The Role Of RIG-I Signaling Pathway In HCC Immunotherapy And The Underlying Mechanisms

Hepatocellular caicinoma (HCC)is one of the cancers with poor pronostic, causing about half a million deaths each year. Up to now, there is no effective approach for the treatment of HCC, especially advanced metastatic HCC. Liver is a unique organ prone to be tolerogenic. HCC is also not sensitive to the current immunotherapy. How HCC escapes the immunological attack or induces immunosuppression of host remains to be fully understood to date. How to reverse the HCC-induced immunosuppression may be useful to improve the effectiveness of the immunotherapy.Recent studies show that there is a positive relation of HCC and infection of HBV and HCV. It is now known that viruses are initially recognized by host pattern-recognition receptors (PRRs). Viral infection of host cells leads to the initiation of antiviral innate immune responses, which results in the induction of the type I interferons (IFNs) IFN-αand IFN-βand pro-inflammatory cytokines. Retinoic-acid-inducible gene I (RIG-I), as a member of the retinoic-acid-inducible gene I(RIG-I)-like receptors (RLRs), plays an important role in the induction of antiviral innate responses including induction of type I IFNs. Type I IFNs have been demonstrated to be useful for the treatment of some kinds of cancers including HCC. However, what the expression of RIG-I in HCC and what is the role of RIG-I in HCC development and treatment remain unclear. We also wonder whether RIG-I is involved in the responsiveness of HCC to the immunotherapy.In this study, we investigated the expression and the role of RIG-I in HCC, and demonstrated that decreased expression of RIG-I may be one of the mechanisms for HCC escape of immune control and overexpression of RIG-I in HCC can increase the effectiveness of IFN-αin the suppression of HCC growth by promoting IFN-α-induced cell arrest and apoptosis. Part I. Decreased expression of RIG-I in human hepatocellular carcinomaFirst, with tissue array, we analyzed the expression of RIG-I in HCC, and found that 90%(27/30) of para-tumor samples were positive for RIG-I expression, but only 20% (6/30) of HCC samples were RIG-I positive. We also found that the expression of RIG-I decreased significantly in tumor samples of 73% cases of HCC compared to that of para-tumor samples. The results of IHC showed the expression of RIG-I was located mostly in cytoplam in para-tumor samples, while negative in HCC samples. These data indicated that the expression of RIG-I was reduced significantly in HCC. In addition, we showed that expression of RIG-I was also decreased significantly in other kinds of cancers we detected through tumor tissue array, which included bladder cancer, renal clear cell carcinoma, endometrial carcinoma and esophagus cancer.In order to further confirm the results of tissue array, we isolated the total RNA and protein of clinical tissue samples of HCC and examined the expression of RIG-I by real time PCR and Western blot. Similar results were observed as those of tissue array, showing that the expression of RIG-I was reduced significantly in HCC compared with that in para-tumor and normal liver tissue samples.Part II. Suppression of RIG-I expression in hepatocellular carcinoma by Basic FGF and the underlying mechanismsThen we investigated the underlying mechanisms for the decreased expression of RIG-I in HCC. It has been shown that acyclic retinoid (ACR) could inhibit HCC cell growth by suppressing FGF-mediated signaling pathways. Also, RIG-I is induced by ACR and bFGF is highly expressed in HCC. Therefore, we speculated if bFGF might cause the decrease of RIG-I expression in HCC. We used different doses of bFGF to stimulate several kinds of HCC cell lines and 24h later, the expression of RIG-I was analyzed by real time PCR. The results showed that the SMMC-7721 cells were most sensitive to bFGF stimulation as compared to HCC HepG2 cells, Hep3B cells and normal liver (Changliver) cells. The expression