IL-6 is a multifunctional inflammatory cytokine, a small molecular polypeptide consisting of four α helices with a molecular weight of 19-228 kD, with 184 amino acid residues located in the p21 region of chromosome 7 (20). Studies have shown that bone marrow stem cells secrete IL-6, and tumor cells themselves and tumor-associated macrophages (TAMs) also release IL-6. Meanwhile, IL-6 can be subjected to upregulation of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and stress reactions. The expression of IL-6 is very low in normal human cells, with increased serum concentration in patients with hepatitis and liver cancer (21, 22). The most fundamental action of IL-6 and plays multipotent functions due to bind with its receptor. The IL-6 receptor(IL-6R) system is mainly composed of the IL-6 ligand binding chain and signal transduction chain, namely, IL-6R and gp130. IL-6R is usually found in many cells, such as hepatocytes, monocytes, macrophages and neutrophils, and is generally divided into membrane-bound IL-6R (mIL-6R) and soluble IL-6R (sIL-6R). mIL-6R is located on the cellular membrane surface, and sIL-6R is formed by protein hydrolysis of mIL-6R on the cellular membrane or directly by splicing mRNA during the translation phase (23). In the classical signaling transduction pathway, IL-6 contacts mIL-6R on the membrane, causes dimerization and then starts transduction of signaling, mainly participating in autoimmunity, metabolism, tumor development, etc. During signal transduction, first, IL-6 binds with sIL-6R, and then the complex binds with membrane gp130. This binding pattern plays a main role in inducing inflammatory reactions (24–26) (see Figure 1 ).

Signal transducers and activators of transcription proteins (STATs) are important in cellular signaling and include seven families: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 (27). STAT3 was originally found by Shi et al (28) as an acute phase reaction factor (APRF) in IL-6 signaling when studying interferon-induced gene transcription in 1996. STAT3 is a family of cytoplasmic proteins, and its encoding gene is located on chromosome 12, consisting of 750-795 amino acid residues with a molecular weight of 89-99 kDa. Its activation sites are primarily the SH2 domain (Src homology 2 domain) as well as tyrosine phosphorylation site 705 (Tyr705) and serine phosphorylation site 727 (Ser727) in the transcriptional activation region. The core structure of STAT3 mainly consists of a coiled-coil domain (CCD), DNA binding domain (DBD), linker domain (LD), Src homology 2 (SH2), amino acid terminal region, and carboxy-end trans activation region (29), where Tyr705, SH2 and DBD play a key role in STAT3 functions (30). The DBD is structurally an immunoglobulin folding domain that binds to DNA in the form of a dimer and is involved in the transfer of STAT3 from the cytoplasm to the nucleus. The SH2 region is the most conserved domain of the STAT3 protein and is mainly involved in the phosphorylation of tyrosine residues, promoting protein interactions with tyrosine phosphorylation proteins (31). This region has three binding pockets, the phosphorylated Tyr705 (pTyr705) binding site, a side pocket, and a hydrophobic binding pocket, where STAT3 participates in phosphorylation and plays an important role in the phosphorylation of STAT3. During STAT3 activation, the tyrosine and serine residues are phosphorylated by the upstream kinase and identified by the SH2 domain (32) (see Figure 2 ).

IL-6, as a classic extracellular stimulation factor of this signaling pathway, during the hepatocarcinogenesis and HBV or HCV stimulates initiation of HCC, the secretion of IL-6 is emerged in the microenvironment of liver tissue cells, then IL-6 interaction with its receptor, conformation of IL-6 changes after binding with its receptor, and then activation of gp130 on the cell membrane surface to trigger isodimer formation of gp130, thus leading to activation of Janus kinases(JAKs). After the activation process of JAKs, the binding sites interacting with STAT3 in the cytoplasm are exposed, wherein STAT3 acts primarily with the binding site of the corresponding tyrosine receptor through its SH2 domain. STAT3 binding to the tyrosine binding site to trigger phosphorylation of C-terminal domain tyrosine residues(Tyr705) on STAT3, and simultaneous activation followed by substantial aggregation and activation of STAT3 within the cytoplasm. Phosphorylated STAT3 forms homologous dimers through their SH2 domains, and the dimers are transported from the cytoplasm into the nucleus with the participation of the DBD. Then, the dimers bind to the promoter region of the downstream effector targeted genes, lead to change in the transcript activity of numerous genes, including antiapoptotic genes, angiogenic genes, proliferating genes, transformational genes, and the immune response factors. The expression of apoptosis-, growth- and metastasis-related proteins, such as Bcl-xL, Bcl-2, VEGF, Src, CXCR4, and MMP2/9 were also regulated by phosphorylated STAT3 forms, thus promoting the growth, development and inhibition apoptosis of cancer cells (33) (see Figure 3 ).

