Tumor Suppressor Genes: Retinoblastoma

Tumor Suppressor Genes and Proto-Oncogenes

• Cancer-Critical Genes are broadly categorized into two types:

  • Proto-oncogenes:

    • Mutation typically leads to overactive or overexpressed versions of the protein.

    • Exhibit dominant inheritance, meaning a mutation in a single copy of the gene is sufficient to contribute to cancer development.

    • Can be conceptualized as a "gas pedal stuck" in an 'ON' position, promoting cell proliferation.

    • Examples include genes like phosphatases which, if mutated to be overactive, might inappropriately remove inhibitory phosphates, thereby activating downstream pathways.

  • Tumor Suppressor Genes:

    • Mutations result in a loss-of-function.

    • Exhibit recessive inheritance, meaning that both copies of the gene must be mutated or inactivated for their tumor-suppressing function to be lost.

    • Can be conceptualized as a "brake pedal broken," leading to uncontrolled cell growth.

Retinoblastoma: A Model for Tumor Suppressor Gene Inactivation

• Retinoblastoma is a childhood eye tumor.

• It results from mutations in the $Rb$ gene, which is a classic tumor suppressor gene.

• Forms of Retinoblastoma:

  • Familial form:

    • Characterized by multiple tumors, typically affecting both eyes (bilateral).

    • Patients with the familial form have a $3$ to $6$% chance of developing other cancers later in life, indicating a germline predisposition.

  • Sporadic form:

    • Typically presents as a single tumor in one eye (unilateral).

• Dynamics of Retinoblastoma Formation (Knudson's Two-Hit Hypothesis):

  • The probability of inactivating one copy of a gene in a somatic cell is approximately $10^{-6}$. Both copies must be inactivated for the tumor suppressor effect to be lost.

  • In the sporadic form, both copies must acquire independent somatic mutations. The probability of both copies becoming inactivated in the same cell is roughly $(10^{-6}) imes (10^{-6}) = 10^{-12}$. This low probability explains why sporadic cases are rare and usually unilateral.

  • In the familial form, an individual inherits one already mutated (inactive) $Rb$ copy. Therefore, only one additional somatic inactivating event (a "second hit") is required in any retinal cell to initiate tumor formation. This higher probability results in multiple, bilateral tumors and an earlier age of onset.

Mechanisms of Tumor Suppressor Gene Inactivation (Loss of Heterozygosity - LOH)

• For a tumor suppressor gene like $Rb$ to cause cancerous growth, both copies of the gene must be inactivated. When one copy is wild-type ($WT$) and the other is mutated, the cell is heterozygous. Inactivation of the $WT$ copy is often achieved through Loss of Heterozygosity (LOH).

• Mitotic Recombination:

  • This is a mechanism by which a wild-type $Rb$ gene copy can be eliminated from a cell.

  • It refers to the exchange of non-sister chromatids between homologous chromosomes during mitosis.

  • Mitotic recombination is thought to occur at a frequency of approximately $10^{-5}$ to $10^{-4}$ per cell generation.

  • If recombination occurs between the centromere and the gene locus, it can lead to a cell inheriting two copies of the chromosome carrying the mutated allele and no copies of the chromosome carrying the wild-type allele, resulting in LOH.

• Promoter Methylation:

  • Inactivates tumor suppressor genes without altering the DNA sequence, making it an epigenetic modification.

  • DNA methylation typically involves the addition of a methyl group ($CH_3$) to a cytosine base when it is followed by a guanine (a CpG site).

  • Methylation in the promoter region of a gene leads to chromatin condensation and compaction, making the promoter region inaccessible to transcription factors and RNA polymerase. This results in the silencing of gene transcription (no transcription).

  • This methylation pattern can be inherited through cell divisions.

  • Significance:

    • Promoter methylation is considered as important as somatic mutations in the inactivation of tumor suppressor genes.

    • Some tumor suppressor genes are rarely inactivated by mutations but are frequently silenced by promoter methylation.

