Oncogenes: Comprehensive Notes (Video Lecture)
Historical context and overarching questions
In the 1970s, viral oncogenesis was considered a leading explanation for cancer; viruses provided mechanisms for transformation without requiring immediate cellular mutations.
Ludwig Gross asserted that the viral origin of the majority of malignant tumors had been documented beyond reasonable doubt, extrapolating from known human cancer viruses (Epstein–Barr virus in Burkitt's lymphoma; human papillomaviruses in cervical cancer) to a universal viral etiology. This view proved incorrect; the vast majority of cancers are not caused by viruses (roughly >80% are due to other factors).
Even if viruses contribute in some cancers, many viruses exploit cellular genes to drive oncogenesis, rather than acting as independent oncogenic drivers.
The central question: where do oncogenes come from? Are oncogenes viral in origin, or are they derived from normal cellular genes (proto-oncogenes) that become cancerous via alterations?
Key experimental milestones: from viruses to cellular oncogenes
Early 1980s landmark experiments by Weinberg and Kopper used DNA from tumor cells to transform non-tumor cells, demonstrating that cellular genes can carry oncogenic potential independent of exogenous viral oncogenes.
Model system:
Mouse fibroblast line NIH 3T3 used because it readily takes up DNA (transfection).
A chemically induced mouse tumor cell line (from 3-methylcholanthrene) is transformed in culture.
DNA extracted from transformed cells is introduced into new fibroblasts via calcium phosphate precipitation; transformed foci, altered morphology, soft agar growth, and tumor formation in immunodeficient mice indicate transformation.
Transfection vs transformation distinction:
Transfection: transfer of DNA from one cell to another.
Transformation: conversion of a normal cell into a tumorigenic cell.
In bacteria, the term transformation has a different meaning (DNA uptake by bacteria); in mammalian cells, transfection is the appropriate term.
Methods and nomenclature in oncogene discovery
Southern blotting (DNA):
DNA from tumor and normal tissue is digested with restriction enzymes, separated by gel electrophoresis, and transferred to a nitrocellulose membrane (Southern blot).
Radiolabeled probes (often RNA or DNA) complementary to viral oncogene sequences are hybridized to the immobilized DNA. A detected band indicates a fragment containing the oncogene or homologous sequences.
The pattern allows identification of common fragments across transformed cells, pointing to candidate oncogenic fragments.
Northern blotting (RNA) and Western blotting (proteins):
Northern blot uses RNA separated on a gel and probed to detect transcripts.
Western blot detects proteins separated by gel and probed with antibodies.
Experimental readouts and interpretation:
A consistent band across transformed tumors suggests the oncogene is on that fragment.
Comparing different restriction digests (EcoRI, HindIII, etc.) helps localize the fragment that harbors the oncogene.
Transfection-driven oncogene identification workflow:
Transfect tumor-derived DNA into NIH 3T3 cells and identify transformed lines.
Isolate DNA fragments from the transforming region and clone into bacterial plasmids to produce ample DNA for re-testing.
Test whether the normal proto-oncogene fragment vs. the cancer-derived fragment can transform cells; the transforming fragment identifies the oncogene region.
Narrow the transforming region by creating chimeric constructs (left half of proto-oncogene with right half of oncogene, and vice versa) to localize the transforming mutation to a small region (< a few hundred nucleotides).
Sequence the critical region to identify specific mutations: insertion, deletion, or base substitution.
Case study: discovery and implications of RAS oncogenes
RAS family and proto-oncogenes:
In normal cells, RAS genes encode small GTPases (HRAS, KRAS, NRAS) that act as molecular switches in signaling pathways.
RAS cycles between active GTP-bound and inactive GDP-bound states; activity is regulated by GAPs (GTPase-activating proteins) and GEFs (guanine nucleotide exchange factors).
Key experimental observation:
Using HRAS/RAS probes in bladder carcinoma and NIH 3T3 transfected cells, investigators found that transformed cells harbored HRAS sequences on acquired DNA fragments, while NIH 3T3 cells contributed a common fragment from their own genome.
The transfected tumor DNA often forms tandem repeats, leading to multiple copies of RAS oncogenes and variable signal intensities across cell lines.
Mechanistic insight: the first discovered mutation activating a proto-oncogene in human cancer
A single nucleotide substitution in the RAS gene (e.g., glycine codon -> valine codon) impairs GTP hydrolysis, locking RAS in an active, GTP-bound state and driving uncontrolled signaling and proliferation.
