Cancer Cell Biology (BC.504) Lec. 3 PDF

# Cancer Cell Biology (BC.504) ## Cell Cycle Control in Cancer (Lec. III) - Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms to ensure the production of two genetically identical cells. - Cell cycle control is focused mainly on two events: the re...

# Cancer Cell Biology (BC.504) ## Cell Cycle Control in Cancer (Lec. III) - Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms to ensure the production of two genetically identical cells. - Cell cycle control is focused mainly on two events: the replication of genomic DNA and its subsequent segregation between daughter cells, which in eukaryotic cells occur during distinct cell cycle phases. - It is often assumed that cancer cells undergo uncontrolled cell cycle progression and that most, if not all, cell cycle checkpoints need to be defective for a cell to become cancerous. - In unicellular and multicellular eukaryotes, cell division is controlled by a complex network of regulatory mechanisms, checks, and balances to ensure that no mistakes are made before a cell is allowed to enter and progress through the cell cycle to divide. ## Cell Cycle Phases - The mitotic cell cycle is divided into two distinct stages: interphase and M phase. - This allows the temporal separation of the duplication of cellular content during interphase and its separation into two genetically identical daughter cells in mitosis. - The complex network of regulatory elements that form the cell cycle has one goal: the timely and accurate duplication and segregation of the genomic DNA. - DNA replication occurs in interphase during S phase (synthesis phase), defined as the time in interphase during which DNA replication is initiated but not completed. - The periods of interphase that separate S phase from M phase have historically been named 'gap phases', or G1 before S phase and G2 after S phase, on the basis of the evident observation that these are gaps in between the two main events, duplication and segregation of the DNA. - However, these phases are key periods for cell cycle regulation and include the crucial decision to enter the cell cycle during G1, and to initiate the process that leads to chromosome segregation during G2. ## Cell Cycle Entry & Progression - The eukaryotic cell cycle is the process during which a cell duplicates its entire cellular content during interphase, and through division in M phase creates 2 genetically identical cells. - Before S phase, in the Pre-replicative G1 phase, there is a decision window during which cells can commit to initiate DNA replication and enter the cell cycle or stay in G1 phase. - During G1 cells can also exit the cell cycle into a non-proliferative state known as quiescence, or G0. - The vast majority of cells in an adult body is in a non-proliferative state and would need to transit into G1 before being able to initiate DNA replication and enter the cell cycle. Upon completion of DNA replication, there is another decision window during the post-replicative G2 phase. - During this window, cells can commit to enter M phase by initiating chromatin condensation and the central alignment of chromosomes. - M phase serves the dual function of both accurately separating the duplicated DNA (mitosis) and dividing the entire cellular content into two new daughter cells (cytokinesis). - During M phase, cells commit to the segregation of the genetic material and reset the cell cycle to return to interphase. ## CDKs and Cell Cycle-regulated Transcription drive Cell Cycle Progression - The key regulator of cell cycle processes is CDK activity. Cell cycle progression is driven by the accumulation of cyclin-dependent kinase (CDK) activity during interphase and M phase. - The mammalian cell cycle is a highly organized and regulated process that ensures duplication of genetic material and cell division. This regulation involves growth-regulatory signals as well as signals by proteins monitoring the genetic integrity to ascertain the absence of any genetic damage. - The key regulator of cell cycle processes is CDK activity. Specific cyclins accumulate during different stages of the cell cycle, driven by cell cycle-regulated transcription and the inhibition of protein degradation. In turn, cell cycle-regulated transcription depends on CDK activity for activation. - Cell cycle-dependent changes in CDK activity drive cell cycle entry, progression and completion. CDK activity in the pre-replicative G1 phase is required for initiation of DNA replication and thereby commitment to cell cycle entry. - Subsequently, during the post-replicative G2 phase, CDK activity plays a key role in preparing for chromosome segregation, via initiation of condensation, permeabilization of the nuclear envelope and central alignment of the replicated chromosomal