Exhaustive Notes on Apoptosis and Cancer Biology

Distinctions Between Necrosis and Apoptosis

• Cells undergo two primary modes of death: necrosis and apoptosis.

• Necrosis occurs due to physical injury, such as cell burning, UV light damage, or chemical burns from acids.

  • Characterized by cells bursting open and spilling their contents.

  • Results in an inflammatory response that can damage surrounding healthy tissue.

• Apoptosis is a programmed cell death induced by internal or external signals.

  • A controlled, genetically determined process.

  • The cell is eventually phagocytosed by specialized cells such as macrophages.

  • Organic components of the cell are recycled and reused.

  • Does not result in an inflammatory response.

Morphological Hallmarks of Apoptosis

• Apoptotic cells undergo distinct morphological changes, which include:

  • Membrane blebbing: The plasma membrane bulges outward, forming bulges called blebs that separate from the cell, carrying pieces of cytoplasm with them.

  • Nuclear and cytoplasmic shrinkage: The entire cell and its organelles decrease in size.

  • Chromosomal condensation: Chromatin becomes highly condensed.

  • DNA fragmentation: DNA is cleaved into specific fragments of $180$ base pairs or multiples of $180$.

  • Translocation of Phosphatidylserine ($PS$): This phospholipid typically has an asymmetric distribution and is located on the cytoplasmic side of the membrane. During apoptosis, it flips to the exoplasmic (extracellular) orientation.

  • Increased caspase activity: Activation of digestive enzymes essential for inducing cell death.

• Morphological sequence observed via electron microscopy:

  • Initially, the cell undergoes mild convolution and chromatin compaction.

  • The nuclear envelope (the double membrane separating the nucleus from the cytoplasm) breaks down.

  • The nucleus fragments as chromatin and DNA are destroyed.

  • Blebbing leads to cell fragmentation into apoptotic blebs.

Physiological Roles of Programmed Cell Death

• Apoptosis is essential in both the developing embryo and adult multicellular organisms.

• Functions include:

  • Sculpting body parts: For example, the webbing between digits (fingers/toes) in a developing mouse paw undergoes apoptosis to form individual digits.

  • Removing unneeded structures or cells.

  • Regulating cell numbers to prevent overgrowth.

• Most cells are poised to die unless they receive survival factors known as trophic factors.

  • Growth factors are a primary example of trophic factors.

  • In the absence of trophic factors, cells undergo programmed cell death.

  • Other inducing signals include DNA damage, cellular stress, or signals from the immune system.

• Interaction-dependent survival:

  • In vertebrates, the number of neurons innervating peripheral cells depends on the size of the target tissue.

  • Removing a limb bud during development leads to the apoptosis of the motor neurons that would have innervated that limb.

  • Adding an extra limb bud increases the survival rate of motor neurons (e.g., survival increases from $50\%$ to $75\%$ when the number of target cells is doubled).

Mechanisms of Apoptotic Activation

• Extrinsic Signals:

  • Transmembrane death receptors can be triggered to induce death.

  • Viral infections.

  • Ionizing radiation damaging the cell.

  • Cytotoxic T cells or Natural Killer ($NK$) cells.

• Immune System Induction:

  • T cells can activate death receptors on the target cell surface.

  • T cells can introduce granzymes (serine proteases) into the cell through cytoplasmic granules, triggering death.

Evolutionary Conservation: C. elegans and Mammals

• Apoptosis was heavily studied using the nematode C. elegans, which has a well-mapped cell lineage.

• The $CED$ genes (cell death abnormal) were discovered via mutational analysis; mutations in these genes block programmed cell death.

• The C. elegans pathway:

  • $EGL-1$ blocks $CED-9$.

  • This disruption releases $CED-4$ from the $CED-4/CED-9$ complex.

  • Free $CED-4$ activates $CED-3$.

  • $CED-3$ is a protease that undergoes autoproteolysis to target cellular components for destruction.

• The Mammalian Pathway (Analogs):

  • $BH3$-only proteins ($BID$, $BIM$) are analogs to $EGL-1$.

  • $BCL-2$ is the analog to $CED-9$.

  • $APAF-1$ is the analog to $CED-4$.

  • Caspase $9$ is the analog to $CED-3$.

Caspases: The Executioners

• Caspases are a family of proteases characterized by a conserved cysteine residue in their active site.

