DNA methylation in cancer

From Wikipedia, the free encyclopedia

DNA methylation in cancer plays a variety of roles, helping to change the healthy regulation of gene expression to a disease pattern.

All mammalian cells descended from a fertilized egg (a zygote) share a common DNA sequence (except for new mutations in some lineages). However, during development and formation of different tissues epigenetic factors change. The changes include histone modifications, CpG island methylations and chromatin reorganizations which can cause the stable silencing or activation of particular genes. Once differentiated tissues are formed, CpG island methylation is generally stably inherited from one cell division to the next through the DNA methylation maintenance machinery.

In cancer, a number of mutational changes are found in protein coding genes. Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations that silence protein expression in the genes affected. However, transcriptional silencing may be more important than mutation in causing gene silencing in progression to cancer. In colorectal cancers about 600 to 800 genes are transcriptionally silenced, compared to adjacent normal-appearing tissues, by CpG island methylation. Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs.

CpG islands are frequent control elements

CpG islands are commonly 200 to 2000 base pairs long, have a C:G base pair content >50%, and have frequent 5' → 3' CpG sequences. About 70% of human promoters located near the transcription start site of a gene contain a CpG island.

Promoters located at a distance from the transcription start site of a gene also frequently contain CpG islands. The promoter of the DNA repair gene ERCC1, for instance, was identified and located about 5,400 nucleotides upstream of its coding region. CpG islands also occur frequently in promoters for functional noncoding RNAs such as microRNAs and Long non-coding RNAs (lncRNAs).

Methylation of CpG islands in promoters stably silences genes

Genes can be silenced by multiple methylation of CpG sites in the CpG islands of their promoters. Even if silencing of a gene is initiated by another mechanism, this often is followed by methylation of CpG sites in the promoter CpG island to stabilize the silencing of the gene. On the other hand, hypomethylation of CpG islands in promoters can result in gene over-expression.

Promoter CpG hyper/hypo-methylation in cancer

In cancers, loss of expression of genes occurs about 10 times more frequently by hypermethylation of promoter CpG islands than by mutations. For instance, in colon tumors compared to adjacent normal-appearing colonic mucosa, about 600 to 800 heavily methylated CpG islands occur in promoters of genes in the tumors while these CpG islands are not methylated in the adjacent mucosa. In contrast, as Vogelstein et al. point out, in a colorectal cancer there are typically only about 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations.

DNA repair gene silencing in cancer

In sporadic cancers, a DNA repair deficiency is occasionally found to be due to a mutation in a DNA repair gene. However, much more frequently, reduced or absent expression of a DNA repair gene in cancer is due to methylation of its promoter. For example, of 113 colorectal cancers examined, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region. Similarly, among 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, 6 had a mutation in the PMS2 gene, while for 103 PMS2 was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). In the remaining 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.

Frequency of hypermethylation of DNA repair genes in cancer

Twenty-two DNA repair genes with hypermethylated promoters, and reduced or absent expression, were found to occur among 17 types of cancer, as listed in two review articles. As listed in one of the reviews, promoter hypermethylation of MGMT occurs frequently in a number of cancers including 93% of bladder cancers, 88% of stomach cancers, 74% of thyroid cancers, 40%-90% of colorectal cancers and 50% of brain cancers. That review also indicated promoter hypermethylation of LIG4, NEIL1, ATM, MLH1 or FANCB occurs at frequencies between 33% to 82% in one or more of head and neck cancers, non-small-cell lung cancers or non-small-cell lung cancer squamous cell carcinomas. The article Werner syndrome ATP-dependent helicase indicates the DNA repair gene WRN has a promoter that is frequently hypermethylated in a number of cancers, with hypermethylation occurring in 11% to 38% of colorectal, head and neck, stomach, prostate, breast, thyroid, non-Hodgkin lymphoma, chondrosarcoma and osteosarcoma cancers.

Likely role of hypermethylation of DNA repair genes in cancer

As discussed by Jin and Roberston in their review, silencing of a DNA repair gene by hypermethylation may be a very early step in progression to cancer. Such silencing is proposed to act similarly to a germ-line mutation in a DNA repair gene, and predisposes the cell and its descendants to progression to cancer. Another review also indicated an early role for hypermethylation of DNA repair genes in cancer. If a gene necessary for DNA repair is hypermethylated, resulting in deficient DNA repair, DNA damages will accumulate. Increased DNA damage tends to cause increased errors during DNA synthesis, leading to mutations that can give rise to cancer. 

If hypermethylation of a DNA repair gene is an early step in carcinogenesis, then it may also occur in the normal-appearing tissues surrounding the cancer from which the cancer arose (the field defect). See the table below. 

Frequencies of hypermethylated promoters in DNA repair genes in sporadic cancers and in adjacent field defects

Cancer	Gene	Frequency in Cancer	Frequency in Field Defect
Colorectal	MGMT	55%	54%
Colorectal	MSH2	13%	5%
Colorectal	WRN	29%	13%
Head and Neck	MGMT	54%	38%
Head and Neck	MLH1	33%	25%
Non-small cell lung cancer	ATM	69%	59%
Non-small cell lung cancer	MLH1	69%	72%
Stomach	MGMT	88%	78%
Stomach	MLH1	73%	20%
Esophagus	MLH1	77%-100%	23%-79%

While DNA damages may give rise to mutations through error prone translesion synthesis, DNA damages can also give rise to epigenetic alterations during faulty DNA repair processes. The DNA damages that accumulate due to hypermethylation of the promoters of DNA repair genes can be a source of the increased epigenetic alterations found in many genes in cancers. 

In an early study, looking at a limited set of transcriptional promoters, Fernandez et al. examined the DNA methylation profiles of 855 primary tumors. Comparing each tumor type with its corresponding normal tissue, 729 CpG island sites (55% of the 1322 CpG island sites evaluated) showed differential DNA methylation. Of these sites, 496 were hypermethylated (repressed) and 233 were hypomethylated (activated). Thus, there is a high level of promoter methylation alterations in tumors. Some of these alterations may contribute to cancer progression.

DNA methylation of microRNAs in cancer

In mammals, microRNAs (miRNAs) regulate the transcriptional activity of about 60% of protein-encoding genes. Individual miRNAs can each target, and repress transcription of, on average, roughly 200 messenger RNAs of protein coding genes. The promoters of about one third of the 167 miRNAs evaluated by Vrba et al. in normal breast tissues were differentially hyper/hypo-methylated in breast cancers. A more recent study pointed out that the 167 miRNAs evaluated by Vrba et al. were only 10% of the miRNAs found expressed in breast tissues. This later study found that 58% of the miRNAs in breast tissue had differentially methylated regions in their promoters in breast cancers, including 278 hypermethylated miRNAs and 802 hypomethylated miRNAs.

One miRNA that is over-expressed about 100-fold in breast cancers is miR-182. MiR-182 targets the BRCA1 messenger RNA and may be a major cause of reduced BRCA1 protein expression in many breast cancers.

microRNAs that control DNA methyltransferase genes in cancer

Some miRNAs target the messenger RNAs for DNA methyltransferase genes DNMT1, DNMT3A and DNMT3B, whose gene products are needed for initiating and stabilizing promoter methylations. As summarized in three reviews, miRNAs miR-29a, miR-29b and miR-29c target DNMT3A and DNMT3B; miR-148a and miR-148b target DNMT3B; and miR-152 and miR-301 target DNMT1. In addition, miR-34b targets DNMT1 and the promoter of miR-34b itself is hypermethylated and under-expressed in the majority of prostate cancers. When expression of these microRNAs is altered, they may also be a source of the hyper/hypo-methylation of the promoters of protein-coding genes in cancers.

Genome instability

From Wikipedia, the free encyclopedia

Genome instability (also genetic instability or genomic instability) refers to a high frequency of mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy. Genome instability does occur in bacteria. In multicellular organisms genome instability is central to carcinogenesis, and in humans it is also a factor in some neurodegenerative diseases such as amyotrophic lateral sclerosis or the neuromuscular disease myotonic dystrophy. 

The sources of genome instability have only recently begun to be elucidated. A high frequency of externally caused DNA damage can be one source of genome instability since DNA damages can cause inaccurate translesion synthesis past the damages or errors in repair, leading to mutation. Another source of genome instability may be epigenetic or mutational reductions in expression of DNA repair genes. Because endogenous (metabolically-caused) DNA damage is very frequent, occurring on average more than 60,000 times a day in the genomes of human cells, any reduced DNA repair is likely an important source of genome instability.

The usual genome situation

Usually, all cells in an individual in a given species (plant or animal) show a constant number of chromosomes, which constitute what is known as the karyotype defining this species (see also List of number of chromosomes of various organisms), although some species present a very high karyotypic variability. In humans, mutations that would change an amino acid within the protein coding region of the genome occur at an average of only 0.35 per generation (less than one mutated protein per generation).

Sometimes, in a species with a stable karyotype, random variations that modify the normal number of chromosomes may be observed. In other cases, there are structural alterations (chromosomal translocations, deletions ...) that modify the standard chromosomal complement. In these cases, it is indicated that the affected organism presents genome instability (also genetic instability, or even chromosomic instability). The process of genome instability often leads to a situation of aneuploidy, in which the cells present a chromosomic number that is either higher or lower than the normal complement for the species.

