Epithelial-to-Mesenchymal Transition of Murine Liver Tumor... : Hepatology (original) (raw)

Hepatobiliary Malignancies

Epithelial-to-Mesenchymal Transition of Murine Liver Tumor Cells Promotes Invasion†,‡

Ding, Wei1; You, Hanning1; Dang, Hien1; LeBlanc, Francis1; Galicia, Vivian2; Lu, Shelly C.3; Stiles, Bangyan2; Rountree, Bart C.*,1,§

1 Department of Pediatrics and Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA

2 Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA

3 Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Disease, The Southern California Research Center for ALPD and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, CA

*Address reprint requests to: C. Bart Rountree, M.D., Department of Pediatrics and Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, H085, Hershey, PA 17033.

E-mail:[email protected]

Received January 08, 2010; accepted April 26, 2010; previously published online June 16, 2010

†Potential conflict of interest: Nothing to report.

‡C. Bart Rountree was supported by National Institute of Diabetes and Digestive and Kidney Disorders (NIDDK) Grant K08DK80928; American Cancer Society Grant RSG-10-073-01-TBG; the Office for the Advancement of Telehealth, Health Resources and Services Administration, Department of Health and Human Services Grant D1BTH06321; the Children's Miracle Network; and a Penn State College of Medicine Barsumian Trust Award. Shelly Lu was supported by NIDDK Grant DK51719 and National Center for Complementary and Alternative Medicine Grant AT1576. Bangyan Stiles was supported by the NIDDK.

§fax: 717-531-0653

Abstract

Epithelial-to-mesenchymal transition (EMT) is predicted to play a critical role in metastatic disease in hepatocellular carcinoma. In this study, we used a novel murine model of EMT to elucidate a mechanism of tumor progression and metastasis. A total of 2 × 106 liver cells isolated from Pten loxp/loxp/ Alb-Cre + mice, expanded from a single CD133+CD45− cell clone, passage 0 (P0), were sequentially transplanted to obtain two passages of tumor cells, P1 and P2. Cells were analyzed for gene expression using microarray and real-time polymerase chain reaction. Functional analysis included cell proliferation, migration, and invasion in vitro and orthotopic tumor metastasis assays in vivo. Although P0, P1, and P2 each formed tumors consistent with mixed liver epithelium, within the P2 cells, two distinct cell types were clearly visible: cells with epithelial morphology similar to P0 cells and cells with fibroblastoid morphology. These P2 mesenchymal cells demonstrated increased locomotion on wound healing; increased cell invasion on Matrigel basement membrane; increased EMT-associated gene expression of Snail1 , Zeb1 , and Zeb2 ; and down-regulated E-cadherin . P2 mesenchymal cells demonstrated significantly faster tumor growth in vivo compared with P2 epithelial counterparts, with invasion of intestine, pancreas, spleen, and lymph nodes. Furthermore, P2 mesenchymal cells secreted high levels of hepatocyte growth factor (HGF), which we propose acts in a paracrine fashion to drive epithelial cells to undergo EMT. In addition, a second murine liver cancer stem cell line with methionine adenosyltransferase 1a deficiency acquired EMT after sequential transplantations, indicating that EMT was not restricted to Pten-deleted tumors.

Conclusion:

EMT is associated with a high rate of liver tumor proliferation, invasion, and metastasis in vivo, which is driven by HGF secreted from mesenchