Lecture: 1 Molecular Basis of Cancer. What Is Cancer? Cancer is a group of diseases caused by the uncontrolled multiplication of abnormal cells in the.

Lecture: 1Molecular Basis of Cancer

What Is Cancer?• Cancer is a group of diseases caused by the uncontrolled

multiplication of abnormal cells in the body, a process calledneoplasia.

• Abnormal new tissues called neoplasms are formed.• Neoplasms usually form masses called tumors that may be

benign (non cancerous) or malignant (cancerous).• Malignant or cancerous tumors grow rapidly,are invasive

(to surrounding tissue) and metastatic (traveling viablood/lymph to invade distant tissues).

• Cancers destroy healthy tissues causing loss of function anddeath.

• Cancer is the 2nd major killer in populations of developedcountries & the leading cause of death in children 3-15 (US).

• Cancers are genetic disorders caused by accumulation ofsomatic mutations (gene & chromosome) in a person’s cells.

• Inherited mutations give a predisposition for certain cancers.

Characteristics of Cancer Cells• Cancer cells are genetically altered via gene or

chromosome mutations so:- lack normal controls over cell division or apoptosis.- may express inappropriate genes (e.g. for telomerase,enzyme that maintains length of DNA for continued division)- are genetically unstable due to loss of DNA repairmechanisms (so are more susceptible to radiation damagethan normal cells).

• Divide excessively (proliferate) & indefinitely producingneoplasms.

• Live indefinitely (do not show apoptosis).• Lose the normal attachment to other cells so become

metastatic (travelling via blood/lymph to invade distantsites).

• Secrete signals for angiogenesis (growth of blood vesselsinto tumor).

Cancer Cells are Undifferentiated & Malignant

• Cancer cells areundifferentiated tovarying degrees(even anaplastic, likestem cells) so divide& do not perform thenormal function ofmature cells.

• The lessdifferentiated thecancer cell the moremalignant thecancer (the morerapidly growing is thetumor).

What Causes Cancer?

Inherited mutations in genes that affect cell cycle,DNA repair, or apoptosis: these mutations give agenetic predisposition for cancer.Somatic mutations to these same genes caused by:

• Exposure to risk factors- environmental mutagens (carcinogenic chemicals,radiation)- hormones- weakening of immune system (as in AIDS).

• Oncogenic (tumor) Virus infections- Epstein Barr virus (causes Burkitt lymphoma)- Human Papilloma Virus (causes cervical cancer).

Tumor viruses transform human cells into cancer cells by:•Introducing viral cancer - causing oncogenes into host cell DNA•Causing Translocation and overexpression of host protooncogenes.

Normal cell cycle is controlled by signal transduction:

• Growth factors bind to surface receptors on the cell; transmembrane proteins relay signals into the cell.

• Two types of growth factors:

1. Growth factors stimulate cell division.

2. Growth-inhibiting factors inhibit cell division.

• Healthy cells divide only when growth factor and growth-inhibiting factor balance favors cell division.

• Cancer cells divide without constraint(e.g., mutations in growth and growth-inhibiting factor genes).

Regulation of cell division by signal transduction.

The Cell Cycle

M (mitosis)M (mitosis)GG22

(cell growth)(cell growth)

REPAIRS AHEAD

S (synthesis)S (synthesis)

GG00 (resting) (resting)

OncogenesOncogenes

Tumor suppressor genesTumor suppressor genes

GG11DNA repairDNA repair

genesgenes

CDK

p53 is known as the ‘ guardian of the genome ‘

Cell cycle

CONTROL of CELL CYCLE

The Cell has 3 major“checkpoints” that aresensitive to signals, theG1, the G2 and the Mcheckpoints.

• If a cell does notpass G1 checkpointit enters a non-dividing GO Phase.

• Most somatic cellsare in GO.

• Some cells in GO

(e.g. liver cells) canreenter cell cycle ifneeded.

G0

G1

checkpoint

DifferentiatedCells enternon dividingG0 phase-

G1 Controlsystem S

M G2

M checkpointG2 checkpoint

In cancer cells genes that control cell cycle have mutatedso cells divide excessively producing neoplasms.