This mutation was the first reported instance where a proto-oncogene mutation directly contributes to human cancer, establishing a concrete molecular mechanism linking normal signaling to oncogenesis.
RAS gene family and cancer associations:
HRAS, KRAS, and NRAS encode closely related proteins; mutations in different isoforms are prevalent in distinct cancers.
KRAS mutations are especially common in pancreatic cancer (~ ext{≈}90 ext{ ext{%}})); papillary thyroid cancers show KRAS/NRAS involvement in ~ ext{≈}60 ext{ ext{%}} ; follicular thyroid cancers show RAS involvement in ~ ext{≈}50 ext{ ext{%}}.
Pancreatic cancers rarely harbor HRAS or NRAS mutations, reflecting tissue-specific expression and reliance on particular RAS isoforms for oncogenic signaling.
Mechanistic summary:
Normal RAS function: RAS-GTP active; intrinsic GTPase activity slowly hydrolyzes to RAS-GDP, turning signaling off; GAPs accelerate this inactivation; GEFs promote GDP-to-GTP exchange to re-activate.
Oncogenic mutation: impaired GTP hydrolysis or impaired GAP interaction yields constitutively active RAS, sustaining proliferative signaling.
Implications across cancers:
RAS mutations are among the most common oncogenic events across tumors; different cancers preferentially mutate specific RAS family members (HRAS, KRAS, NRAS) consistent with tissue-specific expression and function.
Other mechanisms to generate oncogene activity: amplification, translocations, rearrangements, and insertional mutagenesis
Gene amplification (increased gene dosage):
Duplications yield more mRNA and protein, enhancing signaling output.
Two major forms of amplified DNA in cancer:
Double minutes: extrachromosomal, circular DNA fragments that harbor amplified gene copies.
Homogeneously staining regions (HSRs): contiguous amplified gene sequences visible as uniform staining within a chromosome.
Consequence: higher protein levels without altering gene sequence; example implications include MYC amplification in neuroblastoma and HER2 amplification in breast/ovarian cancers (prognostic impact increases with higher copy number).
Clinical correlate: more copies of an oncogene often correlate with worse prognosis and reduced disease-free survival, e.g., MYC amplification is associated with increased aggressiveness in neuroblastoma; HER2 amplification correlates with poorer outcomes when copy number is high.
Chromosomal translocations (gene relocation and/or fusion):
Common in leukemias and lymphomas due to active V(D)J recombination and double-strand breaks in lymphocytes.
Two major outcomes of chromosomal translocations:
1) Overexpression via juxtaposition of an oncogene to a highly active promoter/enhancer (e.g., Burkitt's lymphoma: MYC moved next to immunoglobulin enhancer, driving MYC overexpression).
2) Formation of a fusion protein with novel constitutive activity (e.g., Philadelphia chromosome: BCR-ABL fusion kinase; ABL fused to BCR, yielding constitutively active tyrosine kinase that drives CML; BCR-ABL target for therapy with imatinib/Gleevec).Notable examples:
Burkitt's lymphoma: MYC translocated to Ig heavy chain locus, leading to MYC overexpression.
Chronic myelogenous leukemia (CML): BCR-ABL fusion protein with constitutive tyrosine kinase activity; targeted by ABL inhibitors (e.g., imatinib) with high cure rates.
TRK gene activation via inversion creating TPM-TRK fusion with constitutive TRK kinase signaling (TRK fusion driving oncogenesis via aberrant dimerization and signaling).
Internal DNA rearrangements: deletions, insertions, and inversions within a chromosome that alter gene regulation or create fusion proteins
Inversions can join two different genes, creating a fusion protein that is constitutively active (e.g., TRK-TPM inversion leading to a TRK-TPM fusion with constitutive activity).
Deletions can remove regulatory regions or create novel regulatory contexts that activate oncogenes; insertions can similarly disrupt regulation.
Insertional mutagenesis by retroviruses: promoter/enhancer activation near a proto-oncogene
Some retroviruses lack their own oncogene but carry powerful long terminal repeats (LTRs) that act as strong promoters/enhancers.
Insertion of viral DNA near a proto-oncogene can upregulate its expression (e.g., MYC overexpression via LTR insertion).
This mechanism demonstrates how slow-acting retroviruses can generate oncogenic events by hijacking host gene regulation.
Six major classes of oncogenes: functional categories
Growth factors (secreted ligands):
Examples: PDGF, EGF, TGF-α, others.
Mechanisms of activation:
Overexpression of the growth factor itself.
