GENETIC ABERRATIONS IN CANCER

Most tumors are characterized by changes in chromosomal structure and number. Chromosomal aberrations are generally more numerous in malignant than in benign tumors and the karyotypic complexity and cellular heterogeneity observed is often associated with poor prognosis ³. Genomic changes observed in cancer can be numerical or structural and can often coexist in complex karyotypes. Numerical aberrations comprise aneuploidy, DNA copy number (CN) changes, and “unbalanced” translocations, whereas structural aberrations include “balanced” translocation and inversions (Figure 1.1). Structural changes are defined balanced if they involve equal exchange of material between two chromosomal regions, whereas the term unbalanced refers to a non reciprocal exchange leading to gain or loss of genome portions. Translocations are given by exchange of chromosomal segments between non-homologous chromosomes, and can be balanced or unbalanced. Spatial proximity and sequence homology are factors probably contributing in increasing the propensity of translocated chromosome partners to rearrange ³.

DNA copy number changes comprise deletions, gains or amplifications of genomic material (discussed in details in the next sections). CN aberrations can be also referred to as gene dosage alterations, i.e. alterations in the number of copies of a given sequence found in a cell. It was demonstrated that changes in DNA copy number are a mechanism that lead to altered gene expression in cancer cells ⁴.

Further genomic anomalies involved in cancer are somatic point mutations, loss of heterozygosity (LOH) (discussed in section 1.5) and epigenetic modifications.

The result of somatic point mutations, in which one or a few nucleotides are altered by sequence error and or deletions or insertion, can be nonsense

mutations, missense mutations or frameshift mutations. In nonsense mutations the new codon causes the protein to prematurely terminate, leading to a shorter and usually non functional product. Missense mutations can determine an incorrect amino acid into the protein sequence. The effect on protein function depends on the site of mutation and the nature of the amino acid replacement. In general, mutations that do not affect the protein sequence or function are called silence or synonymous mutations. Frameshift mutations cause the affected codon to be misread and subsequently also all the following codons, leading to a much different and often non functional product⁵.

Differential DNA methylation patterns exist in normal and cancer cells. DNA methylation involves the addition of a methyl group to the cytosine ring (carbon 5 position) by DNA methyltransferases. In the human genome, epigenetic modification to DNA by methylation is primarily found in repetitive DNA elements where it is thought to play a role in protecting against recombination events and to silence potentially destabilizing transposable elements ⁶. On the contrary, hypermethylation of gene promoter regions causes their transcriptional repression. In tumors, hypomethylation of the genome results in an increased mutation rate, including genome deletions and chromosomal copy number changes^{7, 8}. Methylation studies in various types of tumors have found it to be tightly connected to cancer development through local hypermethylation of tumor-suppressor genes causing transcriptional repression and/or global genomic hypomethylation resulting in the expression of oncogenes.

Another relevant epigenetic modification involved in chromatin remodeling is histone acetylation, which consist of the addition of acetyl groups to lysines from amino-terminal tail of core histones. Lysine acetylation and deacetylation reactions are catalyzed by histone acetyltranferase (HAT) and histone deacetylase (HDAC) respectively. Acetylated histone enhances chromatic decondensation and DNA accessibility, therefore the acetylation states correlates with transcriptional activation. The disruption of

acetylation/deacetylation balance due to the alterations of HATs and HDACs has been detected in many cancers, including lymphoma ⁹.

Figure 1.1 Acquisition of numerical and/or structural chromosome aberrations in cancer cells.

(modified by Bayani J et al 2007)³

Importantly, malignant upregulation of histone deacetylation process is being targeted by therapeutic interventions with HDAC inhibitors.

In general, cancer genes are characterized by a redundancy of deregulation mechanisms. Oncogene can be activated by gene dosage alterations, point mutations, translocations, promoter hypomethylation, constitutive active transcription factors, or absence of control by a tumor suppressor gene, if the latter is inactivated. Tumor suppressor genes can be inactivated by gene dosage alterations, LOH events, point mutations or epigenetic silencing by promoter hypermethylation. The identification of genetic alterations can lead to

the discovery of genes, transcripts and pathways that could play a relevant role in cancer.

1.1.1 GENOMIC GAINS

Most recurrent genomic gains probably contribute to tumorigenesis by enhancing the activity of genes in the affected chromosomal regions. Genomic gains commonly arise from chromosomal non-disjunction or unbalanced translocations, which cause complete or partial chromosomal trisomies, or from amplification events affecting DNA segments of different size. Numerous examples of large-scale genomic gains are associated with specific types of cancer. Since such aberrations involve multiple genes, the identification of their functionally relevant targets has proved to be difficult ¹⁰.

1.1.2 GENOMIC AMPLIFICATION

Gene amplification, defined as a high copy number increase of a restricted region of a chromosome arm, can also occur. Amplification is a reiterative process, in which multiple copies of a genome region are accumulated.

