01-CellCyle-2024-REVISED (3) - Copy
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Cell Cycle Control 1 Cell Cycle Under and Out of Control September 23, 2024 Martin Schmidt, PhD LEARNING OBJECTIVES: The student should be able to: 1. Compare and contrast different mechanisms of DNA repair including base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining. 2. Describe the contribution of cyclin dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CDKIs) to cell cycle checkpoints and growth control. 3. Describe the contributions of the following proteins to growth factor signaling pathways: receptor tyrosine kinases (RTKs), protein tyrosine phosphatases (PTPs), mitogen activated protein kinases (MAPKs), Ras, non-receptor tyrosine kinases. 4. Describe the role of human epidermal growth factor receptor 2 (HER2) in the development, progression and treatment of breast cancer. 5. Compare and contrast the role of oncogenes and tumor suppressor genes to the development of cancer 6. Describe the normal cellular role and how loss or change in function contributes to oncogenesis for genes listed in Table 1. 7. Explain how epigenetic changes regulate gene expression and contribute to oncogenesis. KEYWORDS:Base excision repair, Nucleotide excision repair, Mismatch repair, Double Strand Break repair, protein phosphorylation, RTKs, PTPs, MAPKs, EGF, HER, CDKs, CDKIs, restriction point, Single Nucleotide Polymorphism (SNP), Tandem Repeats, Copy number variants (CNVs), S phase, M phase, Interphase, G-0, G-1, G-2 phases, 1. Overview of Oncogenesis Oncogenesis, the process by which normal cells transform into cancerous ones, is driven by a series of genetic and molecular changes that disrupt key cellular functions. Central to this transformation are mutations in genes that regulate cell growth, DNA repair, and the cell cycle. These mutations often lead to the activation of oncogenes, loss of tumor suppressor genes, and defects in DNA repair mechanisms. The result is uncontrolled cell proliferation, resistance to cell death, and the potential for metastasis. Understanding the mechanisms of oncogenesis is crucial Additional Resources: Thompson and Thompson, 9th edition, Chapter 16, Cancer Genetics and Genomics (eBook on reserve for KF at HSLS)
Cell Cycle Control 2 for developing targeted therapies to prevent and treat cancer effectively. 2. DNA Repair Pathways DNA repair pathways are critical for maintaining genomic integrity. Cells are continuously exposed to DNA damage from both internal and external sources. DNA repair pathways, such as nucleotide excision repair and homologous recombination, correct these damages, preventing mutations from being passed on during cell division. Mutations in genes like BRCA1 and BRCA2, which are critical for DNA repair, can lead to an increased risk of cancer as damaged DNA accumulates and promotes oncogenesis. Base excision repair (BER) is a DNA repair process that removes and replaces damaged bases, primarily fixing small, non-helix-distorting lesions. BER involves DNA glycosylases that recognize different bases (deaminated or alkylated bases) and excise them, followed by repair of the resulting abasic sites (sites that have no bases attached to the deoxyribose) to restore DNA integrity (Figure 1). Nucleotide excision repair (NER): is a DNA repair mechanism that removes bulky, helix-distorting lesions, such as thymine dimers caused by UV light. NER excises a short single-stranded DNA segment containing the damage, and the gap is filled in using the complementary strand as a template. Thymine dimers (caused by UV exposure) are an example of a bulky lesion that is repaired by NER (Figure 1). Defects in NER underlie the diseases xeroderma pigmentosum (XP) and Cockayne syndrome (CS). See genes listed inTable 1. Mismatch repair (MMR) is a DNA repair mechanism that corrects errors occurring during DNA replication, such as mismatched bases or small insertion-deletion loops. Using a mechanism similar to NER, MMR proteins recognize and excise the incorrect DNA segment, and DNA polymerase fills in the correct sequence, ensuring high fidelity in DNA replication. Mutations in MER genes (MLH1, MSH2, MSH3 and MSH6) drive genome instability and are commonly found in cancers (Table 1). 3. Double-strand DNA break repair Double-strand DNA breaks can occur following exposure to ionizing radiation, reactive oxygen species, chemotherapy agents and other toxins. These must be repaired for cell survival. The two mechanisms for double strand breaks are homologous recombination (HR) and non-homologous
