One of the premises for the study of relatively rare inherited cancers is the assumption that understanding the molecular basis of these malignancies will result in better understanding and treatment strategies of the more common malignancies. While this has not yet resulted in new therapies, the management at diagnosis of many patients with an inherited predisposition has been radically altered. Thus, a working knowledge of cancer genetics will allow the clinician not only to appropriately manage patients, but also to comprehend the major advances in the field of oncology (1).
At the molecular level, cancer is caused by mutations in DNA, which result in aberrant cell proliferation. Most of these mutations are acquired in age-dependent manner and occur in somatic (uninherited) cells. However, some people inherit mutations in the germline (the gamets and their precursors). Inherited or genetic forms of cancer may be due to mutations in genes that directly control cell growth or apoptosis (1). Cancer results from an accumulation of multiple genetic alterations that either inhibit or enhance normal cellular processes. Through a process of continual genetic evolution a cell requires new phenotypes able to proliferate, invade, disseminate throughout the body, escape immune surveillance and resist treatment. Many genetic alterations have been recognized in human tumors. Some of these confer a survival advantage and hence lead to clonally expansion of the founder cell. Most are frequent (e,g. p53 mutation) found in a wide variety of tumors. Other genetic aberrations are clearly related to individual tumors and either reflects the predisposition in such tissues to these genetic changes or the alterations required to overcome that tissue’s normal regulatory control (2).
Many genes whose alteration is associated with carcinogenesis have been identified. Although initially known as oncogenes, the identification of inhibitory genes has led to the classification of cancer genes as either oncogenes or tumor suppressor genes.
Oncogenes are derived from normal cellular genes called proto-oncogenes. Proto-oncogenes were first elucidated in RNA tumor virus, and are now known to encode proteins that are crucial for normal cellular growth regulation including growth factors, components of the intercellular signaling pathways, DNA binding proteins, cell surface receptors and components of the cell cycle progression pathways. Oncogenes have been identified in human tumors which affect most components of signal transduction pathways such as growth factors (ERB-B2), GTP-binding proteins (ras) and nuclear transcription factors (myc). Proto-oncogenes become activated by:
Alterations of the nucleotide sequence lead to either an alteration in the amino acid sequence, or premature termination of translation may produce a protein with abnormal function.
- Chromosomal rearrangement.
Chromosomal translocation may lead to the formation of a novel fusion protein or loss of normal control of proto-oncogene expression.
- Amplification .
Multiple copies of a proto-oncogene, may result from dysregulated chromosomal replication. This in turn leads to inappropriate high levels of [removed]2).
As well as initiating an oncogenic process, oncogenes are thought to be important in the maintenance of solid tumors. Inherited mutations of oncogenes are rare- it is thought that most of these would be lethal. However, mutations in the RET oncogene and the MET oncogene have been described and cause inherited susceptibility to malignancy (multiple endocrine neoplasia type 2 and familial papillary renal cell carcinoma ) (1). The ras pathway is central to the transmission of a growth factor signal from cell membrane to nucleus. Activation of three ras genes (Ha-ras, Ki-ras, N-ras) by point mutation is the most frequent dominant oncogene abnormality in human cancer. The ras mutations are frequently seen in bladder, lung and colorectal carcinoma (2).
The myc family genes (c-myc, L-myc, N-myc) are frequently activated by amplification to form oncogenes. The myc in combination with associated proteins (MAX, MAD) acts as a transcription factor controlling the expression of genes associated with many tumors such as small-cell lung carcinoma and neuroblastoma. A chromosomal translocation in Burkitt’s lymphoma leads to c-myc activation (2).
The mdm-2 oncogene does not produce a component of the transduction pathway of a growth signal. The mdm-2 protein acts by binding and inactivating genes such as p53 (an example of an oncogene affecting a tumor suppressor gene). The mdm-2 over expression has been identified in soft tissue sarcomas (2).
