Emerging Role of Plant-Based Dietary Components in Post-Translational Modifications Associated with Colorectal Cancer

Colorectal cancer (CRC) is one of the most common cancers worldwide. Its main modifiable risk factors are diet, alcohol consumption, and smoking. Thus, the right approach through lifestyle changes may lead to its prevention. In fact, some natural dietary components have exhibited chemopreventive activity through modulation of cellular processes involved in CRC development. Although cancer is a multi-factorial process, the study of post-translational modifications (PTMs) of proteins associated with CRC has recently gained interest, as inappropriate modification is closely related to the activation of cell signalling pathways involved in carcinogenesis. Therefore, this review aimed to collect the main PTMs associated with CRC, analyse the relationship between different proteins that are susceptible to inappropriate PTMs, and review the available scientific literature on the role of plant-based dietary compounds in modulating CRC-associated PTMs. In summary, this review suggested that some plant-based dietary components such as phenols, flavonoids, lignans, terpenoids, and alkaloids may be able to correct the inappropriate PTMs associated with CRC and promote apoptosis in tumour cells.


Introduction
Colorectal cancer (CRC) is currently the second type of cancer with the highest mortality rate in the population according to Global Cancer Statistics 2020 [1]. Metastatic CRC has a poor prognosis, with less than a 15% of five-year survival rate [2]. Its carcinogenesis is a process of many years of development and some early life risk factors are important contributors [3]. Among them, cigarette smoking, obesity, and a sedentary lifestyle are closely related to CRC incidence [4,5]. However, its quickly increasing incidence is mainly due to lifestyle westernization associated with changes in dietary behaviour such as heavy alcohol consumption and diets rich in sugars, saturated fats, and red and processed meat [6]. Thus, some protective lifestyle factors against CRC include a diet rich in minerals and vitamins, dairy, dietary fibre, fish, vegetables, and fruits. An alternative strategy for CRC prevention is the use of a chemopreventive supplement providing greater individual exposure to some nutrients than can be obtained from the diet (such as phytochemicals) [7].
The pathogenesis of CRC is a complex multi-stage process which includes gut microbiota imbalances, cell DNA disruption, and carcinogenic signalling pathways activation [8]. The aetiology underlying the mechanism of action of specific nutrients in CRC has been mainly attributed to their anti-inflammatory and antioxidant properties, and their modulation of gut microbiota populations, maintaining gut homeostasis and regulating the host immune response [9,10]. However, their effects on epigenetic modulation associated with

SUMOylation
Small ubiquitin-like modifiers (SUMO) are covalently attached to lysine residues [30]. The downregulated SUMOylation in lysine 138 of Rho GDP-dissociation inhibitor 1 has been observed in CRC cell lines (Table 1). This protein is involved in Rho GTPases signalling regulation [31].

Glycosylation
A carbohydrate is attached to specific proteins. In mammals, there are two types: (1) O-glycosylation, where glycosyl groups are connected to tyrosine, hydroxylysine, serine, or threonine side chains with glycosidic linkages by glycosyltransferases, and (2) N-glycosylation, where glycosyl groups are connected to Asn side chains with amide linkages by oligosaccharyltransferase [32,33]. The upregulation of this PTM in complement decayaccelerating factor and cathepsin B has been identified in tumour tissue samples of CRC patients (Table 1) [34,35].

O-GlcNAcylation
There is a covalent attachment of N-acetylglucosamine residue O-linked to the hydroxyl group of threonine and serine residues of multiple cytosolic and nuclear proteins [36,37]. The upregulation of O-GlcNAcylation in ATP-dependent RNA helicase DDX5 has been associated with CRC in cell lines and murine models (Table 1) [38].

O-GlcNAcylation
There is a covalent attachment of N-acetylglucosamine residue O-linked to the hydroxyl group of threonine and serine residues of multiple cytosolic and nuclear proteins [36,37]. The upregulation of O-GlcNAcylation in ATP-dependent RNA helicase DDX5 has been associated with CRC in cell lines and murine models (Table 1) [38].

Ubiquitination
There is an attachment of ubiquitin molecules to the lysine residue of the substrate proteins. This process is based on an enzymatic cascade of ubiquitin-activating, ubiquitinconjugating, and ubiquitin-ligase enzymes [39,40]. There have been two ubiquitinationsusceptible proteins identified that are related to CRC (Table 1)

Methylation
Methylation occurs mainly in arginine or lysine residues. One of the most biologically important roles of methylation is in histone modification [43]. Among the different proteins that suffer dysregulated post-translational methylation associated with CRC (Table 2), the one that is involved in cell growth suppression has downregulated methylation (putative insulin-like growth factor 2 antisense gene protein) [44,45]. The other proteins identified have an upregulated methylation, among them are (1) BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 that is involved in apoptosis [46]; (2) homeobox protein CDX-2 that is involved in the transcriptional regulation of different genes expressed in the intestine [47]; (3) C-X-C motif chemokine 14 that is involved in immunoregulatory and inflammatory processes [48]; (4) transcription factor E2F1 that participates in the cell cycle [49]; (5) DNA mismatch repair protein Mlh1 that participates in DNA repair [50]; (6) nuclear factor NF-kappa-B p105 subunit that is a pleiotropic transcription factor involved in several signal transduction events which are initiated by stimuli such as oxidative stress or inflammation [51]; and (7) 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 that is an essential protein for cell cycle progression and apoptosis prevention [52].  NA: data not available; K: lysine; R: arginine.

Serine Phosphorylation
Serine phosphorylation includes proto-oncogene c-Ak and Fos-related antigen 1 that regulates many processes including proliferation cell survival, growth, and angiogenesis [75,76]; apoptosis regulator Bcl-2 that is a regulator of apoptosis [77]; COP9 signalosome complex subunit 6 which is a component of the COP9 signalosome complex [78]; ELAV-like protein 1 that stabilizes mRNAs and regulates gene expression [79]; fascin-2 that acts as an actin bundling protein [80]; histone H3.1 which plays a central role in transcription regulation and DNA repair [81]; Kirsten rat sarcoma virus which is involved in the propagation of growth factors [82]; MAP kinase kinase 4 and 5 that are dual specificity protein kinase which act as an essential component of the MAP kinase signal transduction pathway [83,84]; NFKB1 and NFKB3 which are pleiotropic transcription factors involved in several signal transduction [85,86]; PHD finger protein 20 that contributes to p53 stabilization after DNA damage [87]; cellular tumour antigen p53 that acts as a tumour suppressor [88]; nuclear receptor ROR-alpha which is a key regulator of glucose metabolism [89]; sirtuin 1 that is an intracellular regulatory protein [90]; DNA topoisomerase 1 that releases the supercoiling tension of DNA introduced during the DNA replication [91]; tropomyosin-1 which is a member of the tropomyosin family of highly conserved proteins [92]; TP53-regulating kinase which is a protein kinase that phosphorylates 'Ser-15' of p53/TP53 protein [93]; SUMO-protein ligase that is essential for nuclear architecture and chromosome segrega-tion [94]; and vimentin which is responsible for maintaining cell shape and stabilizing cytoskeletal interactions [95]. Table 4. Post-translational serine, threonine, and tyrosine phosphorylation associated with CRC.

Protein
Gene Name PTM Site Type Ref

Threonine Phosphorylation
Threonine phosphorylation includes Aurora kinase B which is a serine/threonineprotein kinase component of the chromosomal passenger complex [96]; probable ATPdependent RNA helicase DDX5 which is involved in the alternative regulation of pre-mRNA splicing [97]; ETS domain-containing protein Elk-1 which is a transcription factor that binds to purine-rich DNA sequences [98]; dual specificity mitogen-activated protein kinase kinase 4 which is an essential component of the MAP kinase signal transduction pathway [83]; MAP kinase kinase 5 that acts as a scaffold for the formation of a ternary MAP3K2/MAP3K3-MAP3K5-MAPK7 signalling complex [84]; and 5 -AMP-activated protein kinase catalytic subunit alpha-1 which is the catalytic subunit of AMP-activated protein kinase that plays a key role in regulating cellular energy metabolism [99].

