A Nexus model of cellular transition in cancer

The Nexus model

This model proposes that the probable transition route opens with the primary inducers (established primary causes) such as physical, chemical, biological and lifestyle related causes (Fig. 1). Such primary inducers then interact with the cellular biochemical pathways and generate reactive oxygen and nitrogen species (RONS) along with other free radicals, also known as cellular impeders (Fig. 1). The RONS, free radicals and viruses can also bring random genetic aberrations, which then generates structurally and functionally altered regulatory molecules (biomolecules) involved in metabolic pathways [14–16]. The interference of the cellular impeders thus results in the accumulation of initial substrates, intermediates or partial pathway products. Such an accumulation of the biologically insignificant metabolites congest cellular traffic thereby leading to a cellular environment that hampers the breakdown of normal metabolic pathways. This further develops an overload of residual metabolites in the cellular environment. Such a scenario results in the loss of intercellular signaling in a tissue and ultimately cause prolonged cellular biochemical stress that continues through many cell cycles, and eventually alters the cellular microenvironment. Such a complete alteration of the cellular microenvironment and the loss of intercellular signaling then creates perfect platform to initiate chain of events responsible for epigenetic and genetic changes [17]. Such events cause prolonged biochemical stress, thereby inducing considerable changes in stressed cells and marking the beginning of cellular events leading to cancer. Such events are hereby called as the Nexus. Initially, such mutations are random and result into expression of biomolecules which may either add to or reduce the biochemical stress (the Nexus) [18], better known as positive or negative cellular feedback. While the “positive feedback” refers to the survival of the mutations that reduces cellular stress, “negative feedback” refers to the mutations that might contribute to the increase of substrates, intermediates and partial products. The positive feedback is evidently repeated in the forthcoming cycles featuring mutational selection, resulting into newly evolved genetic machinery powered by such selective mutations [13, 18, 19]. On the other hand, such mutations are also capable of consuming residual metabolites via rewired metabolic pathways and have high rates of proliferation and turn out to be cancerous [20–27]. Over time, survival and accumulation of selective mutations which aid to positive feedback result in cancer phenotype (cancer evolution) of a healthy cell. The word ‘Nexus’ justifies the role of biochemical stress as the junction where strings originated from primary inducers further travel to epigenetics and genetics in cancer evolution.

In order to substantiate the Nexus Model, experimental and established evidences have been composed below under Phase I, Phase II and Phase III.

Phase I: primary inducers and cellular impeders

Oxidative stress is a condition which results due to the production of oxidative radicals, mostly reactive oxygen species (ROS) and reactive nitrogen species (RNS) that exceeds the quenching limit of cells [28]. RONS can be generated by a number of inflammatory reactions, physical and chemical factors [28–30] (Fig. 2). It is an established fact that lifestyle factors, which include cigarette smoking, sun exposure, workplace, diet etc., affect the chances of acquiring cancer [31, 32].

According to the National Cancer Institute, obesity has been found to be prominently associated with the risk of cancer, wherein the United States alone, in 2012, 28,000 newer cancer cases in men and 72,000 newer cases in women were linked to obesity and being overweight [33]. Various factors released by the adipose tissues result in insulin resistance and consequent production of pro-inflammatory factors like tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and cytokines which end up in unusual production of ROS [34, 35]. Another factor is UV radiation that has a prominent role in causing skin cancer [36]. Most of the Ultra Violet (UVA) energy is taken by the photosensitizers in the cells which are believed to generate ROS [37]. Tobacco, cigarette smoke, alcohol, naturally occurring fibrous substance-asbestos and metal toxicity are some chemical factors which are found to be responsible for the production of RONS. Tobacco contains nicotine and structurally similar alkaloids consisting of secondary and tertiary amines which react with nitrite forming nitrosamines [38]. In case of secondary amines, nitrosation is an exceptionally fast process, in which –H atom attached to nitrogen is replaced by –NO [39, 40]. The –NO group being a potential reactive species causes oxidative stress [41]. Exposures to smoke produced by cigarettes can be blamed for the oxidative stress as it persuades the aggregation of misfolded proteins and endoplasmic reticulum (ER) stress and consequently enhances the production of ROS [42–46]. Ethyl alcohol is converted into acetaldehyde in the body which is a budding cause of ROS production in the cells [47, 48]. Asbestos fibers are known to induce the cells to produce ROS due to the iron present on the fibrous silicates [49, 50]. Exposure to lethal waste sites, mines and construction sites may also subject the workers to high intensity metal toxicity of mercury, lead, arsenic etc. [51, 52]. Accumulation of these metals can then lead to the generation of ROS in cells.

Red meat, high salt consumption, viral infections and physical inactivity encompass the biological factors. Antibodies are produced in response to Glycolylneuraminic acid [Neu5Gc], which acts as an antigen to the body, present in red meat. This interaction results in ignition of inflammatory cells thus producing ROS [53]. High salt consumption is also found to be a potential cause of ROS production [54]. The increased salt concentrations alter the expression of virulence factor CagA (cytotoxin-associated gene A) in Helicobacter pylori strain 26695, which is a highly acknowledged factor for cancer [55]. The infections due to Human papillomavirus (HPV) cause oxidative stress which in turn damage the cell DNA [56, 57]. Exercise is found to decrease the ROS production in body [58].