Cancer and genesCancer and genes::

Three classes of genes are frequently mutated in cancer:Three classes of genes are frequently mutated in cancer:

• Proto-oncogenes (Proto-oncogenes ( oncogenes) oncogenes)

• Tumor suppressor genesTumor suppressor genes

• Mutator genesMutator genes

Proto-oncogenes oncogenes:

Proto-oncogenes

• Proto-oncgenes are genes that possess normal gene products and stimulate normal cell development.

Oncogenes

• Oncogenes arise from mutant proto-oncogenes.

• Oncogenes are more active than normal or active at inappropriate times and stimulate unregulated cell proliferation.

Some tumor viruses that infect cells possess oncogenes:

• RNA tumor viruses = possess viral oncogenes (derived form cellular proto-oncogenes) capable of transforming cells to a cancerous state.

• DNA tumor viruses = another class of tumor viruses; do not carry oncogenes, but induce cancer by activity of viral gene products on the cell (no transformation per se).

Types & effects of different types of mutations:

1. Point mutations: occur in protein coding or controlling sequences.

2. Deletion: frameshifts may lead to defective proteins.

3. Gene amplification: random over-replication of small segments of DNA results in extra copies (up-regulates cell growth).

Mutator genes:Mutator genes:

• Mutator gene increases spontaneous mutation rate of other genes.

• Mutator gene products are involved in DNA replication and repair; mutations make the cell error prone.

Proteins Control Cell Division

The cell cycle is controlled by proteinsfrom inside & outside the cell.

• Intracellular Cyclins and Cyclin Dependent Kinases(CDKs) control the checkpoints.

• Hormones or extracellular proteins from other cells(called Growth Factors) signal target cell to divide.

- Hormones (e.g. Growth Hormone) or Growth Factorsbind to receptor proteins of target cell membrane.

- This triggers a molecular signaling pathway.

- A series of linked proteins activate Cyclin- CDKs whichAllows Cells to Pass Cell Cycle Checkpoints & divide.

How Growth Factors Trigger Cell Division

Growth Factoror Hormone

Plasmamembrane

Receptorprotein

Relay•G1 checkpoint prevents

Proteins opendamaged DNA from replicating

checkpoints•Checkpoint controlled byCyclin - CDK

SignalTransductionPathway G1

Controlsystem S

M G2

Tumor Suppressor Proteins Inhibit CellDivision & Prevent Cancer

Tumor suppressor proteins are proteins thatbind to checkpoint proteins to stop the cellcycle & prevent cell division if DNA is damaged.

• Tumor suppressor proteins stop division ofmutated cells until mistakes in DNA arerepaired by enzymes.

• TS proteins keep most mutations from beingpassed on to daughter cells & developing intocancer.

• If the genes for TS proteins mutate the brakeon cell division is removed cancers may result.

• Two important TS proteins are the p53 protein& the RB protein.

The p53 Tumor Suppressor Protein

The p53 tumor suppressor protein is activatedwhen DNA is damaged. The p53 gene is called the“guardian angel of the genome”

P53 proteinactivatesgenes forproteins that

•Prevent cellentering Sphase

•Repair DNA•Causeapoptosis (ifDNA isirreparable)

Internalsignallingpathway

DNA repairApoptosis

Cell cannot enter S phase

Gene Mutations That Cause Cancer

Mutations in 4 types of genes cause Cancer• Proto - oncogenes: genes that code for normal proteins

used in cell division

–Growth factors

–Membrane Receptors for Growth Factors

–Signaling Proteins (e.g. ras proto- oncogenemutates in 30% of cancers).

• Tumor Suppressor genes: gene that code for proteins thathelp prevent uncontrolled cell division by blocking key steps(e.g. DNA replication).

- Retinoblastoma susceptibilty (RB) gene- p53 gene mutates in >50% of cancers.

