Dapito DH, Mencin A, Gwak GY, Pradere JP, Jang MK, et al. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 2012;21:504-516. (Reprinted with permission.)
Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.
Evolution of chronic liver injury to cirrhosis through hepatic inflammation precedes around 80% of hepatocellular carcinomas (HCCs), the third most deadly cancer worldwide and sixth in terms of incidence.1 Cirrhosis is frequently associated with intestinal bacterial overgrowth and functional impairment of the gut barrier, secondary to local hypoxia and edema due to portal hypertension and other factors (e.g., alcohol).2 This increase in permeability of the intestinal wall exposes the liver to translocation of bacteria mainly from the enteric venous system to the portal vein and lymph nodes. As a consequence, bacterial translocation promotes liver inflammation through activation of Toll-like receptors (TLRs),3 whose role in the pathogenesis of liver fibrosis and cirrhosis has been investigated broadly.4
Dapito et al. recently demonstrated that lipopolysaccharide (LPS)-induced TLR4 signaling promotes hepatocarcinogenesis in a mouse model of liver damage induced by a combination of diethylnitrosamine and hepatotoxin carbon tetrachloride.5 This model incorporates different aspects of chronic liver disease preceding HCC development, including liver injury, inflammation, and fibrosis. Using experimental approaches such as genetic TLR4 inactivation, gut sterilization, and prolonged treatment with low-dose LPS, the authors showed that LPS production from the intestinal microbiota induced activation of TLR4 signaling in resident liver cells, particularly hepatic stellate cells. Upon stimulation, TLR4 triggered the activation of the nuclear factor κB (NF-κB) pathway and subsequent production of epiregulin, a member of the epidermal growth factor family (Fig. 1). The secretion of epiregulin and other downstream mediators generated a protumorigenic milieu in a preexisting inflammatory microenvironment and the development of HCC. In particular, TLR4 acted as a promoter of HCC at advanced stages of liver disease, although it was not sufficient to initiate it.
Robust evidence supports a strong link between inflammation and cancer, including HCC.6 This study provides additional support for this connection and persuasively demonstrated the role of TLR4 activation as a promoter. TLR4 activation by LPS and other pathogen-associated molecular patterns (PAMPs) induces NF-κB signaling and subsequent secretion of several cytokines (e.g., interleukin-1) and other inflammatory molecules (e.g., tumor necrosis factor α), which regulate multiple reactions in hepatocytes, Kupffer cells, and hepatic stellate cells. Accordingly, a previous study demonstrated that activated NF-κB acted as a promoter of liver cancer in a mouse model of lymphotoxin-induced HCC,7 supporting the important role of this cascade in the development of this neoplasia.
Because HCC typically occurs in chronically injured livers and bacterial translocation is both a frequent event in patients with advanced cirrhosis and a promoter of tumor growth in animal models, it is reasonable to postulate that strategies interfering with this process might delay or impair promotion and progression of HCC. These novel approaches include the inhibition of bacterial translocation using probiotics and antibiotics and the inhibition of TLR4 pathway by antagonist TLR4 ligands and small molecule inhibitors of downstream signals (Fig. 1). More established alternatives proposed as preventive strategies for HCC chemoprevention include the blockade of epidermal growth factor (EGF) signaling with erlotinib or other EGF receptor inhibitors.8
The use of probiotics and antibiotics has been shown to reduce bacterial translocation in a mouse model of sepsis and decrease liver injury in patients with cirrhosis.9–11 Dapito et al. demonstrated the efficacy of rifaximin, a nonabsorbable antibiotic already used in subjects with advanced liver disease and hepatic encephalopathy, for reducing promotion of HCC in their mouse model. It would have been interesting to explore the effect of norfloxacin in HCC promotion, considering that it is the standard antibiotic used for prevention of spontaneous bacterial peritonitis and infections in high-risk patients who have cirrhosis. Further studies testing these approaches should consider, though that long-term use of antibiotics might alter gut flora balance resulting in dangerous side effects and complications (e.g., infections due to overgrowth of antibiotic-resistant bacteria, malabsorption, and antibiotic- associated diarrhea).
Another avenue that requires further evaluation is the potential role of blocking TLR4 and/or NF-κB signaling in liver cancer prevention or progression. Eritoran tetrasodium (E5564) is a lipid A analogue that binds to an internal pocket of MD-2, a coreceptor of TLR4, and prevents the activation of downstream signals. Preclinical data indicate that E5564 limits inflammation induced by LPS and improves survival in a sepsis model.12 Furthermore, a recent phase III clinical trial enrolling patients with severe sepsis showed benefits of E5564 and minimal side effect profile, but further studies are needed to assess its clinical safety.12 Other inhibitors include CRX-526, an antagonist ligand of TLR4 that inhibits tumor necrosis factor α production after stimulation with LPS in vivo,13 and TAK-242 (resatorvid), a small-molecule inhibitor of the intracellular domain of TLR-4. Studies in mice injected with LPS and treated with TAK-242 showed a decrease of cytokine levels and protection from LPS-induced lethality,14 although no significant difference was observed in patients with severe sepsis enrolled in a randomized clinical trial.15 So far, none of these agents have been tested in the setting of clinical trials with patients with chronic liver disease or HCC. Finally, modulators of NF-κB signaling emerge as a novel class to be explored due to its activation in the majority of liver diseases.16 Bortezomib prevents NF-κB nuclear localization by blocking the proteasomal degradation of inhibitor of κB forms. Despite promising preclinical data, a recent phase II clinical trial showed nonrelevant activity of bortezomib associated with notable adverse events in patients with unresectable HCC.17 All these approaches should be further tested in refined animal models. The model presented by Dapito et al represents an interesting approach since it includes different features of the hepatocarcinogenic process and directs our attention to possible novel inhibitors for evaluation.
Of note, targeting TLR4 signaling might lead to immunosuppressive effects, and its use should be restricted to patients who would likely benefit from these therapies. In this perspective, a gene signature generated from the adjacent nontumor tissue of HCC patients has been proposed to accurately identify patients with cirrhosis who are at high risk of HCC development and poor prognosis.18, 19 This 182-gene signature includes genes involved in inflammation (e.g., interleukin-6 and NF-κB–related genes) and proliferation (e.g., EGF), providing additional evidence linking this biological process to tumorigenesis. Once validated, the implementation of this signature to HCC surveillance would allow trial enrichment with those patients who might benefit from the novel chemopreventative strategies discussed above.
In conclusion, given the current lack of chemopreventive agents and the strong implication of inflammatory pathways in hepatocarcinogenesis, there is a growing rationale to explore the therapeutic benefit of TLR4 and other NF-κB modulators combined or not with antibiotics in patients at high risk of HCC development.