Causes of genome instability

DNA Replication Defects

In the cell cycle, DNA is usually most vulnerable during replication. The replisome must be able to navigate obstacles such as tightly wound chromatin with bound proteins, single and double stranded breaks which can lead to the stalling of the replication fork. Each protein or enzyme in the replisome must perform its function well to result in a perfect copy of DNA. Mutations of proteins such as DNA polymerase, ligase, can lead to impairment of replication and lead to spontaneous chromosomal exchanges. Proteins such as Tel1, Mec1 (ATR, ATM in humans) can detect single and double-stranded breaks and recruit factors such as Rmr3 helicase to stabilize the replication fork in order to prevent its collapse. Mutations in Tel1, Mec1, and Rmr3 helicase result in a significant increase of chromosomal recombination. ATR responds specifically to stalled replication forks and single-stranded breaks resulting from UV damage while ATM responds directly to double-stranded breaks. These proteins also prevent progression into mitosis by inhibiting the firing of late replication origins until the DNA breaks are fixed by phosphorylating CHK1, CHK2 which results in a signaling cascade arresting the cell in S-phase. For single stranded breaks, replication occurs until the location of the break, then the other strand is nicked to form a double stranded break, which can then be repaired by Break Induced Replication or homologous recombination using the sister chromatid as an error-free template. In addition to S-phase checkpoints, G1 and G2 checkpoints exist to check for transient DNA damage which could be caused by mutagens such as UV damage. An example is the Saccharomyces pombe gene rad9 which arrests the cells in late S/G2 phase in the presence of DNA damage caused by radiation. The yeast cells with defective rad9 failed to arrest following radiation, continued cell division and died rapidly while the cells with wild-type rad9 successfully arrested in late S/G2 phase and remained viable. The cells that arrested were able to survive due to the increased time in S/G2 phase allowing for DNA repair enzymes to function fully.

Fragile Sites

There are hotspots in the genome where DNA sequences are prone to gaps and breaks after inhibition of DNA synthesis such as in the aforementioned checkpoint arrest. These sites are called fragile sites, and can occur commonly as naturally present in most mammalian genomes or occur rarely as a result of mutations, such as DNA-repeat expansion. Rare fragile sites can lead to genetic disease such as fragile X mental retardation syndrome, myotonic dystrophy, Friedrich’s ataxia, and Huntington’s disease, most of which are caused by expansion of repeats at the DNA, RNA, or protein level. Although, seemingly harmful, these common fragile sites are conserved all the way to yeast and bacteria. These ubiquitous sites are characterized by trinucleotide repeats, most commonly CGG, CAG, GAA, and GCN. These trinucleotide repeats can form into hairpins, leading to difficulty of replication. Under replication stress, such as defective machinery or further DNA damage, DNA breaks and gaps can form at these fragile sites. Using a sister chromatid as repair is not a fool-proof backup as the surrounding DNA information of the n and n+1 repeat is virtually the same, leading to copy number variation. For example, the 16th copy of CGG might be mapped to the 13th copy of CGG in the sister chromatid since the surrounding DNA is both CGGCGGCGG…, leading to 3 extra copies of CGG in the final DNA sequence.

Transcription-associated instability

In both E. coli and Saccromyces pombe, transcription sites tend to have higher recombination and mutation rates. The coding or non-transcribed strand accumulates more mutations than the template strand. This is due to the fact that the coding strand is single-stranded during transcription, which is chemically more unstable than double-stranded DNA. During elongation of transcription, supercoiling can occur behind an elongating RNA polymerase, leading to single-stranded breaks. When the coding strand is single-stranded, it can also hybridize with itself, creating DNA secondary structures that can compromise replication. In E. coli, when attempting to transcribe GAA triplets such as those found in Friedrich’s ataxia, the resulting RNA and template strand can form mismatched loops between different repeats, leading the complementary segment in the coding-strand available to form its own loops which impede replication. Furthermore, replication of DNA and transcription of DNA are not temporally independent; they can occur at the same time and lead to collisions between the replication fork and RNA polymerase complex. In S. cerevisiae, Rrm3 helicase is found at highly transcribed genes in the yeast genome, which is recruited to stabilize a stalling replication fork as described above. This suggests that transcription is an obstacle to replication, which can lead to increased stress in the chromatin spanning the short distance between the unwound replication fork and transcription start site, potentially causing single-stranded DNA breaks. In yeast, proteins act as barriers at the 3’ of the transcription unit to prevent further travel of the DNA replication fork.

Increase Genetic Variability

In some portions of the genome, variability is essential to survival. One such locale is the Ig genes. In a pre-B cell, the region consists of all V, D, and J segments. During development of the B cell, a specific V, D, and J segment is chosen to be spliced together to form the final gene, which is catalyzed by RAG1 and RAG2 recombinases. Activation-Induced Cytidine Deaminase (AID) then converts cytidine into uracil. Uracil normally does not exist in DNA, and thus the base is excised and the nick is converted into a double-stranded break which is repaired by non-homologous end joining (NHEJ). This procedure is very error-prone and leads to somatic hypermutation. This genomic instability is crucial in ensuring mammalian survival against infection. V, D, J recombination can ensure millions of unique B-cell receptors; however, random repair by NHEJ introduces variation which can create a receptor that can bind with higher affinity to antigens.