of RIG-I in SMMC-7721 cells was significantly reduced after stimulation with 100ng/ml bFGF for 24h.The FGFs mediate their biological effects by binding to their high-affinity cell-surface receptors with protein tyrosine kinase activity. Four receptors of FGF have been identified in human. So, we tested the expression of FGFRs in SMMC-7721 cells, HepG2 cells, Hep3B cells and Changliver cells. We found FGFR1and FGFR3 were highly expressed in SMMC-7721 cells. Furthermore, we examined the expression of RIG-I, bFGF, FGFR1 and FGFR3 in HCC clinical samples by real time PCR, and found that lower expression of RIG-I was correlated with higher expression of bFGF, FGFR1 and FGFR3 in HCC tissue samples.We selected the SMMC-7721 cells as HCC cell model in the following studies. The expression of RIG-I was tested at indicated time in SMMC-7721 cells after bFGF stimulation. The results revealed that bFGF inhibited RIG-I expression in SMMC-7721 cells in a time-dependent manner.To investigate the mechanism for the suppression of RIG-I expression by bFGF, we pretreated SMMC-7721 cells with PD173034, the specific inhibitor of FGFR, and inhibitors of PI3K/Akt and ERK pathways (LY294002 or U0126) for 1h respectively. Then bFGF was added to stimulate SMMC-7721 cells for 24h and RIG-I expression was detected by real time PCR and Western blot analysis. . We found that the inhibition of RIG-I expression by bFGF was reversed by these inhibitors, indicating that activation of PI3K/Akt and ERK pathways was involved in the suppression of RIG-I expression by bFGF.Part III. RIG-I overexpression sensitizes hepatocellular carcinoma cells to the cell arrest and apoptosis induction by IFNalphaIn addition to the critical role in the antiviral response, IFNs can also regulate the immune response, induce cell growth inhibition and cell differentiation. Human recombination IFN-αhas already been widely used in clinical antiviral and antitumor treatments. RIG-I signaling pathway can induce the production of type I IFN, and in turn, type I IFN can improve the RIG-I expression, which may constitute a positive loop. So, we wonder what the effect of RIG-I on the responsiveness of HCC to IFN-αtreatment is.First, we used different dosese of rhIFN-αto stimulate SMMC-7721 cells in vitro according to the previous reports. By MTT analysis, we found there was no obvious difference of cell growth inhibition by different doses of rhIFN-α, indicating that SMMC-7721 cells were insensitive to IFN-αstimulation.Then we overexpressed RIG-I in SMMC-7721 cells. Twenty-four hours post-transfection, 2000IU/ml rhIFN-αwas used to treat SMMC-7721 cells for 24h. Interestingly, the growth of these RIG-I-overexpressing SMMC-7721 cells was inhibited more obviously than that of the SMMC-7721 cells only treated with 2000IU/ml rhIFN-α, suggesting that RIG-I overexpression can sensitize SMMC-7721 cells to the growth inhibition by rhIFN-α.In order to figure out the mechanisms for the increased cell growth inhibition observed above, cell cycle and apoptosis of SMMC-7721 cells were examined by FACS after IFN-αstimulation for 24 hours when SMMC-7721 was transfected with RIG-I full-length plasmid. The results indicated that overexpression of RIG-I could enhance the effect of IFN-αtreatment significantly by inducing a G2/M phase cell arrest and cell apoptosis.In summary, we demonstrate that RIG-I expression is decreased in HCC, however, bFGF and its receptors FGFR1 and FGFR3 are highly expressed in HCC. bFGF can inhibit the expression of RIG-I in HCC cells through activation of PI3K/Akt and ERK pathways. Furthermore, we show that RIG-I overexpression in HCC cells can make HCC cells more sensitive to the G2/M phase cell arrest and cell apoptosis induced by IFN-αtreatment. Thus, FGF-mediated suppression of RIG-I in HCC cells may result in the low responsiveness of HCC to IFN-αtreatment, providing a new mechanistic explanation for the immune escape of HCC and also outling a new approach of RIG-I overexpression for the treatment of HCC.