Cell Cycle Control 3 end joining (NHEJ). HR is the preferred pathway since it maintains sequence fidelity. However, when HR is not possible (lack of homologous sequences or mutations affecting HR genes, such as BRCA1, and BRCA2), the double strand breaks will be repaired via NHEJ, a process that is inherently mutagenic due to the creation of small deletions in the DNA sequence at the repair site (Figure 2). HR is a more complex pathway that requires numerous proteins such as RAD51, RAD52, NBS1, MRE11, BRCA1, BRCA2, DNA polymerase, DNA ligase, etc. Homology-directed repair is most common during S and G2 phasesof the cell cycle, because the two sister chromatids are in close proximity, providing a nearby homology donor. 4. Cell Cycle Control Cell cycle control is fundamental in regulating cell division, ensuring that cells only replicate their DNA and divide when conditions are appropriate. This control is mediated by a complex network of proteins, including cyclins, cyclin-dependent kinases (CDKs), and their inhibitors. When these regulatory mechanisms are disrupted—often due to mutations in genes like TP53 or RB1—cells can bypass the normal checkpoints, leading to uncontrolled proliferation, the accumulation of further genetic damage and oncogenic transformation. The cell cycle is a series of phases that cells undergo to grow, replicate their DNA, and divide. It consists of several key stages: G1(Gap 1), where the cell grows and carries out normal functions while preparing for DNA replication; S(Synthesis), where DNA is replicated, resulting in two complete sets of chromosomes; G2(Gap 2), where the cell continues to grow and prepares for division by producing necessary proteins and organelles; and M (Mitosis), where the cell divides its nucleus and cytoplasm to form two identical daughter cells. Additionally, some cells enter a quiescent state known as G0, where they exit the cell cycle and stop dividing, often performing specialized functions for an extended period (Figure 3). Progression through the cell cycle is under tight control by three checkpoints. The first checkpoint is the G1/S checkpoint(also known as the restriction point) which controls entry into S phase where DNA is replicated. This checkpoint commits the cell to entry into the cell cycle. Passage through this checkpoint is controlled by cyclin-dependent kinases (CDKs). CDKs are serine-
Cell Cycle Control 4 threonine kinases that promote cell cycle progression and cellular proliferation by phosphorylating specific target proteins. CDKs are active only when bound by a specific cyclin. Different combinations of CDKs and cyclin proteins are critical to cell cycle control. Cyclin protein levels vary during specific stages of the cell cycle, thus conferring cell cycle regulation on CDK activity. CDKs are also subject to negative regulation by cyclin-dependent kinase inhibitors (CDKIs or CKIs). Examples of CDKIs are INK4, p21, and p16. These inhibit cell cycle progression by blocking CDK activity. In addition to CDKs and CDKIs, important regulators of the G1/S checkpoint include the retinoblastoma protein (Rb), the E2F transcription factors and p53. The G2/M checkpointis crucial for ensuring that a cell only enters mitosis (M phase) when its DNA is fully replicated and undamaged. The regulation of this checkpoint is controlled by several key factors including CDKs, CDKIs (p21), regulatory kinases (wee kinase) and phosphatases (cdc25), the checkpoint kinases Chk1 and Chk2, p53 protein (a transcription factor that regulates the expression genes involved cell cycle arrest, DNA repair and apoptosis) and the ATM kinase. The M/G1 checkpoint also known as the spindle assembly checkpoint or the