Cancer and Apoptosis BC.504 PDF

# Cancer and Apoptosis ## BC.504 ### Presented by: Dr. Amany H. Adam, Ph.D. Lecturer of Biochemistry, Faculty of Science, Damanhour University. ## Apoptosis in normal & cancer cells - Apoptosis is an essential physiological process of cell death that is meant to occur without the discharge of in...

# Cancer and Apoptosis ## BC.504 ### Presented by: Dr. Amany H. Adam, Ph.D. Lecturer of Biochemistry, Faculty of Science, Damanhour University. ## Apoptosis in normal & cancer cells - Apoptosis is an essential physiological process of cell death that is meant to occur without the discharge of intracellular contents, and subsequent no activation of an inflammatory response as apoptosis is a crucial process in embryonic development, regulation of the immune system, and the response to DNA damage. - However, dysregulation of apoptosis results in significant consequences in carcinogenesis. - The imbalance between cell proliferation and cell death is considered the malignant tumor’s hallmark. - Therefore, maintaining cellular homeostasis between proliferation and cell death is essential for normal physiological processes. - The signaling molecules of the apoptotic pathway played an essential role in the regulation of the apoptosis process. - Therefore, these molecular proteins might be considered potential apoptotic biomarkers that can be targeted in advanced cancer treatment and therapeutics. ## Cancer Progression **Stages of Cancer Progression** - **A. Cancer Initiation:** - DNA mutation - Multiplication of cells - Unchecked growth of cells - **B. Cancer Progression:** - Self-sufficient growth signals - Angiogenesis - Invasion - Insensitivity towards growth/immune modulators inhibitors - **C. Cancer Development:** - Metastasis ## Apoptosis in normal & cancer cells - Apoptosis is a cellular suicide program that organisms have evolved to eliminate unnecessary or unhealthy cells from the body in the course of development or following cellular stress. - It involves a series of cellular events that ultimately leads to activation of a family of cysteine proteases called caspases. - In response to various apoptotic stimuli, “initiator” caspases (caspase-2, -8, -9, or 10) are activated. Initiator caspases, in turn, cleave and activate the zymogenic forms of “executioner” caspases (e.g. caspase-3 or -7), resulting in the proteolytic cleavage of specific cellular substrates and, consequently, cell death. - In this regard, the cleavage (activation) of executioner caspases is a hallmark of apoptosis. There are two routes to apoptosis: extrinsic and intrinsic. # Apoptosis in normal & cancer cells - Apoptosis can be triggered by diverse cellular signals. - These include intracellular signals produced in response to cellular stresses, such as increased intracellular Ca2+ concentration, oxidative damage caused by reactive oxygen species (ROS), and hypoxia. - Extrinsic inducers of apoptosis include bacterial pathogens, toxins, nitric oxide, growth factors, and hormones. ### **Apoptotic Pathways:** - **Intrinsic pathway:** Characterized by mitochondrial outer membrane permeabilization (MOMP) and the release of mitochondrial cytochrome c. - **Extrinsic pathway:** Initiated by death-receptor stimulation. - **Overlap:** There is an overlap between these pathways as the extrinsic pathway usually also activates the intrinsic pathway, and both pathways result in the recruitment and activation of cysteine-aspartic acid proteases (caspases) (Execution Phase). ## The Intrinsic Pathway of Apoptosis - In the intrinsic pathway of apoptosis, mitochondrial outer membrane permeabilization (MOMP) is involved; MOMP triggers the release of a group of pro-apoptotic proteins, including cytochrome c and second mitochondria-derived activator of caspases (SMAC), from the mitochondrial intermembrane space to the cytoplasm. - In the cytoplasm, cytochrome c binds to the adaptor protein APAF-1 (apoptotic protease activating factor 1) to form a caspase-9-activating complex, called the apoptosome. SMAC augments cytochrome c-induced caspase activation by binding and neutralizing XIAP (X-linked inhibitor of apoptosis protein), an inhibitor of caspase-3, -7, and -9. - The release of cytochrome c from the mitochondria is stimulated by pro-apoptotic members of the BCL-2 family, such as BAX, BAK1, BIM, BID and BBC3 (commonly referred to as PUMA), and inhibited by anti-apoptotic members of the same family, such as BCL-2, BCL-XL, BCL-W, BCL-2-A1 and MCL1. ## The Intrinsic Pathway of Apoptosis - **BH3-only proteins** are