91345Biochemistry, Genes and Disease

Lecture 5

CANCER

Dr Najah NassifMedical and Molecular Biosciences

CANCER

Cancer is a disorder of uncontrolled cell growth [imbalance between cell growth and cell death]

Cells are normally programmed to develop, grow, differentiate and die

Cancer results when a cell escapes these programmed constraints

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The Cell Cycle and

Normal Cell Growth

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Occurs in 4 phases:G1SG2M

Normal Cell Growth and DivisionEukaryotic cells have universal regulatory mechanisms for controlling cell division

Protein kinases and protein phosphorylation are central to the timing mechanism that determines entry into cell division and orderly passage through the cycle

In normal cells, cell replication is stopped to repair any DNA damage

If this does not occur, cell replication continues and cells replicate with damaged DNA

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Control of Cell Division

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Major check point

Check pointThe cell cycle and mitosis are subject to many controls, all involving specific proteins

Cell cycle progression is controlled by cyclin-cdk kinase complexes that phosphorylate specific proteins to progress the cell sequentially from one phase to the next

Cell Cycle CheckpointsCheckpoints are mechanisms to halt the progress of replication if chromosomal DNA is damaged or certain processes are aberrant

Checkpoints ensure that each stage of replication is complete and accurate

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Life and Death of a

Cell

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Dormant (G0)

Cell division

Growth

Necrosis

Extrinsic receptor-mediated

IntrinsicGranzyme

cell

Death

Apoptosis Senescence

Autophagy

ApoptosisProgrammed cell death - a genetically controlled method of regulating cell numbers

Pathway of cell death that is induced by a tightly regulated intracellular program

Function in tissue homeostasis

Plays a critical role in development by removing unwanted cells

Contrast to necrosis (cell death due to injury)

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ApoptosisIt is characterised by distinct morphological and biochemical changes including:

- Membrane blebbing- Cell shrinkage- Nuclear Fragmentation- Chromatin condensation- Chromosomal DNA fragmentation

Defective apoptotic processes implicated in a variety of diseases (including cancer)

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Characteristics of Apoptosis

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Active processCell shrinkageMembrane blebbingChromatin condensationDNA fragmentationDoes not induce an inflammatory response

Passive process (?)

Progressive breakdown of cellular structure

Apoptosis Necrosis

Necrosis vs Apoptosis

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Normal Cellular Controls

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nucleus cytoplasmenvironment

mitochondria

GROW REST

DIE

State of DNA

Level of denatured proteins

Energy state Reductive capacity

Membrane integrity

Signals from environment

CANCEROncogenesis or Tumorigenesis

Cancer includes a class of diseases in which cells display uncontrolled growth, invasion and metastasis (spread to other body locations)

Oncogenesis or tumorigenesis is the process whereby normal cells become transformed into cancer cells

It is usually a multi-step process involving genetic mutations/cellular changes at each step

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CANCERA TUMOUR is a population of cells resulting from localised, unregulated growth and division of a single cell

Also called a neoplasm or a cancer (also carcinoma, sarcoma, lymphoma, leukaemia, blastoma)

Tumour cells are characterised by unregulated mitotic activity, under conditions that would normally restrict cell division

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Cancer StatisticsCancer Related Deaths in USA (in 2008)

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Males Females

Properties of Cancer Cells 1. Loss of Contact Inhibition

Cancer cells and normal cells can be cultured in the laboratory

When normal cells are grown on a tissue culture dish, they proliferate until the surface of the dish is covered by a single layer of cells just touching each other. Then mitosis ceases. This phenomenon is called contact inhibition

Cancer cells DO NOT show contact inhibition. Once the surface of the dish is covered, cells continue to divide and grow over each other

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Properties of Cancer Cells 1. Loss of Contact Inhibition

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Normal Cells Transformed Cells

Properties of Cancer Cells 2. Cellular Immortality

Tumour cells placed in culture conditions, in the laboratory, are often immortal

They will grow and divide indefinitely as long as nutrient conditions are provided

