GROWTH FACTORS AND RECEPTORS.docx.pdf

GROWTH FACTORS, RECEPTORS, AND CANCER Growth factor receptors are proteins located on the surface of cells that bind to specific growth factors, which are signalling molecules that regulate growth, differentiation, and survival. TYPES OF GROWTH FACTOR RECEPTORS 1. Tyrosine Kinase receptors...

GROWTH FACTORS, RECEPTORS, AND CANCER Growth factor receptors are proteins located on the surface of cells that bind to specific growth factors, which are signalling molecules that regulate growth, differentiation, and survival. TYPES OF GROWTH FACTOR RECEPTORS 1. Tyrosine Kinase receptors - Most common - Include EGFR (epidermal growth factor receptor), PDGF (platelet-derived growth factor receptor), also known as mitogens, Vascular endothelial growth factor receptor (VEGFR) 2. G-protein-coupled receptors (GPCR) - Receptors bind to various ligands, including growth factor - Also involved in cancer progression 3. Serine/Threonine receptors - These receptors phosphorylate serine or threonine residues and play roles in cellular signalling MECHANISM OF GROWTH FACTOR RECEPTOR 1. Binding of Growth Factor: A specific ligand (growth factor) binds to the receptor's extracellular domain. 2. Dimerization: Ligand binding often causes dimerization (pairing of two receptors), activating the receptor’s intracellular domain. 3. Autophosphorylation: The intracellular domain, which usually has kinase activity, auto-phosphorylates specific tyrosine residues. This activates a series of downstream signaling pathways. 4. Signal Transduction: Activated receptors trigger downstream signaling pathways such as the MAPK/ERK, PI3K/AKT, and JAK/STAT pathways. These pathways control cellular processes like proliferation, differentiation, survival, and migration. 5. Termination: The signaling is terminated through internalization of the receptor or deactivation by regulatory proteins. Mutations in this process can lead to uncontrolled signaling, contributing to cancer. KEY GROWTH FACTOR RECEPTOR IN CANCER 1. Epidermal Growth factor receptor (EGFR) - Role in Normal Cells: EGFR regulates cell growth, survival, and differentiation. - Role in Cancer: Mutations and overexpression of EGFR are implicated in non-small cell lung cancer, colorectal cancer, and glioblastomas. These mutations often lead to constitutive activation of the receptor, driving uncontrolled cell proliferation. - Targeted Therapies: EGFR inhibitors such as gefitinib and erlotinib treat cancers exhibiting EGFR mutations. 2. Platelet-derived Growth Factor Receptor (PDGFR) - Role in Normal Cells: PDGFR regulates cell growth, development, and angiogenesis. - Role in Cancer: Overactivation of PDGFR is observed in glioblastomas, sarcomas, and other solid tumors, contributing to uncontrolled growth and angiogenesis. - Targeted Therapies: Imatinib, a tyrosine kinase inhibitor, targets PDGFR in chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). 3. Human Epidermal Growth Factor Receptor (HER2)  - Role in Normal Cells: HER2 is involved in the growth and repair of cells. - Role in Cancer: Overexpression of HER2 is a significant driver of breast cancer and gastric cancer. HER2-positive breast cancers tend to be more aggressive. - Targeted Therapies: Trastuzumab (Herceptin) is an antibody therapy targeting HER2-positive breast cancer. SRC PROTEIN SRC protein is a non-receptor tyrosine kinase that plays key roles in cell growth, differentiation, adhesion, and migration—the first oncogene to be discovered. SRC is a cytoplasmic protein. SRC activation is tightly regulated under normal conditions, and its deregulation leads to aberrant signaling, a hallmark of cancer. Antibodies against SRC were found to be phosphorylated when incubated with SRC protein and ATP. SRC Activation in Response to Growth Factors - Acts downstream to EGFR and PDGFR - SRC is overexpressed in cancers mainly, breast, colon and lung - role in cancer progression is primarily due to its interaction with overactive or mutated growth factor receptors, leading to dysregulated signaling. SRC and Growth Factor Receptor Crosstalk in Cancer In cancer cells, SRC interacts with multiple growth factor receptors that drive tumorigenesis: EGFR and HER2: In cancers such as lung and breast cancer, overexpression or mutations in EGFR and HER2 activate SRC, contributing to aggressive cancer behavior. For example, in HER2-positive breast cancer, SRC is co-activated with HER2, enhancing cellular proliferation and survival. PDGFR: SRC is also involved in the signaling downstream of PDGFR, contributing to glioblastoma and other cancers by driving cell migration and angiogenesis. GROWTH FACTORS AS ONCOGENES An oncogene is a mutated version of a normal gene called a proto-oncogene that leads to uncontrolled cell division and growth, resulting in cancer. Proto-oncogenes typically play roles in cell growth, differentiation, and survival, but when they acquire mutations or become overactivated, they can drive oncogenesis (tumor formation). How do growth factors become oncogene? - The EGFR (Epidermal Growth Factor Receptor) pathway, activated by binding to its growth factor ligands (like EGF or TGF-α), can become oncogenic when EGFR is mutated. EGFR mutations are found in various cancers, including lung cancer. These mutations cause the receptor to be permanently activated, even without ligand binding. - The BCR-ABL fusion gene in chronic myeloid leukemia (CML) results from a translocation between chromosomes 9 and 22, known as the Philadelphia chromosome. This fusion creates a constitutively active tyrosine kinase that promotes uncontrolled proliferation. While BCR-ABL is not directly a growth factor, it can activate growth factor pathways, promoting cancerous growth. - Autocrine Stimulation: Some cancer cells produce excessive growth factors, which then act on the same cells that produced them. This autocrine signaling leads to continuous activation of growth factor receptors, bypassing the need for external growth stimuli and promoting uncontrolled cell proliferation. Example: Glioblastomas often secrete platelet-derived growth factor (PDGF), which binds to the PDGF receptor (PDGFR) on the same cell, driving cell proliferation and tumor growth. Tumors can produce both growth factors and their receptors In normal cellular biology, growth factors are typically produced by one set of cells and act on nearby or distant cells by binding to their respective receptors. This ensures tightly regulated control over cell growth, differentiation, and survival. However, in cancer, this regulatory mechanism is often disrupted, and tumor cells can produce both the growth factors and their corresponding receptors, creating a self-sustaining loop that promotes uncontrolled cell proliferation and survival. This phenomenon is known as autocrine signaling and plays a crucial role in the development and progression of many types of cancer.  