The isolation of the first human oncogenes and tumor suppressors [1
] led to the prevailing hypothesis during the last decades postulating that the origin of cancer resides in the accumulation of somatic mutations in cancer genes, i.e. proto-oncogenes and tumor suppressors [5
]. In addition to these oncogeneic mutations, the vast majority of solid tumors exhibit aneuploidy and chromosomal rearrangements. Whether somatic chromosomal alterations and aneuploidy are a driving force or a consequence of tumorigenesis remains controversial [7
]. With the exception of the DNA mismatch repair mutator genes that underlie microsatellite instability, diagnostic of a strong mutator phenotype [10
] the search for somatic mutations in genes involved in the preservation of genome integrity has been disappointing [13
]. With the information at hand, it appears that somatic genetic alterations leading to such an active chromosomal instability do not have a great significance in cancer. The chromosomal alterations universally found in solid tumors may be in a greater extent due to clonal selection and evolution of errors of chromosomal segregation that, in some cases, can be originated by age-related epigenetic alterations.
A shift has recently occurred in cancer research with the realization that not only genetic but also epigenetic alterations play a major role in carcinogenesis. To date, three major types of epigenetic mechanisms have been identified in humans: DNA methylation, histone modifications, and, more recently, non-coding RNAs. These mechanisms, responsible for the cell identity and differentiation, have been found substantially altered during cancer development. Consequently, the study of cancer epigenetics has attracted considerable attention.
In vertebrates, DNA methylation occurs almost exclusively at the position 5 of cytosine residues within the dinucleotide CpG. These sites are found concentrated in some genomic regions denominated CpG islands (CGIs), generally associated to gene promoters. While most of the CpG sites outside CGIs are constitutively methylated, the CpG sites inside CGIs are frequently devoid of methylation. The methylation status of the CGIs located in promoters and other gene regulatory regions can exert a drastic effect on the transcriptional levels of downstream genes, providing an epigenetic mechanism to control gene expression. Up to 72% of the human gene promoters are specially rich in CpG sites [18
], consistent with the notion that many genes are susceptible of epigenetic regulation through histone modifications and DNA-methylation.
Global DNA hypomethylation of cancer cells was discovered more than twenty-five years ago [19
]. Soon after, the first example of somatic hypomethylation of cellular oncogenes in human cancer was reported [21
]. Years later, it was found that some sporadic retinoblastoma tumors exhibited hypermethylation of the promoter of the tumor suppressor gene RB1, leading to transcriptional repression [22
]. Since that seminal discovery, numerous genes have been found to undergo promoter hypermethylation in a large variety of cancers [23
]. The role of gene promoter hypermethylation in carcinogenesis has been extensively studied, yielding cancer detection markers and chemotherapy predictors for cancer patients, as well as fostering the development of epigenetic drugs approved for the treatment of hematological malignancies [24
Technologies to detect mutations, chromosomal copy number alterations and DNA methylation alterations have improved exponentially, fostered by the generalization of the microarray platforms in the mid nineties [25
] and, more recently, the development of massively parallel sequencing platforms [26
]. These technologies have reached an impressive throughput: today it is possible to analyze over one million genomic locations in just a single microarray chip and the massively parallel sequencing platforms are capable of delivering more than one hundred million sequences in a single experiment. Very recently, the analysis of colorectal tumors using a comprehensive high-throughput array-based relative methylation (CHARM) method [30
], not biased for CpG island or promoter sequences, yielded a surprising discovery: the authors found that methylation alterations in colon cancer occur not only in promoters and CpG islands, but also in sequences up to 2 kb distant, which they termed 'CpG island shores'. CpG island shore methylation showed an inverse relationship with gene expression [31
However, before these remarkable technological advances were achieved, a handful of ingenious techniques based on the generation of DNA fingerprints of matched normal and tumor tissues were successfully employed to detect genetic and epigenetic alterations and, despite their modest throughput, they were germane to significant discoveries that provided insights in the molecular basis of colorectal cancer. Some of these technologies still have a place in everyday lab research, as they usually require less complex and inexpensive equipment. We will revisit in this review some of the fingerprint technologies employed in colorectal cancer research and their role in the discovery of fundamental oncogenic processes.