The Association between Periodontitis and Human Colorectal Cancer: Genetic and Pathogenic Linkage

Periodontitis has been associated with an increased risk of and mortality associated with human colorectal cancer (CRC). Current evidence attributes such an association to the direct and indirect effects of virulence factors belonging to periodontal pathogens, to inflammatory mediators and to genetic factors. The aims of the study were to assess the existence of a genetic linkage between periodontitis and human CRC, to identify genes considered predominant in such a linkage, thus named leader genes, and to determine pathogenic mechanisms related to the products of leader genes. Genes linking periodontitis and CRC were identified and classified in order of predominance, through an experimental investigation, performed via computer simulation, employing the leader gene approach. Pathogenic mechanisms relating to leader genes were determined through cross-search databases. Of the 83 genes linking periodontitis and CRC, 12 were classified as leader genes and were pathogenically implicated in cell cycle regulation and in the immune-inflammatory response. The current results, obtained via computer simulation and requiring further validation, support the existence of a genetic linkage between periodontitis and CRC. Cell cycle dysregulation and the alteration of the immuno-inflammatory response constitute the pathogenic mechanisms related to the products of leader genes.

In the last decade, a growing body of evidence has reported the association between periodontitis and a variety of systemic inflammatory conditions and diseases, including atherosclerosis, diabetes, rheumatoid arthritis, and inflammatory bowel disease (IBD) [4][5][6]. Most notably, recent findings have Step by step description of the gene clustering analysis procedure, performed via computer simulation, to investigate the existence of a genetic linkage between periodontitis and human colorectal cancer, and to identify leader genes.
Preliminarily, an initial set of genes involved in the above-mentioned phenomenon was built up through various integrated cross-search databases (PubMed, OMIM, Medgen, GeneCards, GenBank, Genedx, GenAtlas) using the search engine Entrez (http://www. ncbi.nlm.nih.gov/). Step by step description of the gene clustering analysis procedure, performed via computer simulation, to investigate the existence of a genetic linkage between periodontitis and human colorectal cancer, and to identify leader genes. Preliminarily, an initial set of genes involved in the above-mentioned phenomenon was built up through various integrated cross-search databases (PubMed, OMIM, Medgen, GeneCards, GenBank, Genedx, GenAtlas) using the search engine Entrez (http://www.ncbi.nlm.nih.gov/). Repeated genes expansions, obtained through the web-available software STRING version 11.0 (https://string-db.org/) ELIXIR infrastructure, Hinxton, UK, and subsequent expanded genes filtrations to eliminate any false positive, through a further search with PubMed, all together defined as "expansion-filtering loops", were performed.
The name as well as the symbol of each gene, derived by the above-mentioned databases, underwent validation by means of the official Human Genome Organization (HUGO) Gene Nomenclature Committee, or HGNC (available at https://www.genecards.org), in order to eliminate previous symbols or aliases.
The combined predicted associations, characterized by the higher level of confidence (that is a result with a score ≥ 0.9), were computed between each single gene and the complete gene dataset, through the free online software STRING (Version 11.0) [22]. The sum of these combined predicted associations scores provided the so-called the "Weighted Number of Links" (WNL) for each gene.
Automatic computations were performed on the whole data related to the genes included in the study. A k-mean algorithm was applied to the input variable WNL, and a partitioning of the overall dataset of genes into mutually-exclusive clusters was automatically performed and a "gap statistic method" was used to estimate the ideal number of clusters for the clusters from 2 to 12, as reported in Figure 1. Significant differences among WNLs of cluster groups obtained by the gap statistic method, were found by the Kruskal-Wallis test (statistical significance at a level of α = 0.01), verifying the accurate estimate of the number of clusters.
The resulting gene clusters were classified and correspondingly named as A, B, C, etc., based on their respective value of WNL centroid. The first genes cluster was identified as a "leader" genes cluster, hypothesizing their possible central role in the phenomenon; in contrast, the last genes cluster, identified as "orphans" genes, included genes without identified predicted associations (WNL = 0).

Determination of the Putative Pathogenic Mechanisms Associating Periodontitis and CRC
Leader genes characterization was performed, via the free online software STRING (Version 11.0) [22], to assess the main function of leader gene products and their involvement in biological processes. A further literature search, using the keywords reported in Table 1, was conducted on PubMed/MEDLINE and ScienceDirect search engines (using the same key words reported in Table 1), to investigate the role of leader genes in the onset and in the progression of CRC as well as of periodontitis and to highlight their putative pathogenic mechanisms in the genetic linkage between periodontitis and CRC.  [16,18,19,21,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41], were employed in the literature search and were logically combined with the boolean operators AND, OR, NOT. (1) gene AND human (2)

Analysis of the Genetic Linkage between Periodontitis and Human Colorectal Cancer
The final set of genes was composed of 137 genes. A complete description of the identified genes, including acronyms, identification numbers, validated names, cluster assignment, and their involvement in biological processes, is shown in Table A1. In compliance with the estimated optimal number of clusters, shown in Figure 2A, the 137 identified genes were divided into 7 clusters, designated as A, B, C, D, E, F, and orphan genes clusters.
WNL computation is reported in Figure 2B. Depending on the WNL score, 54 genes, lacking combined predicted interactions (WNL = 0), were assumed not to be involved in the genetic linkage between periodontitis and CRC, and were, consequently, designated as orphan genes and excluded from the study; the remaining 83 genes, showing a WNL > 0 and the combined predicted interactions mapped in Figure 2C, were hierarchically grouped in descending order of WNL to the six clusters named from A to F, as illustrated in Figure 3.
CBL encodes for an enzyme targeting substrates for proteasomal degradation. CTNNB1 encodes for β-catenin, a subunit of the adherens junctions complex, regulating cell growth and adhesion and Wnt responsive genes (i.e., c-Myc) expression, leading to cell cycle progression. Black arrows are the centroids of the cluster groups: leader genes (in red); cluster B genes (in light blue); cluster C genes (in yellow); cluster D genes (in green); cluster E genes (in purple); cluster F genes (in orange); and 'orphan' genes (in clear); (C) final map of interactions of 137 genes involved in the genetic linkage between periodontitis and CRC according to STRING: leader genes are red; the lines that connect single genes represent predicted functional associations among proteins in the confidence view. genetic linkage between periodontitis and CRC according to STRING: leader genes are red; the lines that connect single genes represent predicted functional associations among proteins in the confidence view. In particular, the 12 genes, belonging to cluster A and defined as leader genes, were: E3 ubiquitin-protein ligase (CBL), catenin beta-1 (CTNNB1), proto-oncogene c-Fos (FOS), growth factor receptor-bound protein 2 (GRB2), interleukins 1B, 4, 6, 10 (IL1B, IL4, IL6, IL10), transcription factor AP-1 (JUN), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA), phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1), and RELA proto-Oncogene NFKB subunit or transcription factor p65 (RELA).
CBL encodes for an enzyme targeting substrates for proteasomal degradation. CTNNB1 encodes for β-catenin, a subunit of the adherens junctions complex, regulating cell growth and adhesion and Wnt responsive genes (i.e., c-Myc) expression, leading to cell cycle progression.
FOS is an oncogene encoding for the c-Fos protein, which heterodimerizes with c-Jun, encoded by JUN (Transcription factor AP-1), to form the transcription factor AP-1, involved in cell proliferation, differentiation, apoptosis, and cancerous transformation.
GRB2 gene encodes for a protein binding the epidermal growth factor (EGF) receptor, activating several signaling pathways.

Determination of the Putative Pathogenic Mechanisms Associating Periodontitis and CRC
The characterization of the 12 leader genes in the genetic linkage between periodontitis and human colorectal cancer is reported in Table 2. Identified leader genes were involved in cell signaling (i.e., CTNNB1, CBL, GRB2, PIK3CA, PIK3R1), transcriptional pathways (i.e., JUN, RELA), cell proliferation/differentiation (i.e., FOS), and immuno-inflammatory processes (i.e., IL1B, IL4, IL6, IL10). Current evidence of the role of leader genes in CRC and in periodontitis onset and progression, as well as the putative pathogenic mechanisms is reported in Table 2. Table 2. Description of the leader genes identified in the genetic linkage between periodontitis and human colorectal cancer: leader genes product(s) main function', as per the free online software FOS is an oncogene encoding for the c-Fos protein, which heterodimerizes with c-Jun, encoded by JUN (Transcription factor AP-1), to form the transcription factor AP-1, involved in cell proliferation, differentiation, apoptosis, and cancerous transformation.
GRB2 gene encodes for a protein binding the epidermal growth factor (EGF) receptor, activating several signaling pathways.

Determination of the Putative Pathogenic Mechanisms Associating Periodontitis and CRC
The characterization of the 12 leader genes in the genetic linkage between periodontitis and human colorectal cancer is reported in Table 2. Identified leader genes were involved in cell signaling (i.e., CTNNB1, CBL, GRB2, PIK3CA, PIK3R1), transcriptional pathways (i.e., JUN, RELA), cell proliferation/differentiation (i.e., FOS), and immuno-inflammatory processes (i.e., IL1B, IL4, IL6, IL10). Current evidence of the role of leader genes in CRC and in periodontitis onset and progression, as well as the putative pathogenic mechanisms is reported in Table 2. Table 2. Description of the leader genes identified in the genetic linkage between periodontitis and human colorectal cancer: leader genes product(s) main function', as per the free online software STRING (version 11.0) [22]; role in CRC development and progression; role in periodontitis onset and progression; putative pathogenic mechanisms related to the effects of the products of leader genes.

CTNNB1 Cell signaling
Mutated in up to 90% of colonic tumors; responsible for initial tissue dysplastic transformation [22]; encodes for β-catenin, a subunit of the adherens junctions complex, regulating cell growth and adhesion and Wnt responsive genes (i.e., c-Myc) expression, leading to cell cycle progression.
Its product, β-catenin, is detectable in periodontal ligament cell nuclei in mice, potentially influencing periodontal ligament homeostasis [23]; regulates Wnt responsive genes. Wnt stimulus induces osteogenic lineage commitment [23], while Wnt depletion is involved in alveolar bone loss.
Cell cycle dysregulation FOS Gene(s) transcription, cell signaling, cell proliferation and differentiation rs7101 and rs1063169 FOS single nucleotide polymorphisms are considered at higher risk of CRC onset [24] and its expression increases in CRC lesions [25]. In addition, a different member of the FOS family, named Fra-1, is over-expressed in colonic cancer cells, particularly in those acquiring motility and invasive ability [25]. Moreover, FOS may participate in the inflammatory microenvironment associated with CRC [25].
May be implicated in periodontitis development and progression through the interaction with prostaglandin-endoperoxide synthase 2, affecting the T-cell receptor (TCR) signaling [26].
Cell cycle dysregulation JUN Gene(s) transcription, cell signaling, cell proliferation, and differentiation inflammation Its product, c-Jun, heterodimerizes with c-Fos protein, encoded by FOS, to form the transcription factor AP-1 (see above). Involved in cell proliferation, differentiation, apoptosis, and malignant transformation [24,27].
Its product, c-Jun, heterodimerizes with c-Fos protein, encoded by FOS, to form the transcription factor AP-1 (see above). Involved in cell proliferation, differentiation, apoptosis, and malignant transformation [24,27].
Cell cycle dysregulation GRB2 Cell signaling Its products stimulate colonic cell proliferation [28]; in particular, the Grb2-associated binding protein 2 (Gab2) has been found responsible for epithelial mesenchymal transition and consequent CRC metastasis development [29].
Its products bind to the epidermal growth factor (EGF) receptor. EGF signaling in the periodontal tissue, indirectly affected by GRB2 expression, is considered essential in tissue regeneration; thus, its interruption may affect healing and regeneration processes. Indeed, EGF ligand alterations, secondary to the effect of the peptidylarginine deiminase enzyme, released by porphyromonasgingivalis, interfere with EGF signaling, and, potentially, favor periodontitis progression [30].
Cell cycle dysregulation PIK3CA Cell proliferation, cell survival The most frequently mutated gene in breast cancer and is centrally involved in other malignancies [22]. n.a. Cell cycle dysregulation PIK3R1 Cell signaling Phosphorylated by PIK3CA, it is downregulated in CRC cells [31]. It is considered as a marker of severe periodontitis [32]. Cell cycle dysregulation IL6 Inflammation Induces CRC cell growth and invasion; and higher levels of IL6 have been detected in the serum from CRC patients compared to controls [33].
Stimulates osteoclastogenesis [34], has been found associated with chronic as well as aggressive periodontitis and, together with IL6R, IL6ST, IL4R, and IL1R1 may link periodontitis to other diseases [20].
Immuno-inflammatory response IL1B Immune response In CRC cells it is produced in higher concentrations compared to healthy surrounding tissues, possibly activating the NFKB signaling pathway [35].
IL1-889 C/T gene polymorphism has been associated with severe periodontitis [34] and its role in periodontitis pathogenesis has long been advocated [36]. Plays a protective role in periodontitis progression, reducing alveolar bone loss. Consequently, IL4 gingivo-crevicular fluid levels are higher in periodontally healthy subjects and after non-surgical periodontal treatment. In addition, the IL4-590 C/T polymorphism has been reported as potentially associated with an increased risk of periodontitis development [38].
Anti-inflammatory cytokine, down-regulating monocyte-macrophage response. Its gene polymorphism has been associated with periodontitis development in Caucasians [34].
Immuno-inflammatory response RELA Cell signaling Its expression is higher in malignant compared to healthy colonic cells, as well as in breast, liver, pancreatic, and gastric cancers, although its role in cancerogenesis, as well as in periodontitis, is still not fully elucidated [40].
It is also classified as leader gene in periodontitis probably because it is functionally related to NFKB pro-inflammatory activity [22].

