Neoplasia : ETIOLOGY AND PATHOGENESIS OF CANCER

CARCINOGENESIS: ETIOLOGY AND

PATHOGENESIS OF CANCER

Carcinogenesis or oncogenesis or tumorigenesis means mechanism of induction of tumours (pathogenesis of cancer);

agents which can induce tumours are called carcinogens (etiology of cancer). Since the time first ever carcinogen was identified, there has been ever-increasing list of agents implicated in etiology of cancer. There has been still greater accumulation in volumes of knowledge on pathogenesis of cancer, especially due to tremendous strides made in the field

of molecular biology and genetics in recent times.

         The subject of etiology and pathogenesis of cancer is discussed under the following 4 broad headings:

A. Molecular pathogenesis of cancer (genes and cancer)

B. Chemical carcinogens and chemical carcinogenesis

C. Physical carcinogens and radiation carcinogenesis

D. Biologic carcinogens and viral oncogenesis.

A. MOLECULAR PATHOGENESIS OF CANCER

(GENETIC MECHANISMS OF CANCER)

Basic Concept of Molecular Pathogenesis

The mechanism as to how a normal cell is transformed to a cancer cell is complex. At different times, attempts have been

made to unravel this mystery by various mechanisms.Currently, a lot of literature has accumulated to explain the pathogenesis of cancer at molecular level. The general concept of molecular mechanisms of cancer is briefly outlined below and diagrammatically shown in Fig.

1. Monoclonality of tumours. There is strong evidence to support that most human cancers arise from a single clone of cells by genetic transformation or mutation. For example:

i) In a case of multiple myeloma (a malignant disorder of plasma cells), there is production of a single type of immuno￾globulin or its chain as seen by monoclonal spike in serum

electrophoresis.

ii) Due to inactivation of one of the two X-chromosomes in females (paternal or maternal derived), women are mosaics with two types of cell populations for glucose-6-phosphatase

dehydrogenase (G6PD) isoenzyme A and B. It is observed that all the tumour cells in benign uterine tumours (leiomyoma) contain either A or B genotype of G6PD (i.e.the tumour cells are derived from a single progenitor clone

of cell), while the normal myometrial cells are mosaic of both types of cells derived from A as well as B isoenzyme(Fig).

2. Field theory of cancer. In an organ developing cancer, in the backdrop of normal cells, limited number of cells only grow in to cancer after undergoing sequence of changes

under the influence of etiologic agents. This is termed as ‘field effect’ and the concept called as field theory of cancer.

3. Multi-step process of cancer growth and progression.

Carcinogenesis is a gradual multi-step process involving many generations of cells. The various causes may act on the cell one after another (multi-hit process). The same process

is also involved in further progression of the tumour. Ultimately, the cells so formed are genetically and phenotypically transformed cells having phenotypic features of malignancy—excessive growth,invasiveness and distant metastasis. 

4. Genetic theory of cancer. Cell growth of normal as well as abnormal types is under genetic control. In cancer, there 

are either genetic abnormalities in the cell, or there are normal genes with abnormal expression. The abnormalities in genetic 

composition may be from inherited or induced mutations (induced by etiologic carcinogenic agents namely: chemicals, viruses, radiation). The mutated cells transmit their characters to the next progeny of cells and result in cancer. 

5. Genetic regulators of normal and abnormal mitosis. In normal cell growth, regulatory genes control mitosis as well as cell aging, terminating in cell death by apoptosis. 

      •In normal cell growth, there are 4 regulatory genes:

i) Proto-oncogenes are growth-promoting genes i.e. they encode for cell proliferation pathway. 

ii) Anti-oncogenes are growth-inhibiting or growth suppressor genes. 

iii) Apoptosis regulatory genes control the programmed cell death. 

iv) DNA repair genes are those normal genes which regulate the repair of DNA damage that has occurred during mitosis and also control the damage to proto-oncogenes and anti-oncogenes. 

