Tumor suppressor genes (TSGs) are responsible for maintaining homeostasis and thereby preventing a cell from progressing to cancer. Tumor suppressor genes often have an inhibitory or regulatory effect on the cell cycle. They prevent the transformation of normal cells to cancer cells by maintaining growth restrictions and blocking immortalization. TSGs achieve this by regulating cell division, promoting cell death upon DNA damage or senescence, promoting DNA repair pathways, and inhibiting invasiveness or anchorage-free growth leading to metastasis (Fig. 3.1.1). Changes to TSGs, such as hypermethylation, can disrupt the balance of cell growth and arrest and interfere with key checkpoints, which can initiate cancer development (1,2). This chapter will discuss the different classes of TSGs and provide examples of TSGs that are commonly inactivated or altered in different cancers. We will first focus on Rb/p53 to model how TSGs play a role in cancer pathogenesis.
Inactivation of TSGs can occur at many levels. Most often, they occur genetically and epigenetically by affecting transcriptional, translational, and post-translational regulation. This governs the amount and activity of tumor suppressor proteins.
At the molecular level, multiple mechanisms exist whereby a TSG may be inactivated (3).
1. Mutations in the DNA can result in inactive/dysfunctional forms of TSGs.
Mutations in TSGs can result in decreased or non-fuctional proteins or can interfere with other functioning proteins, resulting in a cell that is more likely to escape control mechanisms in place and become cancerous. These mutations can occur within the coding regions of the gene or at splice sites. Mutations at the latter may cause splicing errors that render the protein functionally null.
2. Mutations in cis-/trans-acting elements or in the promoter region of a TSG.
Cis/trans-acting elements and the promotor region of a TSG play critical roles in driving and regulating gene expression. Mutations at these sites may knock out expression.
3. Tumor suppressor mRNA overexpression.
Within a cell, microRNAs (miRNAs) act to suppress the translation of their target mRNAs. They do so by associating with the RISC complex, which promote target mRNA degradation (4). Overexpression of miRNA due to mutations in its promoter regions or through a translocation event that places the miRNA under the control of a stronger promoter or enhancer, may result in a lower level of TSG translation.
4. Competing endogenous RNA (ceRNA) underexpression
ceRNAs act on the same elements which miRNAs recognize. Therefore, ceRNAs competitively inhibit the action of miRNAs, which ultimately results in increased TSG translation and expression. The loss of ceRNAs through mutations, however, allows miRNA to act once again on TSG transcripts, dowrnregulating protein expression.
At the cytogenetic level, inactivation of TSGs may occur as a result of events involving the structure of the chromosome, including:
1. Mitotic recombination events
These create homozygous expression in daughter cells of a heterozygous parent following homologous chromosomes crossover, which may also include missegregation events or nondisjunction.
2. Deletion events of an entire chromosome region containing a normal allele of the TSG.
3. Gene conversion
Here, DNA polymerase starts replicating on the template strand, differentially jumps to the homologous chromosome to continue replication, and then jumps back to the template strand (5).
Despite having nominally identical DNA sequences, clonally-derived cells within a multicellular organism express different subsets of genes within their differentiated states. This non-genetic regulation is the basis of epigenetics, heritable changes to genes that are influenced by the cell’s environment and are not directly coded within the DNA sequence. Eukaryotic cells possess several epigenetic mechanisms responsible for a wide range of effects on gene expression. These epigenetic features include the direct modification of the DNA through the methylation or demethylation of cytosine bases, modification and remodeling of the overlying histone and other protein components of chromatin, and post-transcriptional gene regulation by small RNAs. Taken together, these mechanisms can be influenced by both cellular and environmental factors, from early development to continual modulation within the cell’s differentiated state.
As a disease of dysregulation, many of the aberrations leading to cancer are of an epigenetic nature. Epigenetic marks leading to the silencing of tumor suppressor genes has become the predominant mechanisms, however, cases involving the upregulation of proto-oncogenes, with cancer causing abilities at when overexpressed, have been described. Deposition of these marks as well can be a direct function of the cell’s environment, dismissing the notion that mutagens are the only carcinogen.
In this section we will discuss the importance of promoter methylation, histone modifications and non-coding RNA, going into detail on their individual mechanisms and their possible roles in cancer development.
An important epigenetic mechanism capable of regulating transcription, DNA methylation is the chemical addition of a methyl group to cytosine residues in DNA. In eukaryotic cells, cytosines targeted for methylation are often adjacent to guanosine nucleotides, the two bases constituting a CpG dinucleotide. In the mammalian genome, 60-90% of CpGs are methylated, with the highest density of this mark occurring in open reading frames (14). Among the predominately methylated and scattered CpGs within the genome are genomic stretches of high CpG density, termed CpG islands (CGIs), which show a frequent absence of methylation (15). Averaging 1000 bp, CGIs are have been shown to be localized at the 5’ regulatory regions of transcriptionally active genes, an association that has led to the designation of methylated DNA as a repressive mark (14)(16).