A positive/negative feedback pathway exists in cellular signaling. Negative regulation of IL-6/STAT3 mainly includes three classes of negatively regulated proteins: suppressors of cytokine signaling (SOCS), protein inhibitors of activated STAT (PIAS) and protein tyrosine phosphatases (PTPs) (see Figure 4 ).

The SOCS family consists of SOCS1-SOCS7 and CIS, with an N-terminal region, SH2 region and C-terminal SOCS box region. The SH2 region contains an SH2 domain, which can cooperate with N regions to make different SOCS proteins identify different targets by binding to specific cytokine receptors and then regulate various cytokine signal transduction pathways. SOCS molecules are negative feedback regulatory proteins of the classical IL-6/STAT3 signaling pathway, and they inhibit the phosphorylation of STAT3 and the formation of dimers or directly inhibit the phosphorylation of JAK, thus negatively regulating the IL-6/STAT3 pathway to inhibit the continuous proliferation and differentiation of cancer cells (34, 35). Its inhibitory effect on the IL-6/STAT3 pathway also disappears after SOCS inactivation, resulting in continuous proliferation and invasion of cancer cells. Overexpression of the SOCS proteins can inhibit the activity of STAT3 and thus promote apoptosis of cancer cells. Increasing evidence has shown that SOCS is closely related to the initiation and development of HCC (36), and evidence has shown that the absence of the SOCS protein or knockout of the SOCS3 gene in mice, leads to the disappearance of the negative regulation of IL-6/STAT3 by SOCS, thus causing continuous activation of STAT3 and ultimately promoting the occurrence and development of liver cancer (37).

The PIAS family includes PIAS1, PIAS2 (PIASx), PIAS3 and PIAS4 (PIASy), where PIAS3 is the primary specific suppressor of STAT3 and is naturally present in the cytoplasm. On the one hand, PIAS3 specifically binds to STAT3 dimers, thus concealing the DBD of STAT3, and on the other hand, it can bind with the STAT3 monomer to hinder its dimerization (38, 39). A PIAS3-deta peptide can significantly downregulate the expression of the tumor proliferation-related proteins STAT3, pSTAT3, Bcl-2, Cyclin D1, PCNA and c-myc and effectively inhibit the proliferation of HCC cells (40).

The PTP family includes SHP1, SHP2, CD45PTP1B, T-cell PTP (TC-PTP), PTPRT, and PTPBL, where SHP-1 and SHP-2 are the most representative and can bind to a phosphorylated receptor or JAK to dephosphorylate activated molecules, thus blocking the activation of the IL-6/STAT3 signaling pathway. Bard-Chapeau et al. (41) performed selective silencing of SHP-2 in HCC cells, which significantly increased phosphorylation of STAT3 induced by IL-6, thus promoting the transduction of the IL-6/STAT3 signaling pathway. Additionally, SHP-1 plays a strong inhibitory role in HCC epithelial-mesenchymal transition (EMT) by directly lowering pSTAT3 (Tyr705) by exerting its tyrosine phosphatase activity (42).

Classical IL-6/STAT3 signaling pathways are theoretically simple, but they can also interact with other signaling pathways, these signals interlace interaction to exert complex biological effects. (1) Ras signaling pathway: Activated JAKs can phosphorylate tyrosine residues of their associated receptors, leading to the assembly of sites for proteins containing SH2 domains from other pathways, such as SHP-2, which can recruit a large number of GRB2 molecules, and then continuous activation of cascade events, such as the Ras, Raf, MEK, and ERK signaling pathways (43). The IL-6/STAT3 pathway can also indirectly activate the Ras pathway through SOCS3 (44). (2) RTK signaling pathway: Numerous studies verified that STAT3 can be activated through receptor tyrosine kinases (RTKs) (45), and activation of some RTKs, such as epidermal growth factor receptor (EGFR), can cause STAT3 tyrosine phosphorylation through Src kinase. Activation of the RTK pathway results in the upregulation of mitogen-activated protein kinase (MAPK) activity, and MAPK specifically phosphorylates one serine (Ser) in the C-terminus of most STAT3 proteins. Phosphorylation of Ser greatly enhances the transcriptional activity of STAT3. (3) Tumor necrosis factor (TNF) signaling pathway: the TNF signaling pathway interacts with JAK/STAT3 at multiple levels (46). (4) PI3K/Akt signaling pathway: IL-6 binding with its receptor causes phosphorylation of JAK, thus recruiting phosphatidylinositol 3-kinase (PI3K) to the plasmalemma, and a large accumulation of PI3K produces pleckstrin homology (PH) domain binding of Akt. The phosphorylation at Thr308 and Ser473 sites of Akt molecule, leading to changes in the downstream substrate mTOR and thus playing a crucial role in promoting growth, proliferation, survival, differentiation, invasion and metastasis of cancer cells (47). Therefore, the biological effects of IL-6 family cytokines may be involved in the interactions between many signaling pathways (see Figure 4 ).

