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Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer

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Abstract

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.

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Abbreviations

RNAi:

RNA interference

PDAC:

Pancreatic ductal adenocarcinoma

shRNA:

Short hairpin RNA

ITIH5:

Inter-alpha-trypsin inhibitor heavy chain 5

HMP19:

Hypothalamus Golgi apparatus expressed 19 kDa protein

HPNE:

Human pancreatic duct normal epithelium cells

FBS:

Fetal bovine serum

shRNAmir:

MicroRNA-adapted shRNA

ITIs:

Inter-α-trypsin inhibitors

ITIHs:

ITI heavy chains

HA:

Hyaluronic acid

SHAP:

Serum-derived hyaluronic acid-associated protein

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Acknowledgements

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.

Funding

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).

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Authors and Affiliations

  1. Department of Cancer Biology, The University of Kansas Medical Center, 3901 Rainbow Blvd, Mail Stop 1071, Kansas City, KS, 66160, USA

    Ken Sasaki, Hiroshi Kurahara, Eric D. Young, Tomoo Iwakuma & Danny R Welch

  2. The University of Kansas Cancer Center, 3901 Rainbow Blvd, Kansas City, KS, 66160, USA

    Tomoo Iwakuma & Danny R Welch

  3. Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical Sciences, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima, 890-8520, Japan

    Ken Sasaki, Hiroshi Kurahara, Eric D. Young, Shoji Natsugoe, Asami Ijichi, Tomoo Iwakuma & Danny R Welch

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  1. Ken Sasaki
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  2. Hiroshi Kurahara
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Correspondence to Tomoo Iwakuma or Danny R Welch.

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Sasaki, K., Kurahara, H., Young, E.D. et al. Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer. Clin Exp Metastasis 34, 229–239 (2017). https://doi.org/10.1007/s10585-017-9840-3

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  • DOI: https://doi.org/10.1007/s10585-017-9840-3

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