RESEARCH

Genome, or more specifically chromosome instability (or abnormal chromosome numbers) is associated with virtually all tumor types, including breast, ovarian, prostate, and colorectacl cancer. Colorectal cancer for example, affects both males and females and collectively is the second leading cause of cancer-related deaths in North America. In 2012, the Canadian Cancer Society estimated that ~23,300 Canadians would be newly diagnosed with colorectal cancer and an additional ~9,200 individuals would succumb to the disease. Accordingly, a better understanding of how these tumors develop and the mutated genes that drive the development of colorectal cancer is urgently needed so that new therapeutic strategies and drug targets can be identified to combat the disease.

Mutations in genes that cause genome instability are now recognized as significant genetic factors that drive tumor development. Genome instability is a hallmark associated with virtually all tumor types including both solid (e.g. breast and colon) and liquid (e.g. lymphoma and leukemia) and generally arises through 3 mechanisms; 1) Microsatellite Instability (MSI), which arises due to defects in DNA repair and results in a gene mutator phenotype, 2) CpG Island Methylator Phenotype (CIMP), which results in the epigenetic silencing of genes, and 3) Chromosome Instability (CIN), which results in numerical and/or structural defects in chromosomes. Chromosome instability is of particular interest because it associated with up to 85% of randomly arising colorectal cancers. Chromosome instability is also associated with aggressive tumors, multidrug resistance to chemotherapeutics and poor patient prognosis. However, we believe that cancer cells with chromosome instability represent ‘genetically sensitized’ cells that can be selectively targeted. In fact, since chromosome instability is only contained within the tumor cells, we believe it clearly distinguishes cancer cells from normal surrounding tissues and thus will restrict killing to the tumor cells, minimizing many of the adverse side effects associated with many current approaches. Consequently, one of our main goals is to identify and develop the next generation of therapeutic strategies and drug targets designed to combat cancer.

Currently, our research team is highly focused on identifying, characterizing and exploiting the origins of cancer, whether it be colon, breast or ovarian. However, since chromosome instability is found in all tumor types, the results we generate using our colorectal cancer models, will be of general interest to virtually any tumor type. We currently employ biochemistry, genetics, and cell biology to answer four critical questions;

1. What are the genes that when mutated contribute to the development of cancer?

Identifying these players is critical to better understand how these tumors develop so that new therapeutic strategies can be developed.

2. Can we characterize the molecular pathways that are required to maintain chromosome stability?

It is critical to know what pathways drive tumor development to so that new drug targets can be identified.

3. What is the contribution of chromosome instability to Interval colorectal cancers, or tumors that rapidly arise after a clearing or negative colonoscopy?

It is essential to know how these tumors arise so that better screening and treatment options can be identified.

4. Can we identify new drug targets that exploit and selectively kill cancers with defects in specific genes that are normally required to maintain chromosome stability?

This is essential to identify new drug targets and lead chemical compounds for subsequent optimization and pre-clinical testing.

SPECIALIZED EQUIPMENT & TECHNIQUES

Real-time Cellular Analyses (RTCA)

We routinely employ real-time cellular analyses to evaluate novel candidate cancer drug targets that are either silenced using RNAi-based approaches or small molecule inhibitors (drug candidates). The RTCA unit is capable of continually monitoring cell growth and death in real-time without the use of labels or dyes, thus offering a significant advantage over traditional end-point analyses. ACEA Biosciences

High-Content Screening & Image Analysis

We employ high-content analysis to identify novel candidate drug targets and to assess chromosome instability in various tumor types. Cells are generally seeded in 96- or 384-well plates, treated with an appropriate siRNA/shRNA or chemical and permitted to grow. Cells are then fixed, DNA is staind and antibody labeling is used to identify specific cellular features of interestes such as centromeres, centrosomes, DNA damage or apoptosis. BioTek

Indirect Immunofluorescence

We routinely employ indirect immunofluorescence and collaborate with Abcam Inc. to test the applicability of antibodies. In essence, cells are fixed, permeablized and immuno-labeled with a primary antibody that recognizes a specific cellular feature (e.g. centromere, DNA damage, death marker, etc.). The primary antibody is then recognized by a secondary antibody that is bound with a fluorescent marker that can be a variety of fluorescent colors (e.g. green, red, far red, etc.). Cells are often stained with DAPI, which binds to DNA and becomes fluorescent blue.

Cytogenetic Techniques

We routinely employ various cytogenetic approaches in combination with gene silencing (RNAi) to visualize chromosome instability. Mitotic chromosome spreads are generated and various techniques are used to determine if numerical or structural chromosome changes have occurred.

DAPI-counterstained Mitotic Chromosome Spreads

Mitotic chromosome spreads are generated and chromosomes are counterstained with DAPI and fluorescent DNA stain. This approach is ideally suited for enumeration purposes. Under certain conditions this approach can also be employed to characterize persistent DNA breaks.

Fluorescence in situ Hybridization (FISH)

Cells are fixed and processed so that proteins associated with DNA are digested and the double stranded DNA helix is unwound. A DNA sequence specific probe that is fluorescently labeled is then used to determine if that locus has been amplified, delected or translocated.

Whole Chromosome Paints (WCP)

Mitotic chromosome spreads are generated and labeled with a fluorescent tag that specifically recognizes a chosen chromosome. This technique can be used to identify gains or losses in the specifically labeled proteins. In some circumstances, rearrangements (e.g. translocations) involving labeled chromosomes can be readily detected.

Spectral Karyotyping (SKY)

Mitotic chromosome spreads are generated and labeled with five different combinations of fluorescent tags. Based on the unique combinations of fluorescent tags each chromosome can be identified and numerical and/or structural increases in chromosomes can be identified. SKY is particularly useful in identifying recurrent brek points and defining specific karyotypes.

Epigenetic Immuno-Karyotyping

Mitotic chromosomes are spun onto glass microscope slides and immunofluorescently labeled with an antibody against a specific post-translational histone modification. Chromosomes are then fixed and imaged. This approach provides a 'higher-order' global view of the epigenome.