DNA. - Inactivation of CDK activity, via the anaphase-promoting complex/cyclosome (APC/CCDC20)-dependent targeted destruction of cyclins, corresponds with chromosome segregation and return to interphase. - There are several stages during the pre-replicative G1 phase. In early G1 phase, the cell faces a 'choice' to either remain in a cell cycle state or exit the cell cycle into quiescence. - Growth-dependent CDK activity (D-type cyclin–CDK) creates a decision window during which the cell can commit to initiate replication and enter a new cell cycle. - Inhibition or absence of this CDK activity can take the cell out of the G1 state and into quiescence. - Commitment to replication initiation and S phase entry is closely linked to activation of the E2F-dependent transcriptional network. The transcriptional network is activated in G1 phase, drives S phase entry and is subsequently inactivated during S phase. ## E2F-dependent transcriptional network - E2F-dependent transcriptional network includes many genes that encode key proteins in cell cycle and DNA replication control but also genome protection mechanisms and growth. - E2F-dependent transcriptional regulation depends on a family of transcription factors (E2F1–E2F8) and their co-regulators the pocket proteins (retinoblastoma protein (RB), p107 and p130). Activation of E2F-dependent transcription is initiated by growth signals and mitogens that stimulate CDK activity through antagonizing CDK inhibitor activity and by the expression of D-type cyclins. - The E2F inhibitor RB plays a central role in the ‘decision' to enter a new cell cycle. - During the pre-replicative G1 phase, RB keeps E2F-dependent transcription inactive. However, RB is inactivated by CDK-dependent phosphorylation as these enzymes become more active, resulting in E2F-dependent transcription. - This results in the expression of E-type cyclins, which further increases the overall activity of CDKs. - The increased CDK activity induces further phosphorylation of RB, which fully inactivates this E2F inhibitor and allows the expression of E2F-dependent genes. This positive feedback loop creates a decision window to enter S phase by driving the accumulation of E-type cyclin and A-type cyclin-CDK activity required to initiate DNA replication and cell cycle entry. - D-type cyclin–CDK might not be involved in the initial inactivation of RB, but instead ‘primes' a cell for entry into the cell cycle by preventing its exit from, or promoting its entry into, G1 phase, potentially by controlling the metabolic state of the cell. - In this model, mono-phosphorylation of RB by D-type cyclin-CDK activity does not activate E2F-dependent transcription but creates a state between cell cycle exit (quiescence) and the cell cycle entry decision window. - During this state, hyper-phosphorylation of RB by E-type cyclin–CDK activity activates E2F-dependent transcription, initiating a positive feedback loop, which drives cells into a decision window to commit to a new cell cycle. - Once the decision to enter a new cell cycle has been made, the initiation of DNA replication and thereby the 'commitment' to initiate S phase depends on A-type cyclin together with CDK2 activity. - Accumulation of A-type cyclin–CDK activity depends not just on E2F-dependent induction but also on the inactivation of the G1 phase-specific APC/C (APC/CCDH1), which targets A-type cyclins for destruction. - Inactivation of the APC/CCDH1 depends on both the E2F-dependent accumulation of E-type cyclin-CDK activity and the APC/CCDH1 inhibitor EMI1. - Only when E2F-dependent transcription is active and APC/CCDH1 is inactive can a cell pass the commitment point. So activation of E2F-dependent transcription creates a decision window where cells can accumulate enough A-type cyclin–CDK activity to initiate replication and commit to a new cell cycle. - CDK activity plays a key role in the separation of licensing and firing of replication origins to ensure that the genome is replicated in a timely manner and occurs ‘once and only once' per cell cycle. - Activation of CDK1 plays a central role in the decision to enter mitosis. The activity of CDK1 is subject to multiple levels of regulation to ensure rapid and timely mitotic entry. - First, CDK1 activation requires association with A-type or B-type cyclins, which gradually accumulate in the cell from S phase onwards owing to cell cycle-specific transcription. Second, CDK1 is maintained in an inhibited state via phosphorylation by the kinases WEE1 and MYT1. Activation is triggered by removal of these inhibitory phosphorylations by the phosphatase CDC25. - The balance between WEE1/MYT1 and CDC25 activity levels ultimately determines when a cell progresses into mitosis and is itself regulated by multiple regulatory circuits. - The steady increase of the