Increase, Mutations leads to Increase, Cell Proliferation

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).

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. Although the majority of DNA damage is addressed through the activation of repair mechanisms, if the cells fail to prevent DNA synthesis prior to repairing DNA damage (eg. ATM mutant cells), erroneous repair accumulates which could lead to the activation of cell proliferation or cell death (Levine and Holland, 2018). 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; Fernandez-Antoran et al., 2019).

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).

There is moderate empirical evidence supporting the relationship between mutations and the cellular proliferation. The evidence presented below is summarized in table 7, here (click link). There are some available reviews that provide evidence for this relationship in the context of carcinogenesis (Welcker 2008, Kim 2018, Iwakuma 2007, Muller 2011), as one of the hallmarks of this disease is high levels of cellular proliferation (Hanahan and Weinberg 2011). Another review article explores the relationship between mutation accumulation and cellular proliferation through discussion of the stem cell division theory of cancer, and how it compares to the somatic mutation theory of cancer (López-lázaro 2018).  Overall, however, there is little empirical evidence available supporting dose and incidence concordance, little empirical evidence supporting temporal concordance, and strong empirical evidence supporting essentiality for this KER.  Some evidence from human epidemiology association and genetic studies also provides support for this KER.

Dose and Incidence Concordance

There are few studies available that assess the dose and incidence concordance between mutations and cell proliferation. One study providing dose information on this particular relationship analyzed the effect of sequentially adding mutations to mouse lung epithelial cells. Addition of mutations in the form of LT (suppression of p53 and pRB) or Kras(G12V) (an activated oncogene) on their own to lung epithelial cells did not increase tumour volume, but a combination of these genetic manipulations resulted in increasing tumour volume (suggestive of increased cell proliferation) over 40 days. The same results for LT and EGFR(ex19del) genetic manipulations were also achieved. This suggests that addition of multiple mutations increases cell proliferation (Sato et al. 2017). More studies, however, are required to directly assess this particular aspect of the relationship between mutations and cellular proliferation.

Time Concordance

Few studies are available that study the time concordance between mutations and cell proliferation. The timing between these two events is explored in a review that discusses theories of carcinogenesis. The somatic mutation theory of cancer states that accumulation of mutations results in higher rates of cellular proliferation, which eventually leads to cancer. A component of the stem cell division theory of cancer also states that an increased mutation burden may elevate rates of stem cell divisions in late carcinogenesis; however, a high frequency of stem cell division in the initial stages of cancer development is thought to be a key factor that contributes to mutation accumulation (López-lázaro 2018). More research is thus required to definitively determine whether mutations occur prior to increased rates of cellular proliferation.

Essentiality

There is strong evidence for the essentiality component of this KER. Numerous studies indicate that cellular proliferation is increased in biological systems with genetically manipulated TSGs and/or proto-oncogenes. It is important to note that uncontrolled cellular proliferation is a hallmark of human cancers (Hanahan and Weinberg 2011); the Catalogue of Somatic Mutations in Cancer (COSMIC) includes over 136,000 coding mutations in over 500,000 tumour samples (83 major cancer genes and 49 fusion gene pairs) and this number is continually increasing (Forbes et al. 2011). The managers of COSMIC note that key amongst all of these genes is TP53. Several review articles that focussed on genetic manipulations of TP53 demonstrated that mutant or knocked-out p53 increased carcinogenesis across a variety of biological systems (Iwakuma and Lozano 2007; Muller et al. 2011; Kim and Lozano 2018). Furthermore, a number of studies that measured cellular proliferation directly found that both cells and mice lacking p53 had increased rates of cell proliferation (Hundley et al. 1997; Lang et al. 2004; Ventura et al. 2007; Duan et al. 2008; Li and Xiong 2017), in addition to modifications to the cell cycle such that more cells were found in the S- and G2/M phases and less in the G1 phase (Hundley et al. 1997).  Some p53 mutations, including 515A, may also result in increased cellular proliferation (Lang et al. 2004). Further underlining the importance of p53 in controlling cellular proliferation, restoration of p53 in a p53-/- mouse model resulted in a significant size reduction in 7 out of 10 tumours, with some tumours disappearing altogether (Ventura et al. 2007).  

Manipulations to other genes have also been shown to affect cellular proliferation. A review article centred on the tumour suppressor FBW7, which is a ubiquitin ligase that plays a role in degrading proto-oncogene products and thus controlling cellular proliferation, demonstrated that mutations to FBW7 may contribute to carcinogenesis (Welcker and Clurman 2008). Knock-out of prostate SPOP (an E3 ubiquitin ligase adaptor commonly mutated in primary prostate adenocarcinoma) in Spop^fl/fl;PBCre(+) mice resulted in prostates with significantly higher masses, significantly more cellular proliferation, and increased expression of c-MYC protein relative to prostates from Spop^fl/fl;PBCre(-) controls with normal prostate SPOP expression. Furthermore, there was a strong inverse correlation between c-MYC activity and SPOP mRNA levels in two independent prostate cancer patient cohorts, suggesting that c-MYC upregulation in the absence of SPOP may be responsible for the increased cellular proliferation (Geng et al. 2017). Similarly, mouse embryonic fibroblasts lacking Cul9, a scaffold protein for assembly of E3 ubiquitin ligases, had an increased cellular proliferation rate and an increased number of cells in the S-phase of the cell cycle relative to wild-type controls. Cul9 mutant cells also showed similar cellular proliferation rates to Cul9-/- cells. In contrast, Arf-/- cells, p53-/- cells, and Cul9-/-p53-/- double knock-out cells had significantly higher cellular proliferation rates relative to the Cul9-/- and Cul9 mutant cells; all of these mutant cells, however, showed increased proliferation relative to wild-type cells (Li and Xiong 2017).

Inhibitor studies further highlight the role of mutations in increasing cellular proliferation. Mouse lung epithelial cells transformed with both Large T-antigen (LT; suppresses TSGs p53 and pRB) and activated oncogene Kras(G12V) or EGFR(ex19del) resulted in increased tumour volumes, which is suggestive of cell proliferation. Increasing concentrations of MEK inhibitor, which blocks the signalling pathway downstream of both Kras and EGFR, caused declines in cell number in the two transformed cell lines and in the parental lung epithelial cells. An EGFR inhibitor, which blocks signalling downstream of EGFR but upstream of Kras, had no effect on the transformed cells with activated Kras, but caused rapid declines in cell proliferation of transformed cells with activated EGFR. Altogether, these inhibitor studies suggest that the activated oncogene has an important role in promoting high rates of cell proliferation (Sato et al. 2017).

Human epidemiology association and genetic studies

Association studies in humans clearly show the correlation between mutations in specific genes and the proliferative status of human tumours. Human lung adenocarcinoma tumours were assessed for mutational status of KRAS, TP53 and STK11, and cellular proliferation levels were measured in the mutant tumours relative to the wild-type tumours. Overall, mutations in TP53 were associated with significantly increased proliferation levels regardless of the mutational status of KRAS. In contrast, mutations in STK11, either alone or in combination with KRAS mutations, were not associated with increased proliferation (Schabath et al. 2016). Assessment of breast cancer tumours demonstrated that those with low BRCA1 expression displayed increased cellular proliferation relative to those with high BRCA1 expression, as measured by nuclear Ki-67 levels (Jarvis et al. 1998).

Uncertainties in this KER are as follows:

1. 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).
2. 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).\
3. 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).  

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.