1 Molecular Biology of Cancer The genetic basis for the development of cancer

1Molecular Biology of Molecular Biology of CancerCancer

The genetic basis for the development of cancer


Cancers arise through a multistage process in which inherited and somatic mutations of cellular genes lead to clonal selection of variant progeny with the most robust and aggressive growth properties.

Two classes of genes are targets for the mutations:Protooncogenestumor-suppressor genes


The vast majority of the mutations that contribute to the development and behavior of cancer cellsare somatic (ie, arising during tumor development)present only in the neoplastic cells of the patient

A small fraction of all mutations in cancer cells are constitutionalpresent in all somatic cells of affected individualssuch mutations not only predispose to cancer, but

can also be passed on to future generations.


Tumor Suppressor GenesTumor Suppressor Genes


A large number of tumor suppressor A large number of tumor suppressor genes have been hypothesized to existgenes have been hypothesized to exist

Thus far, approximately 20 tumor-suppressor genes have been identified and definitively implicated in cancer development.

The cellular functions of the tumor-suppressor genes appear to be diverse


Cancer-inducing genes, specifically viral Cancer-inducing genes, specifically viral oncogenes, act in a dominant fashiononcogenes, act in a dominant fashion

Viral oncogenes dictate cellular behavior in spite of the continued presence and expression of opposing cellular genes within the virus-infected cell that usually functioned to ensure normal cell proliferation.The viral genes were able to induce a

dominant phenotype—they were bringing about a cell transformation.

Most human cancers do not seem to arise as consequences of tumor virus infections


Cell fusion experiments indicate that Cell fusion experiments indicate that the cancer phenotype is recessivethe cancer phenotype is recessive


fusion of a cancer cell with a wild-fusion of a cancer cell with a wild-type celltype cell

The resulting hybrid cells have lost the ability to form tumors when these hybrid cells were injected into appropriate host animals. This, unexpectedly, mean that

the malignant cell phenotype is recessive to the phenotype of normal, wild-type growth.

Exception: when the transformed parental cell had been transformed by tumor virus infection.


Tumor suppressor genes (TSGs)Tumor suppressor genes (TSGs)HypothesisHypothesis

Normal cells carry genes that constrain or suppress their proliferation.During the development of a tumor, the evolving

cancer cells inactivate one or more of these genes.Once these growth-suppressing genes are lost, the

proliferation of the cancer cells accelerates.As long as the cancer cell lacks these genes, it

continues to proliferate in a malignant fashion.When wild-type, intact versions of these genes

operate once again within the cancer cell (by cell fusion) it will loose its ability to proliferate or to form tumors.


The retinoblastoma tumor is the first The retinoblastoma tumor is the first example of tumor suppressor genesexample of tumor suppressor genes

Sporadic form:unilateral and unifocalonce the tumor is eliminated, no

further risk

Familial formbilateral and often multi-focalcuring the eye tumor does not

protect the children from a greatly increased risk to bone cancers and other cancers


Figure 7.5b The Biology of Cancer (© Garland Science 2007)

Familial formFamilial form

The familial form of retinoblastoma is passed from one generation to the next in a fashion that conforms to the behavior of a Mendelian dominant allele.


Kinetics of Rb: Kinetics of Rb: familial (bilateral) familial (bilateral) vs. vs. sporadic (unilateral)sporadic (unilateral)

the rate of appearance of familial tumors was consistent with a single random event (mutation)

the sporadic tumors behaved as if two random events were required


Two-hit hypothesisTwo-hit hypothesis

Two “hits” or mutagenic events were necessary for retinoblastoma development

In an individual with the inherited form: the first hit is present in the germ line, and thus in

all cells of the body. a second somatic mutation was hypothesized to be

necessary for promoting tumor formation. The second mutation explain the behavior of a Mendelian

dominant allele.In the nonhereditary form: both mutations were proposed to arise somatically

within the same cell.


Two-hit hypothesisTwo-hit hypothesis

Each of the two hits could theoretically be in different genes

Subsequent studies led to the conclusion that both hits were at the same genetic locus, ultimately inactivating both alleles of the retinoblastoma (RB1) susceptibility gene


Loss of Rb heterozygosoty (LOH)Loss of Rb heterozygosoty (LOH)Mitotic recombination: Mitotic recombination: a possible mechanism to

eliminate the wild-type copy of Rb gene

The probability of inactivating a single gene copy by mutation is on the order of 10-6 per cell generation

The probability of silencing both copies is on the order of 10-12 per cell generation.

It seems highly unlikely that both copies of the Rb gene could be eliminated through two recessive mutational event in the relatively small target cell populations in the developing retina (about 106 cells).


Loss of Rb heterozygosoty (LOH)Loss of Rb heterozygosoty (LOH)Mitotic recombinationMitotic recombination: a possible mechanism to : a possible mechanism to

eliminate the wild-type copy of Rb geneeliminate the wild-type copy of Rb gene

This mitotic recombination was found to occur at a frequency of 10–5 to 10–4 per cell generationeasier way for a cell to rid itself of the remaining wild-

type copy of the Rb gene than mutational inactivation of this gene copy, which, as mentioned above, was known to occur at a frequency of about 10–6 per cell generation.


Loss of Rb heterozygosoty (LOH)Loss of Rb heterozygosoty (LOH)Gene conversion: Gene conversion: another possible mechanism to

eliminate the wild-type copy of Rb gene• When gene conversion involve copying of an already inactivated Rb allele, for example, then once again LOH will have occurred in this chromosomal region.

• known to occureven morefrequently per cellgeneration thandoes mitoticrecombination.


