Fundamentals of Cancer

Dr. Rama Rao MallaHead, Dept. of BiochemistryInstitute of ScienceGITAM University

What is cancer ?

Cancer is an abnormal growth of cells caused by multiple changes in gene expression

leading to deregulation of cell

proliferation and cell death

Evolving cell population can invade adjacent tissues and metastasize to distant sites promote the growth of new blood vessels from which the cells derive nutrients. Causing significant morbidity and, if untreated, death of the host

Cancer are usually derived from a single abnormal cell Cancerous (malignant) cells can develop from any tissue within the bodyCancerous cells grow and multiply, form a mass of cancerous tissue—called a tumor

Tumors can be cancerous or noncancerous.

Cancerous cells from the primary (initial) site can spread throughout the body (metastasize).

Over view

General Etiology and Pathogenesis

Cancer is a complex group of diseases with many possible causes.Etiology is the study of causes of a diseaseIt is suggested that every living organism has some inactive cancer-causing genes called proto-oncogenes. A number of physical, chemical or biological agents are known to mutate and activate these proto-oncogenes into active and cancer causing oncogenes. Due to altered gene activity, normal control mechanism is lost and the abnormal cell growth and cell division take place. The physical, chemical and biological agents, which induce cancer growth, are called carcinogens.

Etiology of Cancer or

Causes of cancer

Ionising radiations like X-rays, gamma-rays and particulate radiations from radioactive substances are known to break DNA strands and induce mutations to cause cancers e.g., excessive exposure to sunlight stimulate the development of skin cancer

The evidence of carcinogenic effect of X-rays is the incidence of leukemia in radiologists

Japanese people are exposed to radiations during World War II nuclear explosions and showed the incidence of leukemia.

Carcinogens:

Physical agents

Ultraviolet light (UV) (non-ionizing radiation).

Two nucleotide bases in DNA—cytosine and thymine—are most vulnerable to radiation that can change their properties.

UV light can induce adjacent pyrimidine bases in a DNA strand to become covalently joined as a pyrimidine dimer.

UV radiation, in particular longer-wave UVA, can also cause oxidative damage to DNA

Chemical agents like caffein, polycyclic hydrocarbons, heavy metallic ions etc. are also carcinogenic. Hormones like testosterone and estrogens are known to cause prostate and breast cancer respectively. Chewing of beetles is known to cause mouth cancer. Cigarette and cigar tobacco smoking causes lip, mouth and lung cancers due to presence of a carcinogenic agent, benzpyrene and N-nitroso-dimethylene. Dye workers have a high rate of bladder cancer.Recently high carbohydrate foods like potato chips and French fries are reported to cause cacner due to formation of carcinogenic chemical, called acrylamide by heating

Carcinogens:

chemical agents

Direct-actingDirect-acting carcinogens are already electrophilicElectrophilic (electron-seeking) molecules will bind to nucleophilic (electron-rich) macromolecules in the cell DNA, RNA. Proteinse.g. Nitrogen mustard,NitrosomethylureaBenzyl chlorideIndirect-acting carcinogens are metabolically activated into electrophilic speciese.g. Polycyclic aromatic hydrocarbons (PAH)Produced by incomplete combustion of organic materialsPresent in chimney soot, charcoal-grilled meats, auto exhaust, cigarette smoke

Carcinogens:

Types of chemical carcinogens

Viral infections account for an estimated one in seven human cancers worldwideMajority of these are due to infection with two DNA viruses

HBV - linked to hepatocellular carcinoma

HPV - linked to cervical carcinoma

Very small viruses

Can integrate their viral DNA into host genome

They code for viral proteins which block tumor suppressor proteins in cells

Carcinogens:

Viral Carcinogens:

It contains 70% of proteins Diet, physical inactivity, and obesity are related to approximately 30–35% of cancer deaths. Physical inactivity is believed to contribute to cancer risk not only through its effect on body weight but also through negative effects on immune system and endocrine system. Diets that are low in vegetables, fruits and whole grains, and high processed or red meats are linked with a number of cancers. A high-salt diet is linked to gastric cancer, aflatoxin B1, a frequent food contaminate, with liver cancer, and Betel nut chewing with oral cancer.

Carcinogens:

Diet and exercise:

Cancerous tissues (malignancies) can be divided into two types

Cancer from blood, blood-forming tissues and cells of the immune systeme.g. leukemias and lymphomas

Leukemias arise from blood-forming cells and crowd out normal blood cells in the bone marrow and bloodstream. Cancer cells from lymphomas expand lymph nodes, producing large masses in the armpit, abdomen or chest.