Autocrine Signaling occurs when a tumor cell produces a growth factor that binds to its receptor, leading to a continuous and unregulated loop of activation that drives tumor growth and survival without needing external growth signals.  Paracrine Signaling in tumors can also occur, where tumor cells produce growth factors that act on surrounding cells (such as stromal or endothelial cells) to support tumor progression by inducing angiogenesis or immune suppression. TGF-β (Transforming Growth Factor-Beta) - Transforming Growth Factor-Beta (TGF-β) is a multifunctional growth factor that plays dual roles in cancer progression. - In early cancer stages, TGF-β acts as a tumor suppressor by inhibiting cell proliferation. However, tumor cells can overproduce TGF-β and its receptor in later stages, forming an autocrine loop that promotes tumor cell migration, invasion, and metastasis. - TGF-β signaling also induces epithelial-to-mesenchymal transition (EMT), increasing cancer cell motility and invasiveness.  - Therapeutic Targeting: Strategies to inhibit TGF-β are under investigation, as blocking its signaling in advanced cancers could prevent metastasis and invasion. Kaposi’s Sarcoma (KS) is a type of cancer that forms in the lining of blood vessels and lymphatic vessels. It is caused by Human Herpesvirus-8 (HHV-8) infection, also known as Kaposi's Sarcoma-associated Herpesvirus (KSHV). It produces PDGF, TGF-alpha, IGF-1, and their receptors. MECHANISM OF RECEPTOR ACTIVATION Receptor activation is a process where external signals, often ligands (such as hormones, growth factors, or cytokines), bind to specific receptors on a cell’s surface or inside the cell, triggering intracellular signaling pathways. 1. GENE FUSION Gene fusion refers to the combination of two previously separate genes, producing a hybrid protein. This can lead to abnormal receptor activation. Mechanism of Gene Fusion: o Gene fusion often occurs due to chromosomal rearrangements like translocations or deletions. o A fused gene can lead to a chimeric receptor with new or altered functions, often associated with diseases like cancer. o For example, the BCR-ABL fusion gene in chronic myeloid leukemia (CML) is a product of fusion between the BCR and ABL genes, producing an abnormal tyrosine kinase that continuously signals for cell growth, bypassing the standard regulatory mechanisms. o ROS receptors in glioblastoma. The ectodomain of the ROS gene fuses with the reading frame of the Fig gene that dimerizes. This results in two ROS receptors being dimerized for constitutive activation. Effect on Receptor Activation: o The hybrid receptor may be constitutively active, meaning it does not require ligand binding to become activated. o This unregulated activation can lead to uncontrolled cellular processes like proliferation, differentiation, or survival, often resulting in pathological conditions. 2. JUXTACRINE SIGNALING Juxtracrine signaling is a form of cell communication where signaling occurs between cells in direct contact. This contrasts with other forms of signaling like endocrine (long-distance) or paracrine (nearby cells). Mechanism of Juxtacrine Signaling: o Contact-dependent interaction: The ligand is bound to the membrane of one cell, and the receptor is on the adjacent cell. o Notch-Delta Pathway: The Notch-Delta pathway is a classic example of juxtacrine signaling. The Notch receptor on one cell binds to the Delta ligand on an adjacent cell, triggering cleavage of the Notch intracellular domain, which then translocate to the nucleus and regulates gene expression.  o Juxtacrine signaling often plays a crucial role in tissue development, cell differentiation, and immune responses. RAS PROTEIN - GTP binding proteins, acting as molecular switches - Members of RAS family are HRAS, KRAS, NRAS - Involved in transmission of signals from cell surface receptors to intracellular pathways - Postulated to act downstream Structure of RAS Proteins: GTP/GDP Binding Domain: RAS proteins can bind guanosine triphosphate (GTP) or guanosine diphosphate (GDP). Their activity is determined by whether GTP or GDP is bound. o Active State: RAS is active and can transmit signals when bound to GTP. o Inactive State: When bound to GDP, RAS is in its inactive state. RAS Cycle: Activation: RAS is activated when a growth factor or other extracellular ligand binds to its associated receptor, such as a receptor tyrosine kinase (RTK). This activates a guanine nucleotide exchange factor (GEF), which facilitates the exchange of GDP for GTP on the RAS protein, converting it into its active state. Inactivation: GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP, turning RAS off and terminating the signal. PROTO-ONCOGENE TO ONCOGENE 1. Point Mutation: Proto-oncogenes can become oncogenes when a single point mutation alters their protein product, making it more active or resistant to degradation. Example: KRAS G12C mutation locks KRAS in its active form, continuously promoting signaling for cell growth. 2. Gene Amplification: Amplification increases the number of copies of a proto-oncogene, resulting in overproduction of the oncogenic protein. Example:  HER2 amplification in breast cancer leads to excessive HER2 receptor activation, promoting uncontrolled cell proliferation. 