     •In cancer, the transformed cells are produced by abnormal cell growth due to genetic damage to these normal controlling genes. Thus, corresponding abnormalities in these 4 cell regulatory genes are as under: 

i) Activation of growth-promoting oncogenes causing transformation of cell (mutant form of normal proto-oncogene in cancer is termed oncogene). Many of these cancer associated genes, oncogenes, were first discovered in viruses, and hence named as v-onc. Gene products of oncogenes are called oncoproteins. Oncogenes are considered dominant since 

they appear in spite of presence of normal proto-oncogenes.

ii) Inactivation of cancer-suppressor genes (i.e. inactivation of anti-oncogenes) permitting the cellular proliferation of transformed cells. Anti-oncogenes are active in recessive form 

i.e. they are active only if both alleles are damaged.

iii) Abnormal apoptosis regulatory genes which may act as oncogenes or anti-oncogenes. Accordingly, these genes may be active in dominant or recessive form. 

iv) Failure of DNA repair genes and thus inability to repair the DNA damage resulting in mutations.

 Cancer-related Genes and Cell Growth (Hallmarks of Cancer) 

It is apparent from the above discussion that genes control the normal cellular growth, while in cancer these controlling genes are altered, typically by mutations. A large number of 

such cancer-associated genes have been described, each with a specific function in cell growth. Some of these genes are commonly associated in many tumours (e.g. p53 or TP53), 

while others are specific to particular tumours. Therefore, it is considered appropriate to discuss the role of cancer-related genes with regard to their functions in cellular growth. 

Following are the major genetic properties or hallmarks of cancer:

1. Excessive and autonomous growth: Growth-promoting oncogenes. 

2. Refractoriness to growth inhibition: Growth suppressing anti-oncogenes. 

3. Escaping cell death by apoptosis: Genes regulating apoptosis and cancer. 

4. Avoiding cellular aging: Telomeres and telomerase in cancer. 

5. Continued perfusion of cancer: Cancer angiogenesis.

6. Invasion and distant metastasis: Cancer dissemination.

7. DNA damage and repair system: Mutator genes and cancer. 

8. Cancer progression and tumour heterogeneity: Clonal aggressiveness. 

9. Cancer a sequential multistep molecular phenomenon: Multistep theory. 

10. MicroRNAs in cancer: OncomiRs.

        These properties of cancer cells are described below in terms of molecular genetics and schematically illustrated in 

Fig.

1. EXCESSIVE AND AUTONOMOUS GROWTH:

GROWTH PROMOTING ONCOGENES

Mutated form of normal protooncogenes in cancer is called oncogenes. Protooncogenes become activated oncogenes by following mechanisms as under:

   •By mutation in the protooncogene which alters its structure and function.

   •By retroviral insertion in the host cell.

    •By damage to the DNA sequence that normally regulates growth-promoting signals of protooncogenes resulting in its abnormal activation.

    •By erroneous formation of extra copies of protooncogene causing gene amplification and hence its overexpression or overproduction that promotes autonomous and excessive

cellular proliferation.

          In general, overactivity of oncogenes enhances cell proliferation and promotes development of human cancer.About 100 different oncogenes have been described in

various cancers. Transformation of proto-oncogene (i.e.normal cell proliferation gene) to oncogenes (i.e. cancer cell proliferation gene) may occur by three mechanisms:

i) Point mutations i.e. an alteration of a single base in the DNA chain. The most important example is RAS oncogene carried in many human tumours such as bladder cancer,pancreatic adenocarcinoma, cholangiocarcinoma.

ii) Chromosomal translocationsi.e. transfer of a portion of one chromosome carrying protooncogene to another chromosome and making it independent of growth controls.

This is implicated in the pathogenesis of leukaemias and lymphomas e.g.

      •Philadelphia chromosome seen in 95% cases of chronic myelogenous leukaemia in which c-ABL protooncogene on chromosome 9 is translocated to chromosome 22.

       In 75% cases of Burkitt’s lymphoma, translocation of c-MYC proto-oncogene from its site on chromosome 8 to a portion on chromosome 14.

iii) Gene amplification i.e. increasing the number of copies of DNA sequence in protooncogene leading to increased

mDNA and thus increased or overexpressed gene product.Examples of gene amplification are found in some solid

human tumours e.g.

