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Increase, Mutations leads to Increase, Cell Proliferation
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Direct deposition of ionizing energy leading to lung cancer||adjacent||High||Low||Vinita Chauhan (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
Mutations are defined as changes in the DNA sequence, which could occur in the form of deletions, insertions, missense mutations, nonsense mutations or frameshift mutations (Bertram, 2001; Danesi et al., 2003; Lodish, 2000). Elevated mutation frequencies may impact cellular activities by activating or inhibiting essential processes that control the natural course of cell proliferation (Bertram, 2001; Vogelstein and Kinzler, 2004; Lodish, 2000). Increased rates of cellular proliferation may arise due to mutations that activate proto-oncogenes, which results in sustained signaling for cell growth (Bertram, 2001; Vogelstein and Kinzler, 2004; Larsen and Minna, 2011; Lodish, 2000) and due to mutations that inactivate tumour suppressor genes (TSGs), resulting in the removal of cell cycle inhibition and/or decreased cell death signaling (Bertram, 2001; Vogelstein and Kinzler, 2004; Lodish, 2000). Mutations altering gene expression or protein activity can enable cells to escape growth inhibition by increasing resistance to apoptosis, or other inhibitory signals, or by escape of cell cycle checkpoints. Alternatively, mutations can stimulate growth by activating proliferative pathways such as EGFR.
Evidence Supporting this KER
There is a strong biological plausibility for a relationship between increasing mutation frequencies and increasing cellular proliferation. This relationship is especially evident when examining the molecular biology of carcinogenesis. It is well-known that exposure of cells to a DNA-damaging agent, such as ionizing radiation, may result in damage to the DNA that manifests as genomic instability, including mutations. If enough mutations accumulate in critical genes, cells may begin to proliferate uncontrollably. This, alongside other events, may eventually result in tumourigenesis and cancer (reviewed in Bertram, 2001; Vogelstein and Kinzler, 2004; Panov, 2005; Lodish, 2000). In fact, one of the hallmarks of cancer is sustained proliferative signalling, and one of the enabling characteristics of this increased proliferation is genomic instability/mutations (Hanahan and Weinberg, 2011).
For a mutation to occur, damaged DNA must be passed on to the next generation (Bertram, 2001). To prevent the propagation of erroneous DNA, there are specific cell cycle checkpoints that must be passed before DNA replication and mitosis can proceed. One of the most important checkpoints for committing to cell proliferation occurs during late G1 (Bertram, 2001; Lodish, 2000). This checkpoint is managed by retinoblastoma protein (RB), transcription factor E2F, and transcription factor p53. In a resting cell, RB is tightly bound to E2F; when growth factor signals are present, proteins are activated that phosphorylate RB, resulting in a conformation change and the release of E2F. This transcription factor then initiates transcription of genes required for DNA synthesis and thus cell proliferation. If there is damage to the DNA, p53 is upregulated and binds to unphosphorylated RB, thereby preventing the dissociation of RB and E2F (Bertram, 2001). This gives the cell enough time to repair the damaged DNA prior to DNA replication, and thus minimizes the propagation of the DNA errors. Existing mutations in the checkpoint genes, however, may compromise this process. For example, if mutations in p53 render it non-functional, damaged DNA will not be stopped at the checkpoint and will continue to be synthesized, despite the damage. Accumulation of mutations in this manner may affect genes that impact cell proliferation rates (Bertram, 2001; Lodish, 2000). There are three categories of genes that, if mutated, may allow for uncontrolled cell proliferation: proto-oncogenes, TSGs, and caretaker/stability genes.