3. Chromosomal Translocation: A segment of one chromosome is transferred to another, often placing a proto-oncogene under the control of an active promoter or forming a fusion gene with constitutive activity. Example: The BCR-ABL fusion gene is created in chronic myeloid leukemia (CML), leading to continuous tyrosine kinase activity that drives cell proliferation. 4. Local DNA Rearrangements: Small alterations such as deletions, insertions, or inversions near proto-oncogenes can disrupt gene regulation or create constitutively active gene product forms. Example: RET gene rearrangement creates an oncogenic form of the receptor tyrosine kinase in multiple endocrine neoplasia. 5. Insertional Mutagenesis: Insertion of viral DNA near a proto-oncogene can upregulate its expression or disrupt normal regulatory mechanisms. Example: In retroviral-induced cancers, viral insertion near the MYC gene leads to its overexpression and uncontrolled cell growth. MECHANISM DESCRIPTION EXAMPLE Point Mutation A change in a single nucleotide KRAS G12C mutation in lung leads to a substitution of an cancer, HRAS mutation in amino acid, affecting protein bladder cancer function. Gene Amplification Multiple copies of a MYC amplification in proto-oncogene are created, neuroblastoma leading to the overproduction of the encoded protein. Chromosomal Translocation The rearrangement of BCR-ABL fusion in chronic chromosomes brings a myeloid leukemia (CML) proto-oncogene under the control of a strong promoter or MYC translocation in Burkitt creates a fusion gene. lymphoma Local DNA Rearrangements Changes in the DNA sequence NTRK gene fusion in certain near a proto-oncogene, such cancers as small insertions, deletions, or inversions. Insertional Mutagenesis Insertion of viral DNA near or Retroviral insertion near MYC into a proto-oncogene can gene in avian leukosis disrupt its regulation, virus-induced lymphomas activating it as an oncogene. Frameshift Mutation Frameshift insertion/deletion: EGFR exon 19 deletion in Alters the reading frame, often non-small cell lung cancer leading to non-functional or hyperactive proteins. CELL SIGNALLING PATHWAYS RECEPTOR TYROSINE KINASE (RTK) PATHWAY Receptor tyrosine kinases (RTKs) are cell surface receptors that regulate essential processes like growth, survival, and differentiation. RTKs are often implicated in cancer when they become overactive due to mutations or overexpression. Mechanism: 1. Ligand Binding: Growth factors (e.g., EGF, VEGF) bind to the extracellular domain of RTKs. 2. Dimerization: Ligand binding causes two RTKs to dimerize (pair up). 3. Autophosphorylation: The intracellular tyrosine kinase domain phosphorylates itself on tyrosine residues. 4. Activation of Downstream Pathways: Phosphorylated tyrosine residues recruit adaptor proteins that activate downstream signaling cascades, including the MAPK/ERK and PI3K/AKT pathways. Dysregulation in Cancer: Overexpression: RTKs like HER2 (in breast cancer) are often overexpressed, leading to constant growth signaling. Mutations: Mutations in RTKs like EGFR (in lung cancer) result in constitutive activation without ligand binding. Gene Fusions: ALK fusions in lung cancer can create constitutively active kinases that promote uncontrolled cell proliferation. Cancer Example: EGFR Mutation in non-small cell lung cancer (NSCLC): Mutations in the EGFR gene lead to uncontrolled receptor activation, promoting abnormal growth and division of cancer cells. MAPK/ERK Pathway (Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase) The MAPK/ERK pathway is a key pathway regulating cell growth, proliferation, and differentiation. Mutations in this pathway are common in many cancers. Mechanism: 1. Activation by RTKs: Upon activation of RTKs, the RAS protein is activated through GTP binding. 2. RAF Activation: RAS-GTP recruits and activates RAF kinase. 3. Phosphorylation Cascade: RAF phosphorylates MEK, which in turn phosphorylates ERK. 4. Gene Expression: Activated ERK translocates to the nucleus and activates transcription factors, leading to the expression of genes that drive cell proliferation. Dysregulation in Cancer: RAS Mutations: Mutations in KRAS (e.g., G12D mutation in pancreatic cancer) keep RAS in its active GTP-bound state, continuously driving the MAPK/ERK pathway. BRAF Mutations: Mutations in BRAF (e.g., V600E mutation in melanoma) lead to constitutive activation of the RAF kinase, promoting uncontrolled cell division. Cancer Example: BRAF V600E Mutation in melanoma: This mutation causes continuous activation of the MAPK/ERK pathway, increasing cell proliferation and survival. PI3K/AKT Pathway The PI3K/AKT/mTOR pathway is critical for cell survival, growth, and metabolism. Mutations or overactivation of this pathway are frequently observed in cancers. Mechanism: 1. Activation by RTKs: Upon activation of RTKs or GPCRs, PI3K is activated. 2. Phosphorylation of PIP2: PI3K phosphorylates the lipid PIP2 to form PIP3. 3. AKT Activation: PIP3 recruits and activates AKT (Protein Kinase B), a key player in promoting cell survival and growth.  4. mTOR Activation: Activated AKT promotes mTOR (mechanistic target of rapamycin), a central regulator of protein synthesis and cell growth. Dysregulation in Cancer: PIK3CA Mutations: Mutations in the PI3K catalytic subunit, PIK3CA, lead to increased AKT signaling, promoting cell survival and growth. PTEN Loss: PTEN is a tumor suppressor that dephosphorylates PIP3 to PIP2, negatively regulating the PI3K/AKT pathway. Loss of PTEN function (common in prostate and endometrial cancers) results in overactivation of AKT and mTOR, promoting cancer cell survival. mTOR Activation: Overactivation of mTOR enhances protein synthesis and cell growth, contributing to tumor progression. Cancer Example: PTEN Deletion in prostate cancer: Loss of PTEN function leads to unregulated AKT activation, promoting cell survival and proliferation. Wnt/β-Catenin Pathway The Wnt/β-catenin pathway is involved in cell differentiation, proliferation, and stem cell renewal. Dysregulation of this pathway is linked to cancer, especially in colorectal cancer. - WNT are growth stimulatory factors - 19 wnt genes in mammals - B-catenin is regulated by degradation and have a role in development of heart and carcinogenensis Mechanism: 1. Wnt Ligand Binding: Wnt ligands bind to the Frizzled receptor and co-receptor LRP5/6. 