Bile acid-microbiota crosstalk in gastrointestinal inflammation and carcinogenesis

Abstract

Emerging evidence points to a strong association between the gut microbiota and the risk, development and progression of gastrointestinal cancers such as colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Bile acids, produced in the liver, are metabolized by enzymes derived from intestinal bacteria and are critically important for maintaining a healthy gut microbiota, balanced lipid and carbohydrate metabolism, insulin sensitivity and innate immunity. Given the complexity of bile acid signalling and the direct biochemical interactions between the gut microbiota and the host, a systems biology perspective is required to understand the liver-bile acid-microbiota axis and its role in gastrointestinal carcinogenesis to reverse the microbiota-mediated alterations in bile acid metabolism that occur in disease states. An examination of recent research progress in this area is urgently needed. In this Review, we discuss the mechanistic links between bile acids and gastrointestinal carcinogenesis in CRC and HCC, which involve two major bile acid-sensing receptors, farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5). We also highlight the strategies and cutting-edge technologies to target gut-microbiota-dependent alterations in bile acid metabolism in the context of cancer therapy.

Figure 1. Bile acid biosynthesis, transport and metabolism

The most abundant bile acids in mammals are the primary bile acids CA and CDCA, and the secondary bile acids DCA and LCA. Bile acids are synthesized in hepatocytes via cytochrome P450-mediated oxidation of cholesterol, which occurs through two pathways: the ‘classical’ and ‘alternative’ pathways. The ‘classical’ pathway is initiated by CYP7A1 and produces the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) through the subsequent enzymatic action of CYP8B1, and CYP27A1, The ‘alternative’ pathway is initiated by CYP27A1 and yields CDCA via CYP7B. Expression of CYP8B1 determines the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA) by promoting CA biosynthesis via the ‘classical’ pathway. In the rodent liver, the majority of CDCA is converted to α-muricholic acid (MCA and β-MCA); in the pig, CDCA is primarily converted to hyocholic acid (HCA), whereas in humans it remains as CDCA. In hepatocytes, most bile acids are conjugated to glycine (glyco-, G) or taurine (tauro-, T) through the action of bile acid:CoA synthetase (BACS) and bile acid-CoA:amino acid N-acyltransferase (BAAT) prior to their secretion into bile via bile salt export pump (BSEP). Differences in the types of congugated bile acids produced exist between human and rodents; the solid line and dashed line rectangles list the dominant bile acids in humans and rodents, respectively. At the same time, sulphated (sulpho-) or glucuronidated (glucurono-) bile acids produced by the liver via sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) amidated with a taurine or a glycine are taken up by the multidrug resistance-associated protein 2 (MRP2). In the intestine, microbial enzymes from gut bacteria (dashed arrows) metabolize bile acids; glyco-conjugated and tauro-conjugated CA and CDCA are deconjugated via bile salt hydrolases (BSH) and 7α -dehydroxylated to form secondary bile acids (DCA and LCA). Tα-MCA and Tβ-MCA are deconjugated via BSH to form α-MCA and β-MCA. β-MCA is C-6 epimerized to form ω-MCA and then ω-MCA is 7α-dehydroxylated to from HDCA. CDCA is transformed to UDCA using the hydroxysteroid dehydrogenase (HSDH). Glucurono and sulpho-conjugated BAs are mainly excreted into urine by MRP2^,. The main bacterial genera of the gut microbiota involved in bile acid metabolism include: Bacteroides, Clostridium, Lactobacillus, Bifidobacterium and Listeria in bile acid deconjugation; Bacteroides, Eubacterium, Clostridium, Escherichia, Egghertella, Eubacterium, Peptostreptococcus and Ruminococcus in oxidation and epimerization of hydroxyl groups at C3, C7 and C12; Clostridium and Eubacterium in 7-dehydroxylation; Bacteroides, Eubacterium and Lactobacillus in esterification; and Clostridium, Fusobacterium, Peptococcus and Pesudomonas in desulfatation^,. At the terminal ileum, most of the unconjugated bile acids including CA, CDCA, DCA, UDCA, HDCA, α-MCA, β-MCA, ω-MCA are reabsorbed by apical sodium-dependent BA transporter (ASBT) into the enterocytes, and secreted into the portal circulation via basolateral bile acid transporters organic solute transporter α (OSTα), OSTβ, MRP2 and MRP3. Bile acids are then taken up by sodium-dependent taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide 1 (OATP1) into hepatocytes. Hepatic MRP3, MRP4 and OST-α/β also provide alternative excretion routes for bile acids into the systemic circulation^,. CA, cholic acid; CDCA, chenodeoxycholic acid; α-MCA, α-muricholic acid; β-MCA, β-muricholic acid; ω-MCA, ω-muricholic acid; HCA, hyocholic acid; DCA, deoxycholic acid; HDCA, hyodeoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; T α-MCA, tauro α-muricholic acid; T β-MCA, tauro β-muricholic acid; LCA, Lithocholic acid; UDCA, ursodeoxycholic acid; CYP7A1, cholesterol 7 alpha-hydroxylase; CYP8B1, sterol 12α-hydroxylase; CYP7B1, oxysterol 7α-hydroxylase; CYP27A1, sterol 27-hydroxylase; BACS, bile acid, CoA synthetase; BAAT, bile acid-CoA, amino acid N-acyltransferase; BSEP, bile salt export pump; ASBT, apical sodium-dependent bile acid transporter; OSTα/β, organic solute transporter alpha/beta; MRP2, multidrug resistance-associated protein 2; MRP3, multidrug resistance-associated protein 3; MRP4, multidrug resistance-associated protein 4; NTCP, sodium-dependent taurocholate cotransporting polypeptide; OATP1, organic anion transporter 1; HSDH, hydroxysteroid dehydrogenase

