Oncogenes and tumour suppressor genes

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Oncogenes and tumour suppressor genes

• Dr Orla Sheils,
• Senior Lecturer in Molecular Pathology,
• Department of Histopathology.

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Introduction

• cell division process is dependent on a tightly
  controlled sequence of events.
• dependent on the proper levels of transcription
  and translation of certain genes.
• When this process does not occur properly,
  unregulated cell growth may be the end result.

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Mutation
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Introduction

• Of the 30,000 or so genes that are currently
  thought to exist in the human genome, there is a
  small subset that seems to be particularly
  important in the prevention, development, and
  progression of cancer.
• These genes have been found to be either
  malfunctioning or non-functioning in many
  different kinds of cancer.
• The genes that have been identified to date have
  been categorized into two broad categories,
  depending on their normal functions in the cell.

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• Genes whose protein products stimulate or enhance
  the division and viability of cells. This first
  category also includes genes that contribute to
  tumour growth by inhibiting cell death.
• Genes whose protein products can directly or
  indirectly prevent cell division or lead to cell
  death.

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• The normal versions of genes in the first group
  (whose protein products stimulate or enhance the
  division and viability of cells )are called
  proto-oncogenes.
• The mutated or otherwise damaged versions of
  these genes are called oncogenes.
• The genes in the second group (whose protein
  products can directly or indirectly prevent cell
  division or lead to cell death) are called
  tumour suppressors.

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Definition and discovery

• Cancer is caused by an accumulation of genetic
  alterations that confer a survival advantage to
  the neoplastic cell.
• Genetic Changes affect multiple facets
• Cell proliferation
• Apoptosis
• Tissue invasiveness
• Production of growth and angiogenic factors
• Ability to escape immune surveillance

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Genetic Basis for Cancer

• Reflected in the clonal nature of neoplastic
  cells
• Polyclonal growth/ hyperplasia
• Response to an
• Extrinsic growth factor or
• Internal genetic mutation shared by
• All cells - MEN2a germline ret mutation
• Some cells McCune Albright, postzygotic somatic
  Gsa mutation.
• Hyperplastic cells may subsequently acquire one
  or more somatic mutations and develop clonal
  derivatives.

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Genetic Basis for Cancer

• Reflected in the clonal nature of neoplastic
  cells
• Monoclonal growth reflects the acquisition of
  somatic mutations that confer survival advantage.

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• tumour suppressors function in many key cellular
  processes including the regulation of
  transcription, DNA repair and cellcell
  communication.
• The loss of function of these genes leads to
  abnormal cellular behavior.

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Oncogenes
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Cancer a genetic disease

• Intensive effort to characterise genetic
  alterations associated with different forms of
  cancer
• Landmark advances
• Recognition that some cancers run in families
• 1900s- Rous sarcoma virus (RSV)
• sarcoma transmission in chickens
• Retrovirus
• Harboured a abnormal variant of a normal cellular
  gene src
• Viral gene product referred to as an oncogene
• Greek onkos mass or tumour
• Many viral oncogenes correspond to altered
  versions of normal cellular genes
  (proto-oncogenes) src, ras, raf, kit, jun, fos.

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DNA tumour viruses

• Important role in current understanding of
  neoplasia.
• Viruses produce proteins - target key cellular
  regulatory proteins
• Rb, p53
• SV40 large T Ag associates with and inactivates
  Rb.
• Adenovirus E1A also targets Rb

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Hereditary Cancers

• Transmitted in autosomal dominant manner.
• Based on age dependent appearance of
  retinoblastoma Knudson postulated a two-hit
  model for the disease.
• Hit 1 germline inherited mutation in Allele 1
• Hit 2 somatic event involving remaining normal
  allele.

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Tumour Suppressor Genes

• Historically suspected based on several lines of
  evidence
• Malignant phenotype suppressed by fusion with
  normal cells (presence of tumour suppressor in
  normal implied).
• Chromosomal losses in hybrids caused reversion to
  malignant phenotype.
• Introduction of single chromosomes into malignant
  cells
• e.g. insertion of chromosome 11( WT-1 gene)
  could suppress tumourigenicity in Wilms tumour
  cell line.