mitotic exit checkpoint, ensures that all chromosomes are properly aligned and attached to the spindle apparatus before the cell proceeds from mitosis to the G1 phase. Key regulators of this checkpoint include the anaphase promoting complex (APC), cdc20 (an activator of APC), the mitotic checkpoint complex (MCC) and the aurora kinase that is critical for chromosome separation and cytokinesis. DNA Damage Checkpoint. DNA damage is a crucial regulator of cell cycle progression. The occurrence of DNA double-strand breaks (DSBs) triggers a rapid signaling response mediated by the checkpoint protein kinase ataxia-telangiectasia mutated (ATM). Activated by the DNA damage sensor complex MRN (composed of MRE11, RAD50, and NBS1), ATM phosphorylates key targets, including the protein kinase CHK1 and CHK2 which then phosphorylate the transcription factor p53. This phosphorylation stabilizes p53 by dissociating it from MDM2, preventing its ubiquitination and degradation. Accumulated p53 then promotes transcription of the CDKI p21, which inhibits cyclin-CDK complexes in G1, halting the cell cycle to prevent entry into S phase. ATM, along with ATR (ATM and Rad3-related), plays essential roles in maintaining genome stability by ensuring that cells exit the cell cycle until DNA damage is repaired. The activation of Chk1 and Chk2 kinases further reinforces p53's role in cell cycle arrest and supports the transcription of DNA repair genes such as BRCA1, BRCA2, MLH1, and MSH2. Mutations in these caretaker tumor suppressor genes can impair DNA repair mechanisms, leading
Cell Cycle Control 5 to an increased mutation rate in oncogenes and tumor suppressor genes, thereby promoting cancer development. This coordinated response is vital for preserving genomic integrity and preventing the propagation of damaged DNA. 5. Growth factor signaling pathways Growth factor signaling pathways are crucial in regulating cell growth, survival, and differentiation. These pathways are characterized polypeptide growth factors that act as ligands for extracellular cellular receptors. The receptors then activate downstream kinase cascades, including the MAP kinase pathway, phospholipase C pathway and PI3K-AKT-mTOR pathways. Activation of these kinase cascades transmits signals from the cell surface to the nucleus, promoting cell division and survival. Aberrations in these pathways can lead to the persistent activation of growth signals even in the absence of growth factors, driving the uncontrolled proliferation of cells. A. The Receptor Tyrosine Kinase Gene Superfamily Receptor tyrosine kinases (RTKs) are the largest family of enzyme-linked receptors. These membrane-spanning proteins, upon binding extracellular ligands like growth factors, activate their intracellular kinase domains, leading to the phosphorylation of tyrosine residues. These phosphorylated tyrosines create binding sites for proteins containing SH2 (Src Homology 2) domains, which recognize and bind to specific phosphotyrosine motifs. This interaction activates downstream signaling pathways that regulate cell growth, differentiation, and survival. Receptor tyrosine kinases (RTKs) have a characteristic structure that includes an N-terminal extracellular ligand-binding domain, a single transmembrane domain, and a cytoplasmic C-terminal domain with protein tyrosine kinase activity (Figure 5). Upon ligand binding to the extracellular domain, RTKs form stabilized dimers, activating the receptor's inherent kinase activity. This activation leads to "autophosphorylation" or "cross-phosphorylation" on specific tyrosine residues of the receptor dimers. These phosphorylated tyrosines increase the receptor's kinase activity and create binding sites for specific proteins involved in multiple intracellular signal transduction pathways. Downstream Signaling Pathways Activated by RTKs The phosphorylated tyrosine residues on activated RTKs serve as docking sites for various adaptor proteins and enzymes, activating key signaling cascades that control cell growth. The most relevant pathways for growth control are the MAPK/ERK pathway, PI3K/AKT pathway, and the PLCγ pathway.
