Jingxuan Yang1, Zicheng Zhang2, Yuqing Zhang3, Xiaoling Ni4, Guohua Zhang2, Xiaobo Cui1, Mingyang Liu3, Can Xu5, Qiang Zhang6, Huiyun Zhu7, Jie Yan3, Vivian F Zhu6, Yusheng Luo6, John P Hagan6, Zhaoshen Li8, Jing Fang9, Aminah Jatoi10, Martin E Fernandez-Zapico11, Lei Zheng12, Barish H Edil13, Michael S Bronze14, Courtney W Houchen14, Yi-Ping Li15, Min Li16. 1. Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; The Vivian L. Smith Department of Neurosurgery, the University of Texas Health Science Center at Houston, Houston, Texas. 2. Department of Integrative Biology and Pharmacology, the University of Texas Health Science Center at Houston, Houston, Texas. 3. Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. 4. The Vivian L. Smith Department of Neurosurgery, the University of Texas Health Science Center at Houston, Houston, Texas; Department of General Surgery, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China. 5. The Vivian L. Smith Department of Neurosurgery, the University of Texas Health Science Center at Houston, Houston, Texas; Department of Gastroenterology, Changhai Hospital, Shanghai, China. 6. The Vivian L. Smith Department of Neurosurgery, the University of Texas Health Science Center at Houston, Houston, Texas. 7. Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; Department of Gastroenterology, Changhai Hospital, Shanghai, China. 8. Department of Gastroenterology, Changhai Hospital, Shanghai, China. 9. The Key Lab of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, China. 10. Department of Oncology, Mayo Clinic, Rochester, Minnesota. 11. Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, Minnesota. 12. The Sidney Kimmel Comprehensive Cancer Center and Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. 13. Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. 14. Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. 15. Department of Integrative Biology and Pharmacology, the University of Texas Health Science Center at Houston, Houston, Texas. Electronic address: Yi-Ping.Li@uth.tmc.edu. 16. Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; The Vivian L. Smith Department of Neurosurgery, the University of Texas Health Science Center at Houston, Houston, Texas; Department of Integrative Biology and Pharmacology, the University of Texas Health Science Center at Houston, Houston, Texas. Electronic address: Min-Li@ouhsc.edu.
Abstract
BACKGROUND & AIMS: Cachexia, which includes muscle wasting, is a frequent complication of pancreatic cancer. There are no therapies that reduce cachexia and increase patient survival, so it is important to learn more about its mechanisms. The zinc transporter ZIP4 promotes growth and metastasis of pancreatic tumors. We investigated its effects on muscle catabolism via extracellular vesicle (EV)-mediated stimulation of mitogen-activated protein kinase 14 (p38 MAPK). METHODS: We studied nude mice with orthotopic tumors grown from human pancreatic cancer cell lines (AsPC-1 and BxPC-3); tumors were removed 8 days after cell injection and analyzed by histology. Mouse survival was analyzed by Kaplan-Meier curves. ZIP4 was knocked down in AsPC-1 and BxPC-3 cells with small hairpin RNAs; cells with empty vectors were used as controls. Muscle tissues were collected from mice and analyzed by histology and immunohistochemistry. Conditioned media from cell lines and 3-dimensional spheroid/organoid cultures of cancer cells were applied to C2C12 myotubes. The myotubes and the media were analyzed by immunoblots, enzyme-linked immunosorbent assays, and immunofluorescence microscopy. EVs were isolated from conditioned media and analyzed by immunoblots. RESULTS: Mice with orthotopic tumors grown from pancreatic cancer cells with knockdown of ZIP4 survived longer and lost less body weight and muscle mass than mice with control tumors. Conditioned media from cancer cells activated p38 MAPK, induced expression of F-box protein 32 and UBR2 in C2C12 myotubes, and also led to loss of myofibrillar protein myosin heavy chain and myotube thinning. Knockdown of ZIP4 in cancer cells reduced these effects. ZIP4 knockdown also reduced pancreatic cancer cell release of heat shock protein (HSP) 70 and HSP90, which are associated with EVs, by decreasing CREB-regulated expression of RAB27B. CONCLUSIONS: ZIP4 promotes growth of orthotopic pancreatic tumors in mice and loss of muscle mass by activating CREB-regulated expression of RAB27B, required for release of EVs from pancreatic cancer cells. These EVs activate p38 MAPK and induce expression of F-box protein 32 and UBR2 in myotubes, leading to loss of myofibrillar myosin heavy chain and myotube thinning. Strategies to disrupt these pathways might be developed to reduce pancreatic cancer progression and accompanying cachexia.
BACKGROUND & AIMS:Cachexia, which includes muscle wasting, is a frequent complication of pancreatic cancer. There are no therapies that reduce cachexia and increase patient survival, so it is important to learn more about its mechanisms. The zinc transporter ZIP4 promotes growth and metastasis of pancreatic tumors. We investigated its effects on muscle catabolism via extracellular vesicle (EV)-mediated stimulation of mitogen-activated protein kinase 14 (p38 MAPK). METHODS: We studied nude mice with orthotopic tumors grown from humanpancreatic cancer cell lines (AsPC-1 and BxPC-3); tumors were removed 8 days after cell injection and analyzed by histology. Mouse survival was analyzed by Kaplan-Meier curves. ZIP4 was knocked down in AsPC-1 and BxPC-3 cells with small hairpin RNAs; cells with empty vectors were used as controls. Muscle tissues were collected from mice and analyzed by histology and immunohistochemistry. Conditioned media from cell lines and 3-dimensional spheroid/organoid cultures of cancer cells were applied to C2C12 myotubes. The myotubes and the media were analyzed by immunoblots, enzyme-linked immunosorbent assays, and immunofluorescence microscopy. EVs were isolated from conditioned media and analyzed by immunoblots. RESULTS:Mice with orthotopic tumors grown from pancreatic cancer cells with knockdown of ZIP4 survived longer and lost less body weight and muscle mass than mice with control tumors. Conditioned media from cancer cells activated p38 MAPK, induced expression of F-box protein 32 and UBR2 in C2C12 myotubes, and also led to loss of myofibrillar protein myosin heavy chain and myotube thinning. Knockdown of ZIP4 in cancer cells reduced these effects. ZIP4 knockdown also reduced pancreatic cancer cell release of heat shock protein (HSP) 70 and HSP90, which are associated with EVs, by decreasing CREB-regulated expression of RAB27B. CONCLUSIONS:ZIP4 promotes growth of orthotopic pancreatic tumors in mice and loss of muscle mass by activating CREB-regulated expression of RAB27B, required for release of EVs from pancreatic cancer cells. These EVs activate p38 MAPK and induce expression of F-box protein 32 and UBR2 in myotubes, leading to loss of myofibrillar myosin heavy chain and myotube thinning. Strategies to disrupt these pathways might be developed to reduce pancreatic cancer progression and accompanying cachexia.
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