Molecular Mechanisms Of The Pro-survival Role Of CAMP In Tumor Cells

The second messenger cyclic AMP (cAMP) is an important intracellular mediator, which is produced from ATP by adenylyl cyclases (AC) and can be degraded to 5'-AMP by phosphodiesterases. AC is stimulated by a variety of extracellular stimuli, such as hormones, growth factors and neurotransmitters through G-protein (Gs)-coupled membrane receptors. cAMP regulates many cellular activities, from proliferation to apoptosis, in a cell type-dependent manner. The biological functions of cAMP are mediated by its downstream effectors such as protein kinase A (PKA) and CREB (cAMP element-binding protein). PKA and CREB have been shown to contribute to the tumorgenesis of endocrine tissues. Furthermore, it has been long disclosed that cAMP elevation is associated with impaired cell death of various tumor cells. However, in non-malignant cells cAMP can either promote or suppress cell death, depending on cell type and stimulus used. The prievious studies of our group suggest that, at least in fibroblasts, the crosstalk between the cAMP signaling pathway and MAPKs JNK (c-Jun N-terminal protein kinase) /p38 pathways is the key mechanism by which cAMP plays a dual role in the regulation of cell death.It remains unknown why cAMP can either promote or suppress cell death in non-maligant cells, but always exhibits a pro-survival role in tumor cells. Since resistance to cell death has been implicated in cancer pathogenesis, it is of great importance to elucidate the underlying mechanisms.So we carried out work to investigate the possible mechanisms. We chose the TNF-α-sensitive fibroblastoma L929 cells as the research model. Then we found that elevation of cAMP suppressed TNF-α-induced necrotic cell death in L929 fibroblastoma cells via CREB-mediated transcription. The pro-survival role of cAMP was associated with selective unresponsiveness of L929 cells to the inhibition of p38 activation by cAMP, even though cAMP significantly inhibited the activation of JNK under the same conditions. Further exploration revealed that the induction of DLC, the major mediator of p38 inhibition by cAMP, was impaired in L929 cells. Enforced inhibition of p38 activation by using p38 specific inhibitor or ectopic expression of DLC reversed the protection of L929 cells by cAMP from TNF-α-induced cell death. These data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.On the other hand, our data suggest that cAMP suppresses TNF-α-induced cell death of L929 fibroblastoma cells via, at least partially, inhibiting the activation of JNK pathway. The induction of c-FLIPL by cAMP via CREB-mediated transcription led to the inhibition of JNK activation by targeting MKK7, the upstream kinase of JNK pathway. However, it remains unknown whether there are other negative-regulators in the regulation of JNK by cAMP. So we further designed research to screen the cellular proteins that interact with MKK7 by yeast two-hybrid assay from human fetal brain cDNA library.We identified three new MKK7-interacting proteins by yeast two-hybrid screen. They were ORF60, GNB2L1 and UBA3. Co-immunoprecipitaion assay confirmed the specific interaction with MKK7. Furthermore, immunoblotting analysis and luciferase assay showed that GNB2L1 could enhance the activation of JNK and downstream transcriptional factor AP-1, while the overexpression of ORF60 and UBA3 could inhibit the activation of JNK and AP-1. Our finding of the three new interacting proteins of MKK7 might facilitate the studies to elucidate the complicated mechanisms by which cAMP suppresses JNK pathway and plays a pro-survival role in tumor cells.