• DNA Repair genes• Genes for Apoptosis

HowCarcinogens

CauseCancer Inactivation

of DNARepair Genes

Activation ofOncogenes

Inactivationof Genes forApoptosis

Inactivation ofTumorSuppressorGenes

Oncogenes Are Mutated Proto-oncogenes

A cell can acquire a cancer - causing oncogenefrom•A virus•A mutation in a proto-oncogene.

Oncogenes still code for the proteins needed forcell division but they cause cancer by producing– Too much of the protein– An abnormally active protein, e.g. protein that

activates division by itself– Protein that is made when it is not needed– Protein that should be made by a different (i.e.

dividing) cell.

Cancer causing Mutations

•Proto-oncogenes form active oncogenes by- being misplaced (e.g. by translocation) to a site wherethe gene is continually expressed resulting inoverproduction of a protein that stimulates cell division(e.g. in Chronic Myeloid Leukemia)- By mutating to a form that is over expressed.

•Cancer causing Mutations in Tumor Suppressor genesinactivate the genes so normal protein product is not formed.

oncogeneMutatedTumorSuppressorgene

neoplasm

OverStimulation

of CellDivision byOncogene

Growthfactor

Normal productof ras gene

Relayproteins

MembraneReceptor Hyperactive

relay protein(product ofras oncogene)issues signalson its own.

Transcription factor(activated)

DNA

Transcription

Protein thatstimulatescell division

Translation

Tumor-suppressor gene Mutated tumor-suppressor gene

NormalProteinpreventscelldivisionif DNA isdamaged

Cancerfrom

Mutationof TumorSupressor

Gene

Cell divisionallowed if DNArepaired

Defective,nonfunctioningprotein

Proteinabsent(cell divisionnot blocked)

Mutations accumulatein cancer cells

Multiple Genetic Changes Cause Cancer

Cancers result from a series of genetic changes in a cell lineage

• Inherited (germline) cancers begin with an inherited cancersusceptibility mutation in every cell that is passed on tooffspring.

• Inherited cancers may follow a dominant pattern, e.g. InheritedRetinoblastoma caused by a mutation in the Rb tumorsupressor gene increases cancer risk 10,000 x.

• However, Inherited cancers need at least one more somaticmutation for cancer to develop (“2 hit hypothesis for cancercausation”) .

• Sporadic cancers are caused solely by somatic mutations

occurring in certain body cells so are not passed on tooffspring.

• Accumulation of somatic mutations in a cell over timeeventually leads to uncontrolled cell division and cancer.

• Therefore sporadic cancers tend to appear much later in life thaninherited cancers.

Accumulation of Mutations Cause Cancer

• inheritance of a germ cell mutation acts as a riskfactor for cancers by reducing the number ofsomatic mutations required to cause cancer.

• Early mutations show up in all subsequent stagesof a cancer.

1NormalChromosomes mutation

2mutations

3mutations

4mutations

Normal cell Malignant cell

Oncogenes

Normal genes Normal genes (regulate cell (regulate cell

growth)growth)

1st mutation1st mutation(leads to (leads to

accelerated cell accelerated cell division)division)

1 mutation sufficient for role in cancer development1 mutation sufficient for role in cancer development

Tumor Suppressor Genes

Normal genes Normal genes (prevent cancer)(prevent cancer)

1st mutation1st mutation(susceptible carrier)(susceptible carrier)

2nd mutation or loss 2nd mutation or loss (leads to cancer)(leads to cancer)

The Two-Hit Hypothesis

First hit First hit

First hit in First hit in germline of germline of

childchild

Second hitSecond hit(tumor)(tumor)

Pathogenesis of Colon Cancer

Colon

Colon Cancer is usuallySporadic & develops in a seriesof steps caused by a series ofsomatic cell mutations

Loss oftumor-suppressor

Colon wall gene (e.g. APC)

Removal of polypsprevents cancer

Normal colonepithelial cells

APC: adenomatous polypodsis coli

Small benigngrowth (polyp)= adenoma

Pathogenesis of Colon Cancer -2

Activation ofras oncogene

Loss oftumor-suppressorgene p53

Campbell, & ReeceBiology fig. 19.13

Loss of Additionaltumor- mutations

Malignant tumorsuppressorLarger benign (adenocarcinoma)Small benign genegrowth (adenoma)growth (polyp)

