Ashutosh Tiwari1, Kojiro Tashiro2, Ajay Dixit3, Aditi Soni2, Keianna Vogel2, Bryan Hall2, Iram Shafqat4, Joseph Slaughter4, Nesteen Param4, An Le4, Emily Saunders4, Utkarsha Paithane2, Guillermina Garcia5, Alexandre Rosa Campos6, Jon Zettervall3, Marjorie Carlson3, Timothy K Starr7, York Marahrens8, Aniruddha J Deshpande2, Cosimo Commisso2, Paolo P Provenzano3, Anindya Bagchi9. 1. Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. Electronic address: atiwari@sbpdiscovery.org. 2. Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. 3. Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota. 4. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota. 5. Histology Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. 6. Proteomics Core, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. 7. Department of Obstetrics, Gynecology and Women's Health, University of Minnesota, Minneapolis, Minnesota. 8. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota. 9. Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. Electronic address: abagchi@sbpdiscovery.org.
Abstract
BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. METHODS: We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. RESULTS: EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. CONCLUSIONS: HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.
BACKGROUND & AIMS:Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. METHODS: We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the humanpancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCIDmice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. RESULTS: EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic humanpancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. CONCLUSIONS:HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.
Authors: Justin K Messex; Kiyah L A Adams; William G Hawkins; David DeNardo; Nabeel Bardeesy; Daniel D Billadeau; Geou-Yarh Liou Journal: Cancers (Basel) Date: 2022-04-26 Impact factor: 6.575
Authors: Arjun A Rao; Laura Leonhardt; Sudipta Ashe; Audrey M Hendley; Jennifer A Smith; Simone Giacometti; Xianlu L Peng; Honglin Jiang; David I Berrios; Mathias Pawlak; Lucia Y Li; Jonghyun Lee; Eric A Collisson; Mark S Anderson; Gabriela K Fragiadakis; Jen Jen Yeh; Chun Jimmie Ye; Grace E Kim; Valerie M Weaver; Matthias Hebrok Journal: Elife Date: 2021-05-19 Impact factor: 8.713