Immuno-inflammatory response CBL Cell signaling
It may be related to inflammatory bowel disease (IBD) and CRC [41], as well as to atherogenesis, and neurodegenerative and autoimmune diseases, by a de-regulation in the ubiquitin-proteasome system, with subsequent NFKB activation and immuno-inflammatory response enhancement.
No evidence is available relating CBL to periodontitis [20].

Discussion
Periodontitis and human colorectal cancer are complex multi-factorial disorders, dealing with a multitude of genes, which are interconnected by several heterogeneous networks, and whose products are involved in a wide range of biological pathways [20]. In view of this fact, the present experimental investigation of the genetic linkages between periodontitis and CRC was conducted through a bioinformatic method, called "leader gene approach" [20]. This multi-step procedure, as described above, is especially useful in identifying the highest priority genes in the investigated phenomenon [20,21] and provides the necessary synthesis and analysis of the overwhelming amount of raw bioinformatic data generated. Ranking genes hierarchically and identifying leader genes consistently revealed those genes, and their related products, which are mainly involved in the genetic linkage between periodontitis and CRC ( Table 2). Such bioinformatic data were subsequently integrated with current evidence to reveal cellular functions and biological processes carried out by the gene products, and were interpreted in view of the available clinical and experimental findings to determine the putative pathogenic mechanisms associating periodontitis with CRC.

Genetic Linkages between Periodontitis and Human Colorectal Cancer: Leader Genes and Putative Pathogenic Mechanisms
Among the 137 genes (complete final gene dataset available as metadata) reported in periodontitis and CRC ethio-pathogenesis, 83 were involved and 12 ("cluster A" or "leader" genes) were considered to play a predominant role in the genetic linkage between both disorders. Notably, four of the cluster A genes, specifically, CBL, GRB2, PIK3R1, and RELA, were also ranked among the five leader genes previously identified in periodontitis [20]. Nuclear factor kappa B p105 subunit (NFKB1), instead, which is considered as a leader gene in periodontitis, was assigned to cluster C in the present study. These results may support the existence of a possible genetic linkage between periodontitis and CRC.
Leader genes affecting the immune-inflammatory response, such as IL1B, IL4, IL6, IL10, CBL, and RELA, may underlie a possible bi-directional relationship between the disorders, as described below [20,[33][34][35][36][37][38][39][40]. Moreover, in addition to leader genes affecting the immune-inflammatory response, NFKB, which has been ranked among cluster C genes and is functionally related to RELA, regulates the transcription of several genes, also encoding for pro-inflammatory cytokines. NFKB is constitutively inactivated and its activation, with subsequent immuno-inflammatory response alteration, may be due to a dysregulation in the ubiquitin-proteasome system, which is a mechanism of intracellular protein degradation, occurring in atherogenesis, neurodegenerative and autoimmune diseases, and, possibly, in IBD and CRC [41,42]. Current knowledge about the role of the cellular ubiquitin-proteasome system dysregulation, and subsequent NFKB activation in periodontitis, is still limited, but it may explain the presence of the E3 ubiquitin-protein ligase (CBL) gene among the leader genes in the genetic linkage between periodontitis and CRC, although no evidence is available relating CBL to periodontitis [20].