Environmental Risk factors for Colon Cancer• Low fiber diet• Smoked meats (contain heterocyclic aromatic amines that are

converted to mutagens in the liver)• Low intake of fruits & vegetables (antioxidants)• Low intake of cruciferous vegetables (a chemical in brocolli,

brussels sprouts, cabbage activates enzymes that blockformation of mutagens)

Genetic Abnormalities Associated With Hematologic Malignancies

A- Point MutaionMutaions within the RAS oncogenes or P53 tumor- suppressor gene are common in many haempoietic malignancies. The point mutation may involve several base pairs. In 35% of cases of AML the nucleophosmin gene shows an insertion of 4 base pairs.

B- TranslocationIncludes two main mechanisms:

1- Fusion of parts of two genes to generate a chimeric fusion gene that codes a novel fusion protein. Ex: BCR- ABL in t(9; 22) in chronic myeloid leukaemia.

2- Overexpression of a normal cellular gene. Ex: overexpression of BCL-2 in the t(14; 18) translocation of follicular lymphoma or MYC gene in Burkitt,s lymphoma.

C- Deletions May involve a small part of a chromosome, the short or long

arm or the entire chromosome. Losses most commonly affect chromosomes 5, 6, 7, 11, 20 and Y. The critical event is probably loss of a tumor suppressor gene.

D- Duplication or amplification Gains are common in chromosomes 8, 12, 19, 21 and Y. It is

not a common feature in haematologic malignancy but has been described involving the MLL gene.

E- Epigenetic alterations Means alterations in the mechanism by which genes are

transcribed and are stably inherited with each cell division so they are passed on as the malignant cell divides. The most important mechanisms are methylation of cytosine residues in DNA and enzymatic alterations such as acetylation or methylation of the histone protein that package DNA.

Prostate Cancer

• Prostate Cancer is the most common cancer among men (esp.>65 yrs, African - Americans) & 2nd in cancer deaths in men.

• Risk factors include increasing age, race, family history, fat diet,male hormones over many years.

• Adenocarcinoma occurs in periphery of prostate gland.

• Metastasises to lungs, bones (bone pain often first symptom asearly stage of primary tumor may be asymptomatic).

• Tumors are graded from 1 (well differentiated cells) to 5 (leastdifferentiated cells, high malignancy).

• Manifestations (some similar to Benign Prostatic Hyperplasia):- changes in voiding pattern, dysuria, hematuria,– from metastasis low back pain from bone, wt loss, anemia, &

shortness of breath.

• Screening (important for early asymptomatic cancers) includes:- digital rectal exam (palpation of prostate by DRE detects

nodular lump)- transrectal ultrasonography (measures prostate vol: more

sensitive than DRE) - PSA blood test

PSA Test for Prostate Cancer

• Tumor cells express abnormal genes so form abnormalproteins (antigens) so can act as tumor markers.

• Prostate Specific Antigen (PSA) a glycoproteinreleased by prostate gland into the blood identified as amarker of prostate cancer in 1980.

• PSA is highly specific to prostate gland but not specific toprostate cancer. Elevated blood PSA can also occur withnon cancerous conditions (i.e. false positives occur withprostatitis or with benign prostatic hyperplasia - BPH)

• The need to treat stage 1 cancers detected by PSA test iscontroversial: stage 1 tumors are asymptomatic, not detectedby digital exam & present in 80% of men over 80 yrs.

• Treatment of Prostate Cancer includes surgery,radiation & hormonal manipulation (e.g. androgen inhibitors) ifexpected survival >10yrs, otherwise “watchful waiting” ispreferred in elderly patients.

• PSA test is used to assess treatment (correlates withprostate size & cancer stage).

Etiology of Breast Cancer

Breast cancer is most common cancer in women & 2nd mostcommon in cancer deaths in women (after lung cancer).

Risk Factors for Breast Cancer• Prolonged exposures to estrogens (early menarche & late

menopause). Breast cancers that are estrogen receptor +ve aretreated with drugs (e.g. tamoxifen) that bind to these receptors.

