Genetic heterogeneity of colorectal cancer and the microbiome

Table 1 Features of some bacteria associated with colorectal cancer

Virulence factors, clinical and experimental data	CRC pathways
B. fragilis
- Contains B. fragilis toxin (BFT) that is zinc-dependent metalloprotease toxin[185,186]; -Associated with colitis[186], low-grade dysplasia, tubular adenomas, and serrated polyps[163], Lynch syndrome and familial adenomatous polyposis[187]; - More often associated with left-sided CRC[163]; - Characterized by biofilms formation[38]; - Associated with unfavorable CRC prognosis[123]; - Potentiates oncogenesis in the distal colon in mice[188]	-Induction of stepwise cleavage of E-cadherin and stimulates cell proliferation[185]; - Synthesis of cytokines, incl. IL-17↑[26,123,163,188]; - Activation of the WNT/β-catenin pathway → c-Myc transcription and translation↑ → cell proliferation↑[189]; - STAT3 activation in the mucosal immune cells → mucosal permeability↑[163,186,188]; - NF-κB activation →Th17с↑[163]; - STAT3 and NF-κB activation→ COX-2 and MMP-9 expression↑[123,163]; - BRAF and KRAS mutations, expression of MLH1↓[123]; - APC mutations[187]
E. coli
- Contains Colibactin and Cytotoxic necrotizing factor 1 (CNF1)[190]; - Can carry the pathogenicity island pks (pks + E. coli), which encodes a set of enzymes that synthesize Сolibactin[191]; - Associated with inflammatory bowel disease and CRC[192]; - More frequently found in CRC biopsies than in healthy mucosa[191]; - More common in advanced stages of CRC[193,194]; - Potentiates induction of invasive cancer in mice[192]; – Differences in the frequency of pks + E. coli in patients with CRC, adenomas and in healthy people were not detected[195]	- Induction of DNA double-strand breaks[196]; - DNA alkylation[190]; - Decrease of tumor-infiltrating T lymphocytes (CD3+ and CD8 T cells) and increases colonic inflammation[197]; - Activation of angiogenesis[198] and epithelial-mesenchymal transition[199]; - Modulates activity Rho GTPases (signaling G-proteins of Ras subfamily), thereby affecting the actin cytoskeleton, that contributes to the disruption of cell adhesion (due to the reorganization of the expression of E-cadherin, β-catenin, zonula occludens-1 (ZO-1) and caveolin- 1), the reduction of phagocytosis and improve epithelial cell motility[200,201]; - Induction of cell proliferation[191]; - Depletion of host mismatch repair proteins via bacterially secreted EspF effector protein[142]
F. nucleatum
- Contains adhesion protein FadA, Fap2 и RadD[96]; - Synthesis of toxic metabolites: Secondary bile acids, trimethylamine N-oxide, hydrogen sulfide, heme, nitrosamines, heterocyclic amines and polyaromatic hydrocarbons[96]; - Characterized by biofilms formation[38]; - Often detected in adenomas[38,202], tumor samples with high grade dysplasia[203], carcinoma tissue[44,202,204], distant CRC metastases[125]; - More frequently found in CRC biopsies than in healthy mucosa[205]; - Associated with proximal tumor localization[161,204,206,207], higher depth of invasion[206], higher clinical stage[206,207], low tumor differentiation[206,207], lymph node metastases and low survival rate[204]; - Promotes CRC induction in a traditional experimental model in mice[202]	- Associated with MLH1 methylation[206,207]; MSI-H[161,206-208]; CIMP-H[161,207]; BRAF mutation[206,207]; - FadA-dependent activation of the E-cadherin/β-catenin pathway → cell proliferation↑ and expression of E-cadherin↑[96]; - Activation of p38 MAPK and NF-κB signaling pathways → synthesis IL-6, IL-8 and IL-18↑[204]; - Inhibition of NK cell cytotoxicity by the Fap2 protein and an increase in the number of myeloid suppressor cells[96,204,208]; - Regulates miR21 expression via the TLR4/MYD88/NFκB[96,202,208,209]
Streptococcus gallolyticus (Sg)
- More frequently found in CRC biopsies than in healthy mucosa[210,211]; - Circulation of Sg in the blood in patients with CRC is most likely associated with dysfunction of the epithelial barrier[212]; - In a mouse xenograft model of CRC, Sg promotes tumor growth[211]	- Induction of cell proliferation through WNT/β-catenin pathway[211] and modulation of extracellular matrix[213]; - Increase in expression of c-Myc and cyclin D1 proteins[211]; - Stimulation of proliferation of the intestinal epithelium by some Sg strains which is associated with their ability to adhere to the intestinal epithelium and the genetic characteristics of the host cells[211,214]
Enterococcus faecalis (Ef)
- Contains superoxide[215]- Promotes CRC induction in Il10 -/- mice experimental model[215]	- Promotes chromosome instability[215]; - Ef polarize colon macrophages to produce endogenous mutagens → initiation CIN → expression of progenitor and tumor stem cell markers[216]; - Induces gene mutation and endogenous transformation through microbiome-induced bystander effects[217]; - Activation of the WNT/β-catenin pathway[217]; - Activation of transcription factors c-Myc, Klf4, Oct4 and Sox2[217]