Cell Cycle Control 6 MAPK/ERK pathway is a crucial signaling cascade involved in regulating cell growth, proliferation, and the cell cycle. Upon activation, EGFR phosphorylates itself and recruits the binding of Grb-2 and RAS GEF (guanine nucleotide exchange factor). The docking of a GEF to an activated RTK allows the GEF to stimulate the exchange of GTP for a GDP bound to ras, a monomeric G protein. This pathway operates for many RTKs leading to the activation of specific members of the Ras family. In the case of EGF, activation of RAS leads to activation of RAF kinase, a member of the MAPKKK family. RAF activation leads to the sequential phosphorylation and activation of MEK, and ultimately ERK (Figure 6). Activated ERK translocates to the nucleus, where it regulates the expression of genes essential for cell cycle progression, such as cyclins and growth factors. The MAPK/ERK pathway's precise control ensures proper cell division and growth, with dysregulation often linked to cancer development. MAP kinase pathways: Humans express multiple distinct MAP kinases cascades. These pathways all share three sequential kinases (MAPKKK, MAPKK, MAPK) that transduce extracellular signals via sequential phosphorylation events. These pathways regulate critical processes such as cell growth, differentiation, and survival, with the MAPK/ERK pathway being a key example. Termination of RTK Signaling: RTK-initiated signaling is terminated by protein tyrosine phosphatases and by endocytosis and degradation of ligand-receptor complexes. Protein tyrosine phosphatases (PTPs) remove phosphates from phosphotyrosine residues, reversing the effect of protein tyrosine kinases. They are found in both soluble and membrane-bound forms (i.e. PTP receptors). The Phospholipase C-γ (PLC-γ) pathway plays a significant role in cell cycle control by mediating signals from growth factor-RTKs that influence cellular proliferation. Upon activation, the phosphor-tyrosines on the RTKs recruit PLC-γ to the plasma membrane, where it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2). Hydrolysis of PIP2, a lipid component of the plasma membranes, generates two key second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of calcium ions from intracellular stores, while DAG activates protein kinase C (PKC). These second messengers regulate various downstream effectors involved in the progression of the cell cycle, particularly through the G1 phase, by influencing gene expression, protein activity, and cell growth. This pathway is crucial for ensuring proper cell cycle progression and, when dysregulated, can contribute to uncontrolled cell
Cell Cycle Control 7 proliferation and cancer. Non-Receptor Tyrosine Kinases: Some cell surface receptors (e.g. Cytokines - proteins produced by immune cells that influence immune responses) contain an intracellular domain that lacks intrinsic enzyme activity. Like RTKs, these receptors contain an extracellular ligand binding domain and a transmembrane domain. However, unlike RTKs, cell surface receptors of this type act by stimulating intracellular non- receptor tyrosine kinases that associate with them upon ligand binding. Examples of non-receptor tyrosine kinases are the Janus Kinases (JAKs) and the Src Family Kinases (such as Src, Lck, and Fyn). IL-2, IL-12, interferon alpha and gamma are a few examples of cytokines that activate non-receptor tyrosine kinase pathways. 6. EGFR in Oncogenesis and Cancer Therapy Humans express a family of related growth factors known collectively as the epidermal growth factor (EGF) family proteins. These proteins are synthesized as transmembrane precursors that can signal between attached cells (juxtacrine signaling) or they can be cleaved to release soluble forms that can affect neighboring cells (paracrine signaling). Humans express four receptors for the EGF family of ligands; all are RTKs. They are designated in humans as HERs (Human Epidermal Growth Factor Receptors, i.e., hEGFR, HER2, HER3 and HER4). HER2 (ErbB) Receptors and Cancer: Mutations in the HER genes that lead to overexpression or constitutive activation are found in many human cancers. These contribute to tumorigenesis by enhancing the cells' response to growth factors or making their enhanced proliferation independent of ligand occupancy by the receptors. For example, in breast and ovarian cancers overexpression of HER2 (also known as ErbB) is associated