These findings suggest that phosphorylation of nestin at Thr315 and/or Thr1299 affects cell proliferation, and inhibition of both phosphorylation sites suppresses invasion and metastasis of human pancreatic cancer. nestin or with nestin mutated at Thr315 increased migration and invasion. In contrast, transfection with nestin mutated at both phosphorylation sites (Thr315 and Thr1299) did not enhance cell migration or invasion. In an intra\splenic xenograft experiment using MIA PaCa\2 cells, tumors expressing the nestin double mutant formed fewer liver metastases than tumors expressing wild\type nestin. Nestin phosphorylation at these two sites was decreased upon treatment with inhibitors for AICAR phosphate cyclin dependent kinases, AKT, and Aurora in PANC\1 cells, which express a high baseline level of phosphorylated nestin. These findings suggest that phosphorylation of nestin at Thr315 and/or Thr1299 affects cell proliferation, and inhibition of both phosphorylation sites suppresses invasion and metastasis of human pancreatic cancer. Inhibiting nestin phosphorylation at these two sites may represent a novel therapeutic strategy for pancreatic cancer. and liver metastasis studies, we selected mut\nes3 cells for animal experiments. Eight weeks after splenic injection of mut\nes3 cells, liver metastasis in NOG mice was strongly decreased compared to mice injected with control cells (Fig.?7a). Increases in liver Rabbit Polyclonal to MRPS24 weight due to metastatic tumors were lower in mice injected with mut\nes3 cells compared to mice injected with control cells (Fig.?7b, *suppressed liver metastasis, likely via inhibiting cell migration, invasion, and growth in the liver. Wild type nestin induced cell migration and invasion might affect nestin function. Our study has limitations. We performed animal study using wild type nestin\transfected and both phosphorylated sites mutated nestin\transfected cells; therefore, we did not clarify the roles of each phosphorylate site in?vivo. Further studies are needed to clarify molecular mechanisms of nestin phosphorylation. We previously reported that nestin expression level was higher in metastatic lesions compared to primary lesions.13 Nestin AICAR phosphate was continuously expressed in pancreatic cancer cells, while the phosphorylated form was only observed in the mitotic phase. In the present study, we found that inhibition of both phosphorylation sites suppressed human pancreatic cancer metastasis. These findings suggest that inhibiting nestin phosphorylation is usually more specific than inhibiting total nestin, and is more effective for inhibiting metastasis. Furthermore, most inhibitors of cyclin dependent kinases, Akt, or Aurora utilized in this study decreased nestin phosphorylation at both sites, suggesting that these molecules are upstream regulators of nestin phosphorylation. Molecular targeted therapies that inhibit nestin phosphorylation, such as inhibitors used in the present study, antibodies or small molecules, may be new candidates for pancreatic cancer treatment. In conclusion, phosphorylated nestin regulates proliferation, invasion, and metastasis of pancreatic cancer cells. Inhibiting nestin phosphorylation may represent a novel therapeutic option for pancreatic cancer. Further studies are needed to clarify the mechanisms of nestin phosphorylation in pancreatic cancer, and to develop brokers that inhibit nestin phosphorylation for the treatment of pancreatic cancer. Disclosure Statement The authors declare no conflict of interest. Acknowledgments We thank Drs. Tetsushi Yamamoto and Zenya Naito for helpful AICAR phosphate discussion, and Dr. Masahito Hagio for technical assistance (Department of Integrated Diagnostic Pathology, Nippon Medical School). This work was supported in part by a grant\in\aid from the Japan Society for the Promotion of Science (C, No. 25462127) and grants from the Cancer Research Institute of Kanazawa University and Mitsui Life Social Welfare Foundation to Y. Matsuda, and in part by a grant\in\aid from the Japan Society for the Promotion of Science (C, No. 25461027) to T. Ishiwata. Notes Cancer Sci 108 (2017) 354C361 [PMC free article] [PubMed] [Google Scholar] Notes Y. Matsuda and T. Ishiwata contributed equally to this study. Funding Information The Cancer Research Institute of Kanazawa University, the Japan Society for the Promotion of Science, (Grant / Award Number: C, No. 25461027,C, No. 25462127), Mitsui Life Social Welfare Foundation..