The tumor microenvironment was first formally proposed in 1979, and the microenvironment is a pivotal influence factor when treating cancer (68, 69). The internal environment where the tumor is located, consists of tumor cells themselves, interstitial cells, microvessels, microlymphocytes, tissue fluid, numerous cytokines and a small number of infiltrating cells (70, 71). Hyperactivation of STAT3 is important in the microenvironmental formation of inflammatory tumors and promotes tumor proliferation and metastasis (72). The tumor microenvironment changes dramatically when chronic inflammation and fibrosis occur in liver tissue, and activation of STAT3 can induce the expression and release of cytokines, chemokines and other media associated with chronic inflammation that play a key role in inducing and maintaining the cancer-promoting inflammatory environment. Studies have found that the phagocytosis of macrophages on apoptotic bodies promotes liver fibrosis, thus accelerating the circulation of hepatocyte death and compensatory hyperplasia and eventually leading to the occurrence of HCC. Tumor-associated macrophages (TAMs) promote tumor progression by secreting IL-6 to activate IL-6/STAT3 signals in adjacent HCC stem cells in liver tissue microenvironments (52). Zheng, et al. (73) found that activation of the HCC cells IL-6/STAT3 signaling pathway was possible by upregulating the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) to stimulate the transformation of normal liver fibroblasts (LFs) toward carcinoma-associated fibroblasts (CAFs), thus promoting the initiation of liver cancer.

Apoptosis of HCC cells is mainly achieved by upregulating the expression of anti-apoptotic factors or promoting survival signals. After IL-6-mediated STAT3 activation, promotes the expression of anti-apoptotic protein (Bcl-xL, Bcl-2, survivin and P53, etc.) plays an important role in the anti-apoptosis of HCC cells (73–76). Bcl-2 is particularly important proteins that promotes tumor cell survival. The key factor in apoptosis due to the balance between pro- and anti-apoptotic proteins. Activation of the IL-6/STAT3 signaling pathway may increase the ratio of apoptotic factors to anti-apoptotic factors, and increased IL-6 most likely changes this ratio (77). Meanwhile, phosphorylation of STAT3 can bind directly to the promoter of the survivin gene, upregulate survivin expression and promote the survival of tumor cells; by inhibiting STAT3 activity, survivin gene expression can be downregulated to promote apoptosis of liver cancer cells (78). These findings demonstrate that activation of the IL-6/STAT3 signaling pathway can promote the expression of survival-related proteins that inhibit apoptosis of HCC cells.

Vascular endothelial growth factor (VEGF) also plays an important role in tumor invasion and metastasis, which promotes vascular endothelial cell growth and tumor neoangiogenesis. VEGF is higher expression in liver tumor tissue than in cirrhosis and normal liver tissue (79). Hypoxia is an important factor in regulating the expression of VEGF, which can induce the secretion and expression of VEGF in tumor tissue through hypoxia inducible factor 1 (HIF-1). IL-6 binds with IL-6R to induce the activation of STAT3, and activated STAT3 binds to the promoter region of the VEGF gene to increase transcription, promoting the formation of tumor angiogenesis. The expression of VEGF can also be promoted through HIF-1α to stimulate angiogenesis in tumor tissues. In addition, there may be a positive feedback mechanism during malignant cell transformation between STAT3 and VEGF, namely, STAT3 upregulates VEGF, while VEGF combines with the cellular surface VEGF receptor (VEGFR) to activate the IL-6/STAT3 signaling pathway to further upregulate expression of VEGF and promote the generation of blood vessels in tumor tissues. Studies have shown that STAT3 can inhibit the degradation and increase synthesis of HIF-1α. Therefore, STAT3 is necessary for endothelial cell proliferation, migration, and angiogenesis. Blocking the IL-6/STAT3 signaling pathway can inhibit endothelial cell metastasis and angiogenesis, and hinder the tumorigenesis pathway (80). Additionally, a methylation study of liver cancer indicated that low expression of IL-6 can reduce angiogenesis in HCC tissues (81), suggesting that the IL-6/STAT3 signaling pathway can promote angiogenesis in tumor tissues and provide nutrient guarantees for the development of tumorigenesis.

