Increase, Mutations leads to Increase, Cell Proliferation (Epithelial Cells)

High. Multiple mechanisms limit the proliferation of cells in healthy biological systems. Mutations in many of the genes controlling these mechanisms promote proliferation. Biological mechanisms such as contact inhibition, apoptosis, cell cycle checkpoints, and growth factor availability act to restrain proliferation (Sonnenschein and Soto 1999). Under conditions of proliferation such as ductal branching during development of the mammary gland, selected mechanisms are engaged to permit controlled or directed proliferation. In the case of ductal branching, stromal cells respond to estrogen and growth hormone by releasing IGF1, which activates IGF-1R in epithelial cells to promote survival and proliferation (Hinck and Silberstein 2005; Sternlicht, Sunnarborg et al. 2005; Sternlicht 2006). At puberty, epithelial cells respond to estrogen by signaling to the stroma via EGFR to which the stroma replies with proliferative signals via FGFR (Sternlicht, Sunnarborg et al. 2005; Sternlicht 2006). Multiple additional mechanisms of control include proliferation inhibition by TGF-β, which can both directly inhibit proliferation (Francis, Bergsied et al. 2009) and act through stromal cells to stabilize an inhibitory extracellular matrix (Hinck and Silberstein 2005). When mechanisms controlling proliferation are altered, proliferation can occur outside of the normal biological context (Radice, Ferreira-Cornwell et al. 1997; Davies, Platt-Higgins et al. 1999; Ewan, Shyamala et al. 2002; Lanigan, O'Connor et al. 2007; Croce 2008; de Ostrovich, Lambertz et al. 2008).

Moderate. Mutations that promote proliferation are frequently found in cancers, and both mutation and proliferation occur in response to tumorigenic stressors like ionizing radiation. Mutations appear over the same time frame or prior to the appearance of proliferation. Multiple uncertainties and conflicting evidence weaken this key event relationship. The two key events differ in their dose response- mutation but not proliferation increases with ionizing radiation dose. Furthermore, a single mutation is not necessarily sufficient to increase proliferation- proliferation typically requires multiple mutations or a change in the surrounding environment. In mammary tissue, stromal state – which is modified by hormones - strongly influences the proliferative nature of epithelial cells, and mutated epithelial cells alone appear to be insufficient for tumor growth.

Gene sequencing performed on a wide range of cancers has revealed common mutations (Alexandrov, Nik-Zainal et al. 2013). Some mutations are particularly common in certain cancers, while others are more widely observed. Driver mutations commonly appear in EGFR and other tyrosine kinase signaling pathways (BRAF, EGRF, RAS, PI3K, STK11) and in DNA damage response and cell cycle checkpoint signal pathways (ATM, TP53, CHEK2, CDKN2B (P15), CDK4) (Greenman, Stephens et al. 2007; Croce 2008; Kaufmann, Nevis et al. 2008; Stratton, Campbell et al. 2009; Vandin, Upfal et al. 2012). The same processes are commonly affected in breast cancer. PIK3CA (part of PI3K) is commonly mutated, and HER2 (an EGFR receptor) is amplified in HER2 type cancers. Amplification or mutations of the RB1 checkpoint control pathway and dysfunction in BRCA and homologous recombination related DNA repair processes are also common  (CGAN 2012; Nik-Zainal, Davies et al. 2016; Davies, Glodzik et al. 2017).

While the presence of these mutations in cancers does not prove causality, in vitro studies confirm that these mutations interfere with signals controlling proliferation, and that the mutations increase proliferation, hyperplasias, and tumors (Podsypanina, Politi et al. 2008). Cell cycle regulatory proteins ATM and P53 respond to DNA damage or other growth inhibiting or senescence signals like TGF-β by limiting entry into the cell cycle via RB1 - a process controlled by CHEK2, CDNK2B, CDK4, and other factors - and are also involved in activating apoptosis. Mutations in these checkpoint related genes enable cells to escape mechanisms limiting proliferation in vitro (Tao, Roberts et al. 2011; Higashiguchi, Nagatomo et al. 2016) and mammary hyperplasia in vivo (Francis, Bergsied et al. 2009), and contribute to tumors when combined with other mutations in the same pathway (Francis, Chakrabarti et al. 2011). EGFR, HER2, BRAF, RAS, and PI3K participate in the EGFR (growth factor) signaling pathway. Activating mutations in PI3K generates growth factor independent proliferation of mammary epithelial cells, possibly via the RB1 pathway (Gustin, Karakas et al. 2009). GATA is a transcription factor that maintains luminal epithelial cell differentiation and suppresses proliferation, and mutation results in the proliferation of undifferentiated cells (Kouros-Mehr, Slorach et al. 2006; Shahi, Wang et al. 2017).