APC: Adenomatous polyposis coli; BFT: B. fragilis toxin; BRAF: B-Raf proto-oncogene; CIMP: CpG island methylator phenotype; CIN: Initiate chromosomal instability; CNF: Cytotoxic necrotizing factor 1; COX2: Cyclooxygenase 2; FadA: Fatty acid desaturase A; KRAS: Kirsten ras; MAPK: Mitogen-activated protein kinase; MLH1: MutL homolog 1; MPP-9: Metalloproteinase-9; MSI: Microsatellite instability; MYD88: Myeloid differentiation primary response 88; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NK: Natural killer; PIK3CA: Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; SMAD4: Small mothers against decapentaplegic 4; TLR: Toll-like receptor; TP53: Tumor protein 53; ZO-1: Zonula occludens-1.

Table 2 The main pathways for the development of sporadic CRC and their relationship to gut dysbiosis

Pathway	Clinical features	Genetic and epigenetic disorders
The chromosomal instability (CIN) – 38%-85%[157,158]	- Associated with traditional adenomas[158]; - Associated with Fusobacterium, Escherichia/Shigella и Leptotrichia[63]; - CIN-H tumors are more frequently located on the left colon (87%), are associated with lymphocytic infiltration (82%), are more common in older patients (73%) and are associated with worse prognosis[157]	- Alteration in chromosome number or structure, loss of heterozygosity and aneuploidy[158]; - Mutations in the APC gene, a negative regulator of β-catenin-dependent Wnt signaling pathway (in 70%-80% of cases)[158]; - Mutations in TP53, KRAS, and PIK3CA genes and tumor suppressor genes SMAD2, SMAD4, and DCC[157,158]; - 20%-60% of tumors are CIN-H[158]
The CpG island methylator phenotype (CIMP) – 15%-30%Three groups based on the degree of gene methylation: High CIMP (CIMP -H); low CIMP (CIMP -L) and negative (CIMP-)[157,158]	- Associated with sessile serrated polyps[158,161,218]; - Associated with F. nucleatum[161,208], and E. faecalis[219]; - Data on the clinical features of CIMP-H tumors are controversial:- CIMP-H tumors are more frequently located in the left colon (67%) and are more common in men (68%) and in older patients (73%)[157,158]; - CIMP-H tumors are more common in females (68%) and in older patients (73%) and are associated with smoking, alcohol consumption, overweight, Western diet, right colon cancer, tumor lymphocytic infiltration, and poor differentiation[159,220,221]; - CIMP-H tumors are associated with worse prognosis[157,158,198,221,222];- CIMP-H tumors with MSI-H and BRAF mutations are more common in the proximal colon, whereas CIMP-L tumors with KRAS mutations are more common in the distal colon[158,159]	- Data on the presence of mutations in BRAF, PIK3CA, KRAS and TP53 genes are contradictory:- KRAS mutation in 27% tumors, PI3KCA mutation in 27%, and BRAF mutation in 7% tumors[157]; - Mutations in BRAF and PIK3CA genes and absence of mutations in KRAS and TP53 genes[154,159,161]; - Methylation of the MINT1, MINT2, MINT31, p14, p16 and MLH1 genes[154,159,161]; - 25% to 60% of tumors are MSI-H[157-159]
The microsatellite instability (MSI) – 15%-20%Three groups based on the number of microsatellites associated: High MSI (MSI-H); low MSI (MSI-L), and microsatellite stable (MSS)[158]	- 20% of cases are hereditary[158]; - Associated with F. nucleatum[124,161,208], E. faecalis[219] and P. micra[124]; - MSI-H are more frequently located on the right colon (86,7%), are more common in older patients(80%), and have a good prognosis at an early stage[157,158]	- High frequency of replication errors in MLH1 and MSH3 mismatch repair (MMR) genes[158]; - Hereditary CRC is associated with germline mutations in MMR genes: MLH1, MSH2, MSH6, and PMS2[158]; - 20%- 70% of tumors are CIMP-H[157,158]; - Often BRAF mutation (40%)[158]; - Mutations in the ACVR2A, TGFBR, MSH3, and MSH6 genes, as well as in the RNF43, RNF213, and ZNRF3 Wnt regulatory pathways[158]; - APC, TP53 and KRAS mutations are rare[158]

APC: Adenomatous polyposis coli; CIMP: CpG island methylator phenotype; CIN: Chromosomal instability; KRAS: Kirsten ras; MMR: Mismatch repair; MSI: Microsatellite instability; PIK3CA: Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; SMAD4: Small mothers against decapentaplegic 4; TP53: Tumor protein 53.