Center of Clinical Comparative Oncology (C3O), Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
All cancers are caused by abnormalities in deoxyribonucleic acid (DNA) sequence or epigenetic modifications. Throughout life, the DNA in cells is exposed to mutagens and suffers mistakes in replication, resulting in progressive, subtle changes in the DNA sequence in each cell. Occasionally, one of these somatic mutations alters the function of a critical gene, providing a growth advantage to the cell in which it has occurred and resulting in the emergence of an expanded clone derived from this cell. Additional mutations in the relevant target genes and consequent waves of clonal expansion produce cells that invade surrounding tissues and metastasize. Cancer is the most common genetic disease: approximately one in four of all dogs will at some stage in their life develop a cancer, and the majority dies from it.
The spontaneous occurrence of canine tumors (such as malignant lymphoma, mammary tumors [MTs], and osteosarcomas) has long been claimed to provide a suitable model for human cancer in a number of diagnoses (such as non-Hodgkin's lymphoma, breast cancer, and pediatric osteosarcoma). The unraveling of the complete canine genome sequence in 2005 has finally permitted investigation of the molecular biological similarities in human and canine tumor entities.
This lecture will give a brief overview of molecular biological approaches towards understanding oncogenesis in dogs and explain some terms that not all veterinary surgeons may be completely familiar with.
Many clinicians are involved in the tissue sampling phase of molecular biology projects trying to explain breed predisposition to certain tumors, but often not knowing what will happen to the sample in the next phase. This is sometimes unlucky, as better understanding of molecular analyses will help the clinician optimize phenotyping and sampling technique and improve the outcome.
The DNA is situated in the nucleus and the double-stranded helix is tightly wound around specific proteins (histones) in the chromosomes, which, when duplicated, consist of two identical subunits (called chromatids or sister chromatids) joined by a centromere. Chromosomal recombination plays a vital role in genetic diversity. If this process becomes aberrant, through processes known as chromosomal instability and translocation, the cell may undergo mitotic catastrophe and die, or it may unexpectedly evade apoptosis, leading to the progression of cancer. The end of the chromosome is called the telomere. An enzyme, telomerase, secures the integrity of the telomeres by allowing replacement of these short end bits of DNA, which are otherwise shortened when a cell divides via mitosis. Despite the telomerase activity, the telomeres are constantly shortened and a normal cell undergoes 40–60 cell divisions (the Hayflick limit or Hayflick phenomenon) before entering a senescent phase and eventually die. This mechanism also appears to prevent genomic instability. Telomere shortening may also prevent the development of cancer in aged human cells by limiting the number of cell divisions. However, shortening of telomeres impairs immune function and consequently might also increase susceptibility to cancer. In certain cancers, the telomerase activity is regained and/or increased, leading to immortality of the cells, being one of the hallmarks of cancer.
Transcription and Translation
The copying of DNA into messenger ribonucleic acid (mRNA) is called transcription and is the first step in the gene expression cascade. The copied single-stranded RNA is further spliced into a continuous sequence only containing the coding sequences of the DNA (exons), leaving the nucleus through pores in the nucleus membrane, and is translated into proteins in the cytoplasm of the cell. Ribonucleic acid can also be synthesized from non-coding DNA sequences and form non-coding RNA genes (such as microRNA, lincRNA, etc.) or ribosomal RNA (rRNA) or transfer RNA (tRNA), other components of the protein-assembly process.
Knowing about this process makes it possible to conduct different analyses of different areas of the genome to detect germ line (reflecting features of the gametes, i.e., the ovum or sperm) and somatic (reflecting features of the cells of the body) changes of the genome.
Candidate Gene Approach
Sometimes a gene is known to cause or increase the risk of getting a disease. Such a gene is called a candidate gene. Several human studies have been performed in this area. Recently, canine studies have shown some of these candidate genes to be active also in dogs with the canine counterpart of human diseases. In 2009, Rivera et al. showed that MT development in dogs is associated with breast cancer-associated genes 1 and 2 (BRCA1 and BRCA2).1 These genes are so-called tumor suppressors. Both BRCA1 and BRCA2 are involved in DNA repair. In mutated cells, this function may be impaired or completely lost, increasing the risk for defect DNA to continue in the cell cycle and eventually cause cancer. The discovery in 2009 cemented the theory that canine MTs and human breast cancer share many similarities and, hence, that the dog is a good comparative model in breast cancer research.
