Oncogenes and tumour suppressors

How do oncogenes arise?

Proto-oncogenes are involved in cell growth, proliferation and differentiation. They become oncogenes when mutated which are cancerous. Oncogenes cause increased cell proliferation and arise from gain-of-function mutations. Thus, cancer is a genetic disease - cell division to spread throughout a population, and inheritance to offspring are linked to cancer.

How were oncogenes discovered?

Chicken DNA is added to a tube where it gets denatured and cut up. Viral DNA (RSV), which causes sarcoma in chickens, was denatured and cut up, and added to the tube. Segments of the chicken and viral DNA are complementary and anneal with each other - these viruses that cause cancer had chicken DNA within their genome. This was the discovery of proto-oncogenes - the DNA in the virus encodes for a protein called Src which is a non-receptor tyrosine kinase involved in regulation of cell growth and differentiation.

• The virus injects its RNA which is reverse transcribed to produce DNA, which gets integrated into the host genome. The DNA will become active which hijacks the cellular machinery to form copies of the viruses. The host cell DNA can be transcribed along with the viral DNA which may get inserted into the new viruses - the viral DNA inserted near cellular-src, which gets transcribed to produce RNA with viral-src which gets packaged into a capsid.

• C-src is the proto-oncogene. V-src has slightly diverged from the analogous sequences in the chicken genome - it has a mutation which means it is now an oncogene. This was the discovery of oncogenes. 

What is common among cancer-causing viruses?

Many cancer-causing viruses are found in chickens, and many of them are non-receptor tyrosine kinases, small G proteins (GTPases etc), receptors and TFs. All of these involve pathways that transmit signals from the surface of the cell to the genes.

What is the Philadelphia chromosome?

The Philadelphia chromosome is the translocation between chromosomes 9 and 22 (9 gets longer, 22 gets shorter). It causes chronic myeloid leukaemia in humans and was the first translocation discovered to cause cancer. Ableson is a non-RTK (phosphorylate tyrosine residues on other proteins) from 9 which fuses with Bcr of 22, resulting in the loss of the Abl N-terminal sequences. This activates the non-RTK which activates the Ras signaling pathway and subsequent repression of apoptosis.

What is c-Abl and how does it change in cancer?

The c-Abl is the proto-oncogene. It has an NLS and a proline rich area. Ableson protein also has SH3 which interacts with proline residues and an SH2 domain which binds to phosphorylated tyrosine residues other proteins or itself - these are important regulatory domains. In Bcr-Abl, the fusion shortens the SH3 domain, and in v-Abl, the viral DNA is inserted between the SH3 and SH2 domains, to remove SH3 completely. v-Abl also inserts a GAG where SH3 is supposed to be, meaning it gets transported to the cell surface becoming hyper-oncogenic - this is called myristylation. Loss of SH3 domain means the protein is constitutively activated. 

In inactive c-Abl, the SH3 is bound to its internal proline residues causing a folding that keeps the SH2 hidden, meaning the protein is inactive. When another protein has phosphorylated tyrosine, the SH2 binds to it which separates SH3 from the prolines meaning Abl is active. In Bcr-Abl, SH3 is prevented from binding to internal prolines. 

How does Bcr-Abl lead to cancer?

Bcr-Abl signalling mimics growth factor activation. It can phosphorylate STAT5 which are TFs that move to the nucleus to activate cell proliferation, regulate the AKT pathway which leads to the activation of p53 that prevents apoptosis etc.

What types of proteins are associated with cancer?

The types of proteins associated with cancer are those that are involved in signalling pathways from the cell surface to the nucleus. These include TFs, kinases, signalling molecules and cell death regulators.

How does Gleevec work?

Gleevec is an inhibitor drug that has a higher affinity for the catalytic cleft than ATP so binds to Abl preventing its activity. This is the first successful treatment of CML. Over years, patients can develop a resistance to Gleevec.

How were tumour suppressors discovered?

Tumour suppressor genes were discovered by Henry Harris. He inserted a normal cell into a mouse and nothing happened, but when he inserted a tumour cell it caused cancer. He then fused the two cells and inserted it, and the cancer was suppressed.

Analogy for oncogenes and tumour suppressors.

You can visualise the oncogenes as an accelerator of a car, and the tumour suppressors as the breaks.

What are the roles of tumour suppressors?

Tumour suppressors negatively regulate the cell cycle, so loss of TSG means that the cells are free to proliferate. There is a subclass of TSGs that are “caretakers” of the genome and repair DNA mutations. Mutation in these repair mechanisms results in a “mutator” phenotype which results in the acceleration of mutation accumulation. 

What is retinoblastoma and how does it work?

Retinoblastoma was the first discovered TSG in which a null mutation of the Rb gene causes a malignant tumour of the developing retina. There are 2 copies of this gene - the loss of one gene increases the likelihood of the second copy to be lost via cooperativity (this is when tumour develops). Offspring can inherit the loss of one copy resulting in haploinsufficiency so there is not enough Rb in the body. There are other Rb-like proteins in the body that prevent cancer, but cells in the retina likely lack them which is why the tumour develops in the eye. Rb is a transcriptional repressor for genes involved in the cell cycle (G0 to G1) - loss leads to proliferation.

Rb is found on chromosome 13. Germline deletion of Rb on one allele predisposes the carrier to retinoblastoma.

How does Rb work?

When Rb is un-phosphorylated, it binds to E2F TF to inhibit it by recruiting histone deacetylase (HDAC) so that the genes E2F activate to trigger the G0/G1 transition are blocked. Rb gets phosphorylated by CDKs, it dissociates from E2F,  allowing histone acetylase to open the gene for transcription.

How does p53 work and what happens in mutation?

p53 is a tumour suppressor as well. It is a TF that suppresses G1/S phase transition via arresting cell cycle in G1 due to DNA damage. It also induces p21 (cell cycle inhibitor), DNA repair genes and apoptosis (if DNA damage is unable to be repaired).

p53 targets specific genes via their promoters which contains a p53 response element. The start site is found within the core promoter downstream. p53 helps recruit RNA polymerase to the start of the gene. The amount of p53 increases in response to DNA damage. It is a tetramer with each part containing a DNA binding domain which all bind to the response element. First, it makes the gene more accessible by recruiting co-activators to remodel the DNA at the core promoter, then it brings RNA polymerase to the start site.

Deletion leads to cell survival and loss of G1 arrest.

How are p53 levels regulated?

Proteasomal degradation regulates p53 levels. p53 is constantly made in the nucleus but it is constantly degraded by the proteasome. The protein responsible for ubiquitination of p53 is the E3 ligase called mdm2 - oncogene (often overexpressed in cancer). This pathway is disrupted by kinases which detect cell stress that phosphorylate p53 (prevent interaction with mdm2) and mdm2 (nuclear translocation - away from p53). This means p53 is not ubiquitylated so it can build up. 

How do RTKs work?

RTKs have an extracellular region which binds a ligand, a transmembrane region (may control location in membrane) and an intracellular TK domain which phosphorylates tyrosine residues. Upon binding a ligand, they dimerise and trans phosphorylate each other’s C-terminal. Transphosphorylation can result in conformation change removing an ‘activation loop’ from the catalytic cleft. It also creates docking sites for ‘adaptor’ proteins which propagate the signal into the cytoplasm from the outside.

RTKs can activate the Ras-MAPK pathway. Activated RTK recruits and phosphorylates GRB2 via its SH2 domain. GRB2 binds to SOS protein via the SH3 domain. SOS is a GEF for Ras, resulting in GDP being displaced by GTP. Active Ras stimulates molecules downstream via the RAF-MEK-ERK pathway which is a MAPK pathway. RAF phosphorylates MEK etc, until it reaches the nucleus to activate TFs that trigger the cell cycle.

How does HER2 cause breast cancer and how is it treated?

HER2 is an RTK overexpressed in breast cancer and is an oncogene. Increasing the RTKs in the membrane means they can interact with each other much more frequently so is essentially constitutively expressed. Patients with HER2+ breast cancer are generally oestrogen receptor negative, so standard treatments do not work. They are treated with antibodies; some can inhibit dimerisation (pertuzumab) or through direct Ab binding (trastuzumab). 

How do oncogenes and tumour suppressors contribute to cancer?

Oncogene and TSG mutations increase with age, each contributing more to a cell becoming cancerous. Eventually, there will be cooperativity between these to cause cancer. Cooperativity was shown in mice which have one of two mutations and it survived for a few hundred days. Together, these mutations caused them to die much quicker.