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tumour Suppressor Genes

• Some genes suppress tumour formation.
• Their protein product inhibits mitosis.
• When mutated, the mutant allele behaves as a
  recessive that is, as long as the cell contains
  one normal allele, tumour suppression continues.
• (Oncogenes, by contrast, behave as dominants one
  mutant, or overly-active, allele can predispose
  the cell to tumour formation).

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Example 1 RB - the retinoblastoma gene

• Retinoblastoma is a cancerous tumour of the
  retina. It occurs in two forms
• Familial retinoblastoma
• Multiple tumours in the retinas of both eyes
  occurring in the first weeks of infancy.
• Sporadic retinoblastoma
• A single tumour appears in one eye sometime in
  early childhood before the retina is fully
  developed and mitosis in it ceases.
• Familial retinoblastoma
• Familial retinoblastoma occurs when the fetus
  inherits from one of its parents a chromosome
  (number 13) that has its RB locus deleted (or
  otherwise mutated). The normal Rb protein
  prevents mitosis.

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• Mechanism. The Rb protein prevents cells from
  entering S phase of the cell cycle. It does this
  by binding to a transcription factor called E2F.
• This prevents E2F from binding to the promoters
  of such proto-oncogenes as c-myc and c-fos.
• Transcription of c-myc and c-fos is needed for
  mitosis so blocking the transcription factor
  needed to turn on these genes prevents cell
  division.

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Retinoblastoma

• A random mutation of the remaining RB locus in
  any retinal cell completely removes the
  inhibition provided by the Rb protein, and the
  affected cell grows into a tumour. So, in this
  form of the disease, a germline mutation plus a
  somatic mutation of the second allele leads to
  the disease.

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Example 2 p53

• The product of the tumour suppressor gene p53 is
  a protein of 53 kilodaltons (hence the name).
• The p53 protein prevents a cell from completing
  the cell cycle if
• its DNA is damaged or
• the cell has suffered other types of damage.
• When
• the damage is minor, p53 halts the cell cycle
  hence cell division until the damage is
  repaired.
• the damage is major and cannot be repaired, p53
  triggers the cell to commit suicide by apoptosis.

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• These functions make p53 a key player in
  protecting us against cancer that is, an
  important tumour suppressor gene.
• More than half of all human cancers do, in fact,
  harbour p53 mutations and have no functioning p53
  protein.

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Loss Of Heterozygosity (LOH)

• Because tumour suppressor genes are recessive,
  cells that contain one normal and one mutated
  gene that is, are heterozygous still behave
  normally.
• However, there are several mechanisms which can
  cause a cell to lose its normal gene and thus be
  predisposed to develop into a tumour. These may
  result in a "loss of heterozygosity" or "LOH".

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Mechanisms of LOH

• Deletion of
• the normal allele
• the chromosome arm containing the normal allele
• the entire chromosome containing the normal
  allele (resulting in aneuploidy).
• Loss of the chromosome containing the normal
  allele followed by duplication of the chromosome
  containing the mutated allele.
• Mitotic recombination. The study of tumour
  suppressor genes revealed (for the first time)
  that crossing over with genetic recombination
  occasionally occurs in mitosis (as it always does
  in meiosis).
• In 2 and 3, the resulting cell now carries two
  copies of the "bad" gene. This is called
  "reduction to homozygosity".

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Methylation

• Mutation is not the only way to inactivate tumour
  suppressor genes.
• Their function can also be blocked by methylation
  of their promoter.

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Mechanism of parental imprinting

• The process of imprinting start in the gametes
  where the allele destined to be inactive in the
  new embryo (either the father's or the mother's
  as the case may be) is "marked". The mark appears
  to be methylation of the DNA in the promoter(s)
  of the gene.
• Methyl groups are added to cytosines (Cs) in the
  DNA.
• occurs at stretches of alternating Cs and Gs
  called CpG islands.
• Methylation of promoters prevents binding of
  transcription factors to the promoter thus
  shutting down expression of the gene.

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Methylation

• Cancer cells often contain a methylated promoter
  on one tumour suppressor gene accompanied by
• a similarly blocked promoter on the other allele
  (producing the same effect as 2 above)
• a loss of that locus on the other chromosome
  (like the LOH in 1 above)
• an inactivating mutation in the other allele.