Altered structure increasing stability, receptor affinity, or extracellular matrix tethering (e.g., PDGF fused to collagen, leading to localized, sustained PDGF signaling and fibroblast proliferation in sarcomas).
Note: Growth factors typically lack intrinsic enzymatic activity; overexpression and/or altered availability drive signaling.
Growth factor receptors (tyrosine kinases and related receptors):
Activation mechanisms:
Ligand-induced dimerization activates intrinsic kinase activity; constitutive activation can arise from point mutations in the receptor, mutations that remove the extracellular domain (which may relieve steric hindrance to dimerization), or receptor overexpression driving spontaneous dimerization.
Mutations in receptors such as RET, MET, EGFR, and ERBB family members; HER2 amplification drives signaling through EGF receptor pathways via heterodimerization.
Notable examples:
HER2/ERBB2 amplification commonly observed in a subset of breast and ovarian cancers; higher copy number correlates with worse prognosis.
TRK fusion proteins (e.g., TPM-TRK) as constitutively active signaling receptors.
Signaling molecules downstream of receptors (non-receptor kinases and G proteins):
Small GTPases (G proteins, e.g., RAS family):
Act as molecular switches; active when bound to GTP, inactive when bound to GDP; cycling regulated by GEFs and GAPs.
Oncogenic mutations often impair GTP hydrolysis, locking RAS in the active, GTP-bound state, promoting ongoing proliferation signals.
Non-receptor serine/threonine and tyrosine kinases: RAF, MEK, ERK (MAPK pathway components), SRC family kinases, and BCR-ABL fusion kinase.
Example consequences: constitutive MAPK signaling leading to transcriptional programs promoting proliferation.
Transcription factors: MYC, FOS, JUN, MYB, etc.
Mechanisms of activation:
Overexpression due to gene amplification or transcriptional upregulation.
Altered degradation leading to stabilization and accumulation.
Mutations changing DNA-binding specificity and downstream targets.
Notable connections: FOS/JUN are downstream targets of the RAS-MAPK pathway; amplification/overexpression of MYC is common in many cancers and can be driven by translocations or insertions (e.g., Burkitt's lymphoma).
Cell cycle regulators: CDKs and cyclins (e.g., CDK4; Cyclin D1)
Mechanisms: overexpression or inappropriate activation of CDKs or cyclins drives cell cycle progression (e.g., phosphorylation of RB to advance cells from G1 to S phase).
Consequences: uncontrolled cell proliferation.
Cell death regulators (apoptosis regulators): BCL-2 family, p53 pathway regulators like MDM2
BCL-2 and related family members inhibit apoptosis, contributing to tumor cell survival when overexpressed (e.g., certain non-Hodgkin lymphomas with BCL-2 overexpression).
MDM2 inhibits p53; overexpression of MDM2 can suppress p53-dependent apoptosis, contributing to tumorigenesis.
Selected concrete examples and cancer associations
Burkitt's lymphoma: MYC oncogene overexpressed due to chromosomal translocation placing MYC under the control of immunoglobulin heavy chain enhancer.
Philadelphia chromosome and CML: reciprocal translocation t(9;22) yields BCR-ABL fusion with constitutive tyrosine kinase activity; imatinib (a targeted therapy) inhibits BCR-ABL and has dramatically improved outcomes.
HER2 (ERBB2) amplification: commonly amplified in breast/ovarian cancers; copy number correlates with prognosis and disease-free survival; higher amplification generally indicates worse outcome.
RAS-driven cancers: KRAS mutant pancreatic cancer is highly prevalent; NRAS and HRAS mutations contribute in other lineages (thyroid, etc.); expression patterns in tissues influence which RAS isoforms drive oncogenesis.
Translocations generating fusion oncoproteins: TRK fusions (e.g., TPM-TRK) create constitutively active receptor tyrosine kinases by leveraging dimerization domains from fusion partners.
Insertional mutagenesis and retrovirus-driven oncogenesis: retroviruses with strong LTR promoters can upregulate host oncogenes (e.g., MYC) when integrated near them.
Quantitative and descriptive data points to remember
Virus-associated cancers in the era of the lecture:
In humans, only a few cancers had clear viral etiologies known at the time (e.g., EBV in Burkitt's lymphoma; HPV in cervical cancer).
The majority of cancers are not virally induced; estimates referenced: > of cancers arise from non-viral mechanisms.
