Cell Cycle & Cancer Development II PDF

BIOL 2006SEF CELLS IN HEALTH AND DISEASE TOPIC 3: CELL CYCLE AND CANCER DEVELOPMENT II Heidi Wong [email protected] Outline Overview of Cell Cycle Tumor Suppressor Gene (TSG) Knudson’s two-hit hypothesis Retinoblastoma Gene p53 Gene Pro-oncogene & Oncogene RAS Gene...

BIOL 2006SEF CELLS IN HEALTH AND DISEASE TOPIC 3: CELL CYCLE AND CANCER DEVELOPMENT II Heidi Wong [email protected] Outline Overview of Cell Cycle Tumor Suppressor Gene (TSG) Knudson’s two-hit hypothesis Retinoblastoma Gene p53 Gene Pro-oncogene & Oncogene RAS Gene 2 Overview of Cell Cycle 3 4  Basic function of the cell cycle: duplicate the vast amount of DNA in the chromosomes and then segregate the copies into two genetically identical daughter cells. Two major phases of the cell cycle:  Chromosome duplication occurs during S phase (S for DNA synthesis), which requires 10–12 hours and occupies about half of the cell-cycle time in a typical mammalian cell.  After S phase, chromosome segregation and cell division occur in M phase (M for mitosis), which requires much less time (less than an hour in a mammalian cell). M phase comprises two major events:  Nuclear division, or mitosis, during which the copied chromosomes are distributed into a pair of daughter nuclei  Cytoplasmic division, or cytokinesis, when the cell itself divides in two 5 Four phases of the Cell Cycle The eukaryotic cell cycle is traditionally divided into four sequential phases: G1, S, G2, and M. G1, S, and G2 together are called interphase Mitosis and cytokinesis are called M phase interphase might occupy 23 hours of a 24-hour cycle, with 1 hour for M phase Cell growth occurs throughout the cell cycle 6 Interphase the longest stage of the cell cycle broken down into 3 parts: G1 phase, S phase and G2 phase G1 phase As soon as the cell divides, it enters the G1 phase. This is the growth phase. In this part of interphase, the cell synthesizes mRNA and proteins in preparation for subsequent steps leading to mitosis. The chromosomes unwind into chromatin and the cell transcribes DNA into RNA. Different kinds of proteins (organelles and other cell machinery) are formed. Cell double in size. G1 phase is a particularly important regulatory period because it determine the cells to either continued division or exit from the cell cycle. 7  Towards the end of the G1 phase is a checkpoint called the restriction point. If the conditions are favorable and the requirements have been met, the cell will commit to cell division and enter the S phase. If the conditions are not favorable or if the cell does not need to divide, the cell exits interphase and enter the resting phase called G0 phase. Some cells spend little time in the G0 phase because they are constantly dividing (i.e skin cells) while other spend their entire life in the G0 phase and never divide (i.e nerve cells). The two gaps (G1 & G2) provide time for the cell to monitor the internal and external environment to ensure that conditions are suitable and preparations are complete before the cell commits itself to the complex events of S phase and mitosis. 8 S phase This is the replication stage. The cells spends much less time producing proteins and organelles. It focuses on replicating the DNA. The chromosomes are present in only single copies and duplicated only once per cycle. In human, all 46 individual chromosomes are replicated. The original and replicated chromosomes are joined together at the centromere. Once replicated, the individual chromosomes are called chromatids. At this point, the cell contains 46 original chromatids and 46 chromatids to make a combined total of 92 chromatids. 9 G2 phase The second growth phase starts to prepare the cells with newly replicated DNA for entry into the mitosis phase by putting in place the necessary organelles for mitosis. Once the chromosomes are replicated and the chromosomes number doubles, the cell enters the G2 phase. The cell prepares for cell division by making sure to contain enough proteins and organelles. Protein synthesis and organelle production continues. Toward the end of the G2 phase is the G2 checkpoint. At this point, the cell checks for a special protein called mitosis promoting factor (MPF). If the levels are high enough, the cell will move on to the division stage. 