Investigation of the Expression of Glucose Transporter Proteins in Human Cancer Cells

Cancer cells are known to display increased glucose uptake and consumption. The glucose transporter (GLUT) proteins facilitate glucose uptake, however, their exact role in cancer metabolism remains unclear. The present study examined mRNA and protein expression of GLUT1, GLUT3, GLUT4 and GLUT12 in lung, breast and prostate cancer cells and corresponding noncancerous cells. Additionally, GLUT expression was determined in tumours from mice xenografted with human cancer cells. Differences in the mRNA and protein expression of GLUTs were found between cancerous and corresponding noncancerous cells. These findings demonstrate abundant expression of GLUT1 in cancer and highlight the importance of GLUT3 as it was expressed in several cancer cells and tumours. GLUT expression patterns in vitro were supported by the in vivo findings. The study of GLUT protein expression in cancer is important for understanding cancer metabolism and may lead to identification of biomarkers of cancer progression and development of target therapies.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

KAIRON PROJECT : assessing the role of expressive arts in community-based breast cancer support systems in the Niagara Region

Research Report Written for the Canadian Breast Cancer Foundation.

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Prostate Cancer and the Search for Novel Biomarkers

The primary objective of this research project was to identify prostate cancer (PCa) -specific biomarkers from urine. This was done using a multi-faceted approach that targeted (1) the genome (DNA); (2) the transcriptome (mRNA and miRNA); and (3) the proteome. Toward this end, urine samples were collected from ten healthy individuals, eight men with PCa and twelve men with enlarged, non-cancerous prostates or with Benign Prostatic Hyperplasia (BPH). Urine samples were also collected from the same patients (PCa and BPH) as part of a two-year follow-up. Initially urinary nucleic acids and proteins were assessed both qualitatively and quantitatively for characteristics either unique or common among the groups. Subsequently macromolecules were pooled within each group and assessed for either protein composition via LC-MS/MS or microRNA (miRNA) expression by microarray. A number of potential candidates including miRNAs were identified as being deregulated in either pooled PCa or BPH with respect to the healthy control group. Candidate biomarkers were then assessed among individual samples to validate their utility in diagnosing PCa and/or differentiating PCa from BPH. A number of potential targets including deregulation of miRNAs 1825 and 484, and mRNAs for Fibronectin and Tumor Protein 53 Inducible Nuclear Protein 2 (TP53INP2) appeared to be indicative of PCa. Furthermore, deregulation of miR-498 appeared to be indicative of BPH. The sensitivities and specificities associated with using deregulation in many of these targets to subsequently predict PCa or BPH were also determined. This research project has identified a number of potential targets, detectable in urine, which merit further investigation towards the accurate identification of PCa and its discrimination from BPH. The significance of this work is amplified by the non-invasive nature of the sample source from which these candidates were derived, urine. Many cancer biomarker discovery studies have tended to focus primarily on blood (plasma or serum) and/or tissue samples. This is one of the first PCa biomarker studies to focus exclusively on urine as a sample source.

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.

Inhibition of human lung cancer cell proliferation and survival by wine

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events.

Inhibition of human lung cancer cell proliferation and survival by wine

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events.

Investigation of the Biological Effects of Rosemary (Rosmarinus Officinalis L.) Extract in Human Lung Cancer Cells

Cancer cells display enhanced growth rates and a resistance to apoptosis. Lung cancer accounts for the most cancer related deaths and non-small cell lung cancer (NSCLC) represents an aggressive form of lung cancer, accounting for almost 80% of all lung cancer cases. The phytochemical rosemary extract (RE) has been reported to have anticancer effects in vitro and in vivo however, limited evidence exists regarding the effects of RE and its polyphenolic constituents carnosic acid (CA) and rosmarinic acid (RA) in lung cancer. The present study shows RE, CA and RA inhibit lung cancer cell proliferation and survival in various NSCLC cell lines and that CA and RA interact synergistically to inhibit cell proliferation. Moreover RE, CA and RA are capable of altering activation and/or expression of Akt, ERK and AMPK, signaling molecules which regulate cell proliferation and survival. RE shows potential as an anticancer agent and should be further investigated.