2. Disruption of Destruction Complex: This binding inhibits the destruction complex (composed of APC, Axin, and GSK3β), which usually degrades β-catenin. 3. Stabilization of β-Catenin: β-catenin accumulates in the cytoplasm and translocates to the nucleus. 4. Activation of Gene Transcription: In the nucleus, β-catenin activates TCF/LEF  transcription factors, driving gene expression in cell proliferation. Dysregulation in Cancer: APC Mutations: Loss-of-function mutations in APC (a tumor suppressor) prevent the degradation of β-catenin, leading to its accumulation and the activation of proliferative genes. CTNNB1 Mutations: Mutations in β-catenin (CTNNB1 gene) can make it resistant to degradation, leading to continuous signaling. Cancer Example: APC Mutation in colorectal cancer: Mutations in APC lead to the accumulation of β-catenin, driving uncontrolled cell division and cancer progression. TGF-β/SMAD Pathway The TGF-β (Transforming Growth Factor-beta) pathway controls cell growth, differentiation, and apoptosis. TGF-β acts as a tumor suppressor in normal cells by inhibiting cell proliferation. However, in advanced cancers, the pathway can promote invasion and metastasis. Mechanism: 1. TGF-β Binding: TGF-β ligands bind to the TGF-β receptor. 2. SMAD Activation: This activates SMAD2/3, which form a complex with SMAD4. 3. Gene Regulation: The SMAD complex translocates to the nucleus and regulates cell cycle arrest and apoptosis genes. Dysregulation in Cancer: TGFBR Mutations: Mutations in the TGF-β receptor can prevent the tumor-suppressive effects of the pathway, leading to unchecked cell proliferation. SMAD4 Loss: Loss of SMAD4 function, commonly seen in pancreatic cancer, allows cells to escape growth inhibition. Pro-Metastatic Role: In later stages of cancer, TGF-β signaling promotes epithelial-mesenchymal transition (EMT), aiding cancer invasion and metastasis. Cancer Example: SMAD4 Loss in pancreatic cancer: Loss of SMAD4 function allows cancer cells to evade growth suppression and facilitates metastasis. NOTCH SIGNALING PATHWAY The Notch signaling pathway is crucial for cell fate determination, differentiation, and proliferation. It plays a dual role in cancer, acting as a tumor suppressor in some contexts and as an oncogene in others. - Highly conserved pathway - Imp in development and homeostasis  - Development of sensory hair cells and branched arterial network - associated with cell death and tumor progression - Notch receptors 1.2.3.4 Mechanism: 1. Notch Receptor Activation: The Notch receptor undergoes proteolytic cleavage Upon binding ligands like Delta or Jagged. 2. Intracellular Domain Release: The Notch intracellular domain (NICD) is released and translocates to the nucleus. 3. Gene Transcription: NICD interacts with transcription factors to regulate genes involved in cell fate and proliferation. Dysregulation in Cancer: NOTCH Mutations: Activating mutations in NOTCH1 can promote proliferation in cancers like T-cell acute lymphoblastic leukemia (T-ALL). Aberrant Notch Signaling: Overactive Notch signaling can increase cell survival and proliferation, contributing to cancer progression. Cancer Example: NOTCH1 Mutations in T-cell acute lymphoblastic leukemia (T-ALL): Mutations in the Notch receptor lead to its constant activation, driving the proliferation of cancerous T-cells. JAK-STAT PATHWAY The JAK-STAT pathway (Janus kinase-signal transducer and activator of transcription) is a key signal transduction mechanism used by various cytokines, growth factors, and hormones to regulate cellular processes like proliferation, differentiation, apoptosis, and immune function. Its dysregulation is closely associated with several cancers, as it plays a critical role in cell growth and survival. Steps of the JAK-STAT Pathway: 1. Cytokine or Growth Factor Binding: The pathway is initiated when a cytokine (such as interleukin or interferon) or a growth factor binds to its specific receptor on the cell surface. These receptors are usually associated with Janus kinases (JAKs), a family of non-receptor tyrosine kinases. 2. Activation of JAKs: Upon ligand binding, the receptor undergoes a conformational change, bringing together the receptor-associated JAKs. These JAKs auto-phosphorylate themselves on tyrosine residues, activating their kinase activity. 3. Phosphorylation of Receptor: The activated JAKs phosphorylate specific tyrosine residues on the receptor's intracellular domain. These phosphorylated residues serve as docking sites for STAT proteins. 4. Recruitment and Activation of STATs: Signal transducers and activators of transcription (STATs) are recruited to the phosphorylated receptors via their SH2 domains. Once bound, JAKs phosphorylate STATs on specific tyrosine residues.  5. Dimerization of STATs: Phosphorylated STATs dissociate from the receptor and form homo- or heterodimers. This dimerization exposes a nuclear localization signal, allowing the STAT dimers to translocate into the nucleus. 6. Transcriptional Activation: In the nucleus, STAT dimers bind to specific DNA sequences in the promoter regions of target genes and activate their transcription. These genes often control cell cycle progression, apoptosis, and immune responses. Role of JAK-STAT Pathway in Cancer: The JAK-STAT pathway is tightly regulated under normal conditions, but its dysregulation can lead to cancer. Several mechanisms by which this pathway contributes to oncogenesis include: 1. Constitutive Activation: Mutations in JAKs, STATs, or receptors can lead to continuous pathway activation, even without a ligand. For example, JAK2 V617F mutation is commonly found in myeloproliferative disorders like polycythemia vera and essential thrombocythemia. This mutation causes JAK2 to be continuously active, promoting uncontrolled cell growth and survival. 