Types of cancer:

leukemias and lymphomas

Solid tumors are solid mass of cells often termed as cancer Cancers can be carcinomas or sarcomas.Carcinomas are cancers of cells that line the skin, lungs, digestive tract, and internal organs.

e.g. skin, lung, colon, stomach, breast, prostate, and thyroid gland.

Typically, carcinomas occur more often in older than in younger people.

Types of cancer:

Solid tumors:

Carcinomas:

Sarcomas are cancers of mesodermal cells.

Mesodermal cells normally form muscles, blood vessels, bone, and connective tissue.

e.g. Leiomyosarcoma - cancer of smooth muscle that is found in the wall of digestive organs and osteosarcoma - bone cancer.

Typically, sarcomas occur more often in younger than in older people.

Types of cancer:

Solid tumors:

Sarcomas :

Normal cells grow and divide, but have many controls on that growth. They only grow when stimulated by growth factors. If they are damaged, a molecular brake stops them from dividing until they are repaired. If they can't be repaired, they commit cell suicide (apoptosis). They can only divide a limited number of times. They are part of a tissue structure, and remain where they belong. They need a blood supply to grow.

Hall marks of cancer:

Several mechanisms are required to transform normal cell to cancer cell. This occurs in a series of steps, which Hanahan and Weinberg refer to as hallmarks.Self-sufficiency in growth signalsInsensitivity to anti-growth signalsEvading apoptosisLimitless replicative potentialSustained angiogenesisTissue invasion and metastasisEach mechanism is controlled by several proteins. These proteins become non-functional or malfunctioning when the DNA sequence of their genes is damaged through acquired or somatic mutations.

Hall marks of cancer:

Normal cells require external growth signals to grow and divide. These signals are transmitted through receptors that pass through the cell membrane. When the growth signals are absent, they stop growing.Cancer cells can grow and divide without external growth signals. Some cancer cells can generate their own growth signals. E.g. glioblastomas produce platelet-derived growth factor and sarcomas can produce tumor growth factor α (TGF-α).Receptors are overexpressed. E.g. Epidermal growth factor receptor is overexpressed in stomach, brain and breast cancers, HER2 receptor is overexpressed in stomach and breast cancer.

Hall marks of cancer:

Self-sufficiency in growth signals

Growth of normal cells is controlled by growth inhibitors present in the surrounding environment or in the extracellular matrix or on the surfaces of neighboring cells.

These inhibitors act on the cell cycle by interrupting cell division (mitosis) in the interphase.

The growth inhibitor signals prevents transition from (G1) to S.

Cancer cells are generally resistant to growth-preventing signals from their neighbours.

Hall marks of cancer:

Insensitivity to anti-growth signals

Apoptosis is a form of programmed cell death, the mechanism by which cells are programmed to die

By apoptotic mechanism mutant cells are continually removed. The apoptotic machinery monitor the cell for abnormal behavior.e.g. Survival signals and their receptors monitor DNA damage, oncogene overexpression, and low oxygen (hypoxia).

The p53 tumor suppressor protein elicits apoptosis in response to DNA damage, and is a major mechanism of cancer control.

Cancer cells are characteristically able to bypass this mechanism.

Hall marks of cancer:

Evading apoptosis

Non-cancer cells die after a certain number of divisions. Cells have an intrinsic program, which limits division to 60–70 doublings and reach senescence.The counting device for cell doublings is the telomere, which decreases in size (loses nucleotides at the ends of chromosomes) during each cell cycle.Most tumor cells are immortalized.Cancer cells escape this limit, indefinitely grow and divide. This limit can be overcome by disabling p53 tumor suppressor proteinsMany cancers involve the upregulation of telomerase, the enzyme that maintains telomeres.

Hall marks of cancer:

Limitless replicative potential

Angiogenesis is the process by which new blood vessels are formed. Angiogenesis is involved in the growth of cervix, breast and melanoma tumors.In order to progress, they must develop a blood supply. New blood vessels continuously supply of oxygen and other nutrients.Angiogenesis is balanced by inducers and inhibitors.Inducers include vascular endothelial growth factor (VEGF) and acetic and basic fibroblast growth factor (FGF 1/2)Inhibitor is thrombospondin-1

Hall marks of cancer:

Sustained angiogenesis

Cancer cells can break away from their site or organ of origin to invade surrounding tissue and spread (metastasize) to distant body parts.