10 M phase The M phase consists of mitosis and cytokinesis. Mitosis is the process in which DNA condenses into visible chromosomes, which is followed by the separation of the chromosomes into two identical sets. Mitosis can be broken down into prophase, prometaphase, metaphase, anaphase and cytokinesis. Cytokinesis can be considered as the last phase of mitosis when the two daughter cells separate, each with a nucleus and cytoplasmic organelles. 11  Three regulatory checkpoints 2 3 1 1 2 3 metaphase-to-anaphase Start G2/M checkpoint transition Pass: - sufficient no. of organelles Pass: - completely replicated genome Pass: - attachment of each - large cell volume kinetochore to a spindle fiber - large cell volume Fail: - DNA damage Fail: - DNA damage Fail: - chromatids are not properly assembled on mitotic spindle 12 Cyclin-dependent kinase activation The cell-cycle control system is based on cyclin-dependent kinases (Cdks) that are activated at specific cell-cycle stages by regulatory subunits called cyclins. cyclin: positive regulatory subunit that binds and activates cyclin-dependent kinases, and whose levels oscillate in the cell cycle. cyclin-dependent kinase (Cdk): protein kinase whose catalytic activity depends on an associated cyclin subunit. Cyclin-dependent kinases are key components of the cell-cycle control system. 13  Cell-cycle control system Levels of the three major cyclin types oscillate during the cell cycle (top), providing the basis for oscillations in the cyclin–Cdk complexes that drive cell-cycle events (bottom). In general, Cdk levels are constant and in large excess over cyclin levels; thus, cyclin–Cdk complexes form in parallel with cyclin levels. The enzymatic activities of cyclin–Cdk complexes also tend to rise and fall in parallel with cyclin levels, although in some cases Cdk inhibitor proteins or phosphorylation introduce a delay between the formation and activation of cyclin–Cdk complexes. Formation of active G1/S–Cdk complexes commits the cell to a new division cycle at the Start checkpoint in late G1. G1/S–Cdks then activate the S–Cdk complexes that initiate DNA replication at the beginning of S phase. M–Cdk activation occurs after the completion of S phase, resulting in progression through the G2/M checkpoint and assembly of the mitotic spindle. Anaphase-Promoting Complex (APC) activation then triggers sister-chromatid separation at the metaphase-to-anaphase transition. APC activity also causes the destruction of S and M cyclins and thus the inactivation of Cdks, which promotes the completion of mitosis and cytokinesis. APC activity is maintained in G1 until G1/S–Cdk activity rises again and commits the cell to the next cycle.  Cell cycle regulation by Cyclin dependent kinases (CDKs). Different cyclins bound to different CDKs promote the transition from one cell cycle phase into another. CDK‐dependent phosphorylation of Rb is required to release active E2F transcription factors, which promotes entry into S phase. 15 Tumor Suppressor Gene (TSG) tumor suppressor gene: a gene that encodes a protein that normally restrains cell proliferation or tumorigenesis, such that loss of the gene increases the likelihood of cancer formation Negative regulatory genes whose mutation promotes cancer are called tumor suppressor genes. A mutation in a tumor suppressor gene usually causes a loss of function that is genetically recessive, so that both copies—or alleles—of the gene must be mutated to promote tumorigenesis. regulate cell growth by applying brakes to cell proliferation (Growth Inhibition) failure of growth inhibition is seen in carcinogenesis loss of function of these genes is a key event in carcinogenesis 16 Knudson’s two-hit hypothesis (1971) 17 Knudson A. PLoS Medicine 2(10):e349, 2005 based on epidemiological studies of retinoblastoma (Rb) cases of retinoblastoma a tumor of the retina that occurs both as an inherited disease and sporadically. two inactivating mutations are necessary for tumor development: for Rb, one could be either germline or somatic, the other is always somatic. Knudson suggested that two "hits" to DNA were necessary to cause the cancer. In the