2. Overexpression of Ligands or Receptors: Overexpression of cytokines or their receptors can lead to excessive activation of the JAK-STAT pathway. For instance, elevated levels of IL-6 in the tumor microenvironment activate the pathway in cancer cells, promoting growth and resistance to apoptosis. This is often seen in cancers like multiple myeloma and some solid tumors. 3. Aberrant STAT Activation: STAT3 and STAT5 are frequently implicated in cancer. Constitutive activation of STAT3 is observed in a wide range of cancers, including breast, lung, and prostate. STAT3 promotes tumor cell proliferation, inhibits apoptosis, enhances angiogenesis, and suppresses immune surveillance. Similarly, STAT5 activation is essential in leukemias, which drives cell proliferation and survival. 4. Resistance to Apoptosis: The JAK-STAT pathway can upregulate anti-apoptotic proteins, such as Bcl-xL and survivin, helping cancer cells evade programmed cell death. This resistance to apoptosis allows cancer cells to survive where normal cells would die. 5. Immune Evasion: In some cancers, the JAK-STAT pathway contributes to immune evasion by modulating the tumor microenvironment. For example, cancer cells can use this pathway to produce immunosuppressive factors like IL-10, reducing the ability of the immune system to recognize and destroy tumor cells. CELL CYCLE AND CANCER The Phases of the Cell Cycle: 1. G1 Phase (Gap 1 Phase): o The cell grows and carries out normal functions. o Checks for sufficient resources and external signals to proceed to the next phase.  o Checkpoint: The G1 checkpoint ensures the cell is ready for DNA synthesis. This checkpoint checks for DNA damage and sufficient growth signals. The cell can enter a quiescent state (G0). 2. S Phase (Synthesis Phase): o DNA replication occurs, ensuring each daughter cell receives a complete set of chromosomes. o Checkpoint: DNA replication is monitored for errors, and any detected damage is repaired. 3. G2 Phase (Gap 2 Phase): o The cell prepares for mitosis by producing necessary proteins and organelles. o Checkpoint: The G2/M checkpoint ensures that DNA replication has been completed successfully and the cell can safely enter mitosis. 4. M Phase (Mitosis Phase): o The cell divides into two daughter cells through mitosis and cytokinesis. o Checkpoint: The spindle checkpoint ensures that chromosomes are correctly aligned and attached to the spindle apparatus, preventing chromosome segregation errors. Key Cell Cycle Regulators: Cyclins: Proteins that regulate different stages of the cell cycle by binding to CDKs. Cyclin-Dependent Kinases (CDKs): Enzymes activated by cyclins that phosphorylate target proteins to drive the cell cycle forward. Tumor Suppressors: Proteins like p53, Rb, and BRCA1 prevent uncontrolled cell division by halting the cell cycle when DNA damage or other abnormalities are detected. Oncogenes: Mutated genes that promote cell proliferation. Normally, proto-oncogenes (non-mutated forms) help regulate cell growth. MATURING PROMOTER FCATOR: ACTIVATION II - Mitotic CDK and Cyclin bind together and form an inactive complex - Two inhibitory phosphate groups are attached to the CDK molecule by enzymes called inhibiting kinases  - An activating kinase adds an activating phosphate group, but the CDK remains inactive as long as the inhibitory phosphates are present - A phosphatase removes the inhibiting phosphates, activating the mitotic CDK-cyclin complex. Functions of the Activated CDK-Cyclin Complex: 1. Chromosome Condensation: o CDK1 phosphorylates condensin complexes, which are involved in chromosome condensation. This prepares the chromosomes for segregation. 2. Nuclear Envelope Breakdown: o CDK1 phosphorylates components of the nuclear envelope, such as lamins, leading to the disassembly of the nuclear envelope. This is essential for spindle microtubules to access the chromosomes. 3. Mitotic Spindle Formation: o CDK1 activates proteins that regulate the formation and stabilization of the mitotic spindle, the structure responsible for segregating chromosomes during cell division. 4. Activation of APC/C (Anaphase-Promoting Complex): o The CDK1-cyclin B complex helps activate the APC/C (Anaphase-Promoting Complex/Cyclosome), a ubiquitin ligase that marks cyclin B and other regulatory proteins for degradation. This allows the transition from metaphase to anaphase and ensures that the cell can exit mitosis. ROLE OF p53 IN CELL CYCLE ARREST Introduction to p53 p53 is a tumor suppressor protein often called the "guardian of the genome." It plays a crucial role in preventing cancer by regulating the cell cycle, inducing apoptosis, and maintaining genomic stability. The p53 gene is located on chromosome 17 in humans and encodes a transcription factor that can activate or repress many genes involved in the cell cycle, DNA repair, apoptosis, and senescence. Mutations in p53 are found in more than 50% of human cancers, making it one of the most studied proteins in cancer biology. p53 and the Cell Cycle The cell cycle consists of several phases: G1, S, G2, and M. Regulatory proteins tightly control the transition between these phases to ensure proper cell division. p53 functions at critical checkpoints, particularly at the G1/S and G2/M transitions, to prevent damaged cells from continuing to divide. Mechanism of p53 Activation Stress Signals: p53 is activated in response to a variety of cellular stress signals, including: o DNA damage (e.g., caused by radiation, UV light, or chemicals) o Hypoxia (low oxygen conditions) o Oncogene activation (e.g., excessive activity of proto-oncogenes)  o Telomere shortening When cells are exposed to these stressors, p53 is stabilized and activated, mainly through post-translational modifications such as phosphorylation, acetylation, and