Genetic Linkages between Periodontitis and Human Colorectal Cancer: Cytokines and Systemic Inflammation
Periodontal tissue destruction, occurring in periodontitis, is microbially initiated and sustained by the dysregulation of the immune-inflammatory processes [5]. A body of evidence has shown that cytokines produced in inflamed periodontal tissues, together with virulence factors from periodontal pathogens and oral microbial agents, may gain access to the circulation, and, consequently, induce systemic inflammation [5,43]. Accordingly, it has been proposed that non-resolving periodontal inflammation may affect systemic inflammatory diseases and that cytokines may be considered as a possible pathogenic link between periodontitis and various systemic diseases, including IBD and CRC [4][5][6][7]38,44].
It is well known that IBD has oral mucosal manifestations, such as pyostomatitis vegetans and aphthous stomatitis, and it has been reported that subjects suffering from Crohn's disease show a higher risk of periodontitis compared to non-IBD subjects [8,45]. In addition, evidence suggests that periodontal pathogens, especially Fusobacterium nucleatum, may be involved in IBD [9,10] and colorectal adenomas [46], and that cytokines induced by periodontal pathogens and released in periodontitis may predispose to neoplastic transformation of chronic colitis, favoring colorectal carcinogenesis [12,46,47]. In more detail, oral fusobacterium nucleatum, which is abundant in the oral cavity and increased in periodontal pockets, is mobile and caplable to bind, through the Fusobacterium adhesin A (FadA), both to vascular endothelial-cadherins, gaining access to systemic circulation, and to (E)-cadherin on epithelial cells, stimulating the growth of tumor cells. Binding to (E)-cadherins on colorectal adenoma and cancer cells, FadA, which is only detectable on oral Fusobacterium species, activates the transcriptions of those oncogenes regulated by b-catenin, which is the product of the leader gene CTNNB1, and of some genes involved into the immune-inflammatory response, including IL6, which is presently ranked as a leader gene, and NFKB, belonging to cluster C genes [46]. From this standpoint, periodontitis may be considered as a possible risk factor for CRC genesis in IBD subjects, as it accounts for poor metabolic control in diabetic patients [48]. Such an inter-relationship may rely on the fact that both IBD and periodontitis share a multifactorial etiology, as well as the pathogenic mechanisms affecting the local immuno-inflammatory response, which leads to the genesis of a systemic inflammation [45]. Analogously, it may be supposed that those periodontal cytokines, which are listed among leader genes products, may enhance colonic tumor-associated inflammation, and may subsequently be considered as a risk factor for cancer progression in CRC subjects. As a counterpart, along with the tumor-associated inflammatory environment, CRC cells themselves release inflammatory mediators, which self-sustain neoplastic cell growth and enhance the cancerous cells' interactions with the surrounding stroma and immune cells, favoring, in turn, CRC progression and invasion [13,49]. Since CRC inflammatory mediators have been identified as leader genes in the present study, it may be hypothesized, as previously proposed for cytokines released in diabetes [43], that CRC cytokines may negatively affect periodontitis onset and development, altering the immune-inflammatory response in periodontal tissues.

