IFN-stimulated Genes IFIT Family Members Feedback Amplify Type ⅠIFN Effector Signaling Via Promoting STAT1and STAT2Activation

Because of the potent antiviral and apoptosis inducing activity of type Ⅰ interferon (IFN), IFN-based therapies, especially IFN-α therapies have been widely used for the clinical management of viral infections and malignances. However, IFN therapies have also showed diverse curative outcomes among different patients in treating malignances, especially hepatocellular carcinoma (HCC), and patients’ compliance are easily affected by the severe flu-like side effect of IFN-α therapy. So there is clearly an urgency to search potential biomarkers for predicting the response to IFN-α adjuvant therapy and to identify the well-responders. Moreover, it is also essential to clarify the underlying molecular mechanisms influencing the response to IFN-α therapy and to open new avenues to improve it.Type Ⅰ IFN receptor is widely expressed in various human cell types. The binding of IFN-α to its receptor activates downstream effector Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling cascade, and then induces the transcription of a set of genes, named IFN-stimulated genes (ISGs). These ISGs are responsible for the diverse biological functions of IFN-α, including antiviral and anti-tumor activities. Currently, more than300ISGs have been identified, but their functions are still incompletely understood. IFN-induced proteins with tetratricopeptide repeats (IFIT) family genes, including four members named IFIT1(also ISG56), IFIT2(also ISG54), IFIT3(also ISG60) and IFIT5(also ISG58), are among the ISGs firstly identified. Recently, IFIT family members are reported to potentiate Type Ⅰ IFN production by interacting with MAVS (mitochondrial antiviral signaling protein), STING (stimulator of interferon genes) and TBK1(TANK-binding kinase1protein). However, whether IFIT family members influence the IFN effector signaling is still unknown. In addition, in previous study, we found that the expression of IFIT family members were significantly decresed in HCC samples of HCC patients, compared to their paratumor normal tissue, indicating a possible role of IFIT family members in HCC development. To address this issue and based on the IFN-α therapy to HCC, we mainly focused on the roles of IFIT family members in the modulation of IFN effector signaling in this study, hoping to identify new potential biomarkers to predict the response to IFN-α therapy and to open new avenues to improve the patients’ response to IFN-α.In HCC cell lines BEL-7402and SMMC-7721, IFN-α inhibited HCC cell growth, induced apoptosis and arrested cell cycle progression, and we found that these effects were reduced by the knockdown of IFIT family members. And in the human HCC-bearing nude mice model SMMC-LTNM, the inhibitory effect of IFN-α was also reduced by intratumoral injection of cholesterol-conjugated siRNAs specific to IFIT family members. These results indicate that IFIT family members can promote responses to IFN-α treatment. Also, we were able to find that knockdown of IFIT family members could inhibit the IFN-α-induced expression of apoptotic ISGs, such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and PML (promyelocytic leukemia protein), revealing that IFIT family members may feedback amplify IFN-α effector signaling pathway. By evaluating IFN downstream JAK-STAT pathway, we found that silence of IFIT family members could inhibit IFN-α-induced STAT1and STAT2phosphorylation and activation in HCC cell lines. Mechanistically, co-immunoprecipitation (co-IP) analysis revealed the interactions between IFIT2, IFIT3, IFIT5and the N-terminal conserved coiled-coiled domain of STAT1, as well as interactions between all the four IFIT family members and the DNA binding domain of STAT2. These results further suggest that IFIT family members may amplify the anti-tumor effect of IFN-α by binding and promoting the IFN effectors STAT1and STAT2activation. Thus, we have provided a potential biomarker for predicting IFN-α response and suggested a new biological target for promoting IFN-α therapeutic effectiveness.