ubiquitination. p53-Induced Cell Cycle Arrest G1/S Checkpoint: o p53 plays a critical role in arresting the cell cycle at the G1/S checkpoint, where the cell decides whether to enter DNA replication (S phase). o Upon DNA damage, p53 induces the expression of p21 (Cip1/Waf1), a cyclin-dependent kinase (CDK) inhibitor. o p21 inhibits CDK2/cyclin E and CDK4/cyclin D complexes, which are necessary for the phosphorylation and inactivation of the retinoblastoma (Rb) protein. o In its unphosphorylated state, Rb binds to and inhibits the E2F transcription factor, preventing the transcription of genes required for S phase entry. o Thus, p53-mediated p21 expression leads to cell cycle arrest in G1, allowing the cell to repair damaged DNA before replication. G2/M Checkpoint: o p53 also controls the G2/M checkpoint, ensuring that cells do not enter mitosis (M phase) with damaged DNA. o It induces the expression of 14-3-3σ, a protein that sequesters cyclin B1/Cdk1 in the cytoplasm, preventing the activation of mitotic events. o Additionally, p53 regulates the expression of genes involved in DNA repair, such as GADD45 (Growth Arrest and DNA Damage-Inducible Protein 45), which promotes DNA repair processes and contributes to cell cycle arrest at G2/M. P53 AND CANCER Introduction to p53 p53, also known as the "guardian of the genome," is a critical tumor suppressor protein that plays a fundamental role in preventing cancer development. It regulates several key cellular processes, including the cell cycle, DNA repair, senescence, and most notably, apoptosis.  p53 ensures that cells with damaged DNA repair the damage or undergo programmed cell death (apoptosis) to prevent the proliferation of potentially cancerous cells. Mutations in the TP53 gene, which encodes the p53 protein, are found in more than 50% of human cancers, underlining its pivotal role in tumor suppression. p53 and Apoptosis Apoptosis, or programmed cell death, is a process that enables organisms to eliminate damaged, unwanted, or potentially harmful cells. p53 is a major apoptosis regulator, especially in response to cellular stress such as DNA damage, oncogene activation, and hypoxia. Induction of Apoptosis by p53: o When p53 detects severe DNA damage that cannot be repaired, it activates the apoptotic machinery. This is critical in preventing the survival and replication of cells with potentially oncogenic mutations. o p53 activates apoptosis by both transcription-dependent and transcription-independent mechanisms. 1. Transcription-Dependent Apoptosis: o p53 functions as a transcription factor, binding to specific DNA sequences and regulating the expression of several pro-apoptotic genes. o Key genes activated by p53 in the apoptotic pathway include: ▪ BAX (Bcl-2-associated X protein): A pro-apoptotic member of the Bcl-2 family that promotes mitochondrial membrane permeabilization, releasing cytochrome c and triggering the intrinsic (mitochondrial) apoptotic pathway. ▪ PUMA (p53 Upregulated Modulator of Apoptosis) and NOXA: These proteins also promote mitochondrial dysfunction and enhance apoptosis by antagonizing anti-apoptotic Bcl-2 proteins. ▪ FAS: p53 enhances the expression of the FAS receptor, which triggers the extrinsic apoptotic pathway through ligand binding, leading to the activation of caspases and cell death. ▪ p53AIP1 (p53-regulated Apoptosis Inducing Protein 1): It is involved in mitochondrial-mediated apoptosis, helping to regulate cell death in response to stress signals. 2. Transcription-Independent Apoptosis: o In addition to its role as a transcription factor, p53 can directly interact with pro-apoptotic proteins at the mitochondria. o p53 can translocate to the mitochondria and bind to anti-apoptotic proteins such as Bcl-2 and Bcl-xL, neutralizing their effects and allowing pro-apoptotic proteins like BAX and PUMA to initiate apoptosis. p53 and the Intrinsic Apoptotic Pathway The intrinsic pathway of apoptosis, also known as the mitochondrial pathway, is one of the primary mechanisms by which p53 induces cell death. This pathway is triggered by internal cellular stress signals such as severe DNA damage, oxidative stress, or oncogene activation.  Mechanism: o In response to these stress signals, p53 activates pro-apoptotic proteins like BAX, PUMA, and NOXA, which lead to mitochondrial outer membrane permeabilization. o Once the mitochondrial membrane is permeabilized, cytochrome c is released from the mitochondria into the cytosol. o Cytochrome c then interacts with Apaf-1 (Apoptotic Protease Activating Factor 1) and procaspase-9 to form the apoptosome, which activates caspase-9. o Caspase-9 initiates the downstream activation of executioner caspases (such as caspase-3), which dismantle the cell by degrading proteins and other cellular components, leading to apoptosis. p53 and the Extrinsic Apoptotic Pathway p53 can also induce apoptosis through the extrinsic (death receptor) pathway. The extrinsic pathway is activated by the binding of ligands (e.g., FAS ligand or TNF-related apoptosis-inducing ligand) to death receptors on the cell surface. p53 upregulates the expression of the FAS receptor (CD95) and DR5 (Death Receptor 5), enhancing the cell’s sensitivity to death ligands. This receptor-ligand interaction activates caspase-8, which subsequently activates downstream executioner caspases, leading to cell death. p53 Mutations and Cancer Loss of p53 Function in Cancer: o Mutations in the TP53 gene result in the loss of p53's tumor-suppressive functions, including its ability to induce apoptosis. o When p53 is mutated, cells with damaged DNA or oncogenic mutations continue to survive and proliferate, contributing to tumor formation and progression. o Mutant p53 proteins often acquire dominant-negative activity, meaning that they can inhibit the function of any remaining wild-type p53 in a cell, further compromising the cell's ability to undergo apoptosis. p53 Mutations and Tumor Resistance: o Many cancer therapies, such as chemotherapy and radiation, aim to induce DNA damage in cancer cells