The above discussed factors summarize how physical, chemical, biological and lifestyle related factors, termed as primary inducers (primary causes) generate RONS and other cellular impeders that hamper cellular metabolic pathways. Such interference then causes accumulation of cellular substrates that eventually converge to forcibly induce biochemical stress thereby facilitating the evolution of cancer. The diagrammatic illustration of the same has been provided in Fig. 2.

Phase II: development of biochemical stress (The Nexus)

Generation of reactive oxygen and nitrogen species (RONS) in the cells cause havoc in the normal functioning of enzymes and other biomolecules participating in various metabolic pathways [32]. At normal levels of RONS, the combat mechanisms are capable enough to maintain homeostasis inside cells, but when their concentration exceeds the threshold level, they impede the normal functioning inside the cell. High concentration of RONS interferes or reacts to cause delay, halt or even total loss in integrated framework of metabolic pathways [59, 60]. As in glycolysis, the elevated concentration of RONS oxidizes and thus inactivates pyruvate kinase monomer 2 (PKM2) which is responsible for the conversion of phosphoenol pyruvate (PEP) to pyruvate [54]. Similarly, high levels of RONS regulate the Hypoxia-Inducible Factor-1 (HIF1) to create hypoxic conditions, which is one of the most common features recorded in almost all type of cancer cells [61]. The HIF modulates the activity of pyruvate dehydrogenase kinase 1 (PDK1) thereby restricting the activity of pyruvate dehydrogenase (PDH) which prevents the conversion of pyruvate into acetyl CoA, hence cause hindrance in the tricarboxylic acid cycle (TCA) [62–64]. Obstruction in the TCA cycle greatly reduces the production of ATP via electron transport chain (ETC) [62]. To maintain the redox homeostasis, glycolysis adopts to pentose phosphate pathway (PPP) which is the principal pathway for de novo synthesis of nucleotides and this shunting of pathway generates excess of nucleotides [63]. Generation of excess nucleotides through PPP pathway causes substrate accumulation in cytoplasm. Generation of excess nucleotides through PPP pathway causes substrate accumulation in cytoplasm. Partial or complete obstruction of glycolysis [65] leads to the accumulation of substrates which were supposed to be consumed under normal conditions [66]. As all the metabolic pathways are interdependent [67], it is safe to say that hindrance in a single pathway leads to the upheaval in the other linked or parallel pathways.

The HIF-1 also triggers the activity of hypoxia inducible factor 2 (HIF-2) which causes amassing of lipids in the form of droplets [68] and simultaneous loss of expression of Von Hippel-Lindau gene (VHL) [69]. The loss of VHL expression further leads to the reduced expression of β-oxidation genes causing the curtailment of the β-oxidation pathway [70]. The reduction in the pathway eventually results in the accumulation of lipids. Nevertheless, de novo lipogenesis continues by using other carbon sources such as acetate and glutamine. The de novo synthesis is mediated by an increased level of fatty acid synthase (FASN) [71, 72].

Apart from the discussed routes that affect the biochemical pathways, RONS can cause direct damage to the DNA thereby causing random mutations [73]. These random mutations may occur in the genes that code the enzymes involved in the cellular metabolism and again lead to the disruption of these pathways, ultimately causing accumulation and biochemical stress. One such example is of isocitrate dehydrogenase (IDH) mutation. IDH is an enzyme which catalyzes conversion of isocitrate into α-ketoglutarate [74] and provides defense against oxidative insults. Genetic alteration in the IDH gene results in the alteration of its enzymatic activity. This mutated form of the enzyme catalyzes the conversion of α-ketoglutarate into 2 hydroxyglutarate (2-HG) which is a well-known oncometabolite [75, 76]. High concentration of the 2-HG thereby results in abnormal DNA hypermethylation in cells [77].

Another metabolite that is involved in creating the biochemical stress (Nexus) is the enzyme fumarate hydratase (FH). The RONS guided mutations inactivate FH, which leads to the blockage of TCA cycle [78, 79]. It has two possible consequences; first, it causes accumulation of fumarate and succinate leading to biochemical stress; second, the accumulated fumarate reacts with reduced glutathione (GSH) producing succinated glutathione which is considered to be an oncometabolite [78]. This oncometabolite is further degraded by glutathione reductase releasing GSH which then combines with fumarate in an aborting manner consuming NADPH, ultimately obstructing RONS detoxifying potential of the mitochondria and thereby increasing the RONS generation [79]. Eventually, it leads to substrate accumulation and hence elevated biochemical stress (The Nexus) [80].