It involves cell adhesion molecules (CAMs) , integrins, E-cadherin and Matrix-degrading proteases.

Specific mutations activate ability of cells to metastasize

Ex. decreased cell to cell adhesion, secretion of preteases that digest surrrounding barriers, and ability to grow in new locations

Hall marks of cancer:

Tissue invasion and metastasis

Genome instability (also “genetic instability” or “genomic instability”) refers to a high frequency of mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy. Genome instability is central to carcinogenesis.e.g. High frequency of externally caused DNA damage Reductions in expression of DNA repair genesEndogenous DNA damage is very frequent, occurring on average more than 60,000 times in human cells, any reduced DNA repair is likely an important source of genome instability.

Genome instability :

Introduction

Chromosomal instability:It involves chromosome abnormalities like deletion and duplication of chromosomes or chromosome parts, chromosome rearrangements and mitotic recombination Microsatillite instability :It is characterized by increased rate of small scale genetic changes Several colorectal and gastric cancer syndromes are known to have defects in the replication of short tandem repeat sequences (microsatellite sequences), knownas microsatellite instability. Mechanism of genomic instability is related to cell cycle regulation, DNA damage and repair. Cell aging and telomere function

Genome instability (GI) :

Types

Genomic instability is caused by cellular metabolism

routine errors in DNA replication recombination.

In addition, exogenous genotoxic agents, such as

ultraviolet light, oxidative stress chemical mutagens, can

lead to a range of nucleotide modifications and DNA breaks.

Genome instability (GI) :

Factors affecting genomic instability

Telomere dysfunction and genomic instability

One of the important source of genomic instability is telomere shortening

Base and nucleotide excision repair

Excise & Repair abnormal bases or nucleotides, such as UV radiation

induced pyrimidine dimersMutations in components of these pathways : Cause genomic instability

Genomic instability (GI) :

Main pathways

Mismatch repair (MMR)

during DNA replication

Loss of function of MSH2 and MLH1, which are required for mismatch repair, results in hypermutation and microsatellite instability

DNA replication

Deregulated DNA replication

Deregulation can occur through oncogene activation , loss of certain tumour suppressors, DNA polymerase inhibition ,

replication stress

Double-strand break repair (DSBR)

Homologous recombination repair of double-strand breaks (DSBs) uses the sister DNA molecule as a template to

repair the break

Defect in recombination leads to chromosomal instability

A tumor supressor gene is a gene that prevents a cell from developing into a cancer cell

Tumor suppressor genes can be grouped into categories including

caretaker genes - stabilize the genomee.g. p53

gatekeeper genes - prevent growth of potential cancer cellse.g. Rb

landscaper genes - create environments that control cell growthE.g. PTEN

Oncogenes and tumor suppressor

genes:

Tumor suppressor genes:

Types :

Both alleles that code for a particular tumor suppressor protein must be affected to cause cancer

This is because if only one allele for the gene is damaged, the second can still produce the correct protein

Tumor-suppressor genes or proteins which hinder cell cycle or promote apoptosis.

The functions of tumor-suppressor proteins are:

Repression of genes that are essential for cell cycle progresson . If these genes are not expressed, the cell cycle does not continue, effectively inhibiting cell division.

Tumor suppressor genes:

Functions :

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 cannot be repaired, the cell should initiate apoptosis (programmed cell death) to remove the threat it poses for the greater good of the organisms produced

Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis. These proteins are known as metastasis suppressors.

Tumor suppressor genes:

Functions :

DNA repair proteins are usually classified as tumor suppressors as well, as mutations in their genes increase the risk of cancerExample: mutations in BRCA

The first tumor-suppressor protein discovered was the Retinoblastoma protein (pRb) in human retinoblastoma

Another important tumor suppressor is the p53 tumor-suppressor protein encoded by the TP53 gene

PTEN is third tumor suppressor protein acts by opposing the action of PI3K

Tumor suppressor genes:

Examples :

An oncogene is a gene that has the potential to cause cancer.

In tumor cells, they are often mutated or expressed at high levels.