Tumor suppressor genes
These are defined as genes in which mutation or other genetic modification leads to a loss of function which is then associated with tumor formation. The normal function of the protein product of these genes is generally central to the control of cell division and differentiation. Typically, both copies of a tumor suppressor gene must be affected to lead to loss of the protein’s function that is, the gene acts recessively.
Inactivation of a tumor suppressor gene may result from:
Deletion of both copies of a tumor suppressor gene (homozygous deletion) may occur. Deletion of a single copy with inactivation of the other allele by alternative means is the most frequent mechanism leading to inactivation of a tumor suppressor gene. The deletion of the single remaining allele by molecular techniques leads to a loss of heterozygosis, the characteristic marker of a region of the genome in which tumor suppressor gene is located.
Mutation of one or both alleles may result in tumor suppressor gene inactivation.
Inactivations of a tumor suppressor gene by means which do not alter the genetic sequence of the genome are known as epigenetic changes. For example, inappropriate methylation may lead to a loss of expression and hence inactivation of the cell cycle regulator p16 (2).
A small number of genes has been identified which act as tumor suppressor genes. These include:
- p53: the protein product of the p53 gene has a molecular weight of 53 kDa and is central to the control of the cell cycle and the response of the cell to stress such as DNA damage. p53 functions principally by controlling transcription of other genes which control pathways such as the cell cycle, DNA repair and apoptosis. Loss of normal p53 function is central to the pathogenesis of many malignancies; p53 mutations are the most common identified in human cancer, present in more than 50% of tumors. Germ-line deletion of one copy of the p53 gene leads to an increase in tumor incidence (Li-Fraumeni syndrome).
- pRb: the retinoblastoma gene (Rb1) produces a protein with an essential role in the control of the cell cycle; loss of normal function leads to a loss of control of the G1/S checkpoint and hence inappropriate proliferation (2).
RNA Interference (RNAi)
Geneticists have been puzzled by long RNA molecules that are made by mammalian genomes but do not code for protein. RNA interference (RNAi) is a system within living cells that takes part in controlling which genes are active and how active they are. It is a precious question what do they do?
Two types of small RNA molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific RNAs (mRNA) and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes – viruses and transposons – but also in directing development as well as gene expression in general. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of 20 nucleotides that are called siRNAs. Each siRNA is unwound into two single-stranded (ss) ssRNAs, namely the passenger strand and the guide strand. The passenger strand will be degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA. The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a Lentiviral Delivery of designed shRNA’s and the mechanism of RNA interference in mammalian cells(3).
microRNAs – the ubiquitous, short noncoding RNAs that regulate gene expression-are known to affect the levels of both messenger RNA(mRNA) and protein. Because protein production is dependent on the presence of mRNA, it has been difficult to establish the relative contributions of miRNA –mediated mRNA cleavage versus translational repression. Guo, et al (4) found that miRNA act mainly by destabilizing target mRNAs, rather than by inhibiting their translation.
The first miRNAs were characterized in the early 1990s. However, miRNAs were not recognized as a distinct class of biologic regulators with conserved functions until the early 2000s. Since then, miRNA research has revealed multiple roles in negative regulation (transcript degradation and sequestering, translational suppression) and possible involvement in positive regulation (transcriptional and translational activation). miRNAs are short ribonucleic acid molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing. The human genome may encode over 1000miRNAs, which may target about 60% of mammalian genes and are abundant in many human cell types. (5).
miRNA and cancer
By affecting gene regulation, miRNAs are likely to be involved in most biologic processes. Different sets of expressed miRNAs are found in different cell types and tissues. Aberrant expression of miRNAs has been implicated in numerous disease states, and miRNA-based therapies are under investigations (5). In many organisms, including humans, miRNAs have also been linked to the formation of tumors and dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and tumor suppressor gene (3).