IL-6 promotes the abnormal proliferation of cancer cells through the activation of the IL-6/STAT3 pathway, and the proliferation genes of cancer cells, such as Ras, Src, and cyclin D1, are the direct targets of STAT3 (82). Studies have found that IL-6-induced TAMs promote the amplification of CD44+ T cells to increase sphere and heterograft formation in culture, and blocking the IL-6/STAT3 signaling pathway can reduce the sphere formation ability of CD44+ T cells in the culture and growth of mouse xenotransplantation tumors (53). STAT3 inhibitors can inhibit the proliferation and development of cancers by blocking IL-6/STAT3 signaling, suppressing cancer cells proliferation and promoting apoptosis (83).

Generally, the destruction of the basement membrane is an important characteristic during cancer cells invasion. Cancer cells must penetrate the basement membrane and the natural tissue barrier formed by the extracellular matrix to undergo invasion and metastasis. Degradation of the basement membrane and the extracellular matrix is a crucial step in the invasion and metastasis of cancer cells. As the extracellular matrix degrades, cancer cells begin to infiltrate normal tissues and metastasize, and the process relies on matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9 (84, 85). MMPs interact with activator protein-1 (AP-1), of which expression is mainly regulated by the IL-6/JAK/STAT3 signaling pathway. Overexpression of MMP-9 and MMP-2 is associated with postoperative tumors in patients with liver cancer (84) and accelerates the invasion and migration capacity of HCC cells by regulating the JAK/STAT3 signaling pathway (86).

In addition, epithelial-mesenchymal transition (EMT) is closely related to primary lesion invasion and distant metastasis of cancer cells (87). The formation of EMT is mainly due to loss of the characteristics of epithelial cells, which is an important manifestation for obtaining the migration and invasion abilities of HCC cells. STAT3 is an important transcription factor in the occurrence of EMT, and studies have shown that STAT3 may play an important role in stimulating EMT through the regulation of many downstream genes (such as Snail and Twist) (88). Activated STAT3 signals are associated with Twist and calcium adhesion protein E (E-cadherin) expression and mediate the invasion and metastasis of HCC cells, and an abnormal pSTAT3/Twist/E-cadherin signal axis leads to poor prognosis in patients with liver cancer (89).

The IL-6/STAT3 signaling pathway is closely related to the immune escape of HCC cells. Studies have shown that IL-6 inhibits the antigenic presentation capability of dendritic cells (DCs) by activating IL-6/STAT3 signaling pathway. IL-6 blocked the antitumor immunity reaction in tumor cells (90, 91). STAT3 activation in DCs significantly reduces tumor immune surveillance. In the tumor-bearing host, STAT3 activation from tumor cells or from normal immune cells can both inhibit the secretion of inflammatory factors and reduce the immune surveillance of tumor cells. One study demonstrated that IL-6 secretion could upregulate programmed cell death-ligand 1 (PD-L1) expression in neutrophils, thus inhibiting the activity of T cells and ultimately accelerating the immune escape of tumor cells (92). Liu et al. (93) argued that IL-6 could promote the development of liver cancer by recruiting immunosuppressive cells and excluding CD8+ T cells in tumor microenvironments, and that IL-6 may also damage the function of infiltrated T cells in tumor tissues, thus inhibiting antitumor immunity.

Tumor cells multidrug resistance (MDR) is a notable reason for the clinical treatment failure of liver cancer. The occurrence of MDR is an extremely complex process involving multiple factors, genes and mechanisms. Most antitumor therapies can induce inflammation by killing tumor cells and normal tissues, and in this process, the expression levels of multiple inflammatory cytokines, including IL-6, IL-8, TNF-α, and other inflammatory factors, are upregulated (94). IL-6/STAT3 is closely related to tumor cells drug resistance, and the upregulation of the tumor inflammatory factor IL-6 can promote mitogen activation of protein kinase (MAPK) through the activation of signaling pathways, such as the IL-6/STAT3, PI3K/Akt, and Ras-MAPK pathways, thus upregulating the expression of various drug-resistant proteins, such as MRP, P-gp and BCRP, and leading to HCC cells resisting drug therapy (95). Relevant studies showed that knockout of IL-6 was able to stimulate expression of E-cadherin in HCC cells, and increased the HCC cells sensitivity to sorafenib (79). These found verified that the activation of IL-6/STAT3 signaling pathway has a capacity of promotion HCC cells resist chemotherapy.