Non-tumour cells have only a limited capacity for cell division in culture

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Properties of Cancer Cells 3. Abnormal Karyotype

Normal cells ordinarily have the normal set of chromosomes of the species (i.e. have a normal karyotype)

Cancer cells almost always have an abnormal karyotype (eg. abnormal chromosome number or structure)

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The Hallmarks of Cancer

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Influence of Genetics and Environment in the Development of Cancer

Cancers result from the interaction of both genetic and environmental factors leading to accumulation of mutations in essential genes

Exposure to certain agents know to increase the risk of cancer (eg. cigarette smoke, asbestos, radiation)

Levels of susceptibility differ between individuals

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Some individuals have a genetic makeup that makes them more susceptible to certain agents

Others can tolerate more of the carcinogen before they will develop cancer

Influence of Genetics and Environment in the Development of Cancer

Occupational Exposure and Cancer Risk

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Occupation Agent Site of Disease

The Relationship Between Age and Cancer

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International Variation in the Incidence of Various Types of Cancers

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Involvement of Genes in Cancer Development

Subsets of genes in the genome found to be important in the prevention, development and progression of cancer

These genes have been found to be either malfunctioning, or non-functional in different tumour types

Categorised into 2 broad categories based on cellular function

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1. Genes whose protein products stimulate or enhance cell division and growth and viability (i.e. oncogenes)

2. Genes whose protein products directly or indirectly inhibit or prevent cell division or promote cell death (i.e. tumour suppressor genes)

3. Genes whose protein products are involved in the correction of acquired mutations in DNA (i.e. repair proteins)

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Involvement of Genes in Cancer Development Classes of Genes in Cancer

Major classes of genes identified:

1. Oncogenes2. Tumour suppressor genes

3. DNA repair genes

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Classes of Cancer Genes1. The Oncogenes

Oncogenes = growth promoting (dominant gain of function)

Normal versions are termed proto-oncogenes and their mutated counterparts are called oncogenes

Protein products of proto-oncogenes stimulate cell division and/or inhibit cell death

Proto-oncogenes are usually growth factors, growth factor receptors, signal transduction molecules or nuclear transcription factors

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Genes in CancerOncogenes and Their Functions

Growth factor receptors – stimulate cell growth. May function as transmembrane protein kinases

Protein kinases – alter function of other proteins by phosphorylation

G-proteins – bind GTP and mediate cell signalling

Transcription factors – proteins that bind to DNA and activate transcription

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Classes of Cancer Genes1. The Oncogenes (cont.)

Mutated oncogenes can lead to unregulated cell division

Cells are able to grow in the absence of normal growth signals

Examples: - ras: a signal transduction molecule- myc: a transcription factor

- src: a protein tyrosine kinase

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Oncogenes Identified in Cancer

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The ras OncogeneRas subfamily is a family of small GTPases involved in cell signal transduction

Ras communicates and translates signals from outside the cell to the nucleus

Activation of ras signalling causes cell growth, and survival

Mutations of ras can permanently activate it and this can lead lead to cancer

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The Ras Signalling Cascade

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Ras transmits signals from receptor tyrosine kinases (RTK to regulate many biological functions

Activated Ras acts by regulating the cellular response through distinct Ras effector proteins and their complex signal transduction cascades

The best-characterised signal transduction pathway of Ras is by the Raf kinases

Another cascade of Ras-activated signalling is by anti-apoptotic PI3-K

Mutated Oncogenes Can Lead to Unregulated Cell Growth

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Normal GrowthGene expression without

correct signals

Classes of Cancer Genes2. The Tumour Suppressor Genes

Tumour Suppressor genes = anti-oncogenes (recessive loss of function)

Tumour suppressor gene products act to inhibit the division of cells if conditions of growth are not met

Conditions triggering the brakes of cell division include: DNA damage, lack of growth factors

It is the absence, or loss of function of a tumour suppressor gene product that leads to tumour formation

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Some examples include:

- p53 (TP53): a transcription factor regulating cell division

- Rb: controls cell division- APC: controls availability of a transcription factor- BRCA: Involved in repair of damaged DNA

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Classes of Cancer Genes2. The Tumour Suppressor Genes (cont.) Tumour

Suppressor Genes

Identified in Cancer

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Genes in CancerThe p53 Tumour Suppressor

p53 (the guardian of the genome)

- gene located on chromosome 17

- mutations lead to loss of function

- functions to prevent cell division of cells with damaged DNA

- p53 mutations observed in approximately 50% of all cancers

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Genes in CancerThe p53 Tumour Suppressor

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p53 is a multifunctional protein which plays a role in:

- modulating gene transcription

- policing cell cycle checkpoints

- activating apoptosis- controlling DNA replication and

repair

- maintaining genomic stability- responding to genetic insults

Genes in Tumorigenesis Tumour Specific Tumour Suppressor Genes

Breast cancer- BRCA1 and BRCA2 genes

- Mutations increase risk of developing breast cancer

Colon cancer- Adenomatous polyposis coli (APC) gene- Deleted in colon cancer (DCC) gene

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Knudson s 2-Hit Hypothesis for the Development of Cancer

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Gene mutations may be inherited or acquired during a person s life time

Sporadic Cancer:Both mutations are acquired

Inherited Cancer:1 mutation inherited1 mutation acquired Tumour

Tumour

Familial vs Sporadic Cancers

Familial cancers have younger age of onset compared to their sporadic counterpart

Usually a strong family history, with multiple family members affected in 2 or more generations is observed

Involvement of tumours in other organs (eg. breast and ovary)

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Cancer Development

is a Multistep Process

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Normal Cell

First Mutation

Second Mutation

Third Mutation

Fourth or later Mutation

Malignant Cell

Mutations are acquired at every step

Cancer Development is a Multistep Process

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The Stages of Colorectal Tumorigenesis

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Loss of the tumour suppressor gene APC

Activation of ras oncogene

Loss of tumour suppressor gene DCC

Loss of tumour suppressor gene p53

Additional mutations

ROLE OF THE TUMOUR

SUPPRESSOR GENE PTEN IN

SPORADIC COLORECTAL

CANCER (CRC)

Colorectal Cancer

Cancer of the colon and/or rectum

Third most commonly diagnosed cancer in men (8%), and the second in women (10%)

Third most common cause of cancer death (9%) (NSWCC 2007)

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Familial Colorectal Cancer(A) Hereditary Non Polyposis Colorectal Cancer

(HNPCC)

• Makes up 5-6% of all CRC cases• DNA mismatch repair genes

(B) Familial Adenomatous Polyposis (FAP)

• Makes up 1% of all CRC cases• APC Tumour suppressor gene

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Sporadic Colorectal Cancer

Accounts for approximately 80% of all CRC

10-15% of tumours have a deficiency in the DNA mismatch repair pathway

Distinct pathway(s) from benign polyp to malignant polyp to metastatic cancer have been identified

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Genetic Changes Identified in Colorectal Cancer

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Non-Genetic Factors in Colon Cancer

Age – 90% of CRCs occur in people over 50

Inflammatory bowel disease (IBD) – inflammatory disorder of the colon can lead to ulcers

Diet – diets high in red meat & fat, and low in fibre increases risk

Exercise – Low level increases risk

Alcohol consumption & smoking – increase risk

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Genes Involved in Colonic Tumorigenesis

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PTEN as a Candidate Gene in Colorectal Cancer

Loss of PTEN gene region observed in 35% of sporadic CRC

Mutations / deletions of PTEN observed in many sporadic cancers (eg. brain, prostate, endometrium)

Mice heterozygous for PTEN develop CRC

Germline (inherited) PTEN mutations give rise to inheritable disorders (Cowden, Bannayan Riley Ruvalcaba syndromes) characterised increased risk of certain cancer types

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The PTEN Tumour Suppressor Gene

PTEN: Phosphatase and tensin homolog deleted on chromosome 10

PTEN gene is located at on chromosome 10q23.3

The gene product is a protein and lipid phosphatase

Regulates cell growth survival, cell adhesion, cell differentiation and death (apoptosis)

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PTEN Gene and Protein Structure

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Cellular Functions of PTENThe major substrate of PTEN is the second messenger lipid PIP-3 (phosphoinositol-3,4,5-triphosphate)

PTEN dephosphorylates PIP-3 to PIP-2, thus keeping PIP-3 levels low

Loss of PTEN leads to increased levels of PIP-3 and consequent activation of Akt/PKB and the PI3K cell survival/anti-apoptic pathway (growth stimulatory effect)

Hyperactivation of Akt leads to protection from apoptotic stimuli and increased cell growth and survival

PTEN is therefore a negative regulator of cell survival

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The Cellular Role(s) of PTEN

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Additional Functions of PTENPTEN has also been shown to be:

- a regulator of cell adhesion

- an inhibitor of cell migration- a regulator of cell size- an inhibitor of angiogenesis- an inhibitor of tumour metastasis

The effect is dependent upon the substrate of PTEN dephosporylation

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Multiple Roles of PTEN in Tumour Suppression

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Detection of PTEN

Gene Mutations

in Sporadic Colorectal

Cancer

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Tumour DNA

BA

Germline DNA

Tumour DNA

BA

Germline DNA CRC 1Exon 5A>G

CRC 2Exon 5 G>A

PTEN Mutation Detected in Sporadic CRC

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319X

E1 E2 E3 E4 E5 E6 E7 E8 E9

D153N

D153Y

V217A N323K

E150Q

K125X

C124SG129E

Results

41 paired tumour samples were screened

Mutations detected in 8/41 (19.5%) primary sporadic colorectal tumours

Overall, alterations (mutations and deletions) of the PTEN gene were present in 15/41 (37%) primary sporadic colorectal tumours

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Project

Evaluation of the Functional Significance of the Detected

PTEN Mutations

Functional Analysis of the Detected PTEN Gene Mutations

The wild type (WT) and each of the mutant PTENs engineered into a mammalian expression vector

The effect of the WT and mutant PTEN on cell cycle distribution and cell proliferation (your project aim) was determined after transfection

The effects of PTEN mutation on PTEN subcellular localisation was also determined

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Engineering The PTEN Mutants

Point Mutations were generated from the WT PTEN clone using in vitro site directed mutagenesis

Truncating mutants were generated by PCR amplification from the WT PTEN clone

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Effect of Mutation on PTEN Subcellular Localisation

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C124S C>NC124S C>N

K62R C=N Y65C C=N K125EC>N

Punctate

G129E C>NWT PTEN C=N

WT PTEN is evenly distributed between the cytoplasm and the nucleus

Some mutants have increased cytoplasmic localisation

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C=ND153Y V217A C=NV217A C=N

319XC>N

Punctate N323K C=N

D153Y C>NK125XC>N

Punctate E150Q C=N

Punctate319XC=N

Effect of Mutation on

PTEN Subcellular Localisation

The Effect of PTEN Mutations on Cell Cycle Distribution

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The Effect of PTEN

Mutations on Cell

Proliferation

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WT PTEN is able to cause a slowing of cell proliferation

Aim to determine the effect of PTEN mutation on cell proliferation

ConclusionsWT PTEN brings about cell cycle arrest in cultured cells

Mutant PTEN is unable to bring about a cycle arrest that is equivalent to that of WT PTEN. The level is lower for the PTEN mutants.

WT PTEN is able to bring about a slowing of cell proliferation of cultured cells

Is mutant PTEN able to slow cell proliferation as effectively as the WT PTEN? (You will find out in your project)

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ReferencesNelson and Cox (2008) Lehninger Principles of Biochemistry 5th Edition

G. Karp (2008) Cell and Molecular Biology: Concepts and Experiments. 5th Edition

MH. Lodish et al. Molecular Cell Biology

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