• They cleave their substrate proteins after a specific aspartic acid ($Asp$) residue.

• Common substrates include:

  • Inhibitor of DNase: Cleavage activates DNase, which fragments DNA.

  • Nuclear lamins: Cleavage disrupts the nuclear envelope.

  • Various enzymes contributing to morphological hallmarks.

The BCL-2 Family of Proteins

• Proteins in the $BCL-2$ family are classified by conserved domains called $BH$ (BCL homology) domains, which mediate dimerization and oligomerization.

• Pro-survival (Anti-apoptotic) members:

  • Includes $BCL-2$, $BCL-XL$, $BCL-W$, $MCL-1$, and $A1$.

  • These contain a $BH4$ domain (not found in pro-apoptotic members) and a transmembrane ($TM$) domain.

• Pro-apoptotic members:

  • $BAX$ and $BAK$: Contain $TM$ domains and form channels in the outer mitochondrial membrane to release Cytochrome C.

  • $BH3$-only proteins ($BID$, $BAD$, $BIM$, $PUMA$): Lack a $TM$ domain but have a hydrophobic C-terminal domain. They act by inhibiting pro-survival members.

Intrinsic vs. Extrinsic Apoptotic Pathways

• Intrinsic Pathway (Mitochondrial):

  • Initiated by the release of Cytochrome C from the mitochondrial intermembrane space into the cytosol through $BAX/BAK$ channels.

  • Cytochrome C binds to $APAF-1$, which then binds procaspase $9$.

  • Procaspase $9$ is cleaved to form active Caspase $9$.

  • Caspase $9$ activates effector caspases (Caspase $3$ and $7$), leading to death.

• Regulation of Intrinsic Pathway by Trophic Factors:

  • Growth factors trigger $RTK$\s, activating $PI3$-kinase and then Protein Kinase B ($PKB$).

  • $PKB$ phosphorylates $BAD$, which then associates with the $14-3-3$ protein, keeping it inactive.

  • Unphosphorylated $BAD$ blocks $BCL-2/BCL-XL$, allowing Cytochrome C release.

  • Other pathways: Microtubule disruption activates $BIM$, and nuclear stress can express $PUMA$; both block pro-survival proteins.

• Extrinsic Pathway (Death Receptor):

  • Extrinsic signals like $TNF-\alpha$ (Tumor Necrosis Factor alpha) bind to death receptors.

  • The cytoplasmic tail of the receptor recruits $TRADD$ and $FADD$ proteins.

  • This recruits and activates Caspase $8$.

  • Active Caspase $8$ cleaves procaspase $3$, $6$, and $7$, bypassing the mitochondria to induce apoptosis.

Cancer Basics and Tissue Origin

• Cancer is defined as the loss of control over cellular growth and proliferation.

• Lifetime risk: $1$ in $2$ for males and $1$ in $3$ for females.

• Classifications based on origin:

  • Carcinomas: Derived from endoderm (gut epithelium) or ectoderm (skin/neural epithelium).

  • Sarcomas: Derived from mesoderm (muscle, blood, connective tissue).

  • Leukemias: A class of sarcomas that grow as individual cells in the blood.

• Neoplasms (Abnormal tissue masses):

  • Benign: Cells remain clustered in a single mass; typically curable by surgical removal.

  • Malignant: Cells acquire the ability to invade surrounding tissue and colonize distant sites.

Hallmarks of Cancer Cells

• Cancer cells acquire specific properties to facilitate tumor development and metastasis:

  1. Resisting cell death (evading apoptotic signals).

  2. Sustaining proliferative signaling.

  3. Evading growth suppressors.

  4. Enabling replicative immortality (unlimited replication without natural breaks).

  5. Activating invasion and metastasis.

  6. Inducing angiogenesis (the formation of new blood vessels to supply oxygen and nutrients).

Metastasis and Invasiveness

• Metastasis is a multi-step process:

  • Cells degrade the basement membrane using Matrix Metalloproteinases ($MMP$s).

  • Cells travel along extracellular matrix ($ECM$) fibers.

  • Epidermal Growth Factor ($EGF$) secreted by macrophages attracts cancer cells toward blood vessels.

  • Cells enter the bloodstream or lymphatic system to form secondary tumors (metastases).

• Invadopodia:

  • Specialized structures related to filopodia and lamellipodia used for invasion.