         Neuroblastoma having n-MYC HSR region.

          ERB-B1 in breast and ovarian cancer.

      Most of the oncogenes encode for components of cell signaling system for promoting cell proliferation. Possible

effects of oncogenes in signal transduction for cell proliferation in human tumours are discussed below in relation to the role of protooncogenes in mitosis in normal

cell cycle and are listed in Table 8.4 and schematically shown in Fig. 

i) Growth factors (GFs). GFs were the first protoonocgenes to be discovered which encode for cell proliferation cascade.They act by binding to cell surface receptors to activate cell proliferation cascade within the cell. GFs are small polypeptides elaborated by many cells and normally act on another cell than the one which synthesised it to stimulate its proliferation i.e. paracrine action.

       However, a cancer cell may synthesise a GF and respond to it as well; this way cancer cells acquire growth self sufficiency. Most often, growth factor genes are not altered

or mutated but instead growth factor genes are overexpressed to stimulate large secretion of GFs which stimulate cell proliferation. The examples of such tumour secreted GFs are

as under:

a) Platelet-derived growth factor- (PDGF-β): Overexpression of SIS protooncogene that encodes for PDGF-β and thus there is increased secretion of PDGF-β e.g. in gliomas and

sarcomas.

b) Transforming growth factor-α (TGF-α): Overexpression of TGF-α gene occurs by stimulation of RAS protooncogene

and induces cell proliferation by binding to epidermal growth factor (EGF) receptor e.g. in carcinoma and astrocytoma.

c) Fibroblast growth factor (FGF): Overexpression of HST-1

protoonogene and amplification of INT-2 protoonogene causes excess secretion of FGF e.g. in cancer of the bowel and breast.

d) Hepatocyte growth factor (HGF): Overexpression by binding to its receptor c-MET e.g. follicular carcinoma thyroidthyroid. 

ii) Receptors for GFs. Growth factors cannot penetrate the cell directly and require to be transported intracellularly by GF-specific cell surface receptors. These receptors are

transmembrane proteins and thus have two surfaces: the outer surface of the membrane has an area for binding growth factor, and the inner surface of the membrane has enzyme￾activating area which eventually activates cell proliferation pathway.

       Most often, mutated form of growth factor receptors stimulate cell proliferation even without binding to growth factors i.e. with little or no growth factor bound to them.

Various forms of oncogenes encoding for GF receptors include other mechanisms: overexpression, mutation and gene rearrangement. Examples of tumours by mutated receptors for growth factors are as under:

a) EGF receptors: Normal EGF receptor gene is ERB B1, and hence this receptor is termed as EGFR or HER1 (i.e. human epidermal growth factor receptor type 1). EGFR (or HER1) acts by overexpression of normal GF receptor e.g. in 80% of squamous cell carcinoma of lung and 50% cases of glioblastomas.

         Another EGF receptor gene called ERB B2 (or HER2/neu)acts by gene amplification e.g. in breast cancer (25% cases),carcinoma of lungs, ovary, stomach.

b) c-KIT receptor: The gene coding for receptor for stem cell factor (or steel factor) is c-KIT, that activates tyrosine kinase pathway in cell proliferation. Mutated form of c-KIT by point

mutation activates receptor for tyrosine kinase e.g. in gastrointestinal stromal tumours (GIST).

c) RET receptor: RET (abbreviation of ‘rearranged during transfection’) protooncogene is a receptor for tyrosine kinase normally expressed in neuroendocrine cells of different tissues. Mutated form by point mutation is seen in MEN type

2A and 2B and in medullary carcinoma thyroid.

iii) Cytoplasmic signal transduction proteins. The normal signal transduction proteins in the cytoplasm transduce

signal from the GF receptors present on the cell surface, to the nucleus of the cell, to activate intracellular growth signaling pathways.

        There are examples of oncogenes having mutated forms of cytoplasmic signaling pathways located in the inner surface of cell membrane in some cancers. These are as under:

a) Mutated RAS gene. This is the most common form of oncogene in human tumours, the abnormality being induced by point mutation in RAS gene. About a third of all human tumours carry mutated RAS gene (RAS for Rat Sarcoma gene where it was first described), seen particularly in carcinoma colon, lung and pancreas. Normally, the inactive form of RAS

protein is GDP (guanosine diphosphate)-bound while the activated form is bound to guanosine triphosphate (GTP).

