13. Oncogenes

Why has the study of tumour viruses been important for understanding human cancer?

Although only a minority of human cancers are virally-induced, the study of tumour viruses has been crucial for cancer research.

Viral genomes are very simple, yet they contain extremely potent genes that can perturb the complex regulatory circuitry of the host cell to drive a tumour phenotype.

By identifying and studying these viral oncogenes, scientists were able to discover the corresponding cellular proto-oncogenes and understand the fundamental molecular mechanisms that become dysregulated in all types of cancer, including non-viral ones.

What were the key findings from experiments with Rous Sarcoma Virus (RSV) in the 1910s?

Experiments demonstrated for the first time that cancer could be caused by an infectious agent. His key findings were:

1. He took a sarcoma (a type of tumour) from a chicken, ground it up, and passed it through a fine-pore filter that would block bacteria.

2. When he injected the cell-free filtrate into a healthy young chicken, it developed a sarcoma at the injection site.

3. This showed that the carcinogenic agent was smaller than a bacterium, which he correctly identified as a virus (later named Rous Sarcoma Virus, RSV).

This provided a unique and predictable model to induce cancer at will, allowing for the recovery of the virus in large amounts for further study.

RSV can transform infected cells in culture.

RSV can transform infected cells in culture. Chicken embryo fibroblasts infected with RSV show traits associated with cancer cells:

• Foci (clusters) appear after infection

• Similar metabolism to cells isolated from tumours

Cell transformation = conversion of a normal cell into a cancer cell; it can be accomplished into a petri dish.

The behaviour of cells in foci → the transformation phenotype was transmitted from an initially infected cell to its descendants.

2 possible explanations:

1. RSV particle transformed the progenitor cells and all their descendant = virus must be present all the time to maintain the transformed phenotype.

2. RSV only needed to transform the progenitor cells, which then somehow transmit the phenotype to the descendants = virus acts in a “hit and run” fashion.

How was it demonstrated that the continued presence of the RSV viral transforming gene is required to both initiate and maintain the transformed phenotype?

This was demonstrated using a temperature-sensitive (ts) mutant of RSV. This mutant virus produces a transforming protein that is partially defective and can only function at a lower, permissive temperature (e.g., 37°C), but is non-functional at a higher, non-permissive temperature (e.g., 41°C).

The experiment showed:

1. At the permissive temperature, infected cells exhibited a transformed phenotype (e.g., rounded morphology, loss of contact inhibition).

2. When the cells were shifted to the non-permissive temperature, they reverted to a normal morphology and behaviour - transformed state is lost.

3. When shifted back to the permissive temperature, they became transformed again.

This demonstrated that the viral transforming protein was not just needed for a "hit and run" event to initiate transformation, but its continuous action was required to actively maintain the cancerous state.

What are the key properties of a transformed cell in culture?

When a normal cell is transformed into a cancer cell, it acquires a number of characteristic properties that can be observed in a petri dish:

1. Altered morphology (rounded shape)

2. Loss of contact inhibition (ability to grown over one another)

3. Ability to grow without attachment (anchorage independence)

4. Ability to proliferate indefinitely (immortalisation)

5. Reduced requirement for mitogenic growth factors

6. High saturation density (large number of cells in culture dish)

7. Inability to halt proliferation in the absence of growth factors

8. Increased uptake of glucose

9. Tumorigenicity

How was the specific gene responsible for RSV-induced transformation identified, and what was the surprising discovery about its origin?

Experiments using deletion mutants of the RSV genome showed that the three main viral genes (gag, pol, env) were required for viral replication, but were not required for cell transformation. This indicated the existence of a fourth, separate gene responsible for transformation.

This gene was named `src` (for sarcoma). The critical discovery came when a DNA probe for the viral `src` gene (v-src) was used:

• The probe hybridized not only to the DNA of RSV-infected cells but also to the DNA of normal, uninfected vertebrate cells.

• This revealed that a closely related version of the `src` gene, called `c-src`, is a normal gene present in the genomes of all vertebrates.

This led to the paradigm-shifting concept of the proto-oncogene: a normal cellular gene that, when altered (e.g., by being captured and mutated by a virus), can become a potent, cancer-causing oncogene.

How were non-viral, cellular oncogenes first identified in human tumours?

It was hypothesized that chemical mutagens could activate endogenous proto-oncogenes in a similar way to viruses. This was tested using a transfection assay:

1. DNA was extracted from chemically-transformed human tumour cells (e.g., bladder carcinoma).

2. This tumour DNA was introduced into normal mouse fibroblast cells in culture using calcium phosphate co-precipitation.

3. A small number of the recipient mouse cells became transformed, forming foci.

4. These transformed mouse cells were then able to form tumours when injected into a host mouse.

This proved that the donor tumour DNA carried a genetic element (an oncogene) that was capable of transforming a normal cell. The low probability of transformation (only ~0.1% of donor DNA becam established in the recipient genome) suggested a single gene was responsible.

How was the H-ras gene identified as the first human cellular oncogene activated by a point mutation?

Following the transfection experiments that showed a gene from human bladder carcinoma could transform mouse cells, researchers sought to identify that gene.

H-ras detected by transfection of human bladder carcinoma DNA.

1. They isolated the human oncogene from the transformed mouse cells using molecular cloning.

2. They compared this oncogene to its corresponding proto-oncogene from normal human DNA.

3. Sequence analysis revealed that the only difference was a single point mutation.

This mutation in the `H-ras` gene caused a single amino acid substitution at position 12, changing a glycine to a valine (G12V). This was the first time a specific mutation in a single gene was proven to be the cause of a human cancer, demonstrating that non-viral oncogenes exist and can be activated by genetic changes.

How does the Ras signaling cycle work, and how do oncogenic mutations disrupt it?

Ras is a small GTPase that acts as a molecular switch in growth factor signaling pathways.

• Normal Cycle: Ras cycles between an inactive GDP-bound state and an active GTP-bound state. In response to a growth factor signal, a GEF (Guanine nucleotide exchange factor) promotes the exchange of GDP for GTP, turning Ras "ON". An "OFF" switch is provided by GAPs (GTPase-activating proteins), which help Ras hydrolyze GTP back to GDP.

• Oncogenic Disruption: The common oncogenic mutations in Ras, such as G12V, occur in the GTP-binding pocket. This mutated Ras protein is unable to hydrolyze GTP, even in the presence of GAPs. As a result, it becomes permanently locked in the active, GTP-bound state, continuously sending pro-proliferative signals downstream, even in the absence of a growth factor.

A large number of human tumours were found to carry point mutations in one of the 3 ras genes present in the human genome: H-ras, K-ras & N-ras

Mutations in K-ras are the most frequent drivers of tumour development across the spectrum of human cancers

KRAS^G12C mutation is most prevalent in lung cancers

Several small molecule irreversible inhibitors have been developed → none had any effect on tumours in patients (despite promising results in animal models)

How was the KRAS inhibitor AMG 510 developed, and what is its mechanism of action?

The KRAS protein was long considered "undruggable" due to its smooth surface and high affinity for GTP. The breakthrough came with the development of AMG 510, a novel inhibitor that specifically targets the KRAS G12C mutant protein.

• Mechanism: AMG 510 is a covalent inhibitor. It exploits the mutant cysteine residue (C12) to form an irreversible covalent bond with the KRAS protein. Importantly, it only binds to KRAS G12C when it is in the inactive, GDP-bound state. This traps the protein in the "OFF" configuration, inhibiting downstream signaling and cell proliferation in lung cancer cell line in vitro.

• Specificity: Because it relies on the C12 mutation, it has very high specificity for the mutant protein and does not affect wild-type KRAS.

AMG 510 inhibits tumour growth in mice à tumour regression in 8/10 mice

AMG 510 efficiency improves in combination with standard chemotherapy (carboplatin) or a drug that inhibits MEK, which acts downstream of Ras

AMG 510 is not effective on mice lacking an immune system

What have clinical trials of the KRAS G12C inhibitor AMG 510 (Sotorasib) revealed about its efficacy and the challenges of targeted therapy?

Clinical trials of AMG 510 have shown both promise and challenges.

• AMG 510 in combination with an immunotherapy called anti-PD1 = harness the immune system to combat cancer → complete tumour regression in 9/10 immunocompetent mice.

• AMG 510 boost the expression of pro-inflammatory cytokines in tumour-bearing animals

• Increase infiltration of the tumours by T cells and dendritic cells

• When KRAS^G12C cancer cells are reintroduced in animals cured by the combination therapy, there was no tumour formation → long term T-cell response to KRAS tumour cells induced by the treatment

• Efficacy: AMG 510 shows significant efficacy in shrinking tumours in a subset of patients, particularly those with KRAS G12C-mutant non-small cell lung cancer. The drug also boosts anti-tumour immunity by increasing T-cell infiltration into the tumour.

• Challenge - Adaptive Resistance: A major problem is that while many tumours initially respond, adaptive resistance often occurs rapidly. Tumours can become resistant through a wide range of mechanisms, including:

  • On-target mutations that disrupt drug binding.

  • Activation of downstream signaling pathways (e.g., the PI3K/MAPK pathway).

  • Activation of alternative receptor tyrosine kinases.

This highlights the need for second-generation inhibitors and combination therapies to overcome resistance.

What are the three main mechanisms by which the myc proto-oncogene can be activated to become an oncogene?

Myc (c-myc, N-myc, L-myc) is a potent growth-promoting transcription factor that is overexpressed in over 70% of human tumours. It can be activated via three main mechanisms that all lead to abnormally high levels of the Myc protein:

1. Gene Amplification: The gene itself is present in multiple copies. This is common for N-myc in childhood neuroblastoma, where high copy number is a marker of very poor prognosis.

2. Chromosomal Translocation: The myc gene is moved to a new chromosomal location, placing it under the control of a powerful foreign promoter. In Burkitt lymphoma, a translocation between chromosomes 8 and 14 places c-myc under the control of an immunoglobulin (Ig) gene promoter, which is very active in lymphoid cells, leading to relentless proliferation.

3. Insertional Mutagenesis: This is typically caused by a retrovirus. In Avian Leukosis Virus (ALV)-induced leukaemia, the viral DNA integrates into the host genome immediately adjacent to the c-myc proto-oncogene. The strong viral promoter then drives extremely high levels of myc expression.

How can structural changes in proteins, such as the Epidermal Growth Factor Receptor (EGFR), lead to the creation of an oncogene?

EGFR is a receptor tyrosine kinase that is normally activated only when it binds its ligand, EGF.

However, structural changes can make it constitutively active.

A common mechanism in cancers like glioblastoma and lung cancer is the deletion of the extracellular ligand-binding domain.

This results in a truncated receptor that emits signals constantly, even in the complete absence of the EGF ligand.

This ligand-independent firing drives uncontrolled proliferation and tumour formation.