Cancer Causation and Development

Udadi Sadhana

CANCER CAUSATION
A wide diversity of biological, chemical, and physical agents have the potential for inducing cancer in man. However, the majority of man's common cancers have no recognized cause (breast and ovary in females, prostate in males, brain and pancreas in both sexes). Many of these spontaneouslydeveloping cancers may result from the effects of endogenous mutagens, i.e. mutagens that originate in the body, such as oxygen free radical.
Each of the various classes of agents known to induce cancer in man or animal (naturally or experimentally differ; each has a different mechanism of action, and the epidemiology and pathogenesis of the disease processes also differ. Thus, an analysis of each of the individual causes of cancer is a perplexing, complicated undertaking. Since in this course little time is available to explore these issues in detail and discuss examples individually, we are obliged to provide you generalization annotated by specific examples.

An enormous body of data accumulated from observations in humans and experiments in animals indicate that host factors are critical to the development of cancer. These involve a wide variety of genetic influences as well as factors unique to the environment and lifestyle of the host. All of us experience DNA injury on a continuous basis in both our somatic and reproductive cells. Fortunately, this injury is usually repaired by natural mechanisms. When it is not, a mutation occurs. The mutation may be silent, it may be lethal, it could cause a developmental abnormality, or it could result in cancer. Usually, however, several mutational changes must accumulate in the cell genome for cancer to develop.
Factors related to host susceptibility may determine whether or not disease develops. One can look at this possibility optimistically or pessimistically. From a pessimistic perspective, the notion that we all experience DNA changes which could result in clinical cancer is frightening. From an optimistic perspective, we can accept the fact that cancer occurs uncommonly, whereas DNA damage is an everyday event.

CANCER DEVELOPMENT
Cancer develops when critical changes in the DNA of cells results in their loss of responsitivity to nornal cellular control mechanisms. 1. DNA Changes Occurring in Humans Associated with Cancer
Addition of "new" DNA acquired and integrated with host DNA.
Examples: Epstein-Barr Virus (EB

Oxidative injury to DNA

Examples: Ionizing irradiation (clinical radiation, weapons, and energy generation)- leukemia, breast, thyroid, bronchogenic carcinoma

Adducts to DNA with secondary effects on DNA structure and makeup

Examples: Metabolism of polycyclic aromatic hydrocarbons results in the generation of electrophils which adduct to DNA in bronchial mucosa cells of cigarette smokers- lung cancer Metabolism of aflatoxin- food contaminants and additives - stomach and liver cancer

Induction of chromosomal gaps, breaks, translocations, and sister chromatid exchanges can result in the loss of "key" genetic information resulting in disorganized function.

The loss of SUPPRESSOR genes from cells by these mechanisms may be one of the key factors allowing cancer to develop and progress.

2. Radiation Types:
Ionizing irradiation
X ray alpha, beta, gamma particles neutrons radon gas - alpha particle emitter

Non-ionizing
UV light

3. Electromagnetic Force A complex topic of contemporary interest but no cause and effect relation to cancer has been established 4. Variables Affecting Outcome Dosage effects Type of irradiation- i.e., alpha, beta, etc. Acute exposure (one time) vs. chronic exposure Age susceptibility factors

5. Chemicals
Direct Acting:
Heavy metals - arsenic, nickel, chromium Polycyclic aromatic hydrocarbons Nitrosamines Chemotherapeutic agents

Indirect Acting:
Hormones- such as estrogen and estrogenic compounds such as polychlorobyphenyls (PCB) and Agent Orange (dioxins) Promoters- many constituents of cigarette smoke are promoter substances. A promoter is not a carcinogen, but acts to enhance the effects of a initiator carcinogen by increasing the likelihood that cancer will develop or shorten the latency period.

Many common chemicals are carcinogenic in animals, but relatively few have been proven to cause cancer in humans. Dosage effects- threshold considerations

6. Viruses
DNA- Papillomaviruses, genital papillomas (condylomas) and carcinoma of cervix
Hepatitis B virus - hepatoma Epstein-Barr Virus

RNA- HTLV-1, the agent causing T Cell Leukemia/lymphoma 7. Physical Agents Asbestos- Mesothelioma- a rare pleura cancer Schistosoma hematobium (a common parasite in Africa that lays eggs in the bladder wall)- resulting in urinary bladder cancer

8. Host Factors Genetic

DNA repair enzyme defects or deficiencies (xeroderma pigmentosa) Chromosomal instability- (Bloom's syndrome, ataxic telangiectasia) Variability in P450 enzyme activity (genetically controlled or chemically induced in target cells altering DNA adduct formation and electrophil generation or inactivation. Heritable deletion of suppressor genes
P53 gene Li-Fraumeni syndrome RB gene- Retinoblastoma (a rare eye cancer of infants)

