2.3.3 Molecular basis of Cancer Cheat Sheet (DRAFT) by Molly

The two charac­ter­istics of cancer cells are their heritable ability to prolif­erate despite the normal constr­aints which inhibit cell prolif­era­tion, and their ability to invade neighb­ouring tissues.

Damage to DNA can be acquired due to exposure to carcin­ogens, or result from mistakes in DNA replic­ation. In addition inherited defects can be passed on from parental sources (germ-line mutati­ons).

Enviro­nmental carcin­ogens include UV light, cigarette smoke, asbestos and food additives.

In somatic cells that frequently divide there is less time for DNA repair to occur, prior to DNA replic­ation.

In addition with increasing age DNA repair mechanisms wane and the cumulative exposure to carcin­ogens and enviro­nmental exposure to various factors results in DNA damage which may not be adequately repaired.

The rate of mutation caused by the limited accuracy of DNA replic­ation and repair alone is estimated at 10-6 mutations per gene per cell division.

When a disruptive mutation occurs in a gene involved in the regulation of cell division, the cell may acquire the ability to prolif­erate indepe­ndently of normal cell cycle controls. As mutations caused by errors in DNA replic­ation and repair accumulate in the somatic tissue, and the ability of the cell to accurately repair DNA damage decreases during aging, the chance of cancer developing increases with age.

Ionizing radiation induces DNA damage directly or indirectly through the production of free radicals.

As compared to other types of DNA damage, double­-strand breaks are intrin­sically more difficult to accurately repair through homologous recomb­ina­tion, or non-ho­mologus end joining as no template exists for correct rejoining.

UV radiation is absorbed by the DNA molecule, which can result in the formation of a thymine dimer. As one of the four bases of the DNA molecule, the dimeri­sation of adjacent thymine nitrog­enous bases disrupts the activity of DNA polymerase during DNA replic­ation and without repair causes induction of mutations.

The most noxious chemical carcinogen is that found in cigare­ttes, which is respon­sible for up to 30% of all human tumours.

The more potent chemical carcin­ogens, including the fungal toxin aflatoxin B1, are not specif­ically reactive with DNA until being activated by metabolic processes involving the cytochrome P-450 oxidases.

Only a limited number of human cancer­-in­ducing viruses have been identi­fied, with these being mainly DNA viruses, with a smaller number of RNA retrov­iruses also described.

DNA viruses can carry genes that cause subversion of the normal cell cycle leading to uncont­rolled host cell prolif­era­tion. Retrov­iruses often cause genetic disruption in genes that promote cancer develo­pment following insertion of the viral gene either upstream of the proto-­onc­ogene, or within the coding sequence, leading to cell transf­orm­ation.

This type of cancer suscep­tib­ility gene is present in the inherited genetic material (germ-­line) of the individual that can be passed down from parent to child. Such cancer­-prone families often present with similar types of cancers in many members of the family over successive genera­tions.

It has been predicted that 5-10% of all cancers are part of defined inherited cancer syndromes.

Familial suscep­tib­ility for cancer can occur as inherited cancer suscep­tib­ility for a single type of cancer, or for a number of different types of cancer, as part of a familial cancer predis­posing syndrome.

 Several close (first degree relatives) with the same cancer;  Several close relatives with related cancers - breast and ovarian and endome­trial;  Two family members with the same rare cancer;  Early age of onset of cancer;  Bilateral tumours in paired organs;  Synchr­onous or successive tumours;  Tumours in two organs in one indivi­dual.

Indivi­duals born with a mutation in any one of these gene types has an inherited predis­pos­ition to cancer.

In acquired sporadic cancers the mutation is found only in the cancer cell and its progeny. In sporadic cancers surrou­nding normal cells and germ line cells show no mutations.

These genes are expressed in normal cells as “proto­-on­cog­enes” whose normal function is to promote cell growth and division.

Over 100 proto-­onc­ogenes have been identi­fied. Much of the original identi­fic­ation of oncogenes has come from studies of tumour viruses.