children with inherited retinoblastoma, the first mutation in what later came to be identified as the RB1 gene, was inherited, the second one acquired. In non-inherited retinoblastoma, instead two mutations, or "hits", had to take place before a tumor could develop, explaining the later onset. recessive nature of cancer susceptibility 18 Retinoblastoma Gene (Rb Gene) First discovered Tumor suppressor gene Located on 13q14 Retinoblastoma: the most intrusive intraocular cancer due to inactivation of this gene among children Knudson’s two-hit hypothesis Two mutations (hits) involving both the alleles of TSG (Rb gene) is a basic requisite for the development of tumor. Retinoblastoma can occur as hereditary or sporadic form. Tumors associated with Rb Gene mutations Retinoblastoma Osteosarcoma Glioblastomas Small cell carcinomas of lung Breast cancers Bladder cancers 19  Mechanism of Rb protein in cell cycle regulation 1. G1 Phase Checkpoint Control: In the G1 phase of the cell cycle, the Rb protein acts as a key regulator at the G1/S checkpoint. Rb binds to and inhibits the activity of transcription factors known as E2F proteins. E2F proteins are essential for the expression of genes required for DNA replication and cell cycle progression. By binding to E2F, Rb prevents the transcription of genes involved in cell cycle progression, thereby halting the cell cycle at the G1 phase. 2. Release at G1/S Transition: When the cell receives the appropriate signals to proliferate, such as growth factors, cyclin-dependent kinases (CDKs) are activated. CDKs phosphorylate the Rb protein, leading to a conformational change that releases E2F transcription factors. Released E2F proteins can now activate the expression of genes necessary for DNA replication and entry into the S phase of the cell cycle. 20 https://www.researchgate.net/publication/330979595_Myeloid_cell_leukemia-1_dependence_in_acute_myeloid_leukemia_A_novel_approach_to_patient_therapy 3. Role in DNA Replication: In the S phase, the Rb protein continues to play a role in coordinating DNA replication. Rb helps to ensure that DNA replication proceeds accurately and that cells progress through the cell cycle in a controlled manner. 4. Tumor Suppressor Function: The Rb protein acts as a tumor suppressor by regulating cell cycle progression and preventing uncontrolled cell division. Mutations or loss of the Rb gene can lead to uncontrolled cell proliferation and the development of cancer. 21  Hypophosphorylated RB in complex with the E2F transcription factors binds to DNA, recruits chromatin remodeling factors (histone deacetylases and histone methyltransferases), and inhibits transcription of genes whose products are required for the S phase of the cell cycle.  When RB is phosphorylated by the cyclin D– CDK4, cyclin D–CDK6, and cyclin E–CDK2 complexes, it releases E2F. E2F activates transcription of S-phase genes.  The phosphorylation of RB is inhibited by CDKIs, because they inactivate cyclin-CDK complexes. 22  The Rb protein plays a critical role in regulating the cell cycle by controlling the progression of cells from the G1 phase to the S phase. RB exerts anti-proliferative effects by controlling the G1-to-S transition of the cell cycle. In its active form, RB is hypophosphorylated and binds to E2F transcription factors. This interaction prevents transcription of genes like cyclin E that are needed for DNA replication, and so the cells are arrested in G1. Growth factor signaling leads to cyclin D expression, activation of cyclin D–CDK4/6 complexes, inactivation of RB by phosphorylation, and thus release of E2F. The Rb protein regulates the cell cycle by inhibiting the activity of E2F transcription factors in the G1 phase, thereby controlling the progression of cells from the G1 phase to the S phase. This mechanism helps to ensure proper cell cycle progression and prevent aberrant cell division that could lead to tumorigenesis. 