RAS mutation prevalence by cancer type (illustrative):
KRAS mutations in pancreatic cancer: ~ ext{≈}90 ext{ ext{%}}
HRAS mutations in papillary thyroid cancers: ~ ext{≈}60 ext{ ext{%}}
RAS mutations in follicular thyroid cancers: ~ ext{≈}50 ext{ ext{%}}
Pancreatic cancers show high KRAS mutation incidence, while other isoforms (HRAS, NRAS) are less frequently mutated in pancreas due to tissue expression patterns.
RAS activation mechanism:
Normal GTPase cycle: RAS-GTP active; RAS-GDP inactive; GTP hydrolysis slowed by GAPs; reactivation by GEFs.
Oncogenic mutation mechanism: impairment of GTPase activity or GAP interaction yields constitutively active RAS.
Specific numbers on prognosis and copy number:
Higher copy numbers of oncogenes via amplification generally correspond to worse prognosis and shorter disease-free survival in various cancers; example: MYC amplification in neuroblastoma correlates with increased metastatic potential; HER2 amplification correlates with poorer prognosis with higher copy numbers.
Therapeutic implications:
BCR-ABL fusion in CML is a validated target for tyrosine kinase inhibitors (e.g., imatinib) with high cure rates; demonstrates the translational value of oncogene biology for targeted therapy.
Mechanistic summary: converting proto-oncogenes to oncogenes (recap)
Point mutations: often create constitutively active signaling proteins or transcription factors (e.g., RAS GTPase domain mutations; receptor kinase activation via kinase domain mutations).
Gene amplification: increases the amount of normal protein; elevates signaling output (e.g., MYC, HER2).
Chromosomal translocations: two main outcomes – overexpression via strong promoter/enhancer juxtaposition or production of an oncogenic fusion protein with constitutive activity (e.g., MYC overexpression in Burkitt's lymphoma; BCR-ABL fusion in CML).
Local DNA rearrangements (deletions, insertions, inversions): create novel fusion proteins or dysregulated gene expression (e.g., TRK-TPM fusion via inversion).
Insertional mutagenesis by retroviruses: integration near proto-oncogenes leads to overexpression driven by viral LTRs (e.g., activation of MYC by retroviral insertion).
Therapeutic and research implications for exams and practice
Understanding oncogene mechanisms informs targeted therapy development (e.g., inhibiting BCR-ABL in CML).
When evaluating a cancer, consider which oncogene class is implicated and the likely mechanism of activation (mutation, amplification, translocation, insertional mutagenesis).
For any given oncogene, ask: is the driver overexpression, a constitutively active protein, or a fusion protein with novel function?
Therapeutic strategy depends on mechanism: tyrosine kinase inhibitors for constitutively active kinases; antibodies or inhibitors targeting overexpressed receptors; strategies to degrade or inhibit fusion proteins; modulation of downstream effectors in signaling cascades.
Quick glossary of key terms
Proto-oncogene: normal gene that can become oncogenic via mutation or misregulation.
Oncogene: mutated or misregulated gene driving cancer progression.
Transfection: introduction of exogenous DNA into a cell.
Transformation: conversion of a normal cell to a cancerous, tumor-forming cell.
Southern blot: DNA-based blotting technique to detect specific DNA sequences.
Northern blot: RNA-based blotting technique.
Western blot: protein detection via antibodies after gel electrophoresis.
Double minutes (DM): extrachromosomal DNA fragments containing amplified genes.
Homogeneously staining regions (HSR): chromosomal regions with amplified gene clusters.
LTR: long terminal repeat; strong promoter/enhancer in retroviral DNA.
BCR-ABL: fusion kinase produced by t(9;22) chromosomal translocation in CML; constitutively active.
TRK-TPM fusion: inversion creating a fusion protein with constitutive TRK signaling.
Dimerization: process by which receptor signaling is activated; constitutive dimerization can bypass ligand dependence.
Final takeaway for the exam
Oncogenes arise from normal cellular genes through multiple mechanisms (point mutations, amplification, translocations, rearrangements, and insertional mutagenesis).
There are six major functional classes of oncogenes, spanning growth signals, receptors, signaling mediators, transcription factors, cell-cycle regulators, and cell-death regulators.
The RAS family remains a central paradigm: a small GTPase; activating mutations lock signaling in an ON state, driving proliferation.
Historical strategies (Southern/Northern/Western blotting and transfection studies) revealed that cellular genes could be oncogenic, laying the groundwork for targeted cancer therapies.
The clinical implications are profound: identifying the oncogene and its mechanism guides prognosis and directs therapeutic strategies (e.g., kinase inhibitors for fusion kinases, receptor-targeted therapies, and strategies that disrupt downstream signaling).