with more aggressive and highly metastatic cancers. Mutations have been found in both the extracellular domain and the catalytic domain of HER2. EGFR-based therapeutics: EGFR family members are approved targets for cancer pharmacotherapy utilizing either small molecules or biologics. For example, trastuzumab(Herceptin®) is a monoclonal antibody directed against HER2, which is used in women with HER2-overexpressing breast cancers. Other anti-EGFR receptor pharmacotherapies for specific cancers include cetuximab(Erbitux®), a monoclonal antibody directed against EGFR, and gefitinib(Iressa®), an EGFR tyrosine kinase inhibitor. Antibodies that bind the extracellular domain of EGFRs (like trastuzumab and cetuximab) block growth factor-receptor interactions and induce immune responses that trigger cell lysis and cell death. They also inhibit the tyrosine kinase activity of EGFRs. Gefitinib is an ATP analogue that competes for ATP binding and thereby blocks EGFR tyrosine kinase activity. Gleevac(Imatinib®) is another ATP analogue used in cancer pharmacotherapy that inhibits the activity of some RTKs and non-receptor tyrosine kinases. 7. Cell Cycle – Out of Control Cell number, size, and shape must be precisely controlled to form the major organs in the body. Cell senescence and death, after a limited number of divisions, is a built-in mechanism in cells. This regulation is lost in cancer cells; cells escape from a limited lifespan and grow in an uncontrolled manner. The cancerous phenotype is not the result of a single defect but results from
Cell Cycle Control 8 the accumulation of abnormalities in multiple cellular regulatory pathways. Cancer cells invade normal tissue, eventually proliferating throughout the body. Mutations confer selective advantages on cancer cells such as: • Uncontrolled proliferation • Reduced requirement for growth factors. • Once a mass of cells forms a tumor, they can secrete angiogenic growth factors that stimulate the formation of new blood vessels supporting tumor growth. • Less restricted by normal cell-cell contact or cell-extracellular matrix interactions and can invade surrounding tissue easily. • Do not respond to apoptotic signals, increasing cellular life span. • Malignant cancer cells secrete proteases allowing invasion of neighboring cells. • Cancer cells generally avoid immune surveillance. A summary table of genes that are often involved in loss of growth control and oncogenesis is provided at the end of this chapter (Table 1). 8. Oncogenes and Proto-oncogenes:Oncogenes are mutated or abnormally expressed forms of normal cellular genes known as proto-oncogenes, which normally regulate cell growth and proliferation. When these proto-oncogenes undergo mutations or misexpression, they become oncogenes that promote uncontrolled cell proliferation, contributing to cancer development. These mutations are dominant, meaning that alterations in only one copy of the gene can disrupt normal cellular growth control. Oncoproteins, the products of oncogenes, often have gain-of-function mutations that enable them to drive cell proliferation more effectively. Viral oncogenes arise when a virus incorporates a proto-oncogene into its genome, leading to its overexpression under the control of the viral promoter. Examples of oncogene activation include the Bcr-Abl fusion gene in chronic myelogenous leukemia, the translocation of the MYC proto-oncogene in Burkitt's lymphoma, and Her2 gene amplification in higher-grade breast tumors. 9. Tumor suppressor genesTumor suppressor genes (TSGs) are crucial antiproliferation genes that work by repressing or interfering with proteins necessary for cell cycle progression. When TSGs are mutated, they can contribute to cancer development, but both copies of a TSG in a cell typically need to be functionally lost for the cell to transform into a cancerous state. TSGs can be altered by various mechanisms, including deletions, point mutations, chromosomal rearrangements, and epigenetic changes such as promoter methylation. These alterations disable the tumor-suppressing functions of the genes, thereby removing critical regulatory controls on cell growth and allowing unchecked proliferation, which can lead to cancer. The RB Tumor Suppressor Gene:Retinoblastoma is an aggressive childhood cancer of the retina and occurs in 1/20,000 live births. It is caused by mutations or deletions in the RB gene and can