23 p53 Gene a tumor suppressor gene ( its activity stops the formation of tumors) located on 17p13, first discovered in 1979 the p53 protein is the product of p53 gene one of the most commonly mutated gene in cancer, e.g breast, colorectal, liver, lung, and ovarian cancers Functions --- Cell cycle arrest --- DNA repair --- Cell apoptosis prevents neoplastic transformation either by cell cycle arrest or by triggering apoptosis. In cases of DNA damage, there is triggering of expression of p53 gene which increases the production of p53 proteins. These proteins prevent cell from entering S phase of cell cycle and allows time for the DNA repair to take place and p53 induces DNA repair genes. If the DNA is repaired, the p53 degrades and the cell cycle continues. If the DNA is not repaired, the p53 induces permanent arrest in the cell or activates pro apoptotic proteins bax and promoting apoptosis. By these mechanisms, it is understood that the p53 conserves the stability of the cell and thus called as “THE GUARDIAN OF THE GENOME” 24  p53-mediated DNA damage response 1. Detection of DNA Damage: Various types of cellular stress, such as DNA damage caused by radiation, chemicals, or errors in replication, can activate signaling pathways that lead to the detection of DNA damage. Sensors like ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-related) are activated in response to DNA damage and initiate a cascade of signaling events. 2. Activation of p53: In response to DNA damage, ATM and ATR phosphorylate and activate the p53 protein. This post-translational modification stabilizes p53, preventing its degradation and allowing it to accumulate in the nucleus. 3. Transcriptional Activation: Once activated, p53 acts as a transcription factor, binding to specific DNA sequences known as p53 response elements in the promoters of target genes. p53 regulates the expression of a wide range of target genes involved in various cellular processes, including DNA repair, cell cycle arrest, apoptosis, and senescence. 4 Pitolli, C.; Wang, Y.; Candi, E.; Shi, Y.; Melino, G.; Amelio, I. p53-Mediated Tumor Suppression: DNA-Damage Response and Alternative Mechanisms. Cancers 2019, 11, 1983. 25 4. DNA Repair: p53 promotes DNA repair by upregulating genes involved in different DNA repair pathways, such as nucleotide excision repair, base excision repair, and homologous recombination. By facilitating DNA repair, p53 helps to maintain genomic stability and prevent the accumulation of mutations that could lead to cancer. 5. Cell Cycle Arrest: p53 induces cell cycle arrest at the G1/S and G2/M checkpoints by activating genes that inhibit cell cycle progression. This allows time for DNA repair mechanisms to fix the damage before the cell continues through the cell cycle. 6. Apoptosis: If the DNA damage is severe and cannot be repaired, p53 can induce apoptosis (programmed cell death) by activating pro-apoptotic genes. Apoptosis eliminates cells with irreparable DNA damage, preventing the propagation of damaged cells that could contribute to tumorigenesis. 26  The p53 protein is a crucial tumor suppressor that plays a central role in the DNA damage response pathway. p53, the central monitor of stress in the cell, which can be activated by anoxia, inappropriate oncogene signaling, or DNA damage. Activated p53 controls the expression and activity of genes involved in cell cycle arrest, DNA repair, cellular senescence, and apoptosis. DNA damage leads to activation of p53 by phosphorylation. Activated p53 drives transcription of CDKN1A (p21), which prevents RB phosphorylation, thereby causing a G1-S block in the cell cycle. This pause allows the cells to repair DNA damage. If DNA damage cannot be repaired, p53 induces cellular senescence or apoptosis p53 plays a critical role in the DNA damage response by coordinating DNA repair, cell cycle arrest, and apoptosis in response to cellular stress. By orchestrating these processes, p53 helps to maintain genomic integrity and prevent the development of cancer. 