to trigger p53-mediated apoptosis. o In cancers where p53 is mutated, these therapies are often less effective, as the apoptotic response is diminished, allowing cancer cells to survive despite treatment. o This leads to therapy resistance, making cancers with p53 mutations more challenging to treat. Types of p53 Mutations: o Most p53 mutations in cancer are missense mutations, which result in a single amino acid change in the p53 protein. o These mutations often occur in the DNA-binding domain of p53, impairing its ability to bind to the promoters of target genes involved in apoptosis and cell cycle arrest. o The resulting mutant p53 protein may lose its tumor-suppressive functions, gain oncogenic properties, or interfere with other tumor-suppressing mechanisms. p53 Reactivation Strategies in Cancer Therapy Given the critical role of p53 in inducing apoptosis, restoring or enhancing its function in cancer cells has become a major focus in cancer therapy. 1. MDM2 Inhibitors: o Under normal conditions, p53 activity is negatively regulated by MDM2, a protein that targets p53 for degradation. o In cancers with wild-type p53, MDM2 is often overexpressed, leading to reduced p53 activity. o MDM2 inhibitors (e.g., Nutlin-3) disrupt the interaction between MDM2 and p53, allowing p53 to accumulate and induce apoptosis in cancer cells. 2. Gene Therapy: o One approach to treating cancers with mutant p53 is to introduce wild-type p53 into cancer cells using gene therapy. o This restores the tumor-suppressive functions of p53, including its ability to trigger apoptosis in response to DNA damage or stress. 3. Small Molecule Activators: o Small molecules that can restore the normal function of mutant p53 have been developed. o These molecules can bind to mutant p53, stabilize its structure, and reactivate its ability to induce apoptosis. o PRIMA-1 and APR-246 are examples of drugs in development that target mutant p53. 4. Immunotherapy: o Targeting cells with p53 mutations through immunotherapy is an emerging field. o Cancer cells with mutant p53 often express specific neoantigens that the immune system can recognize. o By harnessing the body’s immune response, therapies can be designed to target and kill p53-deficient cancer cells specifically. Apoptosis, or programmed cell death, can be triggered by various internal and external signals. These triggers can broadly be categorized into two main pathways: the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway. 1. Intrinsic Pathway (Mitochondrial Pathway) - Cellular stress/damage (DNA damage, hypoxia, etc.) ↓ - Activation of p53 (or other stress-related signals) ↓ - Activation of Bcl -2 family pro-apoptotic proteins (e.g., Bax , Bak) ↓ - Mitochondrial outer membrane permeabilization (MOMP) ↓ - Cytochrome c release from mitochondria ↓ - Formation of the apoptosome (Cytochrome c binds with Apaf -1 and pro caspase -9) ↓ - Activation of caspase -9 (initiator caspase) ↓ - Activation of executioner caspases (e.g., caspase-3, caspase-7) ↓ - Apoptosis (DNA fragmentation, membrane blebbing, cell death) 2. Extrinsic Pathway (Death Receptor Pathway) - External death signal (e.g., FasL, TNF) ↓ - Binding to death receptors (Fas/CD95, TNF receptor) ↓ - Recruitment of adaptor proteins (FADD or TRADD) ↓ - Formation of the death-inducing signaling complex (DISC) ↓ - Activation of caspase -8 (initiator caspase) ↓ - If direct apoptosis: Caspase -8 activates executioner caspases (e.g., caspase -3, caspase -7) - If cross-talk with intrinsic pathway: Caspase -8 cleaves Bid → tBid, which promotes mitochondrial outer membrane permeabilization (MOMP) ↓ - Activation of executioner caspases (e.g., caspase -3, caspase -7) ↓ - Apoptosis (DNA fragmentation, membrane blebbing, cell death) 2. Execution Phase Activation of Executioner Caspases (e.g., caspase-3, caspase-7) ↓ Degradation of Cellular Components: Cleavage of Nuclear Lamins (disassembly of nuclear envelope) DNA Fragmentation (by caspase-activated DNase) Cytoskeleton Disassembly Membrane Blebbing ↓ Formation of Apoptotic Bodies (small membrane-bound fragments) ↓ Phagocytosis of Apoptotic Bodies by macrophages or neighboring cells (no inflammation) 3. Regulation Pro-apoptotic Regulators: Bax, Bak (promote MOMP)  Smac/DIABLO (inhibit IAPs, promoting caspase activation) Anti-apoptotic Regulators: Bcl-2, Bcl-xL (prevent MOMP) Inhibitors of Apoptosis Proteins (IAPs) (inhibit caspases) Cancer cell die by apoptosis or necrosis? Cancer cells can die through both **apoptosis** and **necrosis**, but the mechanisms and outcomes of these processes are quite different: 1. Apoptosis (Programmed Cell Death) - **Cancer cells can die by apoptosis**, especially in response to chemotherapy, radiation, or targeted therapies that induce cellular stress or damage. - Key points about apoptosis in cancer: - Apoptosis is a controlled and regulated process. - In many cancers, the apoptotic pathways are dysregulated. Mutations in genes like p53 (a tumor suppressor) can prevent cancer cells from undergoing apoptosis, allowing them to survive longer and proliferate. - Some cancer therapies are designed to reactivate apoptosis by targeting proteins that inhibit it, such as Bcl-2 inhibitors. 