In neuronal and neuromuscular disease

Of about 200 neurological and neuromuscular disorders, 15 have a clear link to an inherited or acquired defect in one of the DNA repair pathways or excessive genotoxic oxidative stress. Five of them (xeroderma pigmentosum, Cockayne's syndrome, trichothiodystrophy, Down's syndrome, and triple-A syndrome) have a defect in the DNA nucleotide excision repair pathway. Six (spinocerebellar ataxia with axonal neuropathy-1, Huntington's disease, Alzheimer's disease, Parkinson's disease, Down's syndrome and amyotrophic lateral sclerosis) seem to result from increased oxidative stress, and the inability of the base excision repair pathway to handle the damage to DNA that this causes. Four of them (Huntington's disease, various spinocerebellar ataxias, Friedreich’s ataxia and myotonic dystrophy types 1 and 2) often have an unusual expansion of repeat sequences in DNA, likely attributable to genome instability. Four (ataxia-telangiectasia, ataxia-telangiectasia-like disorder, Nijmegen breakage syndrome and Alzheimer's disease) are defective in genes involved in repairing DNA double-strand breaks. Overall, it seems that oxidative stress is a major cause of genomic instability in the brain. A particular neurological disease arises when a pathway that normally prevents oxidative stress is deficient, or a DNA repair pathway that normally repairs damage caused by oxidative stress is deficient.

In cancer

In cancer, genome instability can occur prior to or as a consequence of transformation. Genome instability can refer to the accumulation of extra copies of DNA or chromosomes, chromosomal translocations, chromosomal inversions, chromosome deletions, single-strand breaks in DNA, double-strand breaks in DNA, the intercalation of foreign substances into the DNA double helix, or any abnormal changes in DNA tertiary structure that can cause either the loss of DNA, or the misexpression of genes. Situations of genome instability (as well as aneuploidy) are common in cancer cells, and they are considered a "hallmark" for these cells. The unpredictable nature of these events are also a main contributor to the heterogeneity observed among tumour cells. 

It is currently accepted that sporadic tumors (non-familial ones) are originated due to the accumulation of several genetic errors. An average cancer of the breast or colon can have about 60 to 70 protein altering mutations, of which about 3 or 4 may be "driver" mutations, and the remaining ones may be "passenger" mutations Any genetic or epigenetic lesion increasing the mutation rate will have as a consequence an increase in the acquisition of new mutations, increasing then the probability to develop a tumor. During the process of tumorogenesis, it is known that diploid cells acquire mutations in genes responsible for maintaining genome integrity (caretaker genes), as well as in genes that are directly controlling cellular proliferation (gatekeeper genes). Genetic instability can originate due to deficiencies in DNA repair, or due to loss or gain of chromosomes, or due to large scale chromosomal reorganizations. Losing genetic stability will favour tumor development, because it favours the generation of mutants that can be selected by the environment.

The tumor microenvironment has an inhibitory effect on DNA repair pathways contributing to genomic instability, which promotes tumor survival, proliferation, and malignant transformation.

Low frequency of mutations without cancer

The protein coding regions of the human genome, collectively called the exome, constitutes only 1.5% of the total genome. As pointed out above, ordinarily there are only an average of 0.35 mutations in the exome per generation (parent to child) in humans. In the entire genome (including non-protein coding regions) there are only about 70 new mutations per generation in humans.

Cause of mutations in cancer

The likely major underlying cause of mutations in cancer is DNA damage. For example, in the case of lung cancer, DNA damage is caused by agents in exogenous genotoxic tobacco smoke (e.g. acrolein, formaldehyde, acrylonitrile, 1,3-butadiene, acetaldehyde, ethylene oxide and isoprene). Endogenous (metabolically-caused) DNA damage is also very frequent, occurring on average more than 60,000 times a day in the genomes of human cells (see DNA damage (naturally occurring)). Externally and endogenously caused damages may be converted into mutations by inaccurate translesion synthesis or inaccurate DNA repair (e.g. by non-homologous end joining). In addition, DNA damages can also give rise to epigenetic alterations during DNA repair. Both mutations and epigenetic alterations (epimutations) can contribute to progression to cancer.

Very frequent mutations in cancer

As noted above, about 3 or 4 driver mutations and 60 passenger mutations occur in the exome (protein coding region) of a cancer. However, a much larger number of mutations occur in the non-protein-coding regions of DNA. The average number of DNA sequence mutations in the entire genome of a breast cancer tissue sample is about 20,000. In an average melanoma tissue sample (where melanomas have a higher exome mutation frequency) the total number of DNA sequence mutations is about 80,000.