Figure 2. Enterohepatic circulation of bile acids under normal physiological conditions (a) and during dysbiosis and inflammation (b)

The enterohepatic circulation of bile acids between the intestine (enterocytes) and liver (hepatocytes) under normal physiological conditions (a, solid black arrows), and the alterations that occur in this process during dysbiosis of the gut microbiota and inflammation (b, dashed black arrows), are shown. During intestinal inflammation, which occurs as a result of intestinal barrier dysfunction, expression of intestinal FXR is downregulated, which results in: reduced FGF19-FGFR4 signalling; downregulation of intestinal bile acid transport protein (IBABP); downregulation of the bile acid efflux transporters OSTα an OSTβ^,; and upregulation of the apical sodium-dependent BA transporter (ASBT). Reduced expression of IBABP carrier protein results in a reduction in the transfer of bile acids across the enterocyte to the OSTα/β efflux transporters for entry into the portal vein, thus disrupting enterohepatic circulation. The combination of these events permit increased influx of bile acids into the enterocyte and prevention of bile acid efflux back into the portal vein. Collectively, increased influx and decreased efflux of bile acids might increase inflammation in the intestinal mucosa. Diminished signalling via the FGF19-FGFR4 axis also increases bile acid synthesis in the liver via reduced activation of c-JUN/ERK/CYP7A1 axis^–. During hepatic inflammation caused by bile acid perturbation of hepatocyte membranes and subsequent activation of pro-inflammatory PKC pathways leads to the activation NF-κB which inhibits transcription of hepatic FXR, and also prevents the subsequent activation of SHP that normally inhibits the synthesis of the rate-limiting enzyme for BA synthesis, CYP7A1. Therefore, decreased FXR expression promotes increased bile acid synthesis in combination with increased influx of bile acids due to the fact that OSTα/β, MRP3/4 and all of the cannicular transporters (depicted in grey ovals) are under transcriptional control by FXR. Decreased FXR transcription also leads to decreased expression of NTCP transporters which are involved in bile acid influx; however, OATP transporters are not affected by FXR. FXR also controls bile acid detoxification; decreased transcription of FXR leads to decreased expression of PPARα and its target genes which encode Cytochrome P450 enzymes (CYPs), sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs). Decreased PPARα expression also induces additional decreases in expression of the MDR2/3, MRP3 and MRP4 transporters. Under these conditions, both cholestasis and inflammation are intensified which can lead to the development of liver cancer. Cholestasis causes inflammation via various mechanisms which leads to upregulation and activation of NF-κB which in turn, binds directly to the FXR promoter to inhibit its transcription. BSEP, bile salt export pump; CYP7A1, cholesterol-7α-hydroxylase; MDR3/4, multidrug resistance protein 3/4; MRP2/3/4, multidrug resistance-associated protein 2/3/4; NTCP, sodium taurochlorate co-transporting polypeptide; Ostα/β, organic solute transporter α/β; PC, phosphatidylcholine; PPARα, peroxisome proliferator-activated receptor α; SHP, small heterodimer partner; IBABP, ileal bile acid binding protein; FGF19, fibroblast growth factor 19; OATP, multi-specific orgnanic anion transporters; FGFR4, fibroblast growth factor receptor 4; ABCG5/8 ATP-binding cassette subfamily G, members 5/8; ASBT, apical sodium-dependent bile acid transporter; SULTs, sulfotransferases; UGTs, UDP-glucuronosyltransferases