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tumour suppressor genes anti-oncogenes

• Genes like RB and p53 are also called
  anti-oncogenes. They were first given this name
  because they reverse, at least in cell culture,
  the action of known oncogenes.

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Tumour suppressor genes

• This image (courtesy of Moshe Oren, from Cell
  62671, 1990) shows petri dishes which were
  seeded with the same number of cells that had
  been transformed by two oncogenes myc and ras.
• Many of those on the left have grown into
  colonies of cells.
• However, the cells plated on the right also
  contained the tumour suppressor p53 gene. Only a
  few have been able to grow into colonies.

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Human Papillomavirus (HPV)

• The name anti-oncogene may be even more
  appropriate than originally thought.
• Both the Rb protein and the p53 protein complex
  directly in the cell with an oncogene product of
  some human papilloma viruses.
• Once inside the cells of their host, human
  papilloma viruses synthesise
• a protein designated E7 and
• another designated E6.

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Human Papillomavirus

• The E7 protein of one of these binds to the Rb
  protein preventing it from binding to the host
  transcription factor E2F.
• Result E2F is now free to bind to the promoters
  of genes (like c-myc) that cause the cell to
  enter the cell cycle . Thus this version of E7 is
  an oncogene product.
• The E6 protein of human papilloma virus
  implicated in cervical cancer binds the p53
  protein targeting it for destruction by
  proteasomes and thus removing the block on the
  host cell's entering the cell cycle.

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Oncogenes

• Genes associated with the stimulation of cell
  division.
• Cancers result from only one mutant allele of
  gene.

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Oncogenes

• Growth Factors or Receptors for Growth Factors
• PDGF Platelet Derived Growth Factor (brain and
  breast cancer)
• erb-B receptor for epidermal growth factor (brain
  and breast cancer)
• erb-B2 receptor for growth factor (breast,
  salivary, and ovarian cancers)
• RET growth factor receptor (thyroid cancer)

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Oncogenes

• 2. Cytoplasmic Relays in Stimulatory Signaling
  Pathways
• Ki-ras activated by active growth factor receptor
  proteins (lung, ovarian, colon and pancreatic
  cancer)
• N-ras activated by active growth factor receptor
  proteins (leukaemias)
• c-src is a protein kinase that becomes overactive
  in phosphorylation of target proteins

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Oncogenes

• 3. Transcription Factors that Activate Growth
  Promoting Genes
• c-myc activates transcription of growth
  stimulation genes (leukemia, breast, stomach, and
  lung cancer)
• N-myc (nerve and brain cancer)
• L-myc (lung cancer)
• c-jun and c-fos function as transcription factors

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Oncogenes

• 4. Other types of molecules
• Bcl-2 normal protein blocks cell suicide
  (lymphoma)
• Bcl-1 codes for cyclin D1, stimulatory protein of
  the cell cycle (breast, neck, head cancers)
• MDM2 codes for antagonist of p53 (sarcomas)

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RAS

• Ras gene products are involved in kinase
  signalling pathways that control the
  transcription of genes, which then regulate cell
  growth and differentiation.
• To turn "on" the pathway, the ras protein must
  bind to a particular molecule (GTP) in the cell.
• To turn the pathway "off," the ras protein must
  break up the GTP molecule.
• Alterations in the ras gene can change the ras
  protein so that it is no longer able to break up
  and release the GTP.

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RAS Pathway
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• These changes can cause the pathway to be stuck
  in the "on" position.
• The "on" signal leads to cell growth and
  proliferation.
• ras overexpression and amplification can lead to
  continuous cell proliferation, which is a major
  step in the development of cancer.
• Cell division is regulated by a balance of
  positive and negative signals.
• When ras transcription is increased, an excess of
  the gene's protein is in the cell, and the
  positive signals for cell division begin to
  outweigh the negative signals.

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RAS

• The conversion of ras from a proto-oncogene into
  an oncogene usually occurs through a
  point mutation in the gene.
• The altered function can affect the cell in
  different ways because ras is involved in many
  signaling pathways that control cell division and
  cell death.
• Anti-cancer drugs are now being developed that
  target ras dependent pathways. Much remains to be
  discovered before these drugs can be put into use

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RAS

• Mutant ras has been identified in cancers of many
  different origins, including pancreas (90),
  colon (50), lung (30), thyroid (50), bladder
  (6), ovarian (15), breast, skin, liver, kidney,
  and some leukaemias.