Cell Cycle Control 9 be sporadic (60%) or hereditary (40%). It can occur in one or both eyes; if it is bilateral, it is always hereditary. Inherited retinoblastoma is an autosomal dominant disorder, i.e., a person carrying a mutation in one RB allele is highly likely to acquire a somatic mutation or epigenetic change in the other allele (loss of heterozygosity, LOH). In hereditary retinoblastoma, LOH frequently occurs due to mitotic recombination or missegregation of chromosomes. The Rb protein is regulated through phosphorylation by CDKs. The Rb protein (pRb) 105 kDa nuclear phosphoprotein expressed in every cell. The Rb protein regulation of cell growth at the G1/S checkpoint is shown in Figure 6. In early G1, hypophosphorylated pRb binds to transcription factor E2F, inhibiting gene expression from E2F-responsive genes and blocking cell cycle progression.Hyperphosphorylation of pRB causes release from E2F and induction of the E2F-responsive gene. The phosphorylation of pRB is controlled by the activity of CDK4.The p53 Tumor Suppressor Gene:p53 is encoded by the TP53 gene and is mutated in a majority of human cancers. Certain point mutations in p53 convert it from a tumor suppressor to a dominant-acting or gain of function oncoprotein. p53 is a transcription factor that upregulates genes encoding cell cycle regulatory proteins and pro-apoptotic factors. p53 induces expression of p21Cip, a cell cycle inhibitor (Fig. 6). The activity of p53 is counteracted by Mdm2; binding of the Mdm2 oncoprotein to p53 results in ubiquitination of p53 followed by its degradation (Figure 4). p53 activity is induced by DNA damage and cell stress, resulting in cell cycle arrest and/or apoptosis to protect the organism. Cells with a mutant p53 protein continue to replicate damaged DNA and do not undergo apoptosis. MDM2, a critical regulator of p53 function, is itself an oncogene that is frequently amplified in sarcomas. The NF1 Tumor Suppressor Gene:The neurofibromatosis type 1 (NF1) gene is a large gen (spans 350 kb) that encodes the neurofibromin tumor suppressor protein. Mutations in both copies of the NF1 gene within Schwann cells lead to the formation of neurofibromas, which are benign tumors. However, 10-15% of these neurofibromas can progress to malignant peripheral nerve sheath tumors. NF1 mutations are also associated with a variety of cancers, including neuroblastoma, melanoma, and cancers of the breast, lung, ovary, and skin. Neurofibromin plays a key role in downregulating Ras protein activity (Figure 5), and when the NF1 gene is inactivated, this regulation is lost, leading to increased cell proliferation and potential tumor formation.
Cell Cycle Control 10 10. Epigenetic regulation of oncogenes and tumor suppressor genes. Epigenetic regulation of chromatin plays an important role in oncogenesis by controlling gene expression without altering the DNA sequence. Chromatin, the complex of DNA and histone proteins, can exist in two main states: euchromatin (loosely packed, active in transcription) and heterochromatin (tightly packed, repressive). Epigenetic modifications, such as DNA methylation and post-translational modifications of histones (e.g., acetylation, methylation, phosphorylation), regulate the dynamic state of chromatin and, consequently, gene activity. In cancer, these regulatory mechanisms become disrupted, leading to the activation of oncogenes (genes promoting cell proliferation) and the silencing of tumor suppressor genes (genes preventing uncontrolled cell growth). For example, methylation of cytosine residues and deacetylation of histones are chromatin modifications associated with gene silencing. Hypermethylation of promoter regions can silence tumor suppressor genes like TP53, while hypomethylation can activate oncogenes such as MYC. Similarly, abnormal histone modifications, such as reduced acetylation, can lead to chromatin compaction and suppression of critical anti-cancer pathways. Additionally, enzymes that modify chromatin, like histone deacetylases (HDACs) and histone methyltransferases (HMTs), are often overexpressed or mutated in cancers, driving aberrant epigenetic landscapes. This dysregulation of chromatin epigenetics facilitates uncontrolled cell growth, evasion of apoptosis, and metastasis, making it a central mechanism in oncogenesis. Understanding these pathways offers potential therapeutic targets for reversing malignant epigenetic states.