DNA methylation patterning is thought to be a result of the high mutagenic properties of methylcytosine, which is converted to thymine through spontaneous deamination. Therefore, an underrepresentation of CpG dinucleotides is present outside of CGIs, where they are often methylated and prone to mutation. In organisms such as invertebrates, with little to no DNA methylation, CpGs occur at a much higher frequency throughout the genome. In effect, the whole genome of these organisms is CGI-like, showing the same increased CpG density, suggesting that the nucleotide patterning is a consequence of selective DNA methylation (14)(17).
Apart from the correlation with transcriptional activity, our understanding of functional significance of DNA methylation on gene expression remains incomplete. Studies suggest that unmethylated CGIs themselves act as gene promoters, and are able to recruit general transcription factors regardless of classic promoter motif content, such as the TATA box; in general, protein-binding sites within DNA have been shown to by more GC-rich than non-binding sequence. Further evidence from transient reporter gene assays has shown that these regions when associated with actively transcribed genes are enriched for a number general transcription factors (18)(19).
Recent work has established a direct link between CGIs and the transcriptionally active associated histone modification H3K4me3 (20). Studies show that Cfp1, a member of the histone methyltransferase complex Setd1, binds exclusively to unmethylated CpGs, depositing the H3K4me3 mark at these regions which can further promote transcription through the recruitment of chromatin remodeling complexes, transcription factors and other histone modifiers (21).
Approximately 50% of CGIs are located within the promoter region of genes, while the remaining “orphan” CGIs are shared between intergenic and intragenic regions, and generally possess an increased methylation state (17). It has been shown that when demethylated, these orphan regions do possess transcription regulatory activity, potentially marking prior uncharacterized promoter regions of non-coding RNA molecules. Another functional example of orphan regions is CGI contained within intron 2 of Igf2r, a gene silenced on the parental chromosome through an imprinting mechanism. Here, the non-coding transcript Air is able to interact with the CGI leading to inactivation of Igf2r (22).
Studies into development showed that the methylation of CGI promoters controlling expression of early developmental genes effectively silenced them during later stages of development (23). Two theories exist to describe this silencing mechanism: a steric inhibition model, in which transcription factors are blocked from binding due to the methylated state of the promoter, and a repressor recruitment model, in which the high density of methylated is able to bind factors promoting gene silencing. Both models are not exclusive to the other, and evidence has been suggested for both (14).
DNA methylation has been extensively researched in cancer cells, with many cancer specific methylation sites mapped within the human genome. At the basis of most described mechanisms is the aberrant methylation of CGI promoters functioning tin the regulation of tumor suppressor genes. What remains to be uncharacterized is methylation’s role in cancer initiation, if it is causative or a result of prior aberrations. Much work has been conducted into comparing methylation in cancerous and normal cells to observe whether it is consistent between the two. Differences have been observed in colorectal cancer cells, where steady methylation was documented over both CGIs present at the 5’ end of genes as well as at orphan CGIs, where methylation is not normally present (24).
Cancer associated methylation of CGI promoters includes the silencing of: p53 cell cycle inhibitor, p16 cyclin-dependent kinase inhibitor, MGMT tumor suppressor, APC cell cycle regulator and BRCA1 a DNA-repair gene.
As a separate mechanism of repression, CGI promoters can be silenced by polycomb group proteins (PcGs). Though not fully characterized it is known that two PcGs function together in silencing the mechanism: polycomb-repressive complex 2 possesses histone methylation capabilities and deposits the repressive HrK27me3 mark at CGIs. This mark recruits polycomb-repressive complex 1, which functionally inhibits transcription by an unknown mechanism involve H2A ubiquitination (25). The PcG silencing mechanism has been found to play a significant role during development, where CGI promoters in embryonic stem cells possess both H3K4me3 transcriptional activating marks and H3K27me3 deposited by PcGs. These cells are in a pluripotent state, due to the bivalent nature of the histone marks, which is lost during differentiation, where one of marks is removed either permanently activating or silencing the gene (26). Remarkably, in some cancer cells, a similar mechanism have been described, providing pluripotency to varying degrees, favoring unrestrained proliferation of cancer cells (27).
Methylation of intrageneic CpGs, as well possibly lead to malignancies; methylcytosine’s tendency to spontaneously undergo deamination and convert to thymine mutates the base, the effect of which is variable, depending on where in the gene the CpG is located. UV absorption of cytosine is also altered in its methylated state, potentially leading to pyrimidine dimers, and a need for DNA damage repair (28).