a subclass of pro-apoptotic BCL-2 proteins that contain only one BCL-2 homology domain and can be divided into activators and sensitizers. - **Activators:** BIM, BID and PUMA, bind and directly activate BAX and/or BAK1, the central effectors of apoptosis, which subsequently undergo conformational changes and oligomerize, forming a pore in the mitochondrial membrane and causing MOMP. - **Sensitizers:** BAD, PMA- induced protein 1 (commonly referred to as NOXA), BIK, BMF and HRK) is that of inhibiting anti-apoptotic proteins and/or displacing activators (such as BIM or monomers of BAX or BAK1) that could ultimately lead to apoptosis. - **Anti-apoptotic members of the BCL-2 family** have four BCL-2 homology domains forming a binding groove that sequesters activators or sensitizers as well as BAX and BAK1. The BH3- only proteins PUMA, NOXA, BID and BIM have a strong affinity for and inhibit MCL1, whereas BAD has a stronger affinity for BCL-2, BCL-W and BCL- XL than for MCL1. - **The levels of BCL-2 proteins and their interactions** modulate sensitivity and resistance to apoptosis. - **Cells with high levels of pro- apoptotic BCL-2 proteins** are more primed to apoptosis than cells in which BAX or BAK1 is down-regulated. - **Following the release of cytochrome c from the mitochondria,** the activation of caspase 3 can be negatively regulated by IAP proteins such as XIAP, IAP1 and IAP2. - **Proteins released from the mitochondria along with cytochrome c** (for example SMAC and HTRA2) functionally block XIAP, thus facilitating caspase activity and cell death. - **In mammalian cells, the balance of anti- apoptotic versus pro- apoptotic BCL-2 proteins** has long been considered a cellular rheostat that controls the threshold to initiate apoptosis. ## The Extrinsic Pathway of Apoptosis - **In the extrinsic pathway,** initiator caspase-8 and -10 are activated through the formation of a death-inducing signal complex (DISC) in response to engagement of extracellular ligands (e.g. Fas or TNF [Tumor necrosis factor]) by cell surface receptors. - **Pro-apoptotic death receptors** include Fas (whose physiological ligand is Fas-L), the tumor necrosis factor (TNF) receptors TNFR1 and TNFR2, and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. - **The intracellular domains of the pro- apoptotic death receptors** include a conserved protein-protein interaction domain referred to as the death domain. - **Upon ligand binding, death receptors trimerize and aggregate within the cell membrane** (a phenomenon known as capping). - **This process is followed by the recruitment of adaptor proteins,** such as FADD, and initiator caspases, such as caspase 8 and caspase 10. - **In the extrinsic pathway, the activation of initiator caspases is negatively regulated by CASP8 and FADD- like apoptosis regulator** (commonly referred to as FLIP). ## The Extrinsic Pathway of Apoptosis - **The activation of initiator caspases 8 and 10 leads to the activation of effector caspases and to the cleavage of BID,** which is subsequently myristoylated and translocates to the mitochondria, thus contributing to the release of cytochrome c - **BID is positively regulated by p53 at the transcriptional level.** BID has been proposed as a link between the extrinsic and intrinsic pathways: in the so-called “type II” cells (which primarily rely on the intrinsic pathway), BID amplifies the apoptotic signal through the mitochondria, leading to more efficient apoptosis that ultimately requires the mitochondrial events for cell execution. - **In “type I” cells (which primarily rely on the extrinsic pathway),** the activation of death receptors leads to efficient activation of downstream caspases, leading to cell death without the need to amplify the signal through the mitochondria. - **Signaling through death receptors can be attenuated by decoy receptors that bind to death ligands and compete for them at the cell surface,** thereby reducing the activation of pro- apoptotic death receptors. ## The Extrinsic Pathway of Apoptosis - **For example DcR3, a decoy receptor for Fas-L,** is expressed and amplified in primary lung, colon and breast tumors, and two TRAIL receptor decoys (DcR1 and DcR2) are overexpressed in non-malignant cells, in which their presence has been proposed as a mechanism for limiting the toxicity of TRAIL. - **Similarly, low levels of caspase 8 expression in non-malignant cells result in reduced toxicity of death ligands and receptors.