers (CRC). Aims and Objectives: The aim of the study was to determine the association between miRNA (miRNA-21 and miRNA-34a) expression levels and EMT in CRC. In addition, this investigation aimed to correlate miRNA and EMT data with clinicopathologic features of the study cohort. Methodology: A total of 100 CRC (including 8 known HNPCC cases) Formalin Fixed Paraffin Embedded (FFPE) tissue blocks and their corresponding H&E slides were collected from the archives of the Division of Anatomical Pathology at the University of Cape Town. Subsequently, the FFPE tissue blocks were sectioned at 3μm and IHC analysis of 4 EMT markers (E-cadherin, N-cadherin, snail-1 and vimentin) and 1 stem cell marker (CD44V6) was performed. The stains were then evaluated and scored by a pathologist. The IHC data were then correlated with clinicopathologic features. Furthermore, 59 cases (FFPE tissues and corresponding H&E slides) which included the 8 HNPCCs were randomly selected for miRNA analysis. The H&Es were examined by a pathologist to demarcate normal and tumour regions. RNA was then extracted from 59 tumours and 12 normal tissues using a High Pure FFPET Isolation Kit (Roche). Subsequently, cDNA was synthesized and qRT-PCR was performed to determine the expression levels of miRNA-21 and miRNA-34a. MiRNA-21 and miRNA-34a expression levels were ascertained using the relative quantification method. Moreover, the clinical significance of the two miRNAs was evaluated in relation to MSI status. Therefore, IHC analysis of MLH1, MSH2 and MSH6 mismatch repair proteins was performed on the Ventana platform. Statistical analysis was performed using Fisher's and Pearson's Chi Square tests in Stata 12 to correlate EMT and clinicopathologic data. Additionally, the Mann-Whitney non-parametric test in GraphPad prism 6 was used to determine miRNA-21 and miRNA-34a expression in relation to EMT and MSI data. Results: Our results showed low expression of E-cadherin in 77% of cases. In addition, there was decreased expression of N-cadherin and vimentin in 98% whilst snail-1 expression was decreased in 65% of the cases. Low expression of CD44v6 was also seen in 78% of the cases. There was no correlation between EMT/stem cell markers and clinicopathologic data. Furthermore, increased miRNA-21 expression was significantly associated with grade, lymph node metastasis and age of patients. There was a significant correlation between high miRNA- 21 expression and down-regulated snail-1 and N-cadherin expression. MiRNA-34a expression was not associated with any of the clinicopathologic features. In addition, high miRNA-34a expression was linked with low expression of snail-1 and CD44v6. Increased miRNA-21 expression was related with MSS tumours, whereas there was no relationship between miRNA- 34a and MSI status. Conclusion: Our investigation shows that there is an inverse association between miRNA (miRNA-21 and miRNA-34a) expression and two EMT (N-cadherin and snail-1) markers in our colorectal cancer cohort. Our data also show that both miRNA-21 and miRNA-34a cannot be used as biomarkers to determine progression of the cancer. Contrary to previous studies, our findings indicate that miRNA-21 does not activate EMT in this CRC cohort. However, similar to other studies our results confirm that miRNA-34a may be repressing snail-1 expression, thereby inhibiting EMT in the cancer

QTL mapping of Apc modifiers in an ApcMin/+ mouse model of spontaneous and irradiation-induced intestinal adenomas.

PhDBACKGROUND: Radiation exposure to the abdominal region causes intestinal toxicity and is also capable of inducing colorectal cancers (CRC). Genotype-phenotype studies provide some evidence explaining the variation in familial adenomatous polyposis (FAP) patients caused by modifiers of adenomatous polyposis coli (APC). This study aims to extend our understanding of irradiation-induced modifiers of ApcMin/+ mice and CRC. METHODS: By using a pre-existing backcross between recombinant inbred line of ApcMin/+ mice to the irradiation sensitive inbred BALB/c mouse, we obtained panels of 2Gy-irradiated and sham-irradiated N2 ApcMin/+ mice for genotyping with a genome-wide panel of microsatellites markers. Using the number of adenomas in different intestinal segments to represent polyp multiplicity, we carried out a genome wide quantitative trait loci (QTL) scan followed by statistical epistasis modelling and bioinformatics analysis. RESULTS: We identified five significant QTLs responsible for radiation induced tumour multiplicity in the upper small intestine defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p=0.00001, 6.2, p=0.00001) and two regions within chromosome 16 (LOD 4.1, p=4x10-5 and 4.8, p=0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p,0.005). Two significant QTLs were detected in the 2large intestine on chromosome 2 and 7 (LOD 2.7, p=1.2x10-3 and 2.2, p=1.2x10-3, 12 respectively). Using statistical epistasis modelling and logical selection of target genes though in silico sequence based on BALB/c specific non-synonymous polymorphisms which are predicted deleterious we selected target genes and further eliminated genes by sequencing and mRNA expression. CONCLUSIONS: Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in ApcMin/+ mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Bub1r, and Casc5), Wnt pathway (Tiam1, Rac1), DNA repair (Recc1 and Prkdc) and inflammation (Duox2, Itgb2l and Cxcl5).Cancer Research U

Profiling of DNA copy number in sarcomas by array comparative genomic hybridisation and identification of candidate cancer genes

Cancer is a disease resulting from an accumulation of acquired genetic mutations. The consequence is an uncontrolled growth of cells and disruption of normal control mechanisms. Complex chromosomal aberrations such as amplification and deletion of DNA copy number can lead to the activation and deregulation of oncogenes and tumour suppressor genes respectively, leading to uncontrolled cell growth and giving rise to tumours. In sarcomas, rare malignant tumours of mesenchymal origin, aberrations such as amplifications and losses of DNA are frequently seen. In this project, a panel of 13 leiomyosarcomas (LMS) and seven gastrointestinal stromal tumours (GIST) were analysed by array comparative genomic hybridisation (array CGH). This technique makes it possible to map DNA copy number changes and identify chromosomal regions containing “target genes” responsible for tumour development and/or progression. The most frequent aberrations observed in GISTs were losses of the whole or parts of chromosome 22, seen in all tumours with a minimal recurrent region in 22q12.2-q13.31, as well as chromosome 14, 1p36.32-p13.1, 13q12.11-q33.2, 15q13.2-qtel and 9q13-q34.2. In leiomyosarcomas, the most recurrent aberrations were loss of 10q21.13 and 13q14.2-q14.3. The region in chromosome 17p13.1-p11.2 presented high amplification and its analysis revealed nine candidate genes. Four genomic clones within this region were tested in three LMS samples by fluorescence in situ hybridisation (FISH). LMS1, -10 and -25 showed different levels of DNA copy number although LMS10 was expected to have normal copy number in this region. Only two genes previously cited in literature were contained in the clones tested by FISH although other clones within the amplicon could contain the actual “target” genes; those were MAP2K4 often mutated in many tumour types and SPECC1 involved in juvenile myelomonocytic leukaemia. These genes may be useful in studies of the biology of LMS and should be investigated further