Lecture 2: Cancer Genetics and Genomics – Cancer Driver Genes, Proto-oncogenes 🧬

Aims of this lecture:

  • To describe the origins and evolution of cancer.

  • To explain the distinction between proto-oncogenes and tumour suppressors.

  • To outline the normal functions of proto-oncogenes.

  • To describe how mutations may convert proto-oncogenes into disease-associated oncogenes.

  • To provide examples of different oncogenes.

The Origins and Evolution of Cancer 🌳

What type of cells form cancers?

  • Cancers typically do not arise from fully differentiated cells like neurons or muscle fibers.

  • Instead, cancers tend to originate from mutations occurring in stem cells and/or progenitor cells. These cells have a greater capacity for self-renewal and proliferation compared to differentiated cells.

Cancer as an Acquired Genetic Disease:

  • While environmental factors and viruses (e.g., HPV16 and HPV18 in cervical cancer) can play a role, cancer primarily arises due to an accumulation of somatic mutations. It is essentially an acquired genetic disease.

  • Cancer rates increase with age, correlating with an increased mutational burden over an individual's lifespan.

Evolution of Cancer from Benign Tumours:

  • A benign tumour can evolve into cancer through the accumulation of multiple mutations.

  • For example, colon carcinoma often originates from a benign polyp. The progression involves sequential mutations, such as APC inactivation, KRAS/BRAF activation, inactivation of DNA mismatch repair genes (MLH1/MSH2/MSH6), SMAD4/TGFβR2 inactivation, and TP53 inactivation.

  • While APC mutations are seen in very early benign tumours, functional experiments in vivo have demonstrated that this initial mutation is required in stem cells rather than differentiated cells to drive cancer progression.

Intestinal Stem Cells as Cells of Origin:

  • The gastrointestinal tract contains structures called crypts and villi. Stem cells reside at the base of these crypts.

  • Experiments were conducted to determine if intestinal cancer originates from stem cells or differentiated cells.

    • Deletion of the tumour suppressor gene Apc in non-stem cells resulted in micro-lesions that did not progress.

    • However, deletion of Apc specifically in Lgr5-positive intestinal stem cells led to immediate hyperproliferation and adenoma formation.

Cancer Stem Cells:

  • Experiments in the 1960s showed that a large number of cancer cells were needed to generate a new tumour in vivo.

  • Many cancers consist of a heterogeneous mixture of cells with varying degrees of differentiation and replicative potential.

  • Some cancers may contain cancer stem cells (CSCs), which are a subpopulation of cells within a tumour that possess the unique capacity to generate a new tumour.

  • In colon tumours, for instance, cancer stem cells often express Lgr5, the same marker found on normal intestinal stem cells.

  • When sorted, only these CSCs can form new tumours when injected into immunodeficient mice, while other cancer cells cannot.

Proto-oncogenes vs. Tumour Suppressors 🚦

  • Cancer primarily arises due to somatic mutations, making it an acquired genetic disease.

  • Genes that undergo driver mutations (mutations that promote tumour initiation or progression) are classified into two main categories:

    • (i) Proto-oncogenes

    • (ii) Tumour suppressor genes

A normal gene, when mutated, can lead to either an abnormal protein or no protein at all.

  • If the mutation results in an abnormal, hyperactive protein, the gene is acting as an oncogene (an activating mutation).

  • If the mutation results in no protein or a non-functional protein, and this protein normally restrains cell growth, then the gene is a tumour suppressor (a deactivating mutation).

Proto-oncogenes

  • Proto-oncogenes are normal cellular genes that can be converted (activated) into oncogenes (genes that contribute to carcinogenesis) through mutations.

  • Driver mutations in proto-oncogenes cause a gain of function.

  • These mutations typically act dominantly, meaning a mutation in only one allele is sufficient to cause a phenotype.

  • Proto-oncogenes often encode components of mitogenic (growth-promoting) signal transduction pathways but can also be involved in various other cellular processes.

  • Oncogenes can be good therapeutic targets.

Activation of Proto-oncogenes (Conversion to Oncogenes): There are two general mechanisms for proto-oncogene activation:

  1. Qualitative changes: Production of an abnormal, hyperactive protein. This can result from:

    • (i) Point mutations in the coding sequence.

    • (ii) Translocations (exchange of DNA between different chromosomes) that generate fusion proteins.

  2. Quantitative changes: Overproduction of an unaltered (normal) protein. This can result from:

    • (i) Translocations that place the gene under a stronger promoter or enhancer.

    • (ii) Point mutations in cis-acting regulatory sequences (like promoters or enhancers).

    • (iii) Gene amplification (creation of multiple copies of the gene).