Genome-Wide Association Study
In a genome-wide association study (GWAS), the genome is analyzed at several checkpoints, single nucleotide polymorphisms (SNPs). An SNP is a variation in the DNA sequence that usually occurs in non-coding regions of the genome. By investigating the SNP frequency in different individuals, we can identify subtypes associated with higher risk of getting a specific disease. In a regular GWAS, individuals with a known phenotype (i.e., normal controls vs. a specific disease/phenotype) are analyzed. If the disease is caused by one single gene, the GWAS in itself can be sufficient to determine location of the gene. In cases of more complex disease (e.g., most cancers), the GWAS can identify suspect areas of the genome that predispose an individual to a certain cancer. When the genome of interest is considerably restrained, the next phase, DNA fine mapping, needs to occur, where gene analysis is performed at higher resolution and is more disease-directed. Finally, suspect candidate genes are isolated and tested for functionality to decide if they are likely associated with the disease.
The first GWAS performed in dogs showed the power of the analysis, by helping to discover the gene for white color in Boxers (the MITF gene) and the genetic predisposition for dermoid sinus in Rhodesian Ridgeback (duplication of FGF3, FGF4, FGF19, and ORAOV1).2,3 Recently, a large GWAS in three predisposed breeds (Greyhound, Rottweiler, and Irish Wolfhound) implicated 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B.4 Most of these are reported to predispose to human osteosarcoma (about 100 loci hitherto reported), again showing the dog to be an excellent comparative model for cancer research. With the GWAS information, pathway analyses are now being performed to investigate if many of the predisposed genes identified in the GWAS may occur in already known gene/protein cascades. This will lead to better understanding of the accuracy of the findings of the GWAS.
Gene Expression Analysis
Following the GWAS, either in combination with fine mapping or performed on its own, different kinds of gene expression analyses are made to confirm that findings of germ line mutations/aberrations can result in changed gene expression at the mRNA level. In tumors in particular, direct comparison of germ line changes and mRNA expression can often be challenging. One explanation for this is that somatic mutations and altered mRNA expression will continue to occur in the manifest tumor, often due to germ line mutations causing defects in DNA repairing proteins. Nevertheless, molecular profiling of tumors has shown to be very promising in dogs as earlier reported in human cancers. Recently, a molecular profiling study in canine lymphoma succeeded in correctly identifying three clinical entities of lymphoma with different prognosis.5 In the future, gene expression tools may enhance the possibility to more accurately diagnose tumors, as well as suggest a prognosis and enable personalized/individualized treatment.
1. Rivera P, Melin M, Biagi T, Fall T, Haggstrom J, Lindblad-Toh K, et al. Mammary tumor development in dogs is associated with BRCA1 and BRCA2. Cancer Res. 2009;69(22):8770–8774.
2. Karlsson EK, Baranowska I, Wade CM, Salmon Hillbertz NH, Zody MC, Anderson N, et al. Efficient mapping of mendelian traits in dogs through genome-wide association. Nat Genet. 2007;39(11):1321–1328.
3. Salmon Hillbertz NH, Isaksson M, Karlsson EK, Hellmen E, Pielberg GR, Savolainen P, et al. Duplication of FGF3, FGF4, FGF19 and ORAOV1 causes hair ridge and predisposition to dermoid sinus in Ridgeback dogs. Nat Genet. 2007;39(11):1318–1320.
4. Karlsson EK, Sigurdsson S, Ivansson E, Thomas R, Elvers I, Wright J, et al. Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B. Genome Biol. 2013;14(12):R132.
5. Frantz AM, Sarver AL, Ito D, Phang TL, Karimpour-Fard A, Scott MC, et al. Molecular profiling reveals prognostically significant subtypes of canine lymphoma. Vet Pathol. 2013;50(4):693–703.