c-Jun phosphorylated by JNK is required for protecting Gli2 from proteasomal-ubiquitin degradation by PGE2-JNK signaling axis

Hedgehog (Hh) signaling pathway includes canonical and non-canonical activation manners. In colorectal cancer, we have previously shown that PGE2-JNK could initiate non-canonical activation of the Hh signaling pathway. In this study, we showed that c-Jun, a classic substrate of JNK, increased Gli2 protein stability after phosphorylated by PGE2. Suppressing the function of c-Jun or JNK indicated that c-Jun prevents Gli2 from protease degradation caused by PGE2-JNK. Moreoer, we revealed that less ubiquitination of Gli2 was detected in colorectal cancer cells treated with PGE2 while suppression of c-Jun restored the ubiquitination of Gli2. In addition, we observed that suppression of c-Jun significantly decreased Gli2 expression no matter when Gli2 remained in phosphorylation or non-phosphorylation state. These phenomena were recapitulated, when the endpoint of Gli2 expression was replaced by Gli2 ubiquitination. Furthermore, we demonstrated that restricting c-Jun function ablated the PGE2-provoked Hh activity and proliferation of colorectal cancer cells. These results elucidated that the evasion of Gli2 with phosphorylation from proteasomal-ubiquitin degradation needed the cooperation of phosphorylated c-Jun by kinase JNK, which contributed to promoting Hh activation and the proliferation of colorectal cancer cells. This study provides a theoretical foundation to target PGE2 downstream for the prevention and treatment of colorectal cancer.     

shRNA-mediated silencing of Gli2 gene inhibits proliferation and sensitizes human hepatocellular carcinoma cells towards TRAIL-induced apoptosis

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including hepatocellular carcinoma (HCC). As a key effector of this signaling, Gli2 plays a crucial role in carcinogenesis, including the activation of genes encoding apoptosis inhibitors and cell-cycle regulators. In this study, we examined the role of Gli2 proliferation and survival of HCC cells. First, the expression levels of Hh pathway components were detected in a subset of HCC cell lines. To establish the role of Gli2 in maintaining the tumorigenic properties of HCC cells, we developed small hairpin RNA (shRNA) targeting Gli2 and transfected it into SMMC-7721 cell, which was selected with high level of Hh signaling expression. Next, effects of Gli2 gene silencing, on cell proliferation and on the expression of cell cycle-related proteins were evaluated, then, whether down-regulation of Gli2 renders HCC cell susceptible to TRAIL was examined in vitro. Knockdown of Gli2 inhibited cell proliferation and induced G1 phase arrest of cell cycle in SMMC-7721 cell through down-regulation of cyclin D1, cyclinE2, and up-regulation of p21-WAF1. Also, Gli2 gene siliencing sensitized SMMC-7721 cell to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reducing the expression of the long and short isoform of c-FLIP and Bcl-2, and then augmented the activation of initiator caspases-8/-9 and effector caspases-3, which induces PARP cleavage. In conclusion, our data suggest that Gli2 plays a predominant role in the proliferation and apoptosis resistance of HCC cells, and that knockdown of Gli2 may be a novel anticancer strategy for the treatment of HCC.