Nutrition
Obesity---> hyperestrogenism---> breast cancer Relative vitamin A deficiency---> cell proliferation and metabolic instability--->lung cancer. Antioxidants in diet- natural occurring dietary constituents (Vitamin E, selenium) High fat diets---> change biota of GI tract---> colon cancer

CHEMICAL CARCINOGENESIS
The concept of chemical carcinogenesis was first introduced to medical science during the industrial revolution. In that era, residential and industrial building heating was primarily provided by coal fires.
Sir Percival Potts, a surgeon, noted that many of these young men developed "warts" on the scrotum and later warts of this type sometimes became invasive cancers. He associated their work with the development of the "warts" and concluded that the soot in the chimneys was responsible. Innovatively, Potts, provided an opportunity for these youngsters to bath routinely so that the soot would not accumulate in the corrugated skin of the scrotum.

We know now that coal soot contains coal tar that is comprised of a large number of complex hydrocarbons including the polycyclic aromatic hydrocarbons. The basic precursor chemicals are found in coal, wood and petroleum where there are not carcinogenetic, but after incineration at elevated temperatures the noncarcinogenetic chemical are converted to carcinogens, i.e., they are active carcinogens upon metabolism in epithelial cells on the surfaces of the respiratory tract. Humankind now are exposed to these polycyclic aromatic hydrocarbons in PETROLEUM PRODUCTS, COAL EFFLUENTS such as those from coke ovens, and cigarette smoke.

Everyone knows that cigarettes are the major cause of bronchogenic carcinoma (lung cancer). The mechanisms are clearly outlined in your book. You should have in mind a very specific knowledge as to how the polycyclic aromatic hydrocarbons in cigarette smoke are transformed to electrophils by CYTOCHROME P450 MONOOXYGENASE, the ELECTROPHILS then have the ability to form adducts on the DNA and ultimately to cause DNA damage. This is discussed in your text. I will discuss smoking and disease in the Environmental Pathology lecture. Bronchogenic carcinoma is an example of MULTISTAGE CARCINOGENESIS in which multiple cumulative chemical injuries to the cells of the respiratory tract ultimately result in cancer. It introduces the concept of the initiator and the promoter of cancer. In this concept, the polycyclic aromatic hydrocarbons are the initiator of DNA injury but many additional chemicals in the cigarette smoke stimulate cell proliferation and increase sensitivity to the carcinogenic initiator.

VIRAL CARCINOGENESIS
LEUKEMIA AND LYMPHOMAS occur in approximately 1% of the general population. As noted above, the prevalence of these diseases is increased in individuals exposed to excessive concentrations of irradiation (atomic bomb victims) and certain chemicals, including chemotherapeutic agents used to treat solid tumors such as Hodgkin's disease. The role of RETROVIRUSES in human leukemia has become apparent in recent years with the appearance of HTLV-1, the cause of T-CELL LEUKEMIA. Burkitt's lymphoma is a unique malignancy of children, which is prevalent in the highlands of East Africa. It comprises about 1% of the lymphomas occurring in children in this country and the rest of the western world today. The tumor biology is intimately related to infections with EPSTEIN-BARR (EB) virus, a member of the Herpesvirus group. In East Africa, youngsters almost invariably develop the disease before the age of 10. They exhibit massive tumors having a unique morphology, particularly in the mandible of the face, and in females in the ovaries. These are unusual locations for lymphomas in children. Herpesviruses of other types have been shown to play a role in carcinogenesis. Herpesvirus type II was thought to play a role in carcinoma of the cervix.

FOREIGN BODY CARCINOGENESIS

Mesothelioma In 1960, an inquisitive pathologist and epidemiologist discovered that asbestos, of the amphibole type, is associated with malignant mesotheliomas, an exceedingly rare form of tumor of the pleura. Since that time, countless Americans have developed mesotheliomas and died as a consequence of asbestos exposure. These unique tumors spread in the pleural cavity of the lung of individuals and expand to encroach upon the lung until the individual suffocates. Rarely they metastasize. Occasionally, malignant mesotheliomas occur in the abdominal cavity. The mechanisms whereby asbestos causes mesothelioma remains to be defined. Bladder Cancer Schistosoma hematobium is an exceedingly common parasitic infestation of the venules of the urinary bladder in individuals residing in certain parts of Africa and Asia. As a result, the eggs are deposited in the walls of the urinary bladder where they fester and cause a granulomatous response. This irritates the mucosa of the bladder resulting in a hyperplasia of the transitional epithelium of the bladder. Occasionally it becomes malignant, resulting in squamous cell carcinoma. There is a strong association of cigarette smoking with urinary tract carcinomas in those who live in the western world. In endemic areas of Schistosomiasis, there may also be the superimposed effect of a chemical carcinogen which is excreted through the urine. We just don't know.

Tumor suppressor gene

A tumor suppressor gene is a gene that reduces the probability that a cell in a multicellular organism will turn into a tumor cell. A mutation or deletion of such a gene will increase the probability of the formation of a tumor.

Two-hit hypothesis
Unlike oncogenes, tumor suppressor genes generally follow the 'two-hit hypothesis,' which implies that both alleles that code for a particular gene must be affected before an effect is manifested. This is due to the fact that if only one allele for the gene is damaged, the second can still produce the correct protein. In other words, tumor suppressors are usually not haploinsufficient, although there are notable exceptions (the p53 gene product).

Functions
Tumor suppressor genes, or more precisely, the proteins for which they code, either have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. The functions of tumor suppressor proteins fall into several categories including the following:
Repression of genes that are essential for the continuing of the cell cycle. If these genes are not expressed, the cell cycle will not continue, effectively inhibiting cell division. Coupling the cell cycle to DNA damage. As long as there is damaged DNA in the cell, it should not divide. If the damage can be repaired, the cell cycle can continue. If the damage can not be repaired, the cell should initiate apoptosis, or programmed cell death, to remove the threat it poses for the greater good of the organism. Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis.

Examples
The first tumor suppressor protein discovered was the pRb protein in human retinoblastoma; however, recent evidence has also implicated pRb as a tumor survival factor. Another important tumor suppressor is the p53 tumor suppressor protein produced by the TP53 gene.

p53
p53, also known as protein 53 (TP53), is a transcription factor that regulates the cell cycle and hence functions as a tumor suppressor. It is important in multicellular organisms as it helps to suppress cancer. p53 has been described as "the guardian of the genome", "the guardian angel gene", or the "master watchman", referring to its role in conserving stability by preventing genome mutation. The name p53 is in reference to its apparent molecular mass: it runs as a 53 kilodalton (kDa) protein on SDS-PAGE. Its theoretical molecular mass, based on the sum of its amino acid residues, is in fact only 43.7 kilodalton. This difference is due to the high number of amino-acid proline in the p53 protein. This leads to an aberrant migration on SDS-PAGE. This observation is also found for p53 from other species, such as rodents, frogs, and fish.

p53 (blue) bound to a short DNA fragment (orange); zinc ions (green). Based on PDB id 1TUP. tumor protein p53

p53 has many anti-cancer mechanisms:

It can activate DNA repair proteins when DNA has sustained damage.
It can also hold the cell cycle at the G1/S regulation point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle.) It can initiate apoptosis, the programmed cell death, if the DNA damage proves to be irreparable.

p53 pathway: In a normal cell p53 is inactivated by its negative regulator, mdm2. Upon DNA damage or other stress, various pathways will lead to the dissociation of the p53 and mdm2 complex. Once activated, p53 will either induce a cell cycle arrest to allow repair and survival of the cell or apoptosis to discard the damage cell. How p53 make this choice is currently unknown.

Regulation of p53 activity

p53 becomes activated in response to a myriad of stress types, which include but is not limited to DNA damage (induced by either UV, IR or chemical agents,such as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion and deregulated oncogene expression. This activation is marked by two major events. Firstly, the half-life of the p53 protein is increased drastically, leading to a quick accumulation of p53 in stressed cells. Secondly, a conformational change forces p53 to take on an active role as a transcription regulator in these cells. The critical event leading to the activation of p53 is the phosphorylation of its N-Terminal domain. The NTerminal transcriptional activation domain contains a large number of phosphorylation sites and can be considered as the primary target for protein kinases transducing stress signals.
The protein kinases which are known to target this transcriptional activation domain of p53, can roughly be divided into two groups. A first group of protein kinases belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc... A second group of protein kinases (ATR, ATM, Chk1, Chk2, DNA-PK, CAK) is implicated in the genome integrity checkpoint, a molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress. In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A protein called Mdm2 binds to p53 and transports it from the nucleus to the cytosol where it becomes degraded by the proteasome. Phosphorylation of the N-terminal end of p53 by the above mentioned protein kinases disrupts Mdm2-binding. Other proteins, such as Pin1, are then recruited to p53 and induce a conformational change in p53 which prevents Mdm2-binding even more. Trancriptional coactivators, like p300 or PCAF, then acetylate the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes.