  • Utilize the actin cytoskeleton (involving proteins like Cofilin and $WASP$) and proteases to degrade the $ECM$.

The Multi-Hit Model of Cancer

• Cancer is a somatic mutation disease requiring multiple genetic changes over time.

• Aneuploidy: Cancer cells often have abnormal chromosome numbers.

• Colorectal Cancer Case Study:

  1. Loss of $APC$ (Tumor Suppressor): Leads to a polyp. $APC$ loss results in high $\beta-catenin$ levels, which activates Cyclin $D1$ via $TCF$.

  2. Activation of $KRAS$ (Oncogene): Results in a benign precancerous tumor (Adenoma).

  3. Loss of $p53$ (Tumor Suppressor): Leads to a malignant carcinoma and metastasis.

• Synergistic effects: In mice, expressing both $MYC$ and $RAS^{V12}$ oncogenes results in total tumor development within $150$ days, a much faster rate than expressing either alone.

• Cancer incidence increases with age as mutations accumulate over a lifetime.

Three Classes of Cancer-Associated Genes

• Proto-oncogenes:

  • Normal function: Promote cell survival and proliferation.

  • Mutation: Gain-of-function (better activity or higher expression).

  • Genetic property: Dominant (only one allele needs mutation).

  • Mechanisms: Point mutations, chromosomal translocations (forming fusion proteins or promoter swaps), or gene amplification.

• Tumor Suppressor Genes:

  • Normal function: Inhibit cell survival or proliferation; induce apoptosis; check DNA damage.

  • Mutation: Loss-of-function.

  • Genetic property: Recessive (both alleles must be mutated).

  • Mechanisms: Deletion, point mutation, or epigenetic silencing via DNA methylation.

• Genome Maintenance Genes:

  • Normal function: Repair DNA or prevent damage.

  • Mutation: Loss-of-function.

  • Genetic property: Recessive.

  • Mechanism: Allows further mutations to accumulate in oncogenes and tumor suppressors.

Examples of Oncogenic Activation

• $RAS$ Oncogene:

  • $RAS$ transitions between $GTP$-on and $GDP$-off states.

  • The $RAS^{V12}$ mutation replaces Glycine with Valine at position $12$, making the protein constitutively active regardless of growth factor signaling.

  • $KRAS$ is the most frequently mutated isoform in cancer.

• Receptor Mutations:

  • $HER2$ (RTK): A point mutation ($Val \to Gln$) in the transmembrane domain creates the $Neu$ oncoprotein, which is active without a ligand.

  • $EGF$ Receptor: Deletion of the extracellular ligand-binding domain creates the $ErbB$ oncoprotein, which is constitutively active.

Tumor Suppressors: Retinoblastoma and p53

• Retinoblastoma ($RB$) Protein:

  • Acts as a brake on the cell cycle by blocking the $E2F$ transcription factor.

  • Hereditary Retinoblastoma: One defective allele is inherited; a second somatic mutation leads to tumors.

  • Sporadic Retinoblastoma: Both wild-type alleles must acquire somatic mutations.

• $p53$ Protein:

  • A master regulator that senses DNA damage.

  • It can halt the cell cycle or promote apoptosis.

  • Inhibited by $CKI$ proteins like $p21$ which stop Cyclin-$CDK$ progression.

DNA Repair Defects and Hereditary Cancer

• Mismatch Repair Defect: Associated with colon and ovary cancers; fails to fix point mutations and small insertions/deletions.

• Nucleotide Excision Repair ($NER$) Defect: Leads to Xeroderma Pigmentosum; extreme sensitivity to UV radiation resulting in skin carcinomas/melanomas.

• Double-Strand Break Repair (Homologous Recombination) Defects:

  • Faulty repair proteins lead to leukemias and lymphomas.

  • Mutations in $BRCA1$ and $BRCA2$ are highly linked to breast and ovarian cancers.

Environmental Factors: Tobacco

• Tobacco smoke contains chemicals that cause somatic mutations, including those that disrupt $p53$.

• Statistical analysis shows a roughly $30$-year lag time between smoking trends and lung cancer death rates.

• Male smoking rates peaked and began declining around $1960$, followed by a decline in death rates after $1990$.

• Female smoking rates increased in $1960$, leading to a subsequent rise in lung cancer deaths through the 1990s.