Genetic Linkages between Periodontitis and Human Colorectal Cancer: Possible Clinical Implications
The findings discussed, certainly requiring validation by larger studies, may provide preliminary data for further research, especially considering the beneficial clinical applications potentially offered by the insight into the mechanisms associating periodontitis and CRC. Indeed, if the results presented, which suggest a central role for cytokines and systemic inflammation in the genetic bi-directional linkage between periodontitis and CRC, are validated, periodontitis management may be included in CRC prevention and treatment plans. Complex multi-factorial disorders, such as periodontitis and CRC, significantly impact on the quality of life, present life-threatening risks, and imply a heavy burden on society. Therefore, highlighting the genetic traits of such disorders may pave the way for primary prevention strategies, which are essential to reduce the biological impact as well as the healthcare costs of these disorders. The improved understanding of the putative pathogenic mechanisms associating periodontitis with CRC may encourage a multidisciplinary approach, which is strongly advocated for such complex multifactorial disorders.
From this standpoint, oral health professionals may also become part of CRC screening plans, introducing, in their daily practice, general health promotion and disease prevention goals, and including risk assessment for both oral and systemic diseases. CRC screening might be improved by the provision of broader dental health records, with the potential to identify subjects at risk for CRC development for referral to a physician. In addition, based on the definition of oral health as a component of general health affecting the quality of life [50], oral health professionals may widen their activity in an interprofessional setting, providing oral and periodontal evaluation and necessary treatments, in CRC subjects referred by other health professionals, integrating the patient's medical care in therapeutic and follow-up plans.
Periodontal treatment may be proposed as a CRC primary prevention strategy, in subjects considered at higher risk for CRC development, such as those suffering from IBD, in order to decrease the systemic inflammation and the related pro-carcinogenic environment. However, threshold values of cytokines in inflamed periodontal tissues, capable of inducing systemic inflammation and subsequently increasing the risk for colorectal cancer genesis, in IBD subjects have not yet been defined. Furthermore, the quantitative assessment of periodontal cytokines is even more complicated than the qualitative one, since it may actually be biased by the accidental detection of inflammatory mediators possibly derived from mucosal inflammation and orally administered drugs, in whole saliva analysis, and by the need for full mouth sampling, in gingival crevicular fluid analysis [51]. For these reasons, identifying those IBD subjects potentially exposed to a higher risk of systemic inflammation induced by periodontitis, and of consequent malignant transformation of chronic colitis, may be impracticable. Thus, periodontitis prevention and treatment, which potentially reduces systemic inflammation and, consequently, decreases the risk for malignant transformation of chronic colitis, may be routinely included in all IBD subjects' treatment plans. Moreover, periodontal treatment, reducing the periodontal microbial load and the related cytokine levels, may decrease the systemic spread of inflammatory mediators and of Fusobacterium nucleatum, specifically, presumed to be associated with CRC, beyond IBD, lesions [41,46], and to favor tumor-associated environment, and may, therefore, constitute a secondary and/or tertiary prevention strategy in subjects affected by CRC.

Conclusions
Four out of the five leader genes previously identified for periodontitis (CBL, GRB2, PIK3R1, and RELA) were also listed as leader genes in the investigated phenomenon, carefully supporting the genetic linkages between CRC and periodontitis, and suggesting the need for a multi-disciplinary approach, also involving oral health professionals, to CRC subject management.
IL1B, IL4, IL6, IL10 were also ranked among leader genes, suggesting a central role for systemic inflammation in the genomic relationship between CRC and periodontitis; in particular, periodontitis may be linked to IBD, and, in turn, to CRC, both affecting the inflammatory pro-carcinogenic and tumor-associated environment and acting in an indirect way in the "inflammation-dysplasia" carcinogenic sequence, favoring colorectal cancer development. In this perspective, periodontitis management may be proposed as a CRC primary prevention strategy, especially in patients considered at higher risk for CRC development, such as IBD subjects. Indeed, periodontal therapy would reduce the periodontal microbial charge, and, consequently, the systemic widespread of bacterial toxins and of periodontal pathogens them-selves, including Porphyromonas gingivalis and Fusobacterium nucleatum, supposed to be associated with IBD and CRC lesions. Moreover, periodontal treatment and healthy periodontal conditions would indirectly decrease the systemic inflammation and the related CRC pro-carcinogenic environment, as a part of the CRC treatment strategy.
Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflicts of interest.
Appendix A Table A1. Identified genes acronyms, identification numbers, validated names, cluster assignment, and biological process(es) involvement description, as per the free online software STRING (version 11.0) [22].