Cause of high frequency of mutations in cancer

The high frequency of mutations in the total genome within cancers suggests that, often, an early carcinogenic alteration may be a deficiency in DNA repair. Mutation rates substantially increase (sometimes by 100-fold) in cells defective in DNA mismatch repair or in homologous recombinational DNA repair. Also, chromosomal rearrangements and aneuploidy increase in humans defective in DNA repair gene BLM.

A deficiency in DNA repair, itself, can allow DNA damages to accumulate, and error-prone translesion synthesis past some of those damages may give rise to mutations. In addition, faulty repair of these accumulated DNA damages may give rise to epigenetic alterations or epimutations. While a mutation or epimutation in a DNA repair gene, itself, would not confer a selective advantage, such a repair defect may be carried along as a passenger in a cell when the cell acquires an additional mutation/epimutation that does provide a proliferative advantage. Such cells, with both proliferative advantages and one or more DNA repair defects (causing a very high mutation rate), likely give rise to the 20,000 to 80,000 total genome mutations frequently seen in cancers.

DNA repair deficiency in cancer

In somatic cells, deficiencies in DNA repair sometimes arise by mutations in DNA repair genes, but much more often are due to epigenetic reductions in expression of DNA repair genes. Thus, in a sequence of 113 colorectal cancers, only four had somatic missense mutations in the DNA repair gene MGMT, while the majority of these cancers had reduced MGMT expression due to methylation of the MGMT promoter region. Five reports, listed in the article Epigenetics (see section "DNA repair epigenetics in cancer") presented evidence that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region. 

Similarly, for 119 cases of colorectal cancers classified as mismatch repair deficient and lacking DNA repair gene PMS2 expression, Pms2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). The other 10 cases of loss of PMS2 expression were likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.

In cancer epigenetics (see section Frequencies of epimutations in DNA repair genes), there is a partial listing of epigenetic deficiencies found in DNA repair genes in sporadic cancers. These include frequencies of between 13–100% of epigenetic defects in genes BRCA1, WRN, FANCB, FANCF, MGMT, MLH1, MSH2, MSH4, ERCC1, XPF, NEIL1 and ATM located in cancers including breast, ovarian, colorectal and head and neck. Two or three epigenetic deficiencies in expression of ERCC1, XPF and/or PMS2 were found to occur simultaneously in the majority of the 49 colon cancers evaluated. Some of these DNA repair deficiencies can be caused by epimutations in microRNAs as summarized in the MicroRNA article section titled miRNA, DNA repair and cancer.

Lymphomas as a consequence of genome instability

Cancers usually result from disruption of a tumor repressor or dysregulation of an oncogene. Knowing that B-cells experience DNA breaks through development can give insight to the genome of lymphomas. Many types of lymphoma are caused by chromosomal translocation, which can arise from breaks in DNA leading to incorrect joining. In Burkitt’s lymphoma, c-myc, an oncogene encoding a transcription factor, is translocated after the promoter of the immunoglobulin gene, leading dysregulation of c-myc transcription. Since immunoglobulins are essential to a lymphocyte and highly expressed to increase detection of antigens, c-myc is then also highly expressed leading to transcription of its targets which are involved in cell proliferation. Mantle cell lymphoma is characterized by fusion of cyclin D1 to the immunoglobulin locus. Cyclin D1 inhibits Rb, a tumor suppressor, leading to tumorigenesis. Follicular lymphoma results from the translocation of immunoglobulin promoter to the Bcl-2 gene, giving rise to large amounts of Bcl-2 protein which inhibits apoptosis. DNA-damaged B-cells no longer undergo apoptosis leading to further mutations which could affect driver genes leading to tumorigenesis. The location of translocation in the oncogene shares structural properties of the target regions of AID, suggesting that the oncogene was a potential target of AID, leading to a double-stranded break that was translocated to the immunoglobulin gene locus through NHEJ repair.

Telomerase

From Wikipedia, the free encyclopedia

Tribolium castaneum telomerase catalytic subunit, TERT, bound to putative RNA template and telomeric DNA (PDB 3KYL)

 

RNA-directed DNA polymerase
A conceptual diagram showing the protein component of telomerase (TERT) in grey and the RNA component (TR) in yellow
Identifiers
EC number	2.7.7.49
CAS number	9068-38-6
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / QuickGO

Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of eukaryotic chromosomes in most eukaryotes. Telomeres protect the end of the chromosome from DNA damage or from fusion with neighbouring chromosomes. The fruit fly Drosophila melanogaster lacks telomerase, but instead uses retrotransposons to maintain telomeres.

Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule (e.g., with the sequence 3′-CCCAAUCCC-5′ in Trypanosoma brucei) which is used as a template when it elongates telomeres. Telomerase is active in normal stem cells and most cancer cells, but is normally absent from, or at very low levels in, most somatic cells.

History

The existence of a compensatory mechanism for telomere shortening was first found by Soviet biologist Alexey Olovnikov in 1973, who also suggested the telomere hypothesis of aging and the telomere's connections to cancer. 

