Cancer- LECTURE 5

Genetic basis and molecular themes

Research 1: finding the cuases of chicken sarcomas

• Rous Sarcoma Virus (RSV)

  • retrovirus

  • contains +ssRNA genome

  • yet infection cycle required both DNA and RNA synthesis

• identified as the transfomring agent in extracts of chicken sarcomas

Hypothesis as to why this happened?

• provirus hypothesis:

  • DNA intermediate integrates into host cell genome

  • so messed up the chromosomes and caused cancer??

Research 1: finding the genetic cause?

1. Transformed cells with a virus mutant which was temperature sensitive

2. At normal temperate→ no cancer

3. At restrictive 35C temperature→ cancer

   • because virus manifested here

4. The gene that was for this ts mutation was found

   • v-SRC gene

Research 1: how was SRC mapped

• based on a non-transforming mutant

• carrying a deletion near the 3’ end of the viral RNA

• These findings were exploited to generate a specific DNA probe for SRC

Research 1: Use of the SRC DNA probe

1. identified a gene present in normal cells

   • protooncogene→ c-SRC

   • closely related to viral oncogene v-SRC

THEREFORE: the gene was taken from host genome by cirus, then mutated and then when put back in, still has some host features but a bit wrong so messes up cell cycle→ cancer! So ‘protoncogene’ a gene that is invovled in cell-cycle probably and so if changed and sprad with virus= cancer!

Research 1: What this tells us about how cancer is caused?

• oncogene is ‘activated’ by mutations

  • v-SRC encodes a de-regulated protein

• RNA tumour viruses might capture cellular genes into their genome

• They undergo genetic change

• So when infecting normal cells

  • they drive them to malignant transformation!

    • i.e deregulated proliferation

Research 1: What is known about viral oncogenes

1. dominant

2. gain-of-function alleles

   • activated or over-expressed

3. Originally come from cellular protooncogenes

Research 1: what was SRC shown to encode

non-receptor tyrosine kinase

Research 1: what this helped to do…

• Find many other viral oncogenes

• and their cellular counterparts

Another cause of cancer?

Chromosomal aberrations

• controversial:

  • is it a cause or an effect of cancer??

Research 2: Cytogenetics and Chromosomal aberrations

• cytogenetic studies showed a given chromosomal rearragment was associated with a specific cancer

  • must be causative

Research2: first chromosomal aberration caused cancer found

Chronic myeloid leukaemia (CML)

• found to carry a normal number of chromosomes but

• one is too small

  • the Philadelphia Chromosome

• THERFORE: must be the cause of this cancer?

Research 2: What is Philadelphia chromosome

• product of reciprocal translocation

  • between chromosomes 9 and 22

Note: translocations can easily be viewed with chromosome paining and spectral kayrotyping

Research 2: Further anaysis showed

• Translocation breakpoint was cloned

  • found to encode a fusion of

    • BCR-ABL

    • i.e two genes have kinda fused together when the chromosomes 9 and 22 got mixed up with eachother

Research 2: What are these genes?

1. c-ABL

   • human cellular homolog of

   • transforming sequence of Abelson murine leukaemia

   • encodes for a non-receptor tyrosine kinase

THERFORE: BCR-ABL encodes a deregulated tyrosine kinase

• which will cause cancer!

Bringing together findings from virologists and cytogeneticists

• viral oncogenes

  • found from RNA tumour viruses

• also found at

• sites of chromosomal rearrangements

• or

• actiavted by insertional mutagenesis in cancer cells

THEREFORE: viral oncogenes are also linked with chromosomal aberrations→ so must be some kind of cause of cancer!

Research 3: Isolating human oncogene w/o knowing its sequence or strucuture

1. Humour tumour cell line transformed by transfection into

   • NIH 3T3 cells (immortalized mouse fibroblast cell line)

2. DNA extracted from resulting foci

   • from cells that were cancerous

3. Used in new rounds of transffection and extraction

4. Eventually getting rid of the rest of the human genome

   • leaving only the mouse genome and the human oncogene

5. DNA sequence could be cloned

   • Human genes found with DNA hybridisation

   • using Alu sequence probe

   • recognises human Alu repeats

     • linked to human oncogenic sequence

     • against the backdrop of the mouse genome

OVERALL: find the human oncogenes

Research 3: What this proved

• direct maligant transformation by a ‘human oncogene’

• Isolated gene corresponded to a previously identified viral oncogene

  • RAS

Research 3: What is Ras

• Normal Ras

  1. Mitogen signals

  2. switches to its active GTP-bound form

  3. via tyrosine kinase receptors

• Signal mutations abolishing Ras GTPase

  • oncogenic

  • because signal transduction becomes constitutive

  • even if mitogens are present!