It can be deduced from the above-discussed facts that high RONS concentration resulting from primary inducers can, directly and indirectly, affect the normal cellular metabolic pathways. Any type of delays, layoffs or outright loss in any of the metabolic pathways results in the accumulation of substrates, intermediates and partial products. Accumulation of these components then enhances cellular traffic resulting into an overflowing abundance of such products inside the cytoplasm. Under such a condition where nutrients are no more consumed efficiently, the machinery of bioenergetics (ATP) starts to shutdown and the futile intermediates and partial products simultaneously increase the cellular traffic to generate biochemical stress (The Nexus). The prolonged biochemical stress cuts off the intercellular signaling in the affected tissues thereby bringing the cells into isolation. Cellular isolation and long term biochemical stress can be considered as the perfect conditions to stimulate epigenetics followed by genetic evolution. The above enlisted events can be considered as phase II of cellular transition in cancer described in Fig. 3.

Phase III: epigenetics to genetic evolution

Cancer epigenetics

Cellular transition further continues when these epigenetic abnormalities lead to disturbances in the cellular genetic makeup [89]. Epidermal growth factor receptor (EGFR) is one such example which governs the signaling pathways involved in the regulation of growth, metabolism, differentiation and apoptosis under stressed conditions through its tyrosine kinase (TK) activity. Mutation in the epidermal growth factor receptor-tyrosine kinase (EGFR-TK) domain in ovarian cancer has resulted in the over production of EGFR [90, 91]. This overproduction in turn alters the activity of DNA methyltransferase, an enzyme which is responsible for DNA methylation [92–95]. DNA methylation is the most widely investigated epigenetic modulation in cancer. In normal conditions, it regulates gene expression and inactivation. The methyl group covalently attaches to the cytosine residues in the CpG dinucleotides [96, 97]. These CpG sites are not randomly distributed in the genome; instead the CpG rich regions are known as CpG islands and they generally cluster at the 5′ end of the regulatory region (generally the promoter region) of many genes [89, 96]. These islands are not methylated in normal cells [98]. Hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a major event in the origin of many types of cancer. Hypermethylation of promoter region of CpG-islands out-turns into complete or partial loss of genes involved in the normal functioning of cell including those involved in cell cycle, DNA repair, and metabolism of carcinogens, cell to cell interaction, apoptosis and angiogenesis [96]. The methylated CpG islands are not capable of initiating transcription and hence there is an altered gene function. Thus, hypermethylation at the promoter region favors the mechanism of mutation and helps to accelerate random mutations during cellular transformation [99]. As a result of hypermethylation in the promoter region, the tumor suppressor gene p16, which regulates the proliferation rate of the cell is not transcribed and thus gets inactivated. Inactivation of gene p16 leads to the uncontrolled proliferation of tumor cells [100]. Mutations take place in the mTOR (mammalian Target of Rapamycin) signaling pathway due to the loss of tumor suppressors or activation of oncogenes promotes tumor growth and survival. Activation of mTOR pathway is also reported to take place under stressed conditions such as nutrient deprivation [10, 101]. Reports which have identified hypermethylation of many genes in various cancers are collectively presented in Table 1.

S. N.	Site of hypermethylation	Type of cancer
1	Glutathione S transferase gene (GSTPI, also known as GST3)	Prostate and breast cancer [91]
2	Promoter region of Liver Kinase B1 (LKB1)	Papillary breast cancer [92]
3	Promoter region of cyclin-dependent Kinase inhibitor p15INK4B	Leukemia and glioma [93]
4	Promoter CpG island of the O6-MGMT gene	Brain, colon and lung cancer [94]
5	CpG island of death-associated protein Kinase DAP-Kinase	Lymphomas [95]
6	Promoter region of p73 gene	Lymphoma cancer [96]

Apart from DNA methylation, there are other covalent modifications like histone modifications which control gene activity and play a major role in cancer development [100]. Post translational histone modifications have direct influence on chromatin structure and function. It usually results in rewired gene regulation; it includes histone deacetylation and histone acetylation [102]. Histone acetylation mediated by histone acetyltransferase (HAT) and histone deacetylation mediated by histone deacetylase (HDAC) plays a crucial role in gene expression and silencing. HDAC is found to be related with tumor development as it induces transcriptional inactivation [10, 103]. The deacetylation of lysine residues of histone 3 and histone 4 largely increases the ionic contact between positively charged DNA packaging proteins (histone) and negatively charged DNA which condenses the chromatin and makes transcriptional gene inert [104, 105].

Transcriptional blockage of tumor suppressor gene by upregulation or bizarre recruitment of HDACs to their promoter site is a common feature for emergence and tumor development [106]. The acetylation status of histones H3 and H4 seem to largely dictate the fate of chromatin assembly, transcription, and gene expression [107, 108]. Histone acetylation is governed by the opposing activities of HATs and HDACs [109, 110]. Thus the loss of normal functioning of gene opens a divergent pathway to escape early senescence, leading to genetic changes, which results in the escape of cancer cells from apoptosis [111].

The above evidences conclude that biochemical stress (the Nexus) induces epigenetic events which helps cell to rule out normal gene expression and create a demand of improved genetic makeup which could off load the prolonged biochemical stress. Once these epigenetic alterations mediate genetic changes, the last step in the phase of cellular transition of selection, adaptation and evolution comes into play. These events are depicted in Fig. 4.