Tyrosine Phosphorylation
Tyrosine phosphorylation includes breast cancer anti-estrogen resistance protein 1 which plays a central role in cell adhesion [100,101]; caveolin-1 that act as a scaffolding protein within caveolar membranes [102]; leptin receptor that mediates leptin central and peripheral effects [103]; peroxisome proliferator-activated receptor gamma that is a nuclear receptor [104]; serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform which is the major phosphatase for microtubule-associated proteins [105]; focal adhesion kinase 1 which is a non-receptor protein-tyrosine kinase that plays an essential role in regulating cell migration and apoptosis [106,107]; protein tyrosine phosphatase type IVA 3 that stimulates progression from G1 into S phase during mitosis [108]; paxillin which is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix [109]; proto-oncogene tyrosine-protein kinase Src that is a non-receptor protein tyrosine kinase [110]; signal transducer and activator of transcription 3 which mediates cellular responses to interleukins and other growth factors [111,112]; and signal transducer and activator of transcription 5A that is involved in signal transduction and activation of transcription [113].

Relationship between Post-Translational Modifications Associated with Colorectal Cancer
The results of the analysis showed that there were several interactions between some of the proteins susceptible to inappropriate PTMs associated with CRC ( Figure 2). This analysis showed that there were strong interactions between TP53, AKT1, STAT3, STAT5A, JAK1, MAPK1, MAPK14, MAP2K1, and SRC. In fact, this network had significantly more interactions than expected, which means that proteins have more interactions among themselves than what would be expected from a random set of proteins, demonstrating that the proteins may be partially biologically connected as a group. This group of proteins is mainly involved in the PI3K-Akt, EGF-EFGR, MAPK, and VEGFA-VEGFR2 signalling pathways. On the one hand, PI3K-Akt is the classical signalling pathway involved in glucose metabolism that promotes cancer metabolic reprogramming by elevation of aerobic glycolysis (known as the "Warburg effect") [114,115]. Both EGF-EGFR and MAPK signalling pathways are involved in proliferation, differentiation, and apoptosis. Its regulation in cancer cells allows the maintenance of proliferative signalling, promoting cancer cell survival [60,116]. On the other hand, VEGF and its receptors (such as VEGFR2) develop an important role in tumour-associated angiogenesis. This process is essential for tumour progression because it favours oxygen and nutrient uptake by cancer cells [117,118]. Therefore, the main PTMs identified in CRC are involved in cancer progression and cancer cell survival. The next step was to identify how the inadequate PTMs in these proteins associated with CRC may be modulated by plant-based dietary components. This analysis showed that there were strong interactions between TP53, AKT1, STAT3, STAT5A, JAK1, MAPK1, MAPK14, MAP2K1, and SRC. In fact, this network had significantly more interactions than expected, which means that proteins have more interactions among themselves than what would be expected from a random set of proteins, demonstrating that the proteins may be partially biologically connected as a group. This group of proteins is mainly involved in the PI3K-Akt, EGF-EFGR, MAPK, and VEGFA-VEGFR2 signalling pathways. On the one hand, PI3K-Akt is the classical signalling pathway involved in glucose metabolism that promotes cancer metabolic reprogramming by elevation of aerobic glycolysis (known as the "Warburg effect") [114,115]. Both EGF-EGFR and MAPK signalling pathways are involved in proliferation, differentiation, and apoptosis. Its regulation in cancer cells allows the maintenance of proliferative signalling, promoting cancer cell survival [60,116]. On the other hand, VEGF and its receptors (such as