2. Necrosis (Uncontrolled Cell Death) - Cancer cells can also undergo necrosis, typically in conditions of extreme stress like lack of oxygen (hypoxia) or nutrient deprivation, especially in the center of large tumors. - Key points about necrosis in cancer: - Necrosis is a form of unregulated cell death. - Unlike apoptosis, necrosis is often associated with inflammation and can cause damage to surrounding tissues. - Necrosis in tumors is sometimes seen as a sign of aggressive cancer growth, as the rapidly growing tumor outstrips its blood supply, leading to areas of necrotic tissue Summary - Apoptosis is the preferred and orderly form of cancer cell death in response to treatment, but cancer cells often develop mechanisms to evade it. - Necrosis tends to occur in areas of tumors where there is extreme stress or lack of resources, but it is uncontrolled and can lead to inflammation. Thus, cancer cells can die by either apoptosis or necrosis, depending on the context and external factors. AUTOPHAGY AND APOPTOSIS Process of autophagy Here is a detailed flowchart of the **autophagy process**: 1. Initiation Phase - Cellular Stress/Starvation(e.g., nutrient deprivation, hypoxia) ↓ - Activation of mTOR inhibition (mTOR = mechanistic target of rapamycin, an inhibitor of autophagy in normal conditions) ↓ - Activation of ULK1 complex (initiates autophagy machinery) ↓ - Formation of Phagophore (a double membrane that will engulf cellular components) 2. Nucleation Phase - Recruitment of Autophagy-Related (ATG) Proteins (ATG proteins facilitate autophagosome formation) ↓ - Phagophore elongates and expands to surround cytoplasmic components ↓ - Engulfment of damaged organelles, misfolded proteins, and other cellular debris 3. Maturation Phase - Closure of Phagophore to form an autophagosome (a double-membrane vesicle containing the engulfed material) ↓ - Transport of the Autophagosome to the lysosome 4. Fusion Phase - Fusion of Autophagosome with Lysosome (forming an autolysosome) ↓ - Lysosomal Hydrolases degrade the contents of the autophagosome 5. Degradation and Recycling Phase - Breakdown of Cellular Components (damaged organelles, proteins, lipids, etc.)  ↓ - Release of Breakdown Products (e.g., amino acids, fatty acids) back into the cytoplasm ↓ - Recycling of Cellular Components for energy and survival (especially under conditions of stress) Summary of Key Phases: 1. **Initiation:** Triggered by stress (e.g., nutrient deprivation). 2. **Nucleation:** Formation and expansion of the phagophore. 3. **Maturation:** Phagophore closes, forming an autophagosome. 4. **Fusion:** Autophagosome fuses with lysosome. 5. **Degradation:** Cellular components are degraded and recycled. Role of autophagy in aggressive tumour behaviour Autophagy plays a complex and often paradoxical role in the development and progression of **aggressive tumors**. Its effects can be either tumor-suppressive or tumor-promoting, depending on the stage of cancer development, the tumor microenvironment, and other factors. Below is a detailed explanation of how autophagy contributes to **aggressive tumor behavior**: 1. Early-Stage Tumors: Tumor-Suppressive Role - In the early stages of tumor development, autophagy acts as a **tumor-suppressive mechanism**. - **Prevention of DNA damage and genomic instability:** Autophagy helps remove damaged organelles (like mitochondria) and misfolded proteins that could lead to mutations and cancer initiation. - **Elimination of precancerous cells:** By maintaining cellular homeostasis, autophagy can prevent the accumulation of cellular damage, limiting the progression to malignancy. 2. Advanced Tumors: Tumor-Promoting Role In later stages of cancer, especially in aggressive tumors, autophagy often **promotes tumor survival and growth** under harsh conditions. Promoting Tumor Survival: - **Nutrient Recycling:** Aggressive tumors, particularly in hypoxic and nutrient-poor environments (due to poor vascularization), rely on autophagy to recycle cellular components for energy and nutrients, promoting survival under metabolic stress. - **Therapeutic Resistance:** Autophagy is often upregulated in response to cancer therapies (e.g., chemotherapy, radiation), allowing cancer cells to survive treatment by removing damaged organelles and preventing cell death. Facilitating Tumor Growth: - **Maintenance of Cancer Stem Cells (CSCs):** Autophagy supports the survival and maintenance of **cancer stem cells**, which are associated with tumor initiation, metastasis, and therapeutic resistance. - **Evasion of Apoptosis:** In aggressive tumors, autophagy can act as a defense mechanism that prevents apoptosis by degrading pro-apoptotic factors, allowing cancer cells to avoid programmed cell death. Metastasis and Invasion: - **Autophagy Enhances Metastasis:** Autophagy can promote metastatic potential by enabling cancer cells to survive during detachment from the primary tumor, circulation in the bloodstream, and colonization in distant organs. - **Epithelial-to-Mesenchymal Transition (EMT):** Autophagy has been implicated in the process of EMT, which facilitates the invasive and metastatic capabilities of cancer cells. 3. Hypoxia and Tumor Microenvironment - **Adaptation to Hypoxic Conditions:** Aggressive tumors often grow faster than their blood supply can provide nutrients and oxygen. Under hypoxic conditions, autophagy is upregulated, helping cancer cells adapt to this low-oxygen environment. - **Resistance to Cellular Stress:** By removing damaged mitochondria and proteins, autophagy protects cancer cells from oxidative stress, allowing them to survive in otherwise lethal conditions. 4. Drug Resistance - **Resistance to Cancer Therapies:** In aggressive tumors, autophagy is frequently activated in response to anticancer drugs, creating a survival mechanism for cancer cells. Inhibiting autophagy in combination with chemotherapy or radiation has been explored to overcome drug resistance. - **Autophagy Inhibitors:** Research is focusing on autophagy inhibitors (like chloroquine) to disrupt this protective mechanism in aggressive cancers, aiming to make these tumors more susceptible to conventional therapies. 5. Dual Role in Tumor Progression - **Autophagy as a Double-Edged Sword:** While autophagy can prevent cancer initiation by removing damaged cellular components, it later becomes a **pro-survival mechanism** that promotes tumor growth, aggressiveness, and resistance to therapy. Summary: - In early stages, **autophagy suppresses tumor formation** by maintaining cellular integrity. - In **advanced and aggressive tumors**, autophagy enables tumor cells to survive in hostile environments (e.g., low oxygen, nutrient deprivation), resist therapies, and promote metastasis. - **Targeting autophagy** has become a therapeutic strategy in aggressive cancers, particularly to overcome resistance to treatment.