Unlike tumour suppressor genes, which are respon­sible for a number of familial inherited predis­pos­ition syndromes, only one oncogene, ret, has been identified as the cause of a familial cancer syndrome.

1. An activating mutation in a DNA coding sequence**

For example, activating mutations in the Ras gene have been detected in 30% of all human cancers. Oncogenic Ras mutations arise from a point mutation in the coding sequence which result in increased activity. Another example is the Ret proto-­onc­ogene, which is a cell surface tyrosine kinase receptor. Activating mutations at specific sites in the receptor sequence are associated with the clinical syndrome of multiple endocrine neoplasia type 2.

In this situation, multiple copies of the gene are expressed, resulting increased production of a signalling protein such as the epidermal growth factor receptor in breast cancer. The increase in copy number can reach up to several hundred fold. Amplif­ication of specific genes such as the transc­ription factors encoded by the MYC gene family are observed in specific types of tumours.

3. Chromo­somal rearra­nge­ment.

For example, Bcl-2 is a member of a family of proteins that regulate apoptosis. Bcl-2 was initially discovered by the presence of its transl­ocation in a specific type of human lymphoma. Transl­ocation of the immuno­glo­bulin gene on chromosome 14 with the Bcl-2 gene on chromosome 18, results in persistent overex­pre­ssion of Bcl-2 and inhibition of cell death

Inacti­vating mutations in tumour suppressor genes may be inherited in the germ line, or acquired by somatic mutation.

Although many of the originally described tumour suppressor genes were direct regulators of the cell cycle such as Rb and p53, more recently described genes serve other functions. Approx­imately 20 putative tumour suppressor genes have now been identi­fied, including those that encode cell-cycle regula­tors, transc­ription factors, phosph­atases and a protein that regulates RNA polymerase II elonga­tion.

These genes are normally expressed in all cells and function as “brakes” on the cell cycle and typically act before the synthetic or “S” or synthetic phase of the cell cycle. The S phase checkpoint allows time for DNA repair to occur prior to DNA replic­ation.

This has led to the “two hit hypoth­esis” of cancer develo­pment and has been clearly demons­trated in familial and sporadic cases of Retino­bla­stoma, mediated by mutations in the tumour suppressor gene Rb.

Examin­ation of the chromo­somes of a cell from an affected child led to the discovery that a small piece from a portion of chromosome 13 was missing. This deletion is present in one allele of all the child’s cells and as such likely to result from an inherited deletion.

Both copies of the Rb gene are required to be affected for cancer to develop. Therefore a second mutation in the remaining normal allele must occur after birth leading to develo­pment of cancer.

The RB gene product pRb plays a signif­icant role in the regulation of the cell cycle. During most of the G1 phase of the cell cycle, the E2F transc­ription factor is bound by pRb, which prevents E2F transc­ription factor activity inhibiting expression of S phase proteins. Phosph­ory­lation of pRb by cyclin­-de­pendent kinases releases E2F and allows transition of the cell to S phase. When pRb is not present in the cell, due to deletion or mutation, there is no restri­ction of the cells irreve­rsible entry into S phase and subsequent DNA replic­ation.

Mismatch repair genes encode proteins that cooperate in the removal of errone­ously incorp­orated bases from the nucleotide sequence following DNA replic­ation.

Mutations in DNA mismatch repair genes that result in an altered gene product cause an increase in the mutation rate, and thus increases the risk of mutation in proto-­onc­ogenes and on tumour suppressor genes.

The concept of multi-step carcin­oge­nesis is well supported.

In colon cancer it appears that seven or more genetic events (mutations in APC, ras, DCC, mismatch repair, TGF-b-R, p53 and others) may be required before an invasive carcinoma develops. By the time a patient develops clinical evidence of cancer, the cancer usually has many genetic mutations in biopsy samples. With increasing time more mutations accumulate as the cancer becomes more malignant and spreads.

One of the molecular mechanisms mediating the evasion of apoptosis is the acquis­ition by the cancer cell of a mutation in the tumour suppressor gene p53, which leads to abnormal and ineffi­cient apoptotic responses.