    • Example: Runx3 is an oncogene implicated in stomach cancer development. Its promoter region is found to be methylated in 45% to 60% of stomach cancer cases.

    • Mechanisms of inactivation by promoter methylation:

      1. Independent methylation of both copies of the gene.

      2. Methylation of one copy combined with Loss of Heterozygosity (LOH) of the other copy.

    • Example: $p16INK4a$ is another tumor suppressor gene. Methylation of its promoter region has been observed in 44% of normal-looking bronchial tissue from former and current smokers. LOH of $p16INK4a$ was observed in an even higher percentage ($71$ to $73$%) of normal-looking bronchial tissue from the same group. This highlights how epigenetic changes and LOH can precede visible signs of cancer.

The Cell Cycle and $Rb$'s Role in S-Phase Regulation

• The cell cycle is comprised of four successive phases: $G<em>1$ (Gap 1), S (Synthesis), $G</em>2$ (Gap 2), and M (Mitosis).

• Regulation of S-phase entry is critical for preventing inappropriate cell proliferation.

• $Rb$ (Retinoblastoma protein) acts as a crucial regulator preventing inappropriate entry into S-phase.

• Mechanism of $Rb$ function:

  • $E2F$ transcription factors regulate the expression of various genes required for entry into S-phase (e.g., S-cyclins, DNA replication enzymes).

  • In its active (hypophosphorylated) state, $Rb$ binds to and inhibits $E2F$ transcription factors, thereby preventing the transcription of S-phase genes and arresting the cell in $G_1$.

  • $Rb$ achieves this repression by recruiting HDACs (Histone Deacetylases) to chromatin.

    • HDACs remove acetyl groups from histones, leading to chromatin compaction.

    • This compacted chromatin makes the promoter regions of $E2F$-target genes inaccessible, effectively turning transcription "OFF."

• Inactivation of $Rb$ by Mitogens (Growth Signals):

  • When mitogens (growth factors) stimulate cell proliferation, they activate Cyclin D-CDK4/6 complexes.

  • Sequential phosphorylations inhibit $Rb$:

    1. Cyclin D-CDK4/6 phosphorylates $Rb$ (early phosphorylation). This initial phosphorylation causes HDACs to dissociate (release) from $Rb$, beginning to relieve chromatin repression. This is described as a "first part of $Rb$" being modified.

    2. Further phosphorylation by CycE-CDK2 (active in late $G_1$) completely phosphorylates $Rb$. This complete phosphorylation causes $Rb$ to dissociate entirely from $E2F$ transcription factors.

  • Once $Rb$ is fully phosphorylated and thus inactivated, $E2F$ transcription factors are released from inhibition and become active.

  • Active $E2F$ leads to the transcription of S-phase genes (e.g., S-cyclins and genes for DNA replication), promoting cell cycle progression from $G_1$ to the S-phase.

• Overall Role: $Rb$ serves as a pivotal checkpoint protein at the $G_1/S$ transition, ensuring that cells only progress into S-phase when appropriate growth signals are present and DNA is ready for replication. Loss of functional $Rb$ leads to unchecked $E2F$ activity and premature entry into S-phase.

External Factors Causing $Rb$ Inactivation: HPV and Cervical Cancer

• Human Papillomavirus (HPV) is a DNA virus and a significant cause of cervical cancer.

• Mechanism of DNA viruses in cancer: DNA viruses that cause cancer often do so by inactivating critical tumor suppressor proteins in the host cell.

• Inactivation of $Rb$ by HPV:

  • A key HPV oncoprotein, E7, directly binds to the $Rb$ protein.

  • This binding results in the inactivation of $Rb$.

  • The outcome of E7 binding to $Rb$ is functionally the same as if the cell received an abundance of growth factors or if $Rb$ was mutated and non-functional: $E2F$ is released, leading to uncontrolled transcription of S-phase genes.

  • This forces the cell to prematurely enter and progress through the cell cycle, contributing to the cancerous transformation. Thus, HPV E7 effectively bypasses the $Rb$-mediated $G_1$ checkpoint.