• Late Childbearing (having first child after age 30)• Breasts with a high proportion of lobular (milk producing)

and ductal tissue density.• Not breast feeding babies increases post menopausal BC.• Exposure to radiation.• High alcohol consumption.• Family History of BC & Genetic Predisposition in 5-20% of

cases (inheriting mutated breast cancer susceptibilitygenes, BRCA-1 or BRCA-2).

Genetics of Breast Cancer

• 5 - 20% of breast cancers are Familial .• Most involve mutations in 2 Tumor Suppressor genes

involved in DNA repair so are used as genetic markers.• Both genes also increase the risk of ovarian cancer.

- Breast Cancer Susceptibility Gene 1 (BRCA1) onchromosome 17- Breast Cancer Susceptibility Gene 2 (BRCA2) onchromosome 13.

• Mutated HER-2/neu Gene (Human Epidermal GrowthFactor Receptor 2 gene is an Oncogene for a protein thatstimulates cell division & occurs in 25- 30% of BreastCancers.

• Her-2/neu breast cancers strike early in adulthood & spreadquickly.

• Herceptin is a monoclonal antibody based drug that bindsto Her-2/neu receptors & blocks cell division in tumors.

Pathogenesis of Breast Cancer

Tumor (usuallyadenocarcinomain milk ducts)

Campbell, & Reece,Biology, fig. 12.19

Glandulartissue

Note: lumpectomy a possible treatment in early stages

A tumor grows from asingle cancer cell.

Cancer cells invadeneighboring tissue.

Pathogenesis of Breast Cancer -2

Lymphvessel

Bloodvessel

Cancer cell

• Cancer cells spread vialymph & blood to otherparts of the body.

• “Sentinel lymph node ”biopsy determines ifcancer has spread & iffurther lymph noderemoval required.

Metastatic tumor

Small percentageof metastisised cancercells may survive andestablish a new tumor inaanother part of the body.

Diagnostic methods used to study malignant cells1- Karyotype analysis: It is a direct morphological analysis of

chromosomes from tumor cells under the microscope.

2- Fluorescent in situ hybridization analysis FISH analysis involves the use of fluorescent- labelled genetic probes which

hybridize to specific parts of the genome. This can detect extra copies of genetic material or reveal chromosomal translocation.

3- Southern blot analysis: It involves extraction of DNA from leukaemic cells followed by restriction enzyme digestion, gel electrophoresis and transfer by blotting to a suitable membrane. The DNA is then hybridized to a probe complementary to the gene of interest.

4- Polymerase chain reaction: Can be performed on blood or bone marrow for a number of specific translocations such as t(9; 22) and t(15; 17). It is very sensitive and can detect one abnormal cell in one million normal cells. It is of great value to diagnose minimal residual disease.

5- DNA microarray:Allows rapid and comprehensive analysis of cellular transcription by hybridizing labelled cellular mRNA to DNA probes which are immobilized on a solid support. This approach can rapidly determine mRNA expression from a large number of genes and may be used to determine the mRNA expression pattern of different leukaemia or lymphoma subtypes.

6- Flow cytometry: Normal cells each have a characteristic profile but malignant cells often express an aberrant phenotype that can be useful in allowing their detection.

7- Immunohistochemistry: Antibodies can be used to stain tissue sections with fluorescent markers.

Value of using these methods: a- Initial diagnosis. b- Establishing treatment protocol. c- Monitoring response to therapy.

Cytogenetics• Cytogenetics is the original cancer genetic test

used to identify abnormal mutatedchromosomes by karyotype analysis.

• Cytogenetics identified the Philadelphiachromosome resulting from a translocationerror in chromosome 22 forming an oncogenefor chronic myelogenous leukemia (CML) in1960.

• Karyotype Analysis is done by culturing tumorcells, arresting them in metaphase & spreadingchromosomes via use of hypotonic solutions.

• Chromosomes are stained and interpreted by acytogeneticist.

• Process may take weeks (as many tumors do notgrow in vitro).

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