levels of A-type and B-type cyclins throughout G2 phase is translated into a fast, bistable switch in CDK1 activity at mitotic entry. Once the threshold levels for CDK1 activity are reached, entry into mitosis is triggered by widespread phosphorylation of more than a thousand CDK1 substrates. - In parallel, CDK1 activates the mitotic kinases PLK1, Aurora A and Aurora B, which phosphorylate additional mitotic substrates. - The wave of mitotic phosphorylation triggers structural changes to every cell compartment and primes the cell for DNA separation and division. - During prophase, rising CDK1 activity in the cytoplasm triggers cell rounding and centrosome separation. At the same time, rapid nuclear import of activated CDK1–cyclin B induces condensation of chromosomes, activation of the APC/C, nucleolar disassembly and permeabilization of the nuclear envelope. - During prometaphase, cyclin A is degraded by the 26S proteasome, following ubiquitylation by the APC/CCDC20. - CDK1 activity remains high, owing to its continued interaction with B-type cyclins. Following chromosome alignment at metaphase, mitotic exit is initiated by the APC/CCDC20, which at the same time promotes ubiquitylation of B-type cyclins and targets them for proteolytic destruction. - As with mitotic entry, exit from mitosis is rapid and irreversible. - APC/CCDC20 activation triggers a chain of events including separation of sister chromatids, spindle elongation to pull them apart and formation of an actomyosin contractile ring to divide the cell into two. - At the same time, destruction of the sole remaining cyclin type (B) reduces CDK activity to zero and effectively resets the cell cycle in the two daughter cells to the pre-replicative G1 phase. ## Cell Cycle Checkpoints & Cell Cycle Control - Cells rely on cell cycle checkpoints to prevent the accumulation and propagation of genetic errors during cell division. - These cell cycle control checkpoints depend on evolutionarily conserved signaling pathways that monitor DNA damage during interphase (DNA damage checkpoint), loss of DNA replication fork integrity during S phase (Replication stress Checkpoint) and incomplete spindle assembly during M phase (Spindle assembly Checkpoint). ## Checkpoint-dependent Cell Cycle arrest & exit - The replication stress checkpoint can block mitotic entry during S phase by preventing the accumulation of cyclin-dependent kinase 1/2 (CDK1/2)—cyclin A/B activity, and the spindle assembly checkpoint can block mitotic exit during M phase by preventing the activation of the anaphase-promoting complex/cyclosome (APC/C). - By comparison, the DNA damage checkpoint operates throughout interphase. - Depending on the phase of the cell cycle, the DNA damage checkpoint can either block mitotic entry during and following S phase, much like the replication stress checkpoint, or block replication initiation in pre-S phase by preventing the accumulation of cyclin E/A–CDK2 activity. - It can also block cell cycle entry following mitotic exit, or during a prolonged pre-S phase, by preventing or inhibiting cyclin D–CDK4/6 activity, thereby inducing a reversible cell cycle exit known as quiescence. - In response to high levels of DNA damage, the DNA damage checkpoint can induce an irreversible exit from the cell cycle, through senescence, outside S and M phases, or even cell death through apoptosis throughout the cell cycle. ## Cell Cycle Control in Cancer - Continued cell cycle progression in cancer cells is driven mainly by mutations, or deregulation, of proteins involved in cell cycle-control signaling pathways. - However, these mutations are associated with specific cell cycle control pathways more so than others. Mutations commonly found in cancer cells are shown in red: they affect mainly cell cycle control in response to DNA damage and growth signals in pre-S phase. - These mutations drive S phase entry and prevent cell cycle exit. Very few cancer-associated mutations are found in proteins involved in the response to replication stress or incomplete spindle assembly; proteins that are rarely mutated in cancer are shown in blue. - In the context of cancer treatment, these pathways represent therapeutic opportunities. Pocket proteins include retinoblastoma protein (RB), p107 and p130. E2F includes the activating E2Fs E2F1-E2F3 and indicates E2F-dependent transcription. - G1/S cyclin-dependent kinases (CDKs) include cyclin D-CDK4/6 and cyclin E-CDK2. M phase CDKs include cyclin A/B–CDK1/2. ## Sustained proliferative signaling - Sustained proliferative signaling, which drives continuing and excessive rounds of cell division, is the hallmark of cancer. - Recent insight has revealed that this continuous cell division is driven by mutations that both prevent apoptosis and compromise cell cycle exit, rather than driving