Telomerase in the ciliate Tetrahymena was discovered by Carol W. Greider and Elizabeth Blackburn in 1984. Together with Jack W. Szostak, Greider and Blackburn were awarded the 2009 Nobel Prize in Physiology or Medicine for their discovery.

The role of telomeres and telomerase in cell aging and cancer was established by scientists at biotechnology company Geron with the cloning of the RNA and catalytic components of human telomerase and the development of a polymerase chain reaction (PCR) based assay for telomerase activity called the TRAP assay, which surveys telomerase activity in multiple types of cancer.

The negative stain electron microscopy (EM) structures of human and Tetrahymena telomerases were characterized in 2013. Two years later, the first cryo-electron microscopy (cryo-EM) structure of telomerase holoenzyme (Tetrahymena) was determined. In 2018, the structure of human telomerase was determined through cryo-EM by UC Berkeley scientists.

Human telomerase structure

The molecular composition of the human telomerase complex was determined by Scott Cohen and his team at the Children's Medical Research Institute (Sydney Australia) and consists of two molecules each of human telomerase reverse transcriptase (TERT), telomerase RNA (TR or TERC), and dyskerin (DKC1). The genes of telomerase subunits, which include TERT, TERC, DKC1 and TEP1, are located on different chromosomes. The human TERT gene (hTERT) is translated into a protein of 1132 amino acids. TERT polypeptide folds with (and carries) TERC, a non-coding RNA (451 nucleotides long). TERT has a 'mitten' structure that allows it to wrap around the chromosome to add single-stranded telomere repeats. 

TERT is a reverse transcriptase, which is a class of enzyme that creates single-stranded DNA using single-stranded RNA as a template. 

An image illustrating how telomerase elongates telomere ends progressively.

 

The protein consists of four conserved domains (RNA-Binding Domain (TRBD), fingers, palm and thumb), organized into a ring configuration that shares common features with retroviral reverse transcriptases, viral RNA polymerases and bacteriophage B-family DNA polymerases.

TERT proteins from many eukaryotes have been sequenced.

Mechanism

By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-TTAGGG (in vertebrates, the sequence differs in other organisms) to the 3' strand of chromosomes. These TTAGGG repeats (with their various protein binding partners) are called telomeres. The template region of TERC is 3'-CAAUCCCAAUC-5'.

Telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a new telomere repeat (5'-GGTTAG-3') sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. Telomerase reverses telomere shortening.

Clinical implications

Aging

Telomerase replaces short bits of DNA known as telomeres, which are otherwise shortened when a cell divides via mitosis. 

In normal circumstances, where telomerase is absent, if a cell divides recursively, at some point the progeny reach their Hayflick limit, which is believed to be between 50–70 cell divisions. At the limit the cells become senescent and cell division stops. Telomerase allows each offspring to replace the lost bit of DNA, allowing the cell line to divide without ever reaching the limit. This same unbounded growth is a feature of cancerous growth.

Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is highly expressed only in cells that need to divide regularly, especially in male sperm cells but also in epidermal cells, in activated T cell and B cell lymphocytes, as well as in certain adult stem cells, but in the great majority of cases somatic cells do not express telomerase.

A comparative biology study of mammalian telomeres indicated that telomere length of some mammalian species correlates inversely, rather than directly, with lifespan, and concluded that the contribution of telomere length to lifespan is unresolved. Telomere shortening does not occur with age in some postmitotic tissues, such as in the rat brain. In humans, skeletal muscle telomere lengths remain stable from ages 23 –74. In baboon skeletal muscle, which consists of fully differentiated post-mitotic cells, less than 3% of myonuclei contain damaged telomeres and this percentage does not increase with age. Thus, telomere shortening does not appear to be a major factor in the aging of the differentiated cells of brain or skeletal muscle. In human liver, cholangiocytes and hepatocytes show no age-related telomere shortening. Another study found little evidence that, in humans, telomere length is a significant biomarker of normal aging with respect to important cognitive and physical abilities.

Some experiments have raised questions on whether telomerase can be used as an anti-aging therapy, namely, the fact that mice with elevated levels of telomerase have higher cancer incidence and hence do not live longer. Telomerase also favors tumorogenesis, which leads to questions about its potential as an anti-aging therapy. On the other hand, one study showed that activating telomerase in cancer-resistant mice by overexpressing its catalytic subunit extended lifespan.

A study that focused on Ashkenazi Jews found that long-lived subjects inherited a hyperactive version of telomerase.

Premature aging

Premature aging syndromes including Werner syndrome, Ataxia telangiectasia, Ataxia-telangiectasia like disorder, Bloom syndrome, Fanconi anemia and Nijmegen breakage syndrome are associated with short telomeres. However, the genes that have mutated in these diseases all have roles in the repair of DNA damage and the increased DNA damage may, itself, be a factor in the premature aging. An additional role in maintaining telomere length is an active area of investigation.