GDP/GTP are homologous to G proteins and take part in signal transduction in a similar way just as G proteins act as ‘on-off switch’ for signal transduction. Normally, active RAS

protein is inactivated by GTPase activity, while mutated RAS gene remains unaffected by GTPase, and therefore, continues to signal the cell proliferation.

b) BCR-ABL hybrid gene. ABL gene is a non-GF receptor protooncogene having tyrosine kinase activity. ABL gene from its normal location on chromosome 9 is translocated to

chromosome 22 where it fuses with BCR (breakpoint cluster region) gene and forms an ABL-BCR hybrid gene which is more potent in signal transduction pathway. ABL-BCR hybrid

gene is seen in chronic myeloid leukaemia and some acute leukaemias.

iv) Nuclear transcription factors. The signal transduction pathway that started with GFs ultimately reaches the nucleus

where it regulates DNA transcription and induces the cell to enter into S phase. Out of various nuclear regulatory trans￾cription proteins described, the most important is MYC gene located on long arm of chromosome 8. Normally MYC protein binds to the DNA and regulates the cell cycle by transcriptional activation and its levels fall immediately after

cell enters the cell cycle.

     MYC oncogene (originally isolated from myelocytomatosis virus and accordingly abbreviated) is seen mostcommonly in human tumours. It is associated with persistence of or overexpression of MYC oncoproteins which,in turn, causes autonomous cell proliferation. The examples of tumours carrying MYC oncogene are as under:

a) C-MYC oncogene: Mutated MYC gene due to translocation t(8;14) seen in Burkitt’s lymphoma.

b) N-MYC oncogene: Mutated MYC gene due to amplification seen in neuroblastoma, small cell carcinoma lung.

c) L-MYC oncogene: Mutated MYC gene due to amplification seen in small cell carcinoma lung.

v) Cell cycle regulatory proteins.normally the cell cycle is under regulatory control of cyclins and cyclin-dependent kinases (CDKs) A, B, E and D. Cyclins are so named since they are cyclically synthesised during different phases of the cell cycle and their degradation is also

cyclic. Cyclins activate as well as work together with CDKs,while many inhibitors of CDKs (CDKIs) are also known.

           Although all steps in the cell cycle are under regulatory controls, G1 → S phase is the most important checkpoint for regulation by oncogenes as well as anti-oncogenes (discussed below). Mutations in cyclins (in particular cyclin D) and CDKs (in particular CDK4) are most important growth promoting signals in cancers. The examples of tumours

having such oncogenes are as under:

a) Mutated form of cyclin D protooncogene by translocation seen in mantle cell lymphoma.

b) Mutated form of cyclin E by overexpression seen in breast cancer.

b) Mutated from of CDK4 by gene amplification seen in malignant melanoma, glioblastoma and sarcomas.

2. REFRACTORINESS TO GROWTH INHIBITION:

GROWTH SUPPRESSING ANTI-ONCOGENES

The mutation of normal growth suppressor anti-oncogenes results in removal of the brakes for growth; thus the inhibitory effect to cell growth is removed and the abnormal

growth continues unchecked. In other words, mutated anti￾oncogenes behave like growth-promoting oncogenes.

         As compared to the signals and signal transduction pathways for oncogenes described above, the steps in mechanisms of action by growth suppressors are not so well understood. In general, the point of action by anti-oncogenes is also G1 → S phase transition and probably act either by inducing the dividing cell from the cell cycle to enter into G0(resting) phase, or by acting in a way that the cell lies in the post-mitotic pool losing its dividing capability. Just as with activation of protooncogenes to become oncogenes, the

mechanisms of loss of tumour suppressor actions of genes are due to chromosomal deletions, point mutations and loss

of portions of chromosomes.

