Kivanc Görgülü1, Kalliope N Diakopoulos1, Jiaoyu Ai1, Benjamin Schoeps2, Derya Kabacaoglu1, Angeliki-Faidra Karpathaki1, Katrin J Ciecielski1, Ezgi Kaya-Aksoy1, Dietrich A Ruess1, Alexandra Berninger1, Marlena Kowalska1, Marija Stevanovic1, Sonja M Wörmann1, Thomas Wartmann3, Yue Zhao3, Walter Halangk3, Svetlana Voronina4, Alexey Tepikin4, Anna Melissa Schlitter5, Katja Steiger6, Anna Artati7, Jerzy Adamski8, Michaela Aichler9, Axel Walch9, Martin Jastroch10, Götz Hartleben11, Christos S Mantzoros12, Wilko Weichert5, Roland M Schmid1, Stephan Herzig11, Achim Krüger2, Bruno Sainz13, Marina Lesina14, Hana Algül15. 1. Klinik und Poliklinik für Innere Medizin II, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany. 2. Institute of Molecular Immunology and Experimental Oncology, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany. 3. Klinik für Chirurgie Bereich Experimentelle Operative Medizin, Universitätsklinikum Magdeburg, Magdeburg, Germany. 4. Institute of Translational Medicine, University of Liverpool, Liverpool, UK. 5. Institute of Pathology, Technische Universität München, Munich, Germany and German Cancer Consortium, Munich, Germany. 6. Institute of Pathology, Technische Universität München, Munich, Germany and German Cancer Consortium, Munich, Germany; Comparative Experimental Pathology, Institute of Pathology, Technische Universität München, Munich, Germany. 7. Institute of Experimental Genetics, Genome Analysis Centre, Helmholtz Zentrum München, Neuherberg, Germany. 8. Institute of Experimental Genetics, Genome Analysis Centre, Helmholtz Zentrum München, Neuherberg, Germany; Institute for Diabetes and Cancer, German Center for Diabetes Research, Neuherberg, Germany; Lehrstuhl für Experimentelle Genetik, Technische Universität München, Freising-Weihenstephan, Germany. 9. Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany. 10. Helmholtz Diabetes Center and German Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany. 11. Institute for Diabetes and Cancer, German Center for Diabetes Research, Neuherberg, Germany. 12. Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, Massachusetts. 13. Department of Biochemistry, School of Medicine, Autónoma University of Madrid, Madrid, Spain. 14. Klinik und Poliklinik für Innere Medizin II, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany. Electronic address: marina.lesina@tum.de. 15. Klinik und Poliklinik für Innere Medizin II, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany. Electronic address: hana.alguel@mri.tum.de.
Abstract
BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.
BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Krasmice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Krasmice did not develop any tumors. Cultured A5+/-;Krasprimary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Krasmice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In humanPDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.