** - **P53 directly regulates the transcription of death receptors such as Fas or DR5,** and thus, therapeutic agents that stabilize p53 can induce apoptosis through both the extrinsic and the intrinsic pathways. - **Both extrinsic and intrinsic pathways lead to the final pathway of apoptosis. The execution phase includes activation of execution caspases,** which are characteristic of the final phase of apoptosis that is associated with the activation of cytoplasmic endonucleases and proteases. - **This leads to the degradation of nuclear materials and cytoskeletal proteins.** ## The Execution phase of Apoptosis - **Both morphological and biochemical changes characteristic of apoptotic cells are mediated by effector “executioner” caspases such as caspase-3, caspase-6, and caspase-7.** - **Activation of these caspases results in cleavage of various substrates such as cytokeratins, PARP, plasma membrane cytoskeletal protein alpha fodrin, and nuclear mitotic apparatus protein (NuMA).** - **Phospholipid asymmetry and externalization of phosphatidylserine on the surface of apoptotic cells and membrane fragmentation are characteristic of the execution phase.** - **Moreover, externalization of phosphatidylserine in the execution phase appears to be an essential cellular mechanism for removing apoptotic cells since the appearance of phosphotidylserine on the outer leaflet of apoptotic cells may facilitate non-inflammatory phagocytic recognition, thereby allowing their early uptake and finally their removal** ## Anti-apoptotic BCL-2 Family vs. BH3-Only Proteins The intrinsic pathway to apoptosis is initiated by activation of BH3-only proteins. Among the BH3-only proteins, whose expression is often transcriptionally induced following an apoptotic stimulus, BID is unique in that it is activated by caspase cleavage (by caspases-2 and -8/10); cleaved BID, termed tBID. **BH3 proteins** trigger the activation of multi-BH domain proteins by direct binding or by inhibition of anti-apoptotic BCL-2 (B-cell lymphoma 2) family proteins. **Once activated, multi-BH domain proteins** - form oligomers that induce MOMP. - MOMP prompts pro-apoptotic proteins SMAC and cytochrome c to be released to the cytoplasm, where cytochrome c induces the formation of the Apoptosome. <start_of_image> Schematic diagram illustrating the intrinsic pathway of apoptosis (see the attached document). ## Anti-apoptotic BCL-2 Family vs. BH3-Only Proteins - **Apoptosis is triggered in response to internal or external stimuli.** - **The initiator caspase-9 activated within the Apoptosome initiates the activation of executioner caspase-3 and -7, resulting in apoptosis.** - **Of note, caspase-2 is activated via formation of the PIDDosome,** a complex which consists of the adaptor proteins PIDD (p53-induced death domain protein) and RAIDD (RIP-associated protein with a death domain). - **However, despite identification of this complex,** the precise mechanism of caspase-2 activation remains elusive. - **In addition, only a few cellular substrates of caspase-2, including BID,** have been identified. Colored triangles denote that the indicated gene or gene product may be transcriptionally (blue), translationally (red), and post-translationally (green) up- or down-regulated in cancer cells. - **The basic circuitry of how the BCL-2 family regulates apoptosis.** - In response to many types of perturbations, BH3-only activators such as BIM and BID stimulate the oligomerization of effectors BAX and BAK to induce mitochondrial outer membrane permeabilization (MOMP), release of cytochrome c, and cell death. - Anti-apoptotic multidomain members may inhibit activation by sequestration of activators or activated, monomeric BAX and BAK. BH3-only sensitizers can bind to the BH3 binding sites of and inhibit anti-apoptotic multidomain members, but cannot activate BAX or BAK on their own. - If the anti-apoptotic proteins are already binding to activators or effectors, these can be displaced by sensitizers so that the activators and effectors can proceed with mitochondrial outer membrane permeabilization and commitment to cell death. Note that among BH3-only proteins, activator function appears to be present along a gradient, with BIM and BID being the most potent activators, and PUMA and NOXA also exhibiting activator function, though apparently less potent than BIM or BID. Also, BIM preferentially activates BAX over BAK, and BID preferentially activates BAK over BAX ## Dysregulated Apoptosis & Cancer - **Apoptosis is a stochastic process within a cell population.** Thus, a fraction of apoptosis within a population can be attributed to variations in the levels of proteins involved in apoptosis. - **In cancer, the balance between cell death and cell division and cell death is compromised.** Defects in the apoptotic mechanism play an important role in carcinogenesis, by allowing tumor cells to survive over the intended life span. - **Apoptosis resistance is a key hallmark of cancer.** Hence, reduced apoptosis or its resistance plays a pivotal role in carcinogenesis. - **There are many ways a malignant cell can acquire reduction in apoptosis or apoptosis resistance.** Generally, the mechanisms by which evasion of apoptosis occurs can be broadly dividend into: i) disrupted balance of pro-apoptotic such as BAX and anti-apoptotic proteins such as Bcl-2, which is regulated by the P53 tumor suppressor gene, ii) reduced caspase function and activity and iii) impaired death receptor signaling. - **Thus, successful eradication of cancer cells is achieved by either activating the inhibited apoptotic mechanism or repair the defective ones.** Schematic diagram illustrating the different mechanisms that contribute to the evasion of apoptosis in cancer cells (see the attached document). ## Dysregulated Apoptosis & Cancer - **Cancer cells can modulate apoptotic pathways transcriptionally, translationally, and post-translationally.** - **In some cases, cancer cells may escape apoptosis by increasing or decreasing expression of anti- or pro-apoptotic genes, respectively.** Alternatively, they may inhibit apoptosis by stabilizing or de-stabilizing anti- or pro-apoptotic proteins, respectively. - **Moreover, cancer cells may also prevent apoptosis by changing the functions of anti- or pro-apoptotic proteins through post-translational modifications,** such as phosphorylation. - **Importantly, these mechanisms are not mutually exclusive and cancer cells may employ one or multiple mechanisms to evade apoptosis.** - **Various potential players play an active role in inhibiting cell proliferation.** Here, we highlighted apoptosis regulating proteins/elements and other than apoptosis-inducing factors as a targeted strategy to regulate the process of carcinogenesis or cell proliferation. - **The apoptosis regulating strategy begins with the cell cycle—the regulators involved in cell division, cell arrest, and induction of apoptosis.** ## Dysregulated Apoptosis & Cancer - Any dysregulation in the cell cycle checkpoints results in the rapid division of cells without the hindering. - The rapid division increases cell growth. Similarly, up-regulating the various pro-apoptosis proteins/elements and down-regulating the anti-apoptosis proteins might be a positive regulators of apoptosis. - The p53 role in induction apoptosis could be enhanced by up-regulating the transcription of mRNA and translated product. - The use of inhibitors to suppress the activation of inhibitors of apoptosis (IAPs) also aids in the successful establishment of apoptosis in cancer cells. - The other strategy involves various regulator proteins involved in cell progression and growth, such as MMPs (matrix metalloproteinases), play a vital role in cancer progression. - MMP inhibition or blockade is a critical target for reducing metastatic potential. ## Dysregulated Apoptosis & Cancer - In addition to MMP, metastasis suppressor genes such as MKK4 (mitogen-activated protein kinase 4), BRMS1 (breast cancer metastasis suppressor 1), and NM23 (non-metastasis gene 23) play important roles in metastasis inhibition. - Conventional therapy options are exceedingly tough, particularly in patients with metastatic disease. Invasion, intravasation, and extravasation are part of the metastatic pathway. - The spread of cancer cells to distant places via the circulatory system marks the invasion phase. On the other hand, extravasation necessitates cancer cells penetrating the endothelium and the basement membrane. - Additionally, regulation of uPA and uPAR expression and TIMP expression are critical for metastasis prevention. <start_of_image> schematic diagram illustrating the key regulators in the inhibition of cancer growth. ## Targeting Regulators in Inhibition of Cell Progression Targeting regulators in inhibition of cell progression. It is mainly divided into apoptosis regulators and others. Targeting Apoptosis includes i) up-regulation of pro-apoptotic (caspases protein, Bax, and pro-apoptotic member of Bcl-2 family) ii) down-regulation of anti-apoptotic proteins (IAPs). Also, by targeting the pro-survival signaling pathways, invasion and metastasis proteins dysregulated in cancer cells