27 Pro-oncogene & Oncogene proto-oncogene: a gene that when dysregulated or mutated can promote malignancy Proto-oncogenes generally regulate cell growth, cell division, cell survival, or cell differentiation. oncogene: a gene whose protein product promotes cancer, generally because mutations or rearrangements in a normal gene (the proto- oncogene) have resulted in a protein that is overactive or overproduced. Proto-oncogenes can be converted to cancer-causing oncogenes by multiple mechanisms 28 29 Activation Mechanism of Proto-oncogene 30 1. Point Mutation Errors in DNA replication or repair can generate a single base change in the protein-coding sequence of the gene, resulting in a hyperactive protein Many cancer cells, for example, have a single amino-acid change in the GTPase Ras, a positive regulator of many mitogenic pathways. This mutation blocks the GTPase activity of Ras, thereby locking the protein in the active GTP-bound form. Similarly, point mutations in certain protein kinases, such as the tyrosine kinase Src, not only enhance enzymatic activity but also broaden substrate specificity so that the kinase phosphorylates and activates mitogenic proteins that are not normally its targets. 31 2. Gene Amplification Gene amplification—an increase in the number of copies of a gene in the cell, which leads to the synthesis of the gene product in excessive amounts. The mitogenic gene regulatory protein Myc is sometimes overproduced in cancer cells as a result of massive MYC amplification. 3. Translocation or Transposition Errors in DNA repair or chromosome segregation can cause changes in chromosome structure or number, thereby increasing gene copy number. In addition, errors in DNA replication can lead to genetic recombination events that dramatically increase the number of gene copies. 32 RAS Gene RAS is the most commonly mutated oncogene in human tumors Point mutation of RAS is single most common abnormality of human tumors Multiple growth factor (EGF, PDGF) signal transduction pathways depend on RAS Mutated in 15-20% cancer  90% cholangiocarcinoma, pancreatic adenocarcinoma  50% colon, endometrial and thyroid cancers  30% lung and myeloid leukemias 33  GEF= Guanine nucleotide exchange factor GAP= GTPase-activating protein https://www.researchgate.net/figure/Schematic-representation-of-the-mechanism-leading-to-constitutively-active-forms-of_fig5_342818900 34  Ras is a small GTPase. GTP-bound Ras is active and GDP form is inactive. GAPs such as NF1 inactivate and GEFs such as SOS activate Ras. GTPase-activating proteins (GAPs) inactivate Ras by stimulating it to hydrolyze its bound GTP The inactivated Ras remain stightly bound to GDP. GEFs activate Ras by stimulating it to give up its GDP The concentration of GTP in the cytosol is 10 times greater than the concentration of GDP, and Ras rapidly binds GTP once GDP has been ejected. 35 36  The action of Ras proteins involves a complex network of interactions that influence various downstream signaling cascades. 1.Activation: Ras proteins are activated in response to extracellular signals, such as growth factors, hormones, or cytokines, that bind to cell surface receptors. Upon activation, Ras undergoes a conformational change that allows it to bind GTP (guanosine triphosphate), switching it to its active state. 2. Downstream Signaling: Active Ras proteins interact with and activate downstream effector proteins, such as Raf (a serine/threonine kinase), initiating a signaling cascade. 3. MAPK/ERK Pathway Activation: One of the major downstream pathways activated by Ras is the MAPK/ERK pathway. Active Ras proteins stimulate Raf, which in turn activates MEK (mitogen-activated protein kinase kinase), leading to the phosphorylation and activation of ERK (extracellular signal-regulated kinase). 37 4. Gene Expression Regulation: Activated ERK translocates to the nucleus where it phosphorylates transcription factors, leading to changes in gene expression. This results in the upregulation of genes involved in cell proliferation, survival, and differentiation. 5. Cellular Responses: The activation of Ras signaling pathways influences various cellular responses, including: Cell Proliferation: Ras promotes cell division by stimulating the expression of genes that drive cell cycle progression. Cell Survival: Ras activation can enhance cell survival pathways, preventing apoptosis and promoting cell longevity. Differentiation: Ras signaling is involved in regulating cell differentiation processes in various cell types. 