Cancer

In vitro, when cells approach the Hayflick limit, the time to senescence can be extended by inactivating the tumor suppressor proteins - p53 and Retinoblastoma protein (pRb). Cells that have been so-altered eventually undergo an event termed a "crisis" when the majority of the cells in the culture die. Sometimes, a cell does not stop dividing once it reaches crisis. In a typical situation, the telomeres are shortened and chromosomal integrity declines with every subsequent cell division. Exposed chromosome ends are interpreted as double-stranded breaks (DSB) in DNA; such damage is usually repaired by reattaching (religating) the broken ends together. When the cell does this due to telomere-shortening, the ends of different chromosomes can be attached to each other. This solves the problem of lacking telomeres, but during cell division anaphase, the fused chromosomes are randomly ripped apart, causing many mutations and chromosomal abnormalities. As this process continues, the cell's genome becomes unstable. Eventually, either fatal damage is done to the cell's chromosomes (killing it via apoptosis), or an additional mutation that activates telomerase occurs.

With telomerase activation some types of cells and their offspring become immortal (bypass the Hayflick limit), thus avoiding cell death as long as the conditions for their duplication are met. Many cancer cells are considered 'immortal' because telomerase activity allows them to live much longer than any other somatic cell, which, combined with uncontrollable cell proliferation is why they can form tumors. A good example of immortal cancer cells is HeLa cells, which have been used in laboratories as a model cell line since 1951. 

While this method of modeling human cancer in cell culture is effective and has been used for many years by scientists, it is also very imprecise. The exact changes that allow for the formation of the tumorigenic clones in the above-described experiment are not clear. Scientists addressed this question by the serial introduction of multiple mutations present in a variety of human cancers. This has led to the identification of mutation combinations that form tumorigenic cells in a variety of cell types. While the combination varies by cell type, the following alterations are required in all cases: TERT activation, loss of p53 pathway function, loss of pRb pathway function, activation of the Ras or myc proto-oncogenes, and aberration of the PP2A protein phosphatase. That is to say, the cell has an activated telomerase, eliminating the process of death by chromosome instability or loss, absence of apoptosis-induction pathways, and continued mitosis activation. 

This model of cancer in cell culture accurately describes the role of telomerase in actual human tumors. Telomerase activation has been observed in ~90% of all human tumors, suggesting that the immortality conferred by telomerase plays a key role in cancer development. Of the tumors without TERT activation, most employ a separate pathway to maintain telomere length termed Alternative Lengthening of Telomeres (ALT ). The exact mechanism behind telomere maintenance in the ALT pathway is unclear, but likely involves multiple recombination events at the telomere. 

Elizabeth Blackburn et al., identified the upregulation of 70 genes known or suspected in cancer growth and spread through the body, and the activation of glycolysis, which enables cancer cells to rapidly use sugar to facilitate their programmed growth rate (roughly the growth rate of a fetus).

Approaches to controlling telomerase and telomeres for cancer therapy include gene therapy, immunotherapy, small-molecule and signal pathway inhibitors.

Drugs

The ability to maintain functional telomeres may be one mechanism that allows cancer cells to grow in vitro for decades. Telomerase activity is necessary to preserve many cancer types and is inactive in somatic cells, creating the possibility that telomerase inhibition could selectively repress cancer cell growth with minimal side effects. If a drug can inhibit telomerase in cancer cells, the telomeres of successive generations will progressively shorten, limiting tumor growth.

Telomerase is a good biomarker for cancer detection because most human cancers cells express high levels of it. Telomerase activity can be identified by its catalytic protein domain (hTERT). This is the rate-limiting step in telomerase activity. It is associated with many cancer types. Various cancer cells and fibroblasts transformed with hTERT cDNA have high telomerase activity, while somatic cells do not. Cells testing positive for hTERT have positive nuclear signals. Epithelial stem cell tissue and its early daughter cells are the only noncancerous cells in which hTERT can be detected. Since hTERT expression is dependent only on the number of tumor cells within a sample, the amount of hTERT indicates the severity of a cancer.

The expression of hTERT can also be used to distinguish benign tumors from malignant tumors. Malignant tumors have higher hTERT expression than benign tumors. Real-time reverse transcription polymerase chain reaction (RT-PCR) quantifying hTERT expression in various tumor samples verified this varying expression.

Figure 4:A) Tumor cells expressing hTERT will actively degrade some of the protein and process for presenting. The major histocompatibility complex 1(MHC1), can then present the hTERT epitote. CD8- T cells that have antibodies against hTERT will then bind to the presented epitote. B) As a result of the antigenic binding, the T cells will release cytotoxins, which can be absorbed by the affected cell. C) These cytotoxins induce multiple proteases and results in apoptosis (or cell death).