  • e.g common mutation→ Ras hyperactive (Ras^Gly12→Val)

  • most common events in oncogenesis

  humans:

• H-ras, K ras, N-ras$

Research 3: How do oncogenes work to cause cancer

Involved in growth factor signalling pathways

1. Growth factor

2. Couplers/Adaptors

3. Effectors, inducing intracellular tyrosine or Serine/Threonin kinases

4. Transctiption factors

5. Cell proliferation

i.e found crucial insight into oncogenes affecting signalling pathways to cause cancer!

Research 4: finding info about genes which suppress tumours→ Experiment 1

Cell hybrids:

• Malignant + normal→ hybrid

• Observed: hybrid grows like normal

Continued experiment

• hybrids are unstable

• lose chromosomes

• malignant phenotype returns

Conclusion:

• maligant trait was recessive

BUT:

• This is different to the idea that oncogenes are dominant!

Research 4: Experiment 2- Familial predisposition

Looked at Hereditary retinoblastoma

• mutation is recess

• BUT

• predisposition to the mutation is autosomal dominant trait

  • based on likelihood of inactivation of the remaining gene copy

Research 4: Experiment 2- How was RB locus identified

• mapping deletions in chromosome 13 of patients

• Clues to function of RB protein came from studies of DNA tumour viruses

Reserach 4: Experiment 3- Findinding what causes RB inactivation

• DNA tumour viruses encode oncoproteins

• these inactivate RB and p53

Are essential for viral infection

Research 4: What is RB

• Repressor of E2F

  • which is a transcription factor that controls S-pase gene expression

• SO usuually used to stop moving into S-phase

• member of ‘pocket proteins’ family

  • with p107, p130

• Key cell-cyle target is

  • S-phase transcription factor E2F

Research 4: How RB works

1. RB binds and inhibits E2F

2. Upon mitogen signals

3. RB is a substrate of G1/S CDK

   • so undergoes sequential phosphylation

4. Hyperphosphylation inactivates RB triggering E2F-dependent transcription

5. Among S-phase E2F transcriptional targets are

   • E2F and Cyclins E and A

   • Their up regulation sets up a positive feedback loop

     • for RB inactivation unleashing the S-phase transcriptional programme

OVERALL: mitogens→ G1/S-CDK phosphylation inactivate RB→ go into S-phase

The pathway of RB inactivation for cell proliferation shows

Shows requirement of mitogens to pass restriction point!

Research 4: How RB links to cell cycle progression

• with build-up of G1/S-CDK activity

• in response to a constellation of mitogen signals

  • required to pass the restriction point

• RB inactivation is key to cancer progression!

Research 4: what is p53

• Crucial tumour suppressor

• Protein made from TP53 gene

  • transcription factor

• Plays a role in regulating cell growth, preventing uncrontolled cell division and triggering cell death

  • ‘guardian of the genome’

• first discovered as a target of DNA transforming viruses

• inherited predisposition to cancer

  • one defective copy of TP53

  • Somatic mutation in > 50% cancers

Research 4: How does p53 normally work

1. DNA damage of mitogenic stress

2. Causes inhibition of E3 ubiquitin ligase Mdm2

3. Triggers accumulation of p53 and transcriptional activation

4. Induction of p14^ARF by excess/out-of context mitogen signals

   • due to oncogenic activation

5. This impedes inappropriate proliferation

   • promotes cell death

     • unless p53 has been inactivated by mutation (in oncogenesis)

OVERALL: prevents cell cycle progression upon DNA damage

Research 4: What is p14 ARF?

• protein encoded by CDKN2A locus

• Tumour suppressor

• Binds to MDM2 which stabilises p53!