There is increasing evidence that IL-6 is a therapeutic target for several cancers, reducing phagocytosis and migration mediated by STAT3 phosphorylation and by neutralizing IL-6 or antagonistic IL-6R in cancer cells. Studies have shown that apoptosis of HCC cells is facilitated by interfering the role and expression of IL-6, suggesting that blocking IL-6 can be used as a potential treatment for the sorafenib sensitivity of HCC cells (96). Evidence has also verified that anti-PD-L1 resistance can be reversed by blocking IL-6, which provides a potential strategy for overcoming the resistance of anti-PD-L1 in liver cancer (93). Recently, studies indicated that Madindoline A, a small molecule for inhibiting the dimerization of IL-6/IL-6R/gpl30 trimeric complexes, inhibits the growth of HCC cell line, HepG2 cells (97). Siltuximab and CNTO-136 are able to neutralize the activity of IL-6 (98, 99); Suppressing the activity of IL-6 by ALD518, and blocking IL-6R by monoclonal antibody(Tocilizumab) is an effective strategy for treatment of some cancers, such as non-small cell lung cancer (100), multiple myeloma (101), epithelial ovarian cancer (102), B-cell lymphoma (103), renal cell carcinoma (104) etc. These studies imply that blocking IL-6R is applied to therapeutic of HCC.

Numerous clinical studies have shown that JAK-specific inhibitors can reduce growth in various tumor models in vivo, including liver, pancreatic, brain, colorectal, stomach, lung, ovarian, and breast cancer. The studies revealed the effect of the JAK inhibitor Ruxolitinib on HCC cells, the results showed that ruxolitinib could effectively inhibit the JAK/STAT signaling pathway in HCC cells and significantly reduce the expression of the downstream JAK target pSTAT3 (105). Ruxolitinib also significantly reduces the proliferation and colony formation of HCC cells (105). AG490 is an artificial ester derivative of phenylacrylonitrile that effectively blocks JAK activation by competitively binding tyrosine kinase binding sites and thus suppresses STAT3 activation. By studying AG490, Thompson et al. (106) used it in in vitro experiments and found that the inhibitor AG490 significantly reduced the vitality of hepatoma SMMC-7721 cells, thus inhibiting the growth of xenotransplanted HCC cells. AZG1480 and TG101209 inhibit the activity of JAK to suppress the growth of HCC (106, 107). These results prove that JAK inhibitors are able to inhibit activation of STAT3 to suppress the growth of HCC cells.

STAT3 inhibitors can be classified into small molecular types, oligonucleotide types, peptide analogs, natural product derivatives, etc., according to the structure, the inhibitors can be divided into targeted SH2 domain inhibitors, targeted DNA binding domain inhibitors, targeted N-terminal domain inhibitors, STAT3 oligonucleotide inhibitors, etc. STAT3 region targeting inhibitors can suppress the proliferation, survival, and differentiation of HCC cells by preventing phosphorylation of STAT3, inhibiting the formation of STAT3 dimers, or interfering with their activity of interaction with DNA sequences. Jung et al. (108) showed that the proliferation of HCC cell was blocked by the small molecule STAT3 inhibitor C188-9. Gene expression analysis showed that C188-9-treated HepPten(-) mice had inhibited signaling pathways downstream of STAT3. The STAT3 small-molecule inhibitor LLL12 plays a role in blocking IL-6-induced STAT3 phosphorylation and nuclear translocation, thus inhibiting proliferation and promoting apoptosis of HCC cells (109), C188-9, Curcumin, OPB-31121, S31-201 and AZD9150 inhibit the phosphorylation of STAT3 to block the proliferation of HCC cells (108, 110–113). Studies also indicated that destroyed the structure of STAT3 dimer or inhibited the dimerization of STAT3 by small molecule, S3I-1757, STA-21 also can regress human cancer cells in xenografts animal model and abnormal proliferation disorders (114, 115). These evidences implicate that STAT3 inhibitors play important role in blocking the activation of IL-6/STAT3 signaling pathway, lead to inhibit the proliferation of HCC cells. The effect on inhibiting IL-6/STAT3 signal pathway for treatment of cancer is displayed in below table (see Table 1 and Figure 5 ).