Specific Mechanisms of Proto-oncogene to Oncogene Conversion:

  1. Mutations that lead to a hyperactive gene product.

  2. Mutations in the promoter region of a proto-oncogene that result in increased transcription.

  3. Gene amplification events leading to extra chromosomal copies of a proto-oncogene.

  4. Chromosomal translocation events that relocate a proto-oncogene to a new chromosomal site (near a strong promoter or enhancer) leading to higher expression.

  5. Chromosomal translocations that lead to a fusion between a proto-oncogene and a second gene, producing a fusion protein with oncogenic activity.

Examples of Oncogene Activation Mechanisms 🔬

1. Mutations Leading to a Hyperactive Gene Product (e.g., KRas):

  • KRas (Kirsten Ras) is one of the most frequently mutated oncogenes.

  • KRas is a regulatory G protein involved in signal transduction, often stimulated by growth factor receptors (like EGFR), promoting cell proliferation and migration.

  • Normal Ras function: Ras is INACTIVE when bound to GDP. Growth factor receptor activation brings a guanine nucleotide exchange factor (GEF) into contact with Ras, which replaces GDP with GTP, making Ras ACTIVE. GTPase-activating proteins (GAPs) promote the intrinsic GTPase activity of Ras, converting GTP back to GDP, thus INACTIVATING Ras.

  • Mutant KRas: Cancer-associated mutations in KRas often impede its interaction with GAPs. This results in GTP remaining bound to Ras, locking it in a constitutively active "ON" state, leading to uncontrolled proliferation.

2. Promoter Mutations Leading to Increased Transcription (e.g., TERT):

  • Point mutations in the promoter of the TERT gene (e.g., C → T change 146 bp upstream of the initiation codon) are found in a high percentage of cancers like melanoma (80%), glioma (80-90%), and liver cancer (60%), leading to increased transcription.

  • TERT (Telomerase Reverse Transcriptase) is the catalytic subunit of telomerase, an enzyme responsible for maintaining telomere length. Increased TERT expression contributes to cancer cell immortality, a key hallmark of cancer.

3. Gene Amplification (e.g., MYC, ERBB1, ERBB2):

  • Gene amplification leads to extra copies of a proto-oncogene, resulting in overexpression of its protein product.

    • MYC is amplified in neuroblastoma, lung cancer, and breast cancer.

    • ERBB1 (also known as EGFR) is amplified in glioma (a type of brain tumour).

    • ERBB2 (also known as HER2) is amplified in breast cancer, ovarian cancer, and stomach cancer.

4. Chromosomal Translocation Leading to Higher Expression (e.g., MYC in Burkitt's Lymphoma):

  • In Burkitt's lymphoma, a reciprocal translocation, typically t(8;14), moves the Myc proto-oncogene from chromosome 8 to chromosome 14, placing it under the control of the very active immunoglobulin heavy chain (IgH) promoter/enhancer region. This results in very high and inappropriate transcription of Myc.

5. Chromosomal Translocation Leading to a Fusion Protein (e.g., BCR-ABL in CML):

  • The ABL gene, located on chromosome 9, encodes a tyrosine kinase that regulates white blood cell production.

  • In Chronic Myeloid Leukaemia (CML), a translocation between chromosome 9 and chromosome 22, t(9;22), creates the Philadelphia chromosome.

  • This translocation fuses the ABL gene with the BCR (Breakpoint Cluster Region) gene on chromosome 22.

  • The resulting BCR-ABL fusion protein has increased and constitutively active tyrosine kinase activity.

  • BCR-ABL can activate multiple downstream pathways, including those involving KRas and JAK/STAT, leading to unregulated proliferation, chromosome instability, and the development of leukaemia (CML).

Identifying Cancer Driver Genes 🔍

How are driver mutations identified?

  • Most driver mutations are now identified through large-scale genome sequencing of cancer cells.

  • Genes within cancer cells that show a significantly higher frequency of mutations compared to neighbouring normal tissue are likely to be driver genes.

  • For example, over 90% of pancreatic cancers have a mutation in the KRAS gene, while 80-90% of colon tumours have a mutation in the APC gene.

  • Inherited deletion of one allele of APC is the cause of Familial Adenomatous Polyposis (FAP). In FAP patients, the remaining APC allele is often mutated somatically (via loss-of-heterozygosity), leading to multiple benign intestinal polyps which can progress to cancer.

Distinguishing Proto-oncogenes from Tumour Suppressors by Mutation Patterns:

  • Proto-oncogenes typically undergo gain-of-function mutations.

    • These are often missense mutations that alter a critical amino acid, leading to a hyperactive protein.

    • Such activating mutations may occur at only one or a few specific codons within the gene. (e.g., Protein A in the example, with mutations clustered at specific sites).

  • Tumour suppressor genes usually undergo loss-of-function mutations.

    • These can be of various types, including large deletions, frameshift mutations, nonsense (stop) mutations, splice site mutations, or missense mutations that disrupt protein function.