38 Classification of Oncogenes 1. Growth factors 2. Growth factor receptors 3. Cytoplasmic signal transduction molecules 4. Nuclear transcription activators 5. Cell cycle regulators 39 40 The relationship between Cell Cycle and Cancer The cell cycle is a tightly regulated process by which cells grow, replicate their DNA, and divide into two daughter cells. It consists of distinct phases, including the G1 (Gap 1), S (DNA synthesis), G2 (Gap 2), and M (mitosis) phases. The cell cycle is controlled by a complex network of molecules, including cyclins, cyclin-dependent kinases (CDKs), and checkpoint proteins. The relationship between the cell cycle and cancer is tightly intertwined. Cancer is a disease characterized by uncontrolled cell growth and division. Dysregulation of the cell cycle is a fundamental feature of cancer development. 41 42 4. Disrupted Cell Cycle Checkpoints: Cell cycle checkpoints are control mechanisms that ensure proper DNA replication, DNA damage repair, and accurate chromosome segregation. Loss or dysfunction of these checkpoints can result in genomic instability and contribute to cancer development. For instance, defects in the G1 checkpoint, controlled by the RB1 tumor suppressor, can allow cells with DNA damage or other abnormalities to enter the cell cycle and divide. 5. Genomic Instability: Dysregulation of the cell cycle can lead to genomic instability, which is a hallmark of cancer. Errors in DNA replication, impaired DNA repair mechanisms, and chromosome missegregation can result in the accumulation of genetic alterations, including mutations and chromosomal abnormalities. Genomic instability fuels the genetic diversity of cancer cells, facilitating the acquisition of additional mutations that promote tumor progression. 43 Reference Vinay Kumar, Abul K. Abbas, Jon C. Aster, Andrea T. Deyrup. (2022). Robbins & Kumar Basic Pathology (11th ed.). Elsevier. 44  Concept Check (True or False) 1. Mutations in cell cycle regulatory genes can contribute to the development of cancer. 2. The cell cycle is a tightly regulated process that ensures proper cell growth and division. 3. Overexpression of cyclins and cyclin-dependent kinases (CDKs) can promote uncontrolled cell proliferation in cancer. 4. Loss of cell cycle checkpoints can lead to genomic instability and contribute to cancer development. 5. The S phase of interphase is when DNA replication occurs, resulting in two identical copies of each chromosome. 6. p53 gene is called “THE GUARDIAN OF THE GENOME”. 7. Mutations in the p53 gene, which encodes the p53 protein, are commonly found in human cancers. 8. Cytokinesis is the division of the cytoplasm that occurs after mitosis, resulting in two separate daughter cells. 9. The cell cycle progression is primarily controlled by tumor suppressor genes, while oncogenes have little influence. 10. Dysregulation of the cell cycle is a reversible process and can be easily corrected in cancer cells. 45 11. p53 is a tumor suppressor gene. 12. Mutations in p53 can lead to uncontrolled cell division. 13. Rb is an oncogene. 14. Mutations in Rb can result in retinoblastoma only. 15. RAS is a tumor suppressor gene. 16. Mutations in RAS can promote uncontrolled cell growth and division. 17. p53 is frequently mutated in various cancers. 18. Rb is primarily associated with lung cancer. 19. RAS mutations are commonly found in pancreatic cancer. 20. Mutations in RAS can lead to overactive cell signaling pathways. 46 21. p53 acts as a transcription factor to regulate the expression of genes involved in cell cycle control and DNA repair. 22. Rb protein inhibits the activity of transcription factors necessary for cell cycle progression. 23. Mutations in p53 are only acquired through inheritance and cannot be caused by external factors. 24. p53 plays a critical role in the DNA damage response by coordinating DNA repair, cell cycle arrest, and apoptosis in response to cellular stress. 25. The loss or inactivation of p53 function is associated with a higher risk of developing cancer. 26. Rb is involved in regulating the transition from the G1 phase to the S phase of the cell cycle. 27. Mutations in RAS can lead to abnormal cell proliferation and tumor formation. 28. Both p53 and Rb are involved in preventing the formation of cancerous cells. 29. RAS mutations are more commonly found in breast cancer than in colon cancer. 30. The p53 protein can induce cell cycle arrest to allow for DNA repair. 47