We examined TLR4 expression in 60 patients’ tumor samples and hepatoma cell lines using reverse transcription polymerase chain reaction assays. One study showed that tumor and normal tissues, as well as HepG2 and H22 cells, expressed TLR4 messenger (m)RNA.58 Another recent study demonstrated that HCC patients whose tumors expressed high levels of both TLR4 and TLR9 had a poor prognosis.59 Chronic liver damage caused by excessive inflammation due the exposure to various risk factors often results in the development of fibrosis-associated HCC.60 Since stimulation of the TLR4-signaling pathways results in the production of proinflammatory immune mediators, it is likely that TLR4 is involved in the development and progression of hepatocarcinogenesis as well. Seki et al24confirmed that LPS/TLR4 signaling promoted liver fibrosis through two independent mechanisms. LPS/TLR4 induced HSCs to secrete chemokines that promote the chemotaxis of Kupffer cells, while TLR4-dependent signaling enhanced TGF-β signaling by downregulating bone morphogenetic protein and activin membrane-bound inhibitor.24 Diethylnitrosamine, a carcinogen, was shown to stimulate TLR4 signaling in mice, resulting in increased tumor size and number, both of which were reduced in MyD88-deficient mice.61 Dengue-induced apoptotic hepatocytes may activate myeloid cells such as Kupffer cells, induce inflammatory cytokine production and hepatocyte mitogens via the TLR4–MyD88 pathway, and eventually promote HCC development. LPS-induced TLR4 signaling also promotes cancer cell survival and proliferation in HCC.62,63 HMGB1 was secreted in response to the feeding second phase of NF-κB activation from HSP70 through TLR4 signaling, which may result in a higher invasion potential of hepatocarcinoma cells.64 TLR4- and MyD88-deficient mice had lower incidences of HCC and developed significantly fewer and smaller liver tumors compared to the wild-type controls, which implied that TLR4–MyD88 signaling plays a critical role in the development of HCC.48 Key mediating factors during this process seem to involve TLR–MyD88 signaling and the downstream activation of NF-κB. Collectively, TLR4–MyD88 signaling appears to be essential for hepatocarcinogenesis. Moreover, mutations or polymorphisms of the TLR4 gene are closely associated with liver carcinogenesis or malignancy.65

TLR4 and the HCC microenvironment

Malignant cells develop intricate mechanisms that enable them to secrete specific cytokines that create an immunosuppressive environment.3 Studies have correlated elevated TLR expression and dysfunctional immunity within the tumor microenvironment with cancer progression and reduced patient survival in a number of solid tumors.40,41 TLR signaling in immune cells is critical for the regulation of innate and adaptive immune responses, such as dendritic cell maturation and antigen presentation, as well as CD8+ T-cell cytotoxicity, all of which are important factors in antitumor immunity.10 TLR activation can also enhance regulatory T-cell suppressor function, favoring tumor development. Tumor-associated macrophages comprise as much as 50% of the tumor mass and provide essential support for the protumor microenvironment.66 Our study suggested that, in hepatoma cells, TLR4 may indirectly facilitate the recruitment of regulatory T-cells to the tumor site and promote intrahepatic metastasis through its interaction with macrophages.58


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Use of TLR4 for HCC therapy

Recent developments in TLR-related applications for cancer therapy have made it an attractive therapeutic target. Many clinical applications using TLR agonists have met with disappointing results when used as monotherapies due to the protumor mechanisms of cancer, which uses TLR expression to facilitate immune suppression.67–69 TLR agonists have met with greater therapeutic success when used as adjuvants in combination with radiation, chemotherapy, or cancer vaccines by priming the host immunity and leading to an enhanced T-helper (TH) cell cytotoxic or TH1 response. As discussed earlier, chronic inflammation, which plays a critical role in the progression of liver disease and the development of human cancers, is mediated by TLR activation. Hence, the modulation of TLR4 pathways using various drugs, antibodies, microRNAs (miRNAs), and small molecules that function as TLR4 agonists and antagonists to reduce liver inflammation and prevent the progression of liver diseases toward cancer is a promising strategy to combat HCC.

Lipid A mimetics that bind directly to TLR4–MD2 were shown to inhibit LPS-mediated activation of TLR4 signaling both in vitro and in vivo.67 A new small molecule, TAK-242, can suppress the expression of inflammatory mediators by selectively inhibiting the TLR4-mediated signaling pathway.68 Because the intestinal microbiota and TLR4 promote HCC, Schwabe et al69 found that the nonabsorbable antibiotic rifaximin could moderately reduce hepatocarcinogensis at late stages, and subsequent TLR4 activation in Kupffer cells has been suggested.

Cowden et al70 recently tested two inhibitors of the histamine H4 receptor that interact with TLR4 and found that they reduced TNF-α production and LPS-induced inflammation in mouse livers. It has become well established over the past decade that the aberrant expression of a large number of miRNAs correlate with HCC disease severity and poor prognosis.2,71,72 In bone marrow-derived macrophages and RAW264.7 cells, interleukin-10 inhibits miR-155 expression after LPS stimulation and dampens inflammatory immune responses in a STAT-3-dependent manner.73 Although the potential of TLR4 immunomodulation for HCC immunotherapy has not yet been extensively explored, initial results from preclinical and clinical studies look promising. It is reasonable to imagine the development of a TLR4 immunomodulatory agent that reduces the inflammatory response but promotes antitumor immunity. This could be beneficial in controlling multiple stages of HCC.