Role in disease
If the TP53 gene is damaged, tumor suppression is severely reduced. People who inherit only one functional copy of the TP53 gene will most likely develop tumors in early adulthood, a disease known as Li-Fraumeni syndrome. The TP53 gene can also be damaged in cells by mutagens (chemicals, radiation or viruses), increasing the likelihood that the cell will begin uncontrolled division. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. Increasing the amount of p53, which may initially seem a good way to treat tumors or prevent them from spreading, is in actuality not a usable method of treatment, since it can cause premature aging.[citation needed] However, restoring endogenous p53 function holds a lot of promise. Certain pathogens can also affect the p53 protein that the TP53 gene expresses. One such example, the Human papillomavirus (HPV), encodes for a protein, E6, which binds the p53 protein and inactivates it. This, in synergy with the inactivation of another cell cycle regulator, p105RB, allows for repeated cell division manifestested in the clinical disease of warts. In healthy humans, the p53 protein is continually produced and degraded in the cell. The degradation of the p53 protein is, as mentioned, associated with MDM2 binding. In a negative feedback loop MDM2 is itself induced by the p53 protein. However mutant p53 proteins often don't induce MDM2, and are thus able to accumulate at very high concentrations. Worse, mutant p53 protein itself can inhibit normal p53 protein levels.

History
p53 was identified in 1979 by Arnold Levine, David Lane, and Lloyd Old, working at Princeton University, Imperial Cancer Research Fund (UK), and Sloan-Kettering Memorial Hospital, respectively. It had been hypothesized to exist before as the target of the SV40 virus, a strain that induced development of tumors. The TP53 gene from the mouse was first cloned by Peter Chumakov of the Moscow Academy of Sciences in 1982, and independently in 1983 by Moshe Oren (Weizmann Institute). It was initially presumed to be an oncogene due to the use of mutated cDNA following purification of tumour cell mRNA. Its character as a tumor suppressor gene was finally revealed in 1989 by Bert Vogelstein working at Johns Hopkins School of Medicine. Warren Maltzman, of the Waksman Institute of Rutgers University first demonstrated that TP53 was responsive to DNA damage in the form of ultraviolet radiation. In a series of publications in 1991-92, Michael Kastan, Johns Hopkins University, reported that TP53 was a critical part of a signal transduction pathway that helped cells respond to DNA damage. In 1993, p53 was voted molecule of the year by Science magazine.

Retinoblastoma protein
The retinoblastoma protein, also called pRb or Rb, is a tumor suppressor protein found to be dysfunctional in a number of types of cancer. pRb was so named because retinoblastoma cancer results when the protein is inactivated by a mutation in both alleles of the RB1 gene that codes for it. The "p" in pRb stands for protein and is a way to distinguish it from the gene, Rb. pRb is usually present as a phosphoprotein inside cells and is a target for phosphorylation by several kinases as described below. One highly studied function of pRb is to prevent the cell from dividing or progressing through the cell cycle. Thus, when pRb is ineffective at this role, mutated cells can continue to divide and may become cancerous. pRb is a member of the 'Pocket protein family', because it has a pocket to which proteins can bind. Oncogenic proteins such as those produced by cells infected by high-risk types of human papillomaviruses can bind and inactivate pRb, which can lead to cancer.

Cell cycle suppression

pRb prevents the cell from replicating damaged DNA by preventing its progression through the cell cycle into its S, or synthesis phase or progressing through G1, or first gap phase. pRb binds and inhibits transcription factors of the E2F family. E2F transcription factors are dimers of an E2F protein and a DP protein. The transcription activating complexes of E2 promoter-bindingprotein-dimerization partners (E2F-DP) can push a cell into S phase. As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. When pRb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The pRb-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin, further suppressing DNA synthesis

Activation and inactivation pRb can actively inhibit cell cycle progression when it is dephosphorylated while this function is inactivated when pRb is phosphorylated. pRb is activated near the end of mitosis (M phase) when a phosphatase dephosphorylates it, allowing it to bind E2F. When it is time for a cell to enter S phase, complexes of cyclindependent kinases (CDK) and cyclins phosphorylate pRb, inhibiting its activity.The initial phosphorylation is performed by Cyclin D/CDK4,6 and followed by additional phosphorylation by Cyclin E/CDK2. pRb remains phosphorylated throughout S, G2 and M phases. Phosphorylation of pRb allows E2F-DP to dissociate from pRb and become active.When E2F is freed it activates factors like cyclins (e.g. Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA