bgd cancer lecture 2010
TRANSCRIPT
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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