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MYC

• The myc protein acts as a transcription factor
  and it controls the expression of several genes.
• Mutations in the myc gene have been found in many
  different cancers, including Burkitt's lymphoma,
  B-cell leukemia, and lung cancer.
• The myc family of oncogenes may become activated
  by gene rearrangement or amplification.

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• Gene rearrangements involve the breakage and
  re-sealing of chromosomes.
• This process can involve large amounts of DNA and
  can affect many genes.
• The movement of a gene or group of genes to a
  different location within the same chromosome or
  to a different chromosome often leads to altered
  gene expression and cell function.

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SRC

• The Src protein is a tyrosine kinase.
• Kinases are enzymes that transfer phosphate
  groups onto target molecules.
• The important aspect of this process is that the
  removal/addition of phosphates changes
  biomolecules and is a key way by which the
  activities of cells are regulated.

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SRC

• The phosphate addition/removal process acts like
  an on/off switch to control the activity of the
  target molecules.
• The src proteins alter several target molecules,
  resulting in the transmission of signals to the
  nucleus that help regulate the cell

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Tyrosine Kinases

• MAP kinase (MAPK) signaling is among central
  signaling pathways that regulate cell
  proliferation, cell differentiation and
  apoptosis.
• As MAPK should transmit extracellular signals to
  proper regions or compartments in cells,
  controlling subcellular localisation of MAPK is
  important for regulating fidelity and specificity
  of MAPK signaling.

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Tyrosine Kinases

• The ERK1/2-type of MAPK is the best characterized
  member of the MAPK family. In response to
  extracellular stimulus, ERK1/2 translocates from
  the cytoplasm to the nucleus by passing through
  the nuclear pore by several independent
  mechanisms.

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Tyrosine Kinases

• The MAP kinase (MAPK) pathway is a highly
  conserved pathway involved in diverse cellular
  functions, including cell proliferation, cell
  differentiation and apoptosis.
• A wide variety of extracellular stimuli, such as
  growth factors and environmental stresses, induce
  sequential phosphorylation and activation of
  three protein kinases, MAP kinase kinase kinase
  (MAPKKK), MAP kinase kinase (MAPKK) and MAPK.

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Tyrosine Kinases

• MAPK is a serine/threonine kinase activated by
  MAPKK via phosphorylation on both threonine and
  tyrosine residues in the TXY sequence
• The MAPK family consists of four members, ERK1/2
  (also known as classical MAPK), JNK/SAPK, p38 and
  ERK5/BMK1.
• Each molecule is activated by distinct pathways
  and transmits signals either independently or
  co-ordinately

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Tyrosine Kinases

• MAPK plays an important role in transmitting the
  signals from receptors on cell membrane to
  cytoplasmic targets such as cytoskeleton and
  downstream kinases and nuclear targets such as
  transcription factors.
• Thus, regulation of the subcellular localisation
  of MAPK is important for controlling MAPK
  signaling.

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TSG

• tumour Suppressor Genes
• Genes associated with inhibition of cell
  division.
• Cancers require both alleles of the gene to be
  altered.

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TSG

• Cytoplasmic Proteins
• APC (colon and stomach cancers)
• DPC4 codes for relay molecule in cell division
  inhibitory pathway (pancreatic cancer)
• NF-1 codes for protein that inhibits a
  stimulatory (Ras) protein (brain, nerve, and
  leukemia)
• NF-2 (brain and nerve cancers)

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TSG

• 2. Nuclear Proteins
• MTS1 codes for p16 protein, brake on cell cycle
  clock (many cancers)
• RB codes for pRB protein, master brake on cell
  cycle (retinoblastoma, bone, bladder, lung, and
  breast cancer)
• p53 codes for p53 protein, halts cell cycle in G1
  and induces cell suicide (many cancers)
• p16 inhibits cyclin D-dependent kinase activity
• WT1 (Wilms tumour of the kidney)
• BRCA1 functions in repair of damage to DNA
  (breast and ovarian cancers)
• BRCA2 functions in repair of damage to DNA
  (breast cancer)

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