        Major anti-oncogenes implicated in human cancers are as under (Table):

i) RB gene. RB gene is located on long arm (q) of chromosome 13. This is the first ever tumour suppressor gene identified

and thus has been amply studied. RB gene codes for a nuclear transcription protein pRB. RB gene is termed as master‘break’ in the cell cycle and is virtually present in every human cell. It can exist in both an active and an inactive form:

    •The active form of RB gene, it blocks cell division by binding to transcription factor, E2F, and thus inhibits the cell

from transcription of cell cycle-related genes, thereby inhibiting the cell cycle at G1 → S phase i.e. cell cycle is arrested at G1 phase.

     •Inactive form of RB gene occurs when it is

hyperphosphorylated by cyclin dependent kinases (CDKs)which occurs when growth factors bind to their receptors.This removes pRB function from the cell (i.e. the ‘break’ on

cell division is removed) and thus cell proliferation pathway is stimulated by permitting the cell to cross G1 → S phase.

Activity of CDKs is inhibited by activation of inhibitory signal, transforming growth factor- (TGF-β), on cell through activation of inhibitory protein p16.

      The mutant form of RB gene (i.e. inactivating mutation of RB gene) is involved in several human tumours, most

commonly in retinoblastoma, the most common intraocular tumour in young children. The tumour occurs in two forms:

sporadic and inherited/familial. More than half the cases are sporadic affecting one eye; these cases have acquired simultaneous mutation in both the alleles in retinal cells after birth. In inherited cases, all somatic cells inherit one mutant RB gene from a carrier parent, while the other allele gets mutated later. The latter genetic explanation given by Knudson forms the basis of two hit hypothesis of inherited

cancers. Besides retinoblastoma, children inheriting mutant RB gene have 200 times greater risk of development of other cancers in early adult life, most notably osteosarcoma; others are cancers of breast, colon and lungs.

ii) p53 gene (TP53). Located on the short arm (p) of chromosome 17, p53 gene (also termed TP53 because of molecular weight of 53 kd for the protein) like pRB is inhibitory to cell cycle. However, p53 is normally present in very small amounts and accumulates only after DNA damage.

     The two major functions of p53 in the normal cell cycle are as under:

a) In blocking mitotic activity: p53 inhibits the cyclins and CDKs and prevents the cell to enter G1 phase transiently. This

breathing time in the cell cycle is utilised by the cell to repair the DNA damage.

b) In promoting apoptosis: p53 acts together with another anti￾oncogene, RB gene, and identifies the genes that have damaged DNA which cannot be repaired by inbuilt system.

p53 directs such cells to apoptosis by activating apoptosis inducing BAX gene, and thus bringing the defective cells to

an end by apoptosis. This process operates in the cell cycle at G1 and G2 phases before the cell enters the S or M phase.

         Because of these significant roles in cell cycle, p53 is called as ‘protector of the genome’.

       In its mutated form, p53 ceases to act as protector or as growth suppressor but instead acts like a growth promoter or oncogene. Homozygous loss of p53 gene allows genetically damaged and unrepaired cells to survive and proliferate resulting in malignant transformation. More than 70% of human cancers have homozygous loss of p53 by acquired mutations in somatic cells; some common examples are cancers of the lung, head and neck, colon and breast. Besides,

mutated p53 is also seen in the sequential development stages of cancer from hyperplasia to carcinoma in situ and into

invasive carcinoma.

          Less commonly, both alleles of p53 gene become defective by another way: one allele of p53 mutated by inheritance in germ cell lines rendering the individual to another hit of

somatic mutation on the second allele. This defect like in RB gene predisposes the individual to develop cancers of multiple organs (breast, bone, brain, sarcomas etc), termed Li-Fraumeni syndrome.

iii) Transforming growth factor-βββ (TGF-βββ) and its receptor.

Normally, TGF-β is significant inhibitor of cell proliferation, especially in epithelial, endothelial and haematopoieitc cells 

It acts by binding to TGF-β receptor and then the complex so formed acts in G1 phase of cell cycle at two levels: 

    •It activates CDK inhibitors (CDKIs) with growth inhibitory effect. 

    •It suppresses the growth prmoter genes such as MYC, CDKs and cyclins. 