Amplification requires a DNA double strand break and progression through the cell cycle with this damaged DNA ¹¹. A role for genome context in promoting amplification has also been suggested. Particular genome sequences prone to breakage have also been shown to set the boundaries of amplicons, further suggesting that genome position influences the propensity to amplify ¹¹.

1.1.3 GENOMIC LOSSES

The spectrum of genomic losses ranges from cytogenetically visible alterations, such as complete or partial chromosomal monosomies, to single gene or intragenic deletions that are detectable only by techniques that provide high spatial resolution. Most recurrent genomic losses probably contribute to malignant transformation by reducing the function of specific genes in the affected chromosomal regions ¹⁰. Extensive genomic deletions affecting multiple genes are frequent in tumors, making it difficult to identify which lost

gene contributes to the development of the cancer. The classic approach to identify a tumor-suppressor gene compares multiple tumors with a specific chromosomal deletion to determine the minimal genomic region that is lost in all cases ^{12, 13}. Candidate genes from this region are then screened for deletions, mutations, or epigenetic modifications that inactivate the remaining allele.

Cancer-associated chromosomal losses may also act through inactivation of genes that do not encode proteins. For example, several genomic regions that are recurrently deleted in a variety of tumors contain microRNA genes ¹⁴. These genes encode small RNAs involved in post-transcriptional regulation of gene expression, and there is emerging evidence that the loss of specific microRNAs with tumor-suppressive activity may contribute to tumorigenesis ¹⁴.

1.1.4 LOH AND UNIPARENTAL DISOMY (UPD)

Classic LOH is defined as the loss of one allele of a heterozygous locus. Genetic mechanisms leading to LOH are highly variegated. LOH can be caused by deletion, which can involve whole chromosome or smaller region, by mitotic non disjunction, or by mitotic recombination between two homologous chromosomes, including break-induced replication and gene conversion (interstitial mitotic recombination event). LOH results in homozygosity or hemizygosity for a particular mutation. In addition, one allele can be inactivated through mutation and the other through promoter methylation.

Therefore, LOH can be due to both DNA CN changes (one copy lost) and copy-number-neutral changes ¹⁵.

In many cases, tumor-suppressor genes can be inactivated both when a germline mutation occurs in one allele and somatic inactivation occurs in the other allele at a later stage or when sequential somatic inactivation of both alleles occurs. In both cases, somatic inactivation is often associated with LOH at the tumor-suppressor gene ¹⁵. UPD refers to the situation in which both copies of a chromosome pair have originated from one parent, either as isodisomy, in which two identical segments from one parent homologue are present, or as heterodisomy, where sequences from both homologues of the transmitting

parent are present ¹⁶ (Figure 1.2). This can occur during the transmission of a chromosome from parent to gamete and during early somatic cell divisions in the zygote (germ line recombination resulting in constitutive UPD), but it is also likely to occur simply as a consequence of somatic recombination during mitotic cell divisions (mitotic or somatic recombination).

Two mechanisms are supposed to be involved: in UPD of whole chromosomes, mitotic nondisjunction is followed by duplication of the remaining homologue in the monosomic cell or loss of one chromosome in the trisomic cell; partial uniparental disomy (pUPD) involves mitotic recombination events between chromatids, such as reciprocal exchange of chromosome material between two homologues ^{15, 17}.

In general, UPD can be an important step in cancer development as well as a contributing factor to other late onset diseases. In fact this phenomenon might be a common unrecognized mechanism in cancer development, because in UPD, the karyotype appears ‘normal’ when examined by conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) or aCGH ¹⁶. However, analysis by high-resolution single nucleotide polymorphism (SNP) oligonucleotide genomic microarrays allows the identification of copy-number and copy-number-neutral changes. The major difference between a UPD and LOH might be a difference in the gene dosage; copy number loss leading to LOH mostly follows with mutation in the remaining allele (haploinsufficiency);

however, loss of one allele and duplication of the remaining allele leads to UPD (homozygosity of the mutated or methylated allele) ¹⁵. Differences in gene dosage might have different pathologic results.

Figure 1.2 Model of the mechanism of UPD in cancer. (a) Both maternal and paternal alleles are present in the normal cell. (b) Mutated cell. During mitosis, incomplete segregation of the chromosome can occur as either (c) a trisomic cell (by non-disjunction) or (d) a monosomic cell (by anaphase lag), and (e) the remaining allele is duplicated by mitotic non-disjunction.

Alternatively, (f) (i–iv) one or more mitotic recombination events can occur during mitosis, resulting in segmental UPD. (g) (i) Loss of a chromosome arm and (ii) reduplication by non-disjunction might follow. The white segment indicates deletion ¹⁵.