Figure 3. Bile acid–induced hepatic inflammation and carcinogenesis

Owing to their lipophilic, detergent properties, bile acids can directly disrupt the plasma membrane and cause activation of PKC which in turn activates the p38 MAPK pathway. The resultant activation of p53 and NF-κB lead to induction of apoptosis and increased inflammation. Activated NF-κB translocates to the nucleus and promotes transcription of genes that encode pro-inflammatory cytokines such as TNFα, IL-1β and IL-6, which can positively regulate NF-κB activation and thus promote a continued cycle of inflammation. IL-6 also activates the Janus Kinase (JAK)–Signal Transducer and Activator of Transcription 3 (STAT3) pathway which leads to decreased apoptosis and progression of HCC. IL-1β also activates the PI3K–MDM2 pathway to negatively regulate p53, thus increasing survival of DNA damaged cells which might lead to HCC. The NF-κB p50/p65 heterodimer has also been shown to bind directly to the promoter of FXR and inhibits its transcription, resulting in: reduced expression of bile acid transporters such as OSTα/β, BSEP, MRP2, MDR2/3 which leads to decreased bile acid efflux and cholestasis and increased biosynthesis of bile acids. Inhibition of FXR transcription collectively leads to increased amounts of bile acids in the liver which causes inflammation that can lead to HCC. Membrane perturbation by bile acids can also activate PLA₂ which causes release of arachidonic acid (AA) from the cell membrane via cyclooxygenase (COX) and lipoxygenase (LOX), ultimately resulting in increased levels of reactive oxygen species (ROS) in the hepatocyte^,. ROS can directly activate NF-κB and can also induce direct DNA damage in cells which might lead to HCC. PKC, protein kinase C; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; PI3K, phosphoinositide 3-kinase; TNFα, tumor necrosis factor; JAK, Janus kinase; STAT3, signal transducer and activator of transcription-3; PLA₂, phospholipase A₂; COX, cyclooxygenase; LOX, lipoxygenase; ROS, reactive oxygen species; AA, arachidonic acid

Figure 4. Bile acid-induced TGR5 signalling pathways in macrophages

The M1 macrophage phenotype is pro-inflammatory and M2 phenotype is immunosuppressive. Although bile acids are not able to induce complete macrophage polarization to either the M1 or M2 phenotype, they have been shown to activate TGR5 and produce a ‘mixed phenotype’ that exhibits dominant immunosuppressive behavior as evidenced by an increased IL-10:IL-12 ratio. In response to bile acids, TGR5 can activate both the adenyl cyclase–cAMP and EGFR–SRC pathways. In the pro-inflammatory (dotted arrows) M1-like pathways, TGR5-dependent transactivation of EGFR can induce pro-inflammatory signalling via activation of SRC, which in turn activates ERK1/2 and PKC. This transactivation has been shown to occur when EGFR and a relevant GPCR such as TGR5 co-exist side by side in a lipid raft (signified by cholesterol and sphingolipid). Upon TGR5 activation, metalloproteinase-dependent cleavage of the EGFR ligand HB-EGF occurs. TGR5–dependent activation of PKC results in the activation of NF-κB, which causes increased expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα, along with further autocrine activation of ERK1/2 and continued cycles of pro-inflammatory signaling. SRC-dependent activation of ERK1/2 in the M1-like phenotype results in increased expression of c-MYC which increases cell apoptosis. Pro-inflammatory M1-like signalling also regulates innate immunity by increasing activation of pro-inflammatory responsive Th17 cells (increased IL-1β, IL-6) and Th1 cells (increased IL-12, IFNγ) with concurrent suppression of immunosuppressive T_reg cells (decreased IL-10) via the pro-inflammatory cytokines it produces. The pro-inflammatory M1-like phenotype is not dominant during bile acid-dependent activation of TGR5; concomitantly, SRC activation also activates STAT3, which promotes anti-inflammatory effects (solid arrows) including decreased activation of Th17 and Th1-cells, decreased production of TNFα, IFNβ, IL-6, IL-12, and increased production of IL-10 and TGFβ, which corresponds with an increase in the IL-10:IL-12 ratio^,. Bile acid–activated TGR5 also activates the cAMP pathway independently from EGFR transactivation; cAMP activates PKA which leads to the upregulation of cAMP response element binding protein (CREB) expression and activity. cFos, an important target gene of CREB, binds to the p65 subunit of activated NF-κB to inhibit its translocation to the nucleus, representing an anti-inflammatory effect of TGR5. Another important target gene of CREB is IL-10 which exerts immunosuppressive and therefore anti-inflammatory effects^,,. Bold solid arrows represent M2-like immunosuppressive signaling pathways and dotted arrow represent M1-like signaling pathways. cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; CREB, cAMP response element binding protein; ERK, extracellular receptor kinase; STAT3, signal transducer and activator of transcription-3; G_αs, G-protein alpha-s; ROS, reactive oxygen species; PKC, protein kinase C; EGFR, epidermal growth factor receptor; HB-EGF, heparin-binding epidermal growth factor; MMP, matrix metalloproteinase; SRC, sarc kinase; IL-6,10,12, interleukin-6,10,12; TNFα, tumor necrosis factor-α; TGFβ, transforming growth factor-β; cFos, cFos gene/protein