Oncogenes are activated form of proto-oncogenes

There are three basic methods of activation:

1. Mutation within a proto-oncogene, or within a regulatory region can cause a change in the protein structure, causing an increase in protein (enzyme) activity or loss of regulation

Oncogenes :

Activation:

2. An increase in the amount of a certain protein caused by an increase of protein expression (through misregulation)

an increase of protein (mRNA) stability, prolonging its existence and thus its activity in the cell

gene duplication (one type of chromosome abnormality), resulting in an increased amount of protein in the cell

Oncogenes :

Activation:

3. A chromosomal translocation (another type of chromosome abnormality)

There are 2 different types of chromosomal translocations that can occur:

translocation events which relocate a proto-oncogene to a new chromosomal site that leads to higher expression

translocation events that lead to a fusion between a proto-oncogene and a 2nd gene (this creates a fusion protein with increased cancerous/oncogenic activity)

Oncogenes :

Activation:

Types of Oncogenes:

Category Examples Gene functionsGrowth factors, or mitogens c-Sis induces cell

proliferation.

Receptor tyrosine kinases

epidermal growth factor receptor (EGFR)

transduce signals for cell growth and differentiation.

Serine/threonine kinases 

cyclin-dependent kinases (through overexpression)

cell cycle regulation

Regulatory GTPases Ras protein involved in signalling

Transcription factors

myc geneRegulate transcription of genes that induce cell proliferation.

In humans, mutations leading to gain of functions of proto-oncogenes or loss of functions of tumor-suppressor genes (TSGs) predispose to cancerAnimal models have been instrumental in the study of genes involved in human cancer initiation and progression.

Transgenic and knockout cancer mouse models have been developed, which exhibit many biologic hallmarks of human cancerThe major advantages of these models are (i) initiating genetic event is

known(ii) mice are immunocompetent(iii) tumors develop

spontaneously in their appropriate tissue compartments.

Models of cancer study:

Gene knockouts

A knockout mouse is a genetically modified mouse

In knockout mice an existing gene by replacing it or disrupting it with an artificial piece of DNA.

The loss of gene activity often causes changes in a mouse's phenotype, which includes

appearance, behavior observable physical

and biochemical characteristics

Models of cancer study:

Gene knockouts

procedure of producing knockout mice:

1. The gene to be knocked out is isolated from a mouse gene library.

2. New DNA sequence is engineered into the isolated original gene, which become inactive.

3. Stem cells are isolated from a mouse blastocyst

4. The engineered new DNA sequence is introduced into the stem cells by electroporation.

5. The stem cells are isolated using the marker gene

6. The knocked-out stem cells from are inserted into a mouse blastocyst

7. These blastocysts are then implanted into the uterus of female mice, where they develop.

Models of cancer study:

Gene knockouts

8. Some of the newborn mice will have gonads derived from knocked-out stem cells, and will therefore produce eggs or sperm containing the knocked-out gene.

9. When these mice are crossed with wild type, some of their offspring will have one copy of the knocked-out gene in all their cells. However they are still heterozygous.

10.When these heterozygous offspring are crossed, some of their offspring will inherit the knocked-out gene from both parents

11. They carry no functional copy of the original gene

Many mouse models are named after the gene that has been inactivated. For example, the p53 knockout mouse is named after the p53 gene is inactivated

Models of cancer study:

Gene knockouts

Knockout mice are used in a variety of ways.

One of the most exciting applications of knockout technology is in biomedical research. Gene knockout mouse models are used to study the progression of genetic based diseases at the molecular level.

They used to test the functions of specific gene and to observe the regulation of genes

Gene knockouts :

Uses

A transgenic mouse is a biological model which is genetically modified by the introduction of a foreign DNA sequence into a mouse egg. The insertion of the foreign DNA usually results in a gain of function (expression of a new gene) or in the over-expression of endogenous genes.The classic method used for the generation of transgenic mice is called pronuclear injection. The transgene is injected into a fertilized mouse egg and then integrates at random positions in the genome.

Transgenic mouse models are mainly used in modern cancer research.Used to study tumor initiation and progression, metastasis, and therapy

Transgenic mouse model:

The TET system is used to analyze expression of gene after the onset of a diseaseTET system comprises two complementary circuits: The tTA-dependent circuit (TET-Off system) and the rtTA-dependent circuit (TET-On system).TET-Off system: tetracycline prevents the tTA transcription factor from binding DNA at the promoter. Gene expression is inhibited in the presence of tetracycline.

TET-On system: tetracycline binds the rtTA transcription factor and allows it to bind DNA at the promoter. Gene expression is induced in the presence of tetracycline

Regulatable system in Knock out and transgenic mice:

Tetracycline system

Gene expression is regulated by the presence or absence of tetracycline. Tetracycline binds directly to the transcription factors.