Human Molecular Genetics - Cancer Genes Function and Activation Mechanisms

Overview of Cancer Genomics

• Lecture Objectives:

  • Determine the normal physiological functions of cancer-associated genes.

  • Identify the mechanisms by which cancer genes are either activated (oncogenes) or repressed (tumor suppressor genes).

Functional Categories of Tumor Suppressor Genes (TSGs)

Tumor suppressor genes are broadly classified based on their protective roles within the cell. The two primary categories are Gatekeepers and Caretakers.

• Gatekeepers: Directly regulate cell growth by inhibiting the cell cycle or promoting apoptosis.

  • $Rb1$: Inhibition of cell cycle.

  • $p53$: Regulator of the cell cycle and apoptosis; also serves as a caretaker.

  • $CDKN1B$: Inhibition of the cell cycle.

  • $APC$: Involved in the transcription inhibition of $\beta-\text{catenin}$.

  • $STK11 \, (LKB1)$: Involved in Serine-threonine kinase signaling.

  • $CDH1$: Regulates cell adhesion.

• Caretakers: Protect the integrity of the genome by repairing DNA damage.

  • $BRCA1$: DNA repair.

  • $ATM$: DNA repair.

  • $MLH1$: Mismatch repair.

  • $MSH2$: Mismatch repair.

  • $p53$: DNA repair (Dual role).

Normal Physiological Functions of Cancer Genes

• Cell Cycle Regulators: Control the transitions between phases (e.g., G1/S checkpoint). Key genes include $p53$, $p21$, $p16$,(3 tumour suppressor genes) and $Myc$(oncogene) .

• DNA Repair: Includes damage response genes ($ATM$, $BRCA1$) and direct repair factors ($Rad51$, $Nbs1$).

• Apoptosis Regulation:

  • Pro-apoptotic Factors: Support cell death (e.g., $Bax$, $Bad$, $Bim$, $Apaf-1$).

  • Anti-apoptotic Factors: Prevent cell death (e.g., $Bcl-2$, $Bcl-X$).

• Senescence: Regulating entry and exit from the cell cycle. This is often mediated indirectly through telomerase and telomere maintenance regulators.

• Angiogenesis: Stimulating endothelial cell migration into the tumor via factors like $VEGF$ (Vascular Endothelial Growth Factor), $FGF$, and $HGF$.

• Invasion and Migration:

  • Changes in cell adhesion properties (using cadherins and integrins).

  • Degradation of the Extracellular Matrix (ECM) using Matrix metalloproteases.

  • Alterations in the cellular cytoskeleton.

Case Study: The Ras Gene and Proliferation

• Function: The Ras gene encodes a G protein that relays signals from growth factor receptors on the plasma membrane to a protein kinase cascade.

• Mechanism:

  1. Growth factor binds to a receptor.

  2. Activation of G protein (Ras).

  3. Phosphorylation cascade (Protein kinases).

  4. Activation of transcription factors in the nucleus.

  5. Gene expression resulting in a protein that stimulates the cell cycle.

• Cancer Implication: Mutations that make Ras abnormally active lead to excessive, unregulated cell division.

Progressive Genetic Loss in Melanoma

Melanoma progression illustrates the multi-step nature of cancer mutations:

1. Naevus: Characterized by $100\%$ coverage of $BRAF$ or $NRAS$ mutations.

2. Primary Melanoma: Requires the additional loss of one copy of a Tumor Suppressor Gene and TERT promoter mitation

3. Metastatic Melanoma: Requires the loss of both copies of TSGs and a $TERT$ promoter mutation.

Telomeres and Replicative Immortality

• The Mitotic Clock: Normal cells have a finite number of divisions. Short telomeres elicit a DNA damage signal, leading to senescence or apoptosis.

• Telomerase: A specialized reverse transcriptase that extends the $3'$ end of chromosomes by adding $TTAGGG$ repeats created from telomere capping proteins.

• Cancer Mechanism: Telomerase is frequently activated in cancer cells to bypass the mitotic clock and achieve immortality.

• Genomic Instability: Critically short telomeres that are not repaired can be recognized as double-strand DNA (dsDNA) breaks, leading to massive instability resulting in apoptosis or cancer.

Genes involved in Senescence and Apoptosis

• p53 in feedback loop controlled by MDM2 (oncogene) that regulates p53.

• DNA replication stress etc. would cause MDM2 to allow p53 to be expressed at much higher level.