Figure 5. Effects of intestinal bile acids on colorectal carcinogenesis

Secondary bile acids, in particular deoxycholic acid (DCA), influence several signaling pathways in enterocytes that can lead to the development of colorectal cancer (CRC). Bile acids can activate EGFR which in turn leads to activation of RAS–ERK1/2 signalling. ERK1/2 induces activation and nuclear translocation of c-JUN and c-FOS, which together form the Activator protein 1 (AP-1) transcription factor complex, which promotes the transcription of the AP-1 target gene c-MYC. Expression of c-MYC increases cell proliferation and stemness. Activation of EGFR also leads to the upregulation of cyclooxygenase (COX) and lipoxygenase (LOX) which promote the production of reactive oxygen species (ROS). Bile acids can disrupt the plasma membrane to release arachidonic acid (AA) into the cytoplasm where it is rapidly used as a substrate for the biosynthesis of both COX and LOX enzymes^,. NADP(H) oxidase can be stimulated by bile acids to further increase ROS production. In the enterocyte, ROS can cause DNA damage and inflammation, leading to upregulated expression of the pro-inflammatory cytokine IL-1β. All of these events contribute to the development and progression of CRC. Perturbation of the plasma membrane by bile acids also activates PKC, which subsequently activates p38 MAPK and NF-κB. Activated NF-κB translocates into the nucleus where it transcribes the gene encoding IL-1β. IL-1β can then signal in an autocrine and paracrine fashion to activate the PI3K–MDM2 axis which results in blockage of p53 activity. Decreased p53 activity leads to decreased apoptosis and increased survival of DNA-damaged cells, thus contributing to the potential development of CRC^,. Bile acids also stimulate the binding of Wnt to the low-density lipoprotein receptor-related protein (LRP)–frizzled receptor (Fzr)–E-cadherin-β-catenin complex, which causes release of β-catenin from E-cadherin and into the cytoplasm where it translocates to the nucleus and stimulates transcription factors such as LEF/TCF to increase cell proliferation and cell stemness, thus contributing to the development and progression of CRC. DCA, deoxycholic acid; CRC, colorectal cancer; EGFR, epidermal growth factor; Ras, Ras oncoprotein; ERK1/2, extracellular signal-regulated kinase 1/2; C-Jun, C-Jun protein; C-Fos, C-Fos protein; c-Myc, myc oncoprotein; AA, arachidonic acid; COX, cyclooxygenase; LOX, lipoxygenase; ROS, reactive oxygen species; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; IL-1β, interleukin-one-beta; PI3K, phosphatidylinositol 3-kinase; MDM2, mouse double minute chromosome 2; Wnt, Wingless-related integration site; LRP, low density receptor related protein; Fzr, frizzled receptor; APC, adenomatous polypopsis coli; LEF, lymphoid enhancer factor; TCF, T-cell factor