Nutrigenomic Effects of Plant-Based Dietary Components on Protein Post-Translational Modifications Associated with Colorectal Cancer
The available scientific literature showed several plant-based dietary components that may modulate CRC-associated PTMs (Table 5). These components can be grouped according to bioactive compounds as follows: phenols, flavonoids, lignans, terpenoids/alkaloids, vitamins, phytochemicals, and plant extracts.
The PTMs induced by plant-based dietary components mainly consist of modulating those modifications observed in CRC (Figure 3). Concerning STAT3, tyrosine phosphorylation (Tyr 705) was upregulated in CRC. Several articles showed that some phenols, flavonoids, terpenoids, alkaloids, phytochemicals, and plant extracts were able to downregulate not only this phosphorylation but also STAT3 serine phosphorylation (Ser 727) in some cases [120,121,124,130]. Some of them are also involved in reduced phosphorylation of JAK2, which is upregulated in CRC [120,148,149,155,159]. Both proteins form the JAK/STAT signalling pathway, which has an important role in cytokine receptor signalling. In response to cytokines, its activation promotes immune cell division, survival, activation, and recruitment. This pathway not only participates in the immune response but also in the transcription of several genes involved in cell division and apoptosis regulation such as BCL2 [171,172].
In the case of AKT, serine phosphorylation (S473) was upregulated in CRC. Some studies revealed that harmine [151], ophiopogonin D [154], and luteolin [157] were able to downregulate this specific serine phosphorylation, while others such as coumarins [119], resveratrol [125], quercetin [140], secoisolariciresinol diglucoside [145], lycopene [158], and some plant extracts decrease protein phosphorylation [168,169]. Similarly, in PI3K and BCL2 proteins, quercetin, secoisolariciresinol diglucoside, and iodine-biofortified lettuce extract downregulated their phosphorylation, respectively [140,145,164]. On the other hand, delphinidin [136] and two different plant extracts [163,165] reverse carcinogen-induced phosphorylation of NF-kβ3 (Ser536). These proteins interact within the same pathway. The PI3K/AKT pathway participates in the modulation of cellular metabolism, cell growth, and apoptosis. PIK3K produces conformational changes and phosphorylation of AKT protein, inducing its activation. This cascade triggered the activation of NF-κB by enhancing the transcriptional activity of the p65 subunit, leading to apoptosis inhibition [173]. Likewise, the PI3K/AKT signalling pathway promotes the upregulation of Bcl-2 expression, which is considered an oncogene that inhibits apoptosis [174]. This suggests that plant-based dietary components may promote cancer cell apoptosis through downregulating AKT, PI3K, BCL2, and NF-kβ phosphorylation. In the case of AKT, serine phosphorylation (S473) was upregulated in CRC. Some studies revealed that harmine [151], ophiopogonin D [154], and luteolin [157] were able to downregulate this specific serine phosphorylation, while others such as coumarins [119], resveratrol [125], quercetin [140], secoisolariciresinol diglucoside [145], lycopene [158], Another of the main proteins identified that can be modulated by plant-based dietary components is P53. This is considered a tumour suppressor involved in processes such as apoptosis, senescence, DNA repair, and cell cycle arrest [175]. The P53 pathway is activated against stress signals such as DNA damage. In response to this, P53 suffers from PTM and promotes the transcription of genes involved in cell response against stress [175]. These PTMs are mainly phosphorylation that is downregulated in CRC. However, several studies have shown that some plant-based dietary components such as phenols, flavonoids, terpenoids, and alkaloids could reverse it [123,126,128,135,137,139,140,146,147].
Concerning ERK1 and ERK2, both are part of a structurally related kinases family (MAPKs), whose signalling mechanism depends on an activating phosphorylation cascade. ERKs are central regulators of essential cellular functions such as cell differentiation, proliferation, migration, growth, survival, and metabolism [176]. Both proteins have posttranslational phosphorylation upregulated in CRC, favouring cancer cell survival. As it has been shown in Table 5, some plant-based dietary components may downregulate their post-translational phosphorylation [130,156,160,161,166,169].
It is important to highlight that, due to the lack of human studies, the present review is focused on studies conducted in cells and murine models. Therefore, these findings cannot be transferable to other species. Future studies should address the connection between the modulation of PTMs associated with CRC elicited by plant-based dietary components in patients.

Conclusions
The available literature data suggest that different plant-based dietary components such as phenols, flavonoids, lignans, terpenoids, and alkaloids could prevent CRC development by targeting several molecular mechanisms such as the P53, JAK/STAT, PI3K/AKT, and ERK/MAPK pathways and affecting tumour behaviour through PTMs' modulation in cell lines and murine models. The different signalling pathways affected during CRC development are mainly involved in cancer cell survival, and the main effects of plant-based dietary components are to promote apoptosis in tumour cells. Therefore, this could be a very interesting target for investigating the effect of supplementation based on these components as an adjuvant to chemotherapeutic, radiotherapeutic, and immunotherapeutic treatment in clinical trials. These findings will highlight the potential for precision nutrition strategies and the development of personalized nutritional plans in CRC treatment and may even serve as a basis for the development of dietary supplementation formulations for these patients to improve their prognosis and disease-free survival.