uncontrolled cell cycle progression. - These include mutations in the signaling pathways that initiate exit from the cell cycle or promote S phase entry, but much less frequently in those that prevent mitotic entry and exit. - Cells can exit the cell cycle either reversibly, through initiating quiescence, or non-reversibly, by senescence or apoptosis. - The decision to exit the cell cycle depends on just one of the cell cycle checkpoints—the DNA damage checkpoint. - Throughout interphase, in response to irreparable DNA damage, the DNA damage checkpoint can initiate quiescence, senescence or programmed cell death largely through p53-dependent pathways. - Unsurprisingly, p53 mutations are the most common mutations found in cancer. - Even if cancer-associated mutations prevent cell cycle exit, continuous proliferation can still be prevented by blocking cell cycle entry in the pre-replicative G1 phase, which depends on activation of E2F-dependent transcription. - In line with this, cancer-associated mutations in this pathway have been found in all types of cancer, and include mutation in many oncogenes and tumor suppressors. These mutations induce E2F-dependent transcription, promoting S phase entry and compromising the ability of a cell to exit the cell cycle in the pre-replicative phase. ## DNA Repair Mechanisms in Cancer initiation and Development - Living organisms have the crucial task to preserve their genome and faithfully transmit it across generations. - Transmission of genetic information is constantly in a selective balance between the maintenance of genetic stability versus elimination of mutational change and loss of evolutionary potential. - The DNA molecule is under the continuous attack of a multitude of endogenous and exogenous genotoxic insults and it has been estimated that every cell experiences up to 105 spontaneous or induced DNA lesions per day. - Endogenous damage can result from DNA base lesions like hydrolysis and alkylation (6-O-Methylguanine) or oxidation (8-oxoG) by intracellular free radical oxygen species (ROS) that can occur as by-products of mitochondrial respiration. - Mutations can also arise during normal cellular metabolism for instance by erroneous incorporation of deoxyribonucleotides (dNTPs) during replication. - Environmental sources of damage can be physical [e.g., ultraviolet (UV) light, ionizing radiations (IRs), and thermal disruption] or chemical (drugs, industrial chemicals, and cigarette smoke). ## DNA Repair Pathways - DNA damage is caused by endogenous agent oxygen species (ROS) or exogenous agents such as UV light, IR and chemotherapy agents. DNA damage response (DDR) is induced to deal with the lesions, including signal transduction, transcriptional regulation, cell-cycle checkpoints, induction of apoptosis, multiple DNA repair pathways as well as damage tolerance processes. - DNA repair pathways include nuclear and mitochondrial DNA repair pathways. Direct repair, BER, MMR and recombinational repair (HR and NHEJ) are existence in both nuclear and mitochondrial repair systems. NER has been reported only appearance in nucleus. - In response to Catastrophic levels of DNA damage, the DNA damage checkpoint can induce cell death through apoptosis throughout the cell cycle. ## Table 1 - Base excision repair (BER) : Oxidative lesions - Reactive oxygen species (ROS) - Nucleotide excision repair (NER): Helix-distorting lesions - UV radiation - Translesion synthesis : Various lesions - Various sources - Mismatch repair (MMR): Replication errors - Replication - Single strand break repair (SSBR) : Single strand breaks - Ionizing radiation, ROS - Homologous recombination (HR) : Double-strand breaks - Ionizing radiation, ROS - Non-homologous end joining (NHEJ): Double-strand breaks - Ionizing radiation, ROS - DNA interstrand crosslink repair pathway: Interstrand crosslinks - Chemotherapy - DNA repair pathways. MMR, mismatch repair; TLS, trans-lesion DNA repair; RR, recombination repair; NHEJ, non-homologous end joining; HR, homologous recombination. ## Key Signaling Pathways involved in cell cycle control & Cancer - APC/C, anaphase-promoting complex/ cyclosome; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; MCC, mitotic checkpoint complex (BUB3 together with MAD2 and MAD3 bound to CDC20); RTKs, receptor tyrosine kinases. ## When DNA damage persists and interferes with replication or transcription - When DNA damage persists and interferes with replication or transcription, DNA damage checkpoints trigger cellular senescence or apoptosis that inactivate or eliminate damaged cells and thus suppress tumorigenesis (gray). DNA repair mechanisms prevent cancer by preventing mutations. Chemo-and radio therapy often inflict DNA damage to halt cancer cell proliferation or trigger the apoptotic demise of cancer cells.