Several miRNAs have been found to have links with some types of cancer. miRNA-21 is one of the first miRNAs that was identified as an oncomiR. A study of mice altered to produce excess c-Myc — a protein with mutated forms implicated in several cancers — shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days. Leukemia can be caused by the insertion of a viral genome next to the 17-92 arrays of miRNAs leading to increased expression of this miRNA. Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off. By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer. Transgenic mice that over-express or lack specific miRNAs have provided insight into the role of small RNAs in various malignancies. A novel miRNA-profiling based screening assay for the detection of early-stage colorectal cancer has been developed and is currently in clinical trials. Early results showed that blood plasma samples collected from patients with early, resectable (Stage II) colorectal cancer could be distinguished from those of sex-and age-matched healthy volunteers. Sufficient selectivity and specificity could be achieved using small (less than 1 mL) samples of blood. The test has potential to be a cost-effective, non-invasive way to identify at-risk patients who should undergo colonoscopy (5).
John Rinn and Maite Huarte at the Broad Institute in Cambridge, Massachusetts, and their colleagues reported that one long non-coding RNA is important in a cell’s response to the protein p53. Best known as a tumor suppressor, p53 controls the transcription of many genes. The team showed that it also triggers the production of several long non-coding RNAs and that one of these, lincRNA-p21, stifles the expression of many genes further downstream in the p53 response pathway, and promotes cell suicide. It seems to do this by associating with a second protein, hnRNP-K. The authors propose that other proteins like p53 activate long non-coding RNAs that help to silence genes.
Once, it all seemed so beautifully simple. Our DNA, we thought, consisted of a set of recipes, or genes, for making proteins, and once we had identified them all and worked out what they do, we would be a long way towards understanding what makes us what we are. For starters, rather than each gene coding for one protein, they often code for many. The coding parts of genes come in pieces, like beads, or exons, after RNA copies are made, a single gene can potentially code for tens of thousands of different beads, or exons, after RNA copies are made, a single gene can potentially code for tens of thousands of different proteins, although the average is about five. Recent studies suggest up to 95% of our genes may be alternatively spliced in this way (6).
It is the way in which genes are switched on and off, though, that has turned out to be really mind-bloggling, with layer after layer of complexity emerging. Early studies suggested that gene activity was regulated mainly by transcription factors –protein that binds to DNA, blocking or boosting the production of RNA copies of a gene and thus the amount of protein that genes produces (6).
While transcription factors do play a big role, cells also produces a wide variety of RNAs that, rather than coding for a protein, control gene activity. Some dubbed small interfering RNAs (siRNAs), from complexes that seek out and destroy RNA copies of genes with a complementary sequence, preventing protein production. microRNAs work in a similar way but are not specific, controlling the activity of many genes simultaneously. Piwi-acting RNAS, meanwhile, shutdown the parasitic genes that litter our genome to stop them wreaking havoc, though it’s not clear how (6). John Avise of the University of California, Irvine argued that splicing mistakes and errant miroRNAs play a role in some cancers. On the Brightside, discoveries like siRNA could lead to potent new treatments for all kind of diseases (6).
A large scale genetic analysis of different malignant samples has identified thousands of cancer-related mutations. Of these, most are thought to be significantly mutated, implying possible pathogenic roles in abnormal protein synthesis or gene silencing by the activation of RNAi. siRNA technology is now extensively recognized as a powerful tool for the specific suppression of gene expression and is presently being used by researchers in a wide range of disciplines for the assessment of gene function.
- Lalloo, F. In: Genetics for oncologists, The molecular genetic basis of oncologic disorders, ed. Cold Spring Harbor Laboratory, Remedica Publishing, London, (2002).
- Hall, G.D, Patel, P.M, Protheroe, A.S. Key topics in Oncology, ed. Selby. P. J., Bios Scientific Publishers Limited, (1998).
- Guo, H et al. Nature, 466: 835-840 (2010).
- Le Page, M. June NewScientist. 34-35 (2010).