  • means p53 can do its job and cause cell death

Research 4: How does p53 trigger cell cycle arrest or apoptosis?

1. Cell cycle arrest

   • vis CKI p21^Cip1

2. Apoptosis

   • Via BAX

   MDM2 is also a transcriptional target of p53

   • and forms a negative feedback loop

Whether chose between arrest and apoptosis depends on extent and persistence of the DNA damage!

Research 4: How can you have genetic predisposition to cancer?

A loss-of-function mutation in genes for:

1. negative regulators of cell cycle

2. checkpoint components

3. apoptotic pathways

With one of these mutations inherited, only need one inactivating ‘hit’ to cause cancer (in the other allele?)

• THEREFORE: increased susceptibility

These genes are tumour suppressor genes

Research 4: mutations involved in different cancers

Research 4: Getting cancer from gain-of function mutations

• A state of genomic instability causes

• loss of critical control genes and emergence of gain-of-function mutations

• Gets worse over generations

• translates into gene amplification

  • boosting positive regulators in cell cycle

    • e.g cyclins D and E

  • Or cause even more chromosomal rearragments

    • can cause more oncogenesis

OVERALL: gain-of-function mutations→ cell proliferation and in a positive feedback loop

Research 4: How can this genomic instability occur?

1. Aneuploidy

   • incorrect number of whole chromosomes or portions due to segregation errors

2. instability at the nucleotide sequence level

Linking mitogen/antimitogen signals to core cell cycle controls

Note:

Ink4→group of CKIs

TGFb→ transforming growth factor beta

• used in cell growth, differentiation and apoptosis

Jun, Myc, Fos→ protooncogenes

• crucial role in regulating gene transciption and cell growth

• abnormal expression= cancer

Two ways to view the expansion of cancer

1. Clonal view

2. Hierarchical view

1. Clonal view of cancer

• Viewed as a multistep evolutionary process driven by mutations

• Mutations are the driving force

  • of iterative cycles of selection and clonal expansion

  • Due to proliferative advantage!

constantly changing and evolving with mutations to get better at proliferation

2. Hierarchical View

• cancer organised like normal tissues

• some cells are ‘cancer stem cells’

  • give rise to the diverse set of cells making up a tumour

• but: what is the nature of the cell of origin in this views??

In either model: malignant transformation is enhanced by…

1. Increased rates of DNA damage

2. Loss or rearrangement

3. Disruption of DNA repair

4. loss of checkpoint mechanisms

ALL LEAD TO: increase above the spontaneous rate of mutations in normal cells

Cause of cancer→ Drive by mutations in cell cycle control:

1. upreg core elements for cell cycle progression

   • i.e activate oncogenes

2. inactivate core elements for cell cycle brakes

   • i.e inactivate TSGs

3. altering signalling pathways

   • at receptros, transducer or effector levels

4. Others

Overall causes of these mutations

1. chromosomal instability (CIN)

   • e.g lose, gain, rearrange whole or portion of chromosomes

   • e.g anueloploidy

     • both an outcome and further instigator of CIN

2. Genetic instability (GIN)

   • increased rate of point mutations

overall: These affect protooncogenes and TSGs→ to cause cancer!

What causes GIN and CIN

• mis-regulation of cell-cycle

• through loss of brakes or checkpoints

  → tumourgenesis

E.g (2) Mutations in TSGs

Due to mutations in Tumour suppressor genes (TSGs)

Normally:

• encode cell cycle brakes

• checkpoint elements

• apoptosis stuff

• DNA repair pathways

Cancer when

• recessive loss-of-function mutation occur in both alleles

  →This can be caused by either GIN or CIN

TSGs genetic predisposition to cancer

• May inherit an already defective TSG allele

• only new one further alteration to cause cancer in the other allele

  • it is the predisposition that is dominant over the actual gene that will cause the cancer

Why studying the molecular basis of cancer is important

Lead to novel therapies for

• general or

• unique signatures!

Cancer drugs for general cell division

Cancer drugs which are more specific due to molecular understanding

How make it easier to make new target strategies

• Look at individual hallmarks of cancer

  • holistic view

• see what they affect

• form a drug/action plan which can target these specific things