Histone modifications are epigenetic mechanisms that alter chromatin conformation by changing the accessibility of DNA to various proteins, which in turn regulate the expression genes in these areas. This is accomplished by moving nucleosomes, DNA segments wrapped around histones proteins. Histones can be mono-, di-, or trimethylated at a particular lysine residue (9). Other modifications, such as phosphorylation, acetylation and ubiquitination, also play a role in the histone modification. TSGs can be inactivated by histone acetylation. Histone acetyltransferase (HAT) adds acetyl groups to the tails of histone proteins, neutralizing the inherent positive charge of the histone. Removing the positive charge on the histone reduces its affinity for the negatively charged DNA, resulting decondensation of chromosomes and increased access of transcription factors and DNA polymerase to the local genes. Histone deacetylate (HDAC) enzymes perform the opposite by removing acetate groups on histone proteins, thus blocking transcription (8).
Although noncoding RNAs, such as microRNAs, have long been discovered, it is only recently that its function as an epigenetic biomarker for cancer diagnosis was recognized (9). MiRNAs are thought to play a role in post-transcriptional modification, and change in their expression levels are associated with many cancers (8). MiRNAs typically regulate the expression of target, protein-coding genes through degradation of mRNA transcripts or through inhibition of translation (29). Many of their target genes are involved in cancer development, which highlight the role of these miRNAs in the regulation of normal cellular processes and tumor suppressive functioning (29). One particular study examined this link and found that a significant downregulation of miR-375 was associated with the development of esophageal cancer (29). These results have been replicated in multiple cancer cell lines, and involve numerous miRNAs. The relationship between miRNA levels and pathogenicity renders it a potential therapeutic target.
Another interesting application of miRNA studies, in the context of cancer, is the characterization of miRNA profiles in order to provide functional evidence for the link between expression signatures and stem cell biology (30). For example, researchers have shown that certain miRNAs are downregulated in both normal mammary stem cells and human breast cancer stem cells (30). This evidence that normal stem cells and cancer stem cells share molecular mechanisms aids in the understanding of cancer stem cells and could enable the development of therapeutic targets.
The study of TSGs has illustrated several paradigms for tumour suppression and cancer susceptibility. The extent to which a paradigm of tumour suppression cancer development occurs depends on the context of the gene and the sensitivity of dose responses related to the TSG.
1. Two-Hit Hypothesis:
The two-hit hypothesis suggests that tumor initiation requires two "hits," or mutations, in TSGs--one to each allele. This can occur in one of two ways:
The discovery of the Two-Hit Hypothesis came from the observation of young retinoblastoma patients who inherited RB1 gene mutations but did not display the disease phenotype until a later age. Given this hypothesis, TSG inactivation is an all-or-nothing event that requires biallelic loss. The loss of heterozygosity results from the second hit, whereby the heterozygous individuals become homozygous for the mutated allele.
2. Haploinsufficiency Hypothesis:
The haploinsufficiency or one-hit hypothesis illustrates that the loss of one allele is enough to obliterate TSG function and contribute to tumorigenesis, while the severity of cancer progression increases with further loss. An example of haploinsufficiency can be found in a p53+/- genotype, where tumorigenesis occurs despite the presence of a wild-type allele.
3. Quasi-sufficiency or Obligate Haploinsufficency Hypothesis:
Subtle reduction in PTEN (phosphatase and tensin homolog) protein levels has been observed to lead to the development of cancer without haploinsufficiency. This led to the idea of quasi-sufficiency, where dosage of functional proteins rather than copy number dictate the consequences of cancer susceptibility and progression. As the dosage decreases with downregulation, the disease severity increases and peaks at the obligate haploinsufficiency. Further reduction will result in biallelic loss and homozygosity, which may become lethal leading to cell death or senescence, when fail-safe mechanisms are triggered. APC, a TSG closely related with colorectal cancer, follows this hypothesis and will be discussed later in the Chapter 7. This model is also known as the Continuum Model.
Mutations in Tumor Suppressor Genes are both dominant and recessive:
At the cellular level, TSG mutations are considered to be recessive, because in order for cells to display the mutant phenotype, both copies of the TSG allele need to be mutated (13). This phenomenon is observed in heterozygotes, where the wild type phenotype persists because one normal allele is sufficient to repress tumor formation.
However, at the organismal level and in pedigree analysis, TSG mutations are seen as dominant (13). If an individual inherits a mutant allele generated in a parent's germline, they would only need to acquire a mutation in the second allele to display the mutant phenotype (13). Thus, the increased cancer risk is dominant.
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