The Rb gene often undergoes loss of The Rb gene often undergoes loss of heterozygosity in tumorsheterozygosity in tumors

In a small number of retinal tumors, careful karyotypic revealed interstitial deletions within the long (“q”) arm of Chromosome 13.All of these deletions

caused the loss of chromosomal material in the 4th band of the 1st region of this chromosomal arm (13q14).


A number of genes (including Rb) in this region had been lost simultaneously by the developing retinal tumor cells.

This is precisely the outcome predicted by the tumor suppressor gene theory.

LOH achieved by simply breaking off and discarding an entire chromosomal region without replacing it with a copy duplicated from the other, homologous chromosome is called hemizygosity


Mutations of the Mutations of the Rb geneRb gene


LOH of chromosomal arms in CRCLOH of chromosomal arms in CRC

High frequency of LOH allows the detection of putative TSG



Table 7.1 part 2 of 2 The Biology of Cancer (© Garland Science 2007)


Many familial cancers can be explained by Many familial cancers can be explained by inheritance of mutant tumor suppressor genesinheritance of mutant tumor suppressor genes

These genes specify a diverse array of proteins that operate in many different intracellular sites to reduce the risk of cancer.

An anti-cancer function is the only property that is shared by these otherwise unrelated genes.

Many familial cancers can be explained by inheritance of mutant TSGs.

Inheritance of defective copies of most TSGs creates an enormously increased risk for cancerOften a type of relatively rare tumors


There are two distinct classes of There are two distinct classes of familial cancer genesfamilial cancer genes

Gatekeepers:Tumor suppressor genes that function to directly

control the biology of cells (proliferation, differentiation, or apoptosis)

Caretakers:The DNA maintenance genes affect cell biology

only indirectly by controlling the rate at which cells accumulate mutant genes


Promoter methylation represents an important Promoter methylation represents an important mechanism for inactivating tumor suppressor mechanism for inactivating tumor suppressor

genesgenesDNA molecules can be altered covalently by the

attachment of methyl groups to cytosine bases.This modification of the genomic DNA is as important

as mutation in shutting down tumor suppressor genes.

In mammalian cells, this methylation is found only when these bases are located in a position that is 5’ to guanosines, that is, in the sequence CpG.

Such methylation can affect the functioning of the DNA in this region of the chromosome.

When CpG methylation occurs in the vicinity of a gene promoter, it can cause repression of transcription of the associated gene.


• Analyses of five DNA samples from tumor 232 indicate methylation at almost all CpG sites in the RASSF1A CpG island

• Adjacent, ostensibly normal tissue is unmethylated in most but not all analyses of this CpG island.

• Analyses of control DNA from a normal individual indicate the absence of any methylation of the CpGs in this CpG island.


More than half of the tumor suppressor genes that are involved in familial cancer syndromes because of germ-line mutation have been found to be silenced in sporadic cancers by promoter methylation.Ex. Rb germ line mutations familial

retinoblastoma.Rb somatic mutations or promoter methylation

sporadic retinoblastomas.


The elimination of tumor suppressor gene function by promoter methylation

1. One copy might be methylated and the second might then be lost through a loss of heterozygosity (LOH) accompanied by a duplication of the already-methylated tumor suppressor gene copy

2. Both copies of a tumor suppressor gene might be methylated independently of one another

1st hitMethylation

Methylation

(2nd hit)

LOH


Example:Example:p16INK4A tumor suppressor genep16INK4A tumor suppressor gene

In a study of the normal bronchial (large airway) epithelia of the lungs:p16INK4A methylation:

in 44% of (ostensibly normal) bronchial epithelial cells cultured from current and former smokers

not at all in the comparable cells prepared from those who had never smoked.

LOH in this chromosomal region: in 71 to 73% of the two smoking populations in 1.5 to 1.7% of never-smokers.

Conclusion: methylation of critical growth-controlling genes often occurs early in the complex, multi-step process of tumor formation, long before histological changes are apparent in a tissue



Silencing of genes through promoter Silencing of genes through promoter methylation can also involve the “caretaker” methylation can also involve the “caretaker”

genes genes

Example: the BRCA1 gene:Its product maintaining the

chromosomal DNA in ways that are still poorly understood.

Inheritance of mutant alleles of BRCA1 a high lifetime risk of familial breast and, to a lesser extent, ovarian cancer syndrome.

10 to 15% of sporadic breast carcinomas carry inactive BRCA1 gene copies that have been silenced through promoter methylation.


• Promoter methylation is common during the development of a wide variety of human cancers

• the frequency of methylation of a specific gene varies dramatically from one type of tumor to the next.

• Tumor suppressor genes as well as caretaker genes undergo hypermethylation.

• Perhaps the


The (Neurofibromin Neurofibromin ) NF1 protein acts as a

negative regulator of Ras signaling

• When cells stimulated by growth factor, they may degrade NF1, enabling Ras signaling to proceed without interference by NF1.

• After 60 to 90 minutes, NF1 levels return to normal, and the NF1 protein that accumulates helps to shut down further Ras signaling in a form of negative-feedback control


Neurofibromatosis type 1 is a relatively common familial cancer syndrome, with 1 in 3500 individuals affected on average worldwide.

The primary feature of this disease is the development of benign neurofibromas of the cell sheaths around nerves in the peripheral nervous system

On occasion, a subclass of these neurofibromas, progress to malignant tumors termed neurofibrosarcomas.


In neuroectodermal cells lacking NF1 function, Ras proteins are predicted to exist in their activated, GTP-bound state for longer than- normal periods of times.

In fact, in the cells of neurofibromas, which are genetically NF1–/–, elevated levels of activated Ras and Ras effector proteins can be found

Consequently, the loss of NF1 function in a cell can mimic functionally the activated Ras proteins that are created by mutant ras oncogenes.

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1 Molecular Biology of Cancer The genetic basis for the development of cancer