Molecular Mechanism For STAT3 In Attenuating TGF-β Signaling

Cancer-associated inflammation is always characterized by the presence of specific cytokines, such as IFN-y, IL-1β, IL-6, IL-11, TGF-β, TNFa and VEGF. Among these, TGF-β is a multifunctional cytokine that regulates diverse cellular responses, including apoptosis, cell growth inhibition, and immune surveillance. Due to its potent tumor-suppressive effects, the TGF-β signaling pathway is often inactivated in numerous cancers such as colon, pancreatic and gastric cancers, yet these mutations are rare in other cancers such as breast, prostate and skin cancers. This phenomenon prompts us to study how TGF-β signaling is inhibited via non-mutational events in specific cellular contexts. STAT3 is a common downstream effector of cytokines overwhelmingly expressed in tumor environment such as IL-6, IL-11 and VEGF. Moreover, STAT3 is also a critical mediator of EGFR signaling, which is aberrantly activated in various tumors.We firstly investigated and confirmed the inhibitory effects of EGF and IL-6 in TGF-β signaling. We then determined both transient and stable knockdown of STAT3 could increase TGF-P activity and impair the inhibitory effects of EGF/IL-6 stimulation. These results demonstrate that EGF/IL-6-mediated suppression of TGF-β pathway is dependent of STAT3. We then established STAT3C stable cells and STAT3 knockdown stable cells to examine the inhibitory effect of STAT3 on regulating TGF-β7-induced physiological function, such as cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and apoptosis. We found that cells overexpressing STAT3C behaved less sensitive to TGF-β-mediated activities; in contrast, STAT3 depletion sensitized cells to TGF-β-mediated responses. These further indicate STAT3 can attenuate TGF-P signaling. To elucidate the molecular mechanism underlying STAT3-mediated repression of TGF-β signaling, we first used reporter assays to identify the downstream target of STAT3. The result suggests that the inhibition is dependent of Smad3. Then we performed co-immunoprecipitation (co-IP) analysis and GST Pull-Down assays to analyze and confirm the interaction between STAT3 and Smad3. We also assessed the importance of this interaction and found that binding to Smad3 is essential for STAT3 to inhibit TGF-β signaling.Taken together, our results demonstrate a new molecular mechanism of STAT3 regulation on TGF-β signaling, which provides a new method for cancer treatment. Our study for the first time shows that STAT3 inhibits TGF-β signal transduction pathway through its interaction with Smad3. The results provide a new molecular mechanism underlying STAT3-mediated regulation of TGF-β signaling, which further provides a new strategy for cancer treatment.