However, in highly malignant tumours, the cell death pathways are modified, or signif­icantly inhibited.

Therefore unders­tanding the mechanisms by which a normal cell lives or dies in response to p53, may help us to develop new strategies to promote regulated cancer cell death.

p53 was so named because the molecular mass of the protein is 53 kiloda­ltons.

p53 shuttles in and out of the nucleus, regulated by N-terminal and C-terminal regions.

Two other p53 family members have been identified p63 and p73, these latter 2 proteins regulate normal develo­pment, but are not commonly associated with mutations in cancer. p53 is not required for normal develo­pment, as shown by p53 knockout mice, which develop normally, but start to develop cancer by 3 months of age.

Elevated levels of p53 may drive damaged cells to commit suicide by apoptosis, if DNA damage is severe.

The choice of cell response to p53 depends on specific factors such as the cell type, the cell enviro­nment, and other oncogenic altera­tions sustained by the cell.

Collec­tively these responses prevent tumour develo­pment and progre­ssion. The rapid cellular increase in p53 levels in response to DNA damage stabilises the p53 protein by specific molecular mechan­isms, which lead to an increase in the intrac­ellular p53 levels.

These classi­cally occur at the G1 checkp­oint, which prevents entry of the cell into S-phase and the late G2 checkp­oint, which prevents entry of the cell into mitosis.

Low-level DNA damage occurs during normal cellular life, and if not corrected DNA errors accumu­late. In cells that lack these checkp­oints, accumu­lation of DNA mutations occurs, which may lead to mutation of proto-­onc­ogenes, or tumour suppressor genes, and results in enhanced cellular prolif­era­tion.

In the human genome at least 4,000 potential p53 regulator gene targets have been identi­fied, although it is not clear yet if they will all be genuine physio­logical p53 targets. Many of these genes that are induced by p53 can be divided into specific classes of protein that either inhibit cell growth, regulate DNA repair, regulate apoptosis, or control angiog­enesis, i.e., the formation of new blood vessels.

p53 can also regulate the expression of genes that inhibit cell survival, such as PTEN, a commonly mutated tumour suppressor gene in breast and brain cancers.

Mutation of p53 in cancer cells results in loss of apoptotic function, and cell cycle regula­tion.

Often the mutations comprise a single point mutation. Mutant p53 proteins are often more stable than normal p53 and may be expressed at extremely high levels in the tumour cell. However, mutant p53 cannot function to mediate apoptosis, or cell cycle arrest.

n addition, some of the mutant p53 may acquire new transf­orming (cance­r-p­rom­oting) functions, i.e., a gain-f­unction mutant. As a conseq­uence of mutation in p53, cancer cells escape apoptosis, DNA damage is not repaired, the cell cycle is not halted at the checkp­oints to repair damaged DNA, and therefore cells may survive and prolif­erate with a mutated genome.

The accumu­lative loss of tumour suppressor genes and mutation of proto-­onc­ogenes to form oncogenes results in enhanced cell prolif­era­tion. In addition, gene amplif­ica­tion, which, in turn, may cause the acquis­ition of tumour drug resist­ance. Collec­tively these changes result in a signif­icant prolif­erative advantage to the cell.

If we could reactivate p53 function in cancer cells this could potent­ially be of tremendous therap­eutic value as it would induce cancer cell death.

p53 plays a role in determ­ining sensit­ivity to radiot­herapy. Tumour cells frequently acquire mutations, which decreases the tumour sensit­ivity to radiation. As radiation induces DNA damage, one of the critical molecules that mediate sensit­ivity to radiation is p53, therefore unders­tanding p53 function may help scientists and clinicians improve responses in cancer patients to radiot­herapy and minimise radiation resist­ance.

Cellular response to radiation therapy is tissue specific, i.e., that is cells, which are rapidly growing tend to apoptose, while fibroblast type tissues, the structural components of organs, tend to undergo growth arrest. Normal cells exposed to radiation will apoptose via p53-me­diated pathways.