The Role And Mechanism Of Gadd45a In Tumor Angiogenesis

Angiogenesis is a normal and vital process in embryonic development and homeostasis, as well as wound healing and female menstrual cycle. In contrast, abnormal angiogenesis occurs in many pathological processes, including ischemic disease, chronic inflammation and cancer. As a hallmarker of cancer, angiogenic ability is required for malignant transformation of cell in precancerous tissue. Current achievements in research on tumor angiogenesis provide new insights into cancer treatment. The mechanisms of tumor angiogenesis are complex, and have become a hotspot in cancer research.As a DNA damage responsive gene, Gadd45a is induced by a wide spectrum of genetoxic stress agents, such as UV radiation, ionizing radiation, serum starvation, and chemotherapeutic drugs. Gadd45a participates mediating cell cycle arrest, promoting apoptosis and inducing DNA repair. It is demonstrated that Gadd45a plays a critical role in protecting against tumorigenesis. Disruption of Gadd45a gene results in genomic instability and disturbances in growth control. Gadd45a suppresses cancer invasion and metastasis, via decreasing MMPs expression.In the current study, we provide evidences that Gadd45a suppresses tumor angiogenesis in vivo and in vitro. Endogenously disrupted Gadd45a increases the microvessal density in xenograft tumor and angiogenic response in CAM assay, and enhances the migration and tube formation of endothelial cells. The results of expression profiling arrays indicated that Gadd45a deletion increased expression of five angiogenic factors, such as Mmp3, Ccl2, Cxcl5, Pdgfra and Vegfa. VEGF-A is a key factor in promoting angiogenesis, and MMP3 is involved in extracellular matrix remodeling. Consistent with the results of expression profiling arrays, data from quantitative RT-PCR and western blot shown that VEGF-A and MMP3 were significantly increased in MEF Gadd45a-/-, compared with MEF Gadd45a+/+. Meanwhile, Gadd45a knocked down in HeLa cells also increased VEGF-A and MMP3. Here, our initial observations showed that the biological function of Gadd45a in tumor angiogenesis might be correlated with STAT3. Deletion or knockdown of Gadd45a increased the phosphorylation at Ser727 of STAT3, whereas Gadd45a overexpression reduced the phosphorylation at Ser727. STAT3 is one of pluripotent transcription factors, and its transcriptional activity depends on phosphorylation of two key residues, including Tyr705 and Ser727. In addition, activation of STAT3 stimulates VEGF-A and MMPs expression which are important for switching on angiogenesis. These results indicate that Gadd45a suppresses angiogenesis probably via decreasing Ser727 phosphorylated STAT3.Gadd45a is not a phosphatase and incapable of directly dephosphorylating proteins. The phosphorylation of STAT3 on Ser727 is mediated by a series of kinases, such as ERK, NLK, p38 and mTOR. Here, we identify that Gadd45a is involved in the regulation of mTOR/STAT3 pathway. Our data have shown that Ser727 phosphorylated STAT3 was obviously decreased after rapamycin treatment in MEF Gadd45a+/+ and MEF Gadd45a-/-, whereas Ser727 phosphoryation was not effected by p38 inhibitor. In addition, the results of GST-pulldown assay indicated that Gadd45a associated with mTOR, not STAT3. More interestingly, we further found that Gadd45a deletion enhanced the association between mTOR and STAT3, whereas Gadd45a overexpression reduced this association. Taking together, we demonstrate that Gadd45a suppresses Ser727 phosphorylation of STAT3 and expression of VEGFa and MMP3 probably via decreasing the interaction between STAT3 and mTOR.In summary, we have demonstrated that Gadd45a plays as a negative regulator in tumor angiogenesis via regulating mTOR/STAT3 pathway. Gadd45a defect induces the phosphorylation of STAT3 on Ser727 via enhancing the association between mTOR and STAT3. This process results in increasing VEGFa and MMP3 expression which are critical stimulators for angiogenesis. All these findings demonstrate a novel correlation between Gadd45a and tumor angiogenesis, and further explore the mechanisms of Gadd45a in protecting against carcinogenesis and cell malignancy.