• Mild stress: Growth arrest and DNA repair

• Severe stress: Apoptosis

Adhesion and Angiogenesis in Cancer

• Cell Adhesion: Loss of $E-cadherin$ (normally found in cell adherin junctions) correlates with increased dysplasia and higher tumor grade. For example, pancreatic ductal adenocarcinoma often shows a complete loss of $E-cadherin$.

• Angiogenesis: Tumors secrete growth factors to remodel local tissue and stimulate vascularization. Examples of secreted factors include $SDF1$, $IL8$, $PGE2$, and $VEGF$.

Mechanisms of Oncogene Activation

Oncogenes (activated proto-oncogenes) can be triggered through several molecular pathways:

• Deletion or Point Mutation in Coding Sequence: Results in a hyperactive protein produced in normal amounts.

• Gene Amplification: Results in the overproduction of a normal protein (e.g., $20\%$ of breast cancers have $Her2$ amplification; $30\%$ of tumors have $c-myc$ amplification).

• Chromosome Rearrangement:

  • Regulatory Rearrangement: A nearby regulatory DNA sequence causes the normal protein to be overproduced.

  • Fusion Protein: Fusion to an actively transcribed gene results in the overproduction of a hyperactive fusion protein.

Specific Oncogenic Mutations: Ras and Translocations

• Ras Point Mutations: Prevent GTP hydrolysis, locking the protein in a permanent "on" state.

  • The most common mutation is $G12V$.

  • The substitution of Glycine for Valine at residue $12$ makes Ras insensitive to inactivation by GAP.

  • Found in $20\%$ to $30\%$ of all human tumors.

• Chromosomal Translocations:

  • Burkitt's Lymphoma: $t(8;14)$. The $c-myc$ gene on chromosome $8$ is moved near the rearranged immunoglobulin gene on chromosome $14$, leading to constant $c-myc$ expression.

  • Chronic Myelogenous Leukaemia (CML): $t(9;22)$. The Philadelphia chromosome creates a $bcr-abl$ fusion protein with constitutive kinase activity.

The Breakage-Fusion-Bridge Cycle

This cycle generates chromosomal aberrations via dsDNA breaks:

1. A chromosome fragment is detached after a strand break.

2. The cell replicates DNA in S phase despite the break.

3. Sister chromatids lacking telomeres fuse at their ends.

4. During mitosis, the fused chromatids are pulled apart, creating a new break at a different site.

5. One daughter cell inherits a chromosome with duplicated genes but still lacks a telomere, repeating the cycle.

Mechanisms of Tumor Suppressor Gene Inactivation

TSGs can be silenced through various "hits" consistent with Knudson's Two-Hit Hypothesis:

• Loss of Heterozygosity (LOH): Occurs via chromosome loss, deletion, unbalanced translocation, mitotic recombination, or point mutation.

• Homozygous Deletion (HD): Complete loss of both alleles of a gene (e.g., deletions on Chromosome $9$ or $10$).

• Epigenetic Silencing: Promoter hypermethylation (adding $CH_3$ groups to CpG islands in the promoter) inactivates transcription regardless of the DNA sequence.

• MicroRNA Regulation: Post-transcriptional silencing of TSG transcripts.

• Small Scale Mutations: Point mutations creating nonsynonymous or nonsense (premature stop) codons.

  • Example: BRCA1 mutations include $185delAG$ and $5382insC$.

  • Example: BRCA2 mutations include $6174delT$.

Viruses and Cancer

Viruses are involved in a small fraction of cancers by transforming cells through the introduction of foreign DNA that interferes with growth regulation.

• HPV Mechanism in Cervical Cancer:

  • HPV introduces genes $E6$ and $E7$.

  • $E6$ protein binds to and sequesters $p53$, removing the "safety brake" on the cell cycle.

  • $E7$ protein binds to and sequesters the $Rb$ protein, releasing the active cell proliferation factor (transcription factor).

  • Total Result: Uncontrolled cell proliferation and possible transition to invasive cancer upon viral DNA integration into the host genome.

Summary of Cancer Mechanisms

• Cancer is the result of an accumulation of mutations in genes governing proliferation, adhesion, and DNA repair.

• Oncogene Activation: Driven by activating mutations, gene amplification, and chromosome translocations.

• TSG Inactivation: Driven by point mutations, deletions (small or whole chromosome), unbalanced translocations, and epigenetic promoter methylation.