    • These inactivating mutations can occur at many different sites scattered throughout the gene. (e.g., Protein B in the example, with truncating mutations spread across the gene).

  • Sequencing the same driver gene from many different cancer cases and analyzing the spectrum of mutations allows for the distinction between proto-oncogenes and tumour suppressors.

The most recent cancer gene census (as of 2020) has identified 568 cancer driver genes. Many proto-oncogenes are involved in signalling pathways that stimulate cell division (mitogenic pathways).

Examples of Proto-oncogenes and Their Roles 🚦

The MAP Kinase Pathway:

  • Many growth factors act through the Mitogen-Activated Protein (MAP) kinase pathway.

  • This pathway involves a cascade of protein phosphorylations that ultimately lead to the activation of genes involved in cell division.

  • Key components include:

    • Receptor Tyrosine Kinases (RTKs)

    • G-proteins (like Ras)

    • Cytoplasmic kinases (like RAF, MEK, ERK)

    • Transcription factors (like Myc, Fos)

  • Many proto-oncogenes encode components of this pathway. Examples include:

    • Growth factors (ligands): FGF3, PDGFB

    • Growth factor receptors: ERBB1 (EGFR), ERBB2 (HER2), RET

    • G-proteins: KRAS, HRAS, NRAS

    • Cytoplasmic kinases: BRAF, MEK

    • Transcription factors: MYC, MYCN, FOS

  • Activation of any of these proto-oncogenes can allow cell division in the absence of normal growth-promoting signals, contributing to the hallmark of sustained proliferation.

Oncogenic Myc:

  • Myc is a transcription factor that is not typically mutated in cancer but is rather deregulated (e.g., overexpressed) in over 50-70% of cancers.

  • Myc overexpression can occur due to:

    • Retroviral promoter or enhancer insertion.

    • Chromosomal translocation placing Myc next to a strong promoter like the Immunoglobulin (Ig) promoter.

    • Gene amplification.

    • Deregulated cell signalling pathways (e.g., Ras, Wnt) that have Myc as a target gene.

  • Myc regulates many cellular functions, including cell growth, DNA repair, and apoptosis, and is estimated to regulate ~10-15% of the transcriptome.

  • In the context of intestinal tumours, loss of the tumour suppressor APC triggers tumour initiation. Myc is upregulated in APC mutant cells (acting as an oncogene in this context) and is required for the tumorigenic phenotype in these cells.

Other Examples of Human Proto-oncogenes:

  • BCL2: An apoptosis inhibitor. Translocation to the immunoglobulin heavy-chain enhancer drives its overexpression, allowing cells to evade apoptosis (e.g., in B-cell leukaemia).

  • Beta-Catenin (CTNNB1): A transcription factor in the Wnt signalling pathway. Mutations can prevent its negative regulation (phosphorylation and proteasomal destruction), leading to constitutive Wnt pathway activation, nuclear translocation, and activation of target genes like Myc to promote proliferation. Mutations are often clustered in sites required for its phosphorylation.

  • Cyclin D1 (CCND1): Regulates the cell cycle. Overexpression promotes cancer cell proliferation.

  • TERT: Catalytic subunit of telomerase, regulating telomere maintenance. Amplification can provide cancer cell immortality.

Bcl2 as an Oncogene – Discovery:

  • The function of Bcl2 as an anti-apoptotic protein was discovered by David Vaux in 1988.

  • His PhD project initially aimed to investigate if Bcl2 regulated cell proliferation.

  • Overexpression of Bcl2 did not increase cell growth but made cells very resistant to death when growth factor was removed.

  • Cells overexpressing both Bcl2 (anti-apoptosis) and Myc (pro-proliferation) did not die at all, mimicking transformation.

Mechanism of Bcl2 as a Proto-oncogene:

  • Bcl2 inhibits apoptosis (it's a pro-survival protein) by binding to pro-apoptotic proteins like Bax, Bak, and Bok on the mitochondrial membrane, preventing them from forming pores and releasing cytochrome c.

  • Under cellular stress, BH3-only proteins (like Bim, Bad, Puma, Noxa) inhibit Bcl2, releasing this block on Bax/Bak and allowing apoptosis to proceed.

  • Overexpression of Bcl2 due to translocation (e.g., t(14;18) in follicular lymphoma) leads to excessive Bcl2, which sequesters pro-apoptotic proteins and prevents cell death.

Learning Outcomes 🎓

After this lecture, you should be able to:

  • Discuss the distinction between proto-oncogenes and tumour suppressors.

  • Explain how proto-oncogenes and tumour suppressors can be detected (e.g., by analyzing mutation patterns).

  • Outline the normal function of proto-oncogenes in the stimulation of cell division in response to growth signals.

  • Appreciate that mutations that activate proto-oncogenes can either generate an altered protein or cause inappropriate expression of an unaltered protein.

  • Describe examples of mechanisms which lead to the activation of proto-oncogenes.