       Mutant form of TGF-β gene or its receptor impairs the growth inhibiting effect and thus permits cell proliferation. Examples of mutated form of TGF-β are seen in cancers of pancreas, colon, stomach and endometrium. 

iv) Adenomatous polyposis coli (APC) gene and βββ-catenin

protein. The APC gene is normally inhibitory to mitosis, which takes place by a cytoplasmic protein, β-catenin. β- catenin normally has dual functions: firstly, it binds to cytoplasmic E-cadherin that is involved in intercellular 

interactions, and secondly it can activate cell proliferation signaling pathway. In colon cancer cells, APC gene is lost and thus β-catenin fails to get degraded, allowing the cancer cells to undergo mitosis without the inhibitory influence of 

β-catenin.

      Patients born with one mutant APC gene allele develop    large number of polyps in the colon early in life, while after the age of 20 years these cases start developing loss of second APC gene allele. It is then that almost all these patients invariably develop malignant transformation of one or more polyps. 

v) Other antioncogenes. A few other tumour-suppressor genes having mutated germline in various tumours are as under: 

a) BRCA 1 and BRCA 2 genes: These are two breast (BR) cancer (CA) susceptibility genes: BRCA1 located on chromosoe 17q21 and BRCA2 on chromosome 13q12-13. Women with inherited defect in BRCA1 gene have very high 

risk (85%) of developing breast cancer and ovarian cancer (40%). Inherited breast cancer constitutes about 5-10% cases, it tends to occur at a relatively younger age and more often 

tends to be bilateral.

b) VHL gene. von-Hippel-Lindau (VHL) disease is a rare autosomal dominant disease characterised by benign and malignant tumours of multiple tissues. The disease is 

inherited as a mutation in VHL tumour suppressor gene located on chromosome 3p. This results in activation of genes 

that promote angiogenesis, survival and proliferation; VHL gene is found inactivated in 60% cases of renal cell carcinoma. 

c) Wilms’ tumour (WT) gene: Both WT1 an WT2 genes are located on chromosome 11 and normally prevent neoplastic proliferation of cells in embryonic kidney. Mutant form of 

WT-1 and 2 are seen in hereditary Wilms’ tumour.

d) Neurofibroma (NF) gene: NF genes normally prevent proliferation of Schwann cells. Two mutant forms are  described: NF1 and NF2 seen in neurofibromatosis type 1 

and type 2.

        The contrasting features of growth-promoting oncogenes and growth-suppressing anti-oncogenes are summarised in 

Table.

3. ESCAPING CELL DEATH BY APOPTOSIS:

GENES REGULATING APOPTOSIS AND CANCER

Besides the role of mutant forms of growth-promoting oncogenes and growth-suppressing anti-oncogenes, another  mechanism of tumour growth is by escaping cell death by 

apoptosis. Apoptosis in normal cell is guided by cell death receptor, CD95, resulting in DNA damage. Besides, there is role of some other pro-apoptotic factors (BAD, BAX, BID and 

p53) and apoptosis-inhibitors (BCL2, BCL-X).

In cancer cells, the function of apoptosis is interfered due to mutations in the above genes which regulate apoptosis in the normal cell. The examples of tumours by this mechanism 

are as under:

a) BCL2 gene is seen in normal lymphocytes, but its mutant form with characteristic translocation (t14;18) (q32;q21) was 

first described in B-cell lymphoma and hence the name BCL. It is also seen in many other human cancers such as that of breast, thyroid and prostate. Mutation in BCL2 gene removes 

the apoptosis-inhibitory control on cancer cells, thus more live cells undergoing mitosis contributing to tumour growth. Besides, MYC oncogene and p53 tumour suppressor gene 

are also connected to apoptosis. While MYC allows cell growth BCL2 inhibits cell death; thus MYC and BCL2 together allow cell proliferation. Normally, p53 activates proapoptotic gene BAX but mutated p53 (i.e. absence of p53) reduces apoptotic activity and thus allows cell proliferation. 

b) CD95 receptors are depleted in hepatocellular carcinoma and hence the tumour cells escape apoptosis.