 

The lack of telomerase does not affect cell growth, until the telomeres are short enough to cause cells to “die or undergo growth arrest”. However, inhibiting telomerase alone is not enough to destroy large tumors. It must be combined with surgery, radiation, chemotherapy, or immunotherapy.

Cells may reduce their telomere length by only 50-252 base pairs per cell division, which can lead to a long lag phase.

Immunotherapy

Immunotherapy successfully treats some kinds of cancer, such as melanoma. This treatment involves manipulating a human’s immune system to destroy cancerous cells. Humans have two major antigen identifying lymphocytes: CD8+ cytotoxic T-lymphocytes (CTL) and CD4+ helper T-lymphocytes that can destroy cells. Antigen receptors on CTL can bind to a 9-10 amino acid chain that is presented by the major histocompatibility complex (MHC) as in Figure 4. HTERT is a potential target antigen. Immunotargeting should result in relatively few side effects since hTERT expression is associated only with telomerase and is not essential in almost all somatic cells. GV1001 uses this pathway. Experimental drug and vaccine therapies targeting active telomerase have been tested in mouse models, and clinical trials have begun. One drug, imetelstat, is being clinically researched as a means of interfering with telomerase in cancer cells. Most of the harmful cancer-related effects of telomerase are dependent on an intact RNA template. Cancer stem cells that use an alternative method of telomere maintenance are still killed when telomerase's RNA template is blocked or damaged.

Telomerase Vaccines

Two telomerase vaccines have been developed: GRNVAC1 and GV1001. GRNVAC1 isolates dendritic cells and the RNA that codes for the telomerase protein and puts them back into the patient to make cytotoxic T cells that kill the telomerase-active cells. GV1001 is a peptide from the active site of hTERT and is recognized by the immune system that reacts by killing the telomerase-active cells.

Targeted apoptosis

Figure 5: A) Human telomerase RNA (hTR) is present in the cell and can be targeted. B) 2-5 anti-hTR oligonucleotides is a specialized antisense oligo that can bind to the telomerase RNA. C) Once bound, the 2-5 anti-hTR oligonucleotide recruits RNase L to the sequence. Once recruited, the RNase L creates a single cleavage in the RNA (D) and causes dissociation of the RNA sequence.

 

Another independent approach is to use oligoadenylated anti-telomerase antisense oligonucleotides and ribozymes to target telomerase RNA, reducing dissociation and apoptosis (Figure 5). The fast induction of apoptosis through antisense binding may be a good alternative to the slower telomere shortening.

Small interfering RNA (siRNA)

siRNAs are small RNA molecules that induce the sequence-specific degradation of other RNAs. siRNA treatment can function similar to traditional gene therapy by destroying the mRNA products of particular genes, and therefore preventing the expression of those genes. A 2012 study found that targeting TERC with an siRNA reduced telomerase activity by more than 50% and resulted in decreased viability of immortal cancer cells. Treatment with both the siRNA and radiation caused a greater reduction in tumor size in mice than treatment with radiation alone, suggesting that targeting telomerase could be a way to increase the efficacy of radiation in treating radiation-resistant tumors.

Heart disease, diabetes and quality of life

Blackburn also discovered that mothers caring for very sick children have shorter telomeres when they report that their emotional stress is at a maximum and that telomerase was active at the site of blockages in coronary artery tissue, possibly accelerating heart attacks. 

In 2009, it was shown that the amount of telomerase activity significantly increased following psychological stress. Across the sample of patients telomerase activity in increased peripheral blood mononuclear cells by 18% one hour after the end of the stress.

A study in 2010 found that there was "significantly greater" telomerase activity in participants than controls after a three-month meditation retreat.

Telomerase deficiency has been linked to diabetes mellitus and impaired insulin secretion in mice, due to loss of pancreatic insulin-producing cells.

Rare human diseases

Mutations in TERT have been implicated in predisposing patients to aplastic anemia, a disorder in which the bone marrow fails to produce blood cells, in 2005.

Cri du chat syndrome (CdCS) is a complex disorder involving the loss of the distal portion of the short arm of chromosome 5. TERT is located in the deleted region, and loss of one copy of TERT has been suggested as a cause or contributing factor of this disease.

Dyskeratosis congenita (DC) is a disease of the bone marrow that can be caused by some mutations in the telomerase subunits. In the DC cases, about 35% cases are X-linked-recessive on the DKC1 locus and 5% cases are autosomal dominant on the TERT and TERC loci.

Patients with DC have severe bone marrow failure manifesting as abnormal skin pigmentation, leucoplakia (a white thickening of the oral mucosa) and nail dystrophy, as well as a variety of other symptoms. Individuals with either TERC or DKC1 mutations have shorter telomeres and defective telomerase activity in vitro versus other individuals of the same age.

In one family autosomal dominant DC was linked to a heterozygous TERT mutation. These patients also exhibited an increased rate of telomere-shortening, and genetic anticipation (i.e., the DC phenotype worsened with each generation).