Proto-oncogenes are defined as genes that, when activated, promote cellular proliferation (Bertram, 2001; Lodish, 2000); they have been likened to the gas pedal of the car (Vogelstein and Kinzler, 2004). These genes are particularly dangerous if they are rendered abnormally active by gain-of-function (GOF) mutations; this may result in cellular proliferation being aberrantly activated (Bertram, 2001; Vogelstein and Kinzler,, 2004; Larsen and Minna 2011; Lodish, 2000). Two common examples of mutated proto-oncogenes that contribute to increased cell proliferation rates are EGFR and KRAS. The EGFR gene encodes the epidermal growth factor receptor (EGFR), a trans-membrane protein with tyrosine kinase activity. Binding of growth factors to EGFRs results in receptor dimerization, autophosphorylation, and propagation of pro-proliferative signals to the nucleus (Danesi et al., 2003; Santos et al., 2010; Larsen and Minna, 2011; NIH, 2018 EGFR). KRAS is responsible for making the KRAS protein, which is a G-protein with GTPase activity that is used in the RAS/MAPK signalling pathway. When a signal that promotes cellular growth is detected, KRAS binds to GTP and activates downstream signalling molecules, thus facilitating signal propagation to the nucleus (Adjei, 2001; Panov, 2005; Jancik et al., 2010; NIH, 2018 KRAS). Mutations that render these receptors constitutively active would thus result in increased rates of cellular proliferation (Sanders and Albitar, 2010).
TSGs, which are analogous to the brakes in a car (Vogelstein and Kinzler, 2004; Lodish, 2000), are genes that negatively regulate cellular growth by preventing proliferation and in some cases, promoting apoptosis (Bertram, 2001; Vogelstein and Kinzler, 2004; Panov, 2005; Sanders and Albitar, 2010; Lodish, 2000). Many of the cell cycle checkpoint proteins and proteins controlling cell death are TSGs (Bertram, 2001; Lodish, 2000). Loss-of function (LOF) mutations that result in the inactivation of these TSGs may thus promote cellular proliferation (Bertram, 2001; Vogelstein and Kinzler, 2004; Lodish, 2000). A common example of a mutated TSG is TP53, which encodes the p53 protein. As mentioned above, p53 is a cell checkpoint protein that delays replication when damaged DNA is present; if damage is severe enough, p53 may also activate an apoptotic pathway (Bertram, 2001; Danesi et al., 2003; Panov, 2005; Larsen and Minna, 2011; Lodish, 2000, NIH 2018c). Inactivating mutations in p53 thus allow for unhindered progression through the cell cycle, resulting in higher cell proliferation rates (Danesi et al., 2003).
Finally, caretaker/stability genes encode for proteins involved in the detection, repair and prevention of DNA damage (Vogelstein and Kinzler 2004; Hanahan and Weinberg 2011). Genes involved in mismatch repair (MMR), nucleotide excision repair (NER) and base-excision repair (BER) pathways are examples of caretaker/stability genes (Vogelstein and Kinzler, 2004). Mutations in these genes may compromise aspects of DNA repair—the detection of damage, the initiation of repair, the repair process itself, or the removal of mutagens that could possibly damage DNA—thus allowing for more mutations to accumulate in the genome than usual (Hanahan and Weinberg, 2011). Although all genes may suffer from increased mutation rates when caretaker/stability genes are improperly functioning, mutations in TSGs and proto-oncogenes are the main contributors to increased cellular proliferation (Vogelstein and Kinzler, 2004). Caretaker/stability genes are similar to TSGs in that disruption of both alleles must occur for the gene function to be compromised (Vogelstein and Kinzler, 2004; Hanahan and Weinberg, 2011).
Uncertainties and Inconsistencies
Uncertainties in this KER are as follows:
- The location of the mutation will be critical in determining the downstream effects. This can also be modulated by an individual’s susceptibility (Loewe and Hill 2010).
- Although activating mutations in oncogenes such as RAS and MYC may induce abnormally high rates of cellular proliferation, extremely high levels of these proteins may actually lead to the opposite—cells may enter into a state of senescence and cease proliferation (Hanahan and Weinberg 2011).\
- Cellular proliferation may be impacted by circadian cycles, such that disruptions to this natural circadian rhythm may also affect the cell cycle (Shostak 2017).