While no research directly links specific mutations from IR and chemical DNA-damaging agents with subsequent proliferation and hyperplasia in the same experiment, multiple studies support an increase in proliferation and hyperplasia following exposure to these mutagenic stressors.  Proliferative nodules and hyperplasia appear in mammary terminal end bud, alveolae, and ducts of rats and mice after exposure to chemical carcinogens (Beuving, Faulkin et al. 1967; Russo, Saby et al. 1977; Purnell 1980) and ionizing radiation (Faulkin, Shellabarger et al. 1967; Ullrich and Preston 1991; Imaoka, Nishimura et al. 2006). Based on the time frame of mutation and proliferation measured in different tissue following exposure to IR, mutations precede or occur over the same general timeframe as proliferation and hyperplasia, consistent with a causative mechanism (Ullrich and Preston 1991; Schiestl, Khogali et al. 1994; Sandhu and Birnboim 1997; Wu, Randers-Pehrson et al. 1999; Zhou, Ivanov et al. 2005; Imaoka, Nishimura et al. 2006; Liang, Deng et al. 2007; Ameziane-El-Hassani, Boufraqech et al. 2010; Datta, Hyduke et al. 2012; Snijders, Marchetti et al. 2012; Suman, Johnson et al. 2012; Tang, Fernandez-Garcia et al. 2014; Fibach and Rachmilewitz 2015; Sherborne, Davidson et al. 2015; Zhou, Ma et al. 2015). However, proliferation after IR has also been reported within the first day, before the likely appearance of mutations (Han, Chen et al. 2010; Cho, Kang et al. 2016).

Mutations are clearly not the only events driving proliferation in mammary gland, particularly in female mammary glands after exposure to a stressor like ionizing radiation where proliferation varies with age and microenvironment (Tang, Fernandez-Garcia et al. 2014). In mammary tissue, stromal state strongly influences the proliferative and metastatic nature of epithelial cells, and mutated epithelial cells alone appear to be insufficient for tumor growth.  Stroma exposed to carcinogens can make transplanted unexposed epithelial cells tumorigenic in rats (Maffini, Soto et al. 2004) and transplanted p53 mutant epithelial cells tumorigenic in BALB/c mice (Barcellos-Hoff and Ravani 2000), while neither epithelia exposed to carcinogens nor p53 mutant cells are tumorigenic when transplanted into unexposed animals (Barcellos-Hoff and Ravani 2000; Maffini, Soto et al. 2004). Similarly, post-lactational stroma can make tumor cells more invasive and metastatic than nulliparous stroma (McDaniel, Rumer et al. 2006), and younger and nulliparous stroma makes tumor cells proliferate more than older and multiparous stroma (Maffini, Calabro et al. 2005). Even proliferating tissue and tumors can regress (Haslam and Bern 1977; Purnell 1980), suggesting that proliferation is insufficient for carcinogenesis in some cases.

While mutations increase linearly in response to ionizing radiation or carcinogens, proliferation (or proliferation of stem cell populations) apparently does not (Beuving, Bern et al. 1967; Mukhopadhyay, Costes et al. 2010; Nguyen, Oketch-Rabah et al. 2011; Tang, Fernandez-Garcia et al. 2014). Because we expect only a subset of mutations to affect cell-cycle or proliferation-related genes and because most cells require multiple mutations for proliferation to commence, only a very small number of cells would be expected to proliferate in response to mutation. It is therefore possible that the proliferation typically observed is actually due to a separate mechanism such as the self-renewal of stem-like or senescent-resistant cells and that a delayed mutation-based proliferation is not being measured.

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.

Gorgoulis, V. G., L. V. Vassiliou, et al. (2005). "Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions." Nature 434(7035): 907-913.

Halazonetis, T. D., V. G. Gorgoulis, et al. (2008). "An oncogene-induced DNA damage model for cancer development." Science 319(5868): 1352-1355.

Kiraly, O., G. Gong, et al. (2015). "Inflammation-induced cell proliferation potentiates DNA damage-induced mutations in vivo." PLoS Genet 11(2): e1004901.

Preston-Martin, S., M. C. Pike, et al. (1990). "Increased cell division as a cause of human cancer." Cancer Res 50(23): 7415-7421.