Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer
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The overwhelming majority of pancreatic ductal adenocarcinoma (PDAC) is not diagnosed until the cancer has metastasized, leading to an abysmal average life expectancy (3–6 months post-diagnosis). Earlier detection and more effective treatments have been hampered by inadequate understanding of the underlying molecular mechanisms controlling metastasis. We hypothesized that metastasis suppressors are involved in controlling metastasis in pancreatic cancer. Using an unbiased genome-wide shRNA screen, an shRNA library was transduced into the non-metastatic PDAC line S2-028 followed by intrasplenic injection. Resulting liver metastases were individually isolated from these mice. One liver metastatic nodule contained shRNA for ITIH5 (Inter-alpha-trypsin inhibitor heavy chain 5), suggesting that ITIH5 may act as a metastasis suppressor. Consistent with this notion, metastatic PDAC cell lines had significantly lower protein expression of ITIH5 compared to immortalized pancreatic ductal epithelial cells and non-/poorly-metastatic PDAC cell lines. By manipulating expression of ITIH5 in different PDAC cell lines (over-expression in metastatic, knockdown in non-metastatic) functional and selective regulation of metastasis was observed for ITIH5. Orthotopic tumor growth of PDAC cells was not blocked following orthotopic injection. In vitro ITIH5 over-expression inhibited motility and invasion. Immunohistochemical analysis of a human PDAC tissue microarray revealed that ITIH5 expression inversely correlated with both survival and invasion/metastasis. ITIH5 is, therefore, functionally validated as a PDAC metastasis suppressor and shows promise as a prognostic biomarker.
KeywordsITIH5 Pancreatic cancer Metastasis suppressor RNAi screen
Pancreatic ductal adenocarcinoma
Short hairpin RNA
Inter-alpha-trypsin inhibitor heavy chain 5
Hypothalamus Golgi apparatus expressed 19 kDa protein
Human pancreatic duct normal epithelium cells
Fetal bovine serum
ITI heavy chains
Serum-derived hyaluronic acid-associated protein
The authors are extremely grateful to Dr. Tony Hollingsworth (Eppley Cancer Center) for the gift of the SUIT-2 cell lines and appreciate helpful advice from Drs. Animesh Dhar, Shrikant Anant and Dan Von Hoff. They thank Dr. Christopher Bohl and Carolyn Vivian for technical assistance and other members of our labs for thoughtful suggestions and advice from the University of Kansas Cancer Center Biostatistics Shared Resource. DRW is the Hall Family Professor of Molecular Medicine, a Kansas Bioscience Authority Eminent Scholar and a Komen Scholar. The authors also thank the funding agencies who supported this research.
These studies were funded primarily by a grant from the National Foundation for Cancer Research (DRW), with additional support from U.S. Public Health Service grants CA134981 (DRW), CA168524 (DRW) and CA174735 (TI), the American Cancer Society (RSG-09-169-01-CS to TI), Susan G. Komen for the Cure SAC11037 (DRW) and The Kansas Biosciences Authority (DRW).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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