Data establishing a response-response relationship between mutation frequency and cellular proliferation was not identified. More research is required to establish the response-response relationship between these two events.
Although the time scale is not well-established for this KER, there are a few studies that have examined how cellular proliferation changes overtime in the presence of mutations. In Cul9-/- mouse embryonic fibroblasts, a higher proliferation rate relative to Cul9+/+ cells was evident by 3 days in culture (Li and Xiong 2017). A similar relationship was observed in mouse embryonic fibroblasts with p53 manipulations. Increased proliferation in p53-/-, p53 515A/+ and p53 515A/515A relative to p53+/- and p53+/+ cells was present by the fourth day in culture (Lang et al. 2004). Examination of population doublings in various cell lines found that Cul9-/- and Cul9 mutant cells had higher population doublings than wild-type cells by approximately passage 7; Arf-/-, p53-/-, and Cul9-/-p53-/- cells, however, displayed even higher rates of population doublings by passage 6 (Li and Xiong 2017). Additionally, tumour growth in mice inoculated with lung epithelial cells engineered with LT (suppresses p53 and pRB) and an activated oncogene (either EGFR or KRAS) was monitored over 40 days post-injection. Relative to mice inoculated with either LT-lung epithelial cells or activated oncogene-lung epithelial cells, mice inoculated cells containing both mutations had detectable tumours by approximately day 10 - 12 post-injection; the volumes of these tumours continued increasing until the end of the experiment (Sato et al. 2017).
There were also differences in the rate of DNA synthesis over time, which could possibly indicate higher rates of cell division. In all cell types examined (p53-/-, p53+/- and p53+/+, p53 515A/+, and p53 515A/515A), DNA synthesis declined over the first 6 days in culture, though the mutant p53 lines always had higher synthesis rates than p53-/-, p53+/- and p53+/+ cells. During culture days 6 - 10, DNA synthesis in the mutant p53 lines drastically increased, while the other p53 lines remained at the same relatively low level of synthesis (Lang et al. 2004).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Proliferation increases the likelihood that existing DNA damage will result in mutation and creates new mutations through errors in replication.
It is generally accepted that proliferation increases the risk of mutation and cancer (Preston-Martin, Pike et al. 1990). DNA damage that has not been completely or correctly repaired when a cell undergoes mitosis can be fixed in the genome permanently as a mutation, to be propagated to future daughter cells. Incomplete DNA repair can also cause additional DNA damage when encountered by replicative forks. Therefore, in the presence of any DNA damage (and there is a background rate of damage in addition to any other genotoxic stimuli) mutations will increase with cell division (Kiraly, Gong et al. 2015). Mutation-prone double strand breaks can also arise from replicative stress in hyperplastic cells including hyperplasia arising from excess growth factor stimulation (Gorgoulis, Vassiliou et al. 2005). This relationship between proliferation and mutation is thought to drive a significant portion of the risk of cancer from estrogen exposure since breast cells proliferate in response to estrogen or estrogen plus progesterone and risk increases with cumulative estrogen exposure (Preston-Martin, Pike et al. 1990).
Not all proliferating tissue shows replicative stress and DSBs - tissue with a naturally high proliferative index like colon cells don’t show any sign of damage (Halazonetis, Gorgoulis et al. 2008). Additional factors are therefore required beyond replication for damage and mutation from replicative stress, but replication is essential for the expression of these factors.
Domain of Applicability
The domain of applicability pertains to all multicellular organisms, as cell proliferation and death regulate tissue homeostasis (Pucci et al. 2000).
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NIHa, EGFR gene epidermal growth factor receptor Normal. 2018. Genetics Home Reference EGFR gene.
NIHb, KRAS proto-oncogene, GTPase Normal. 2018. Genetics Home Reference KRAS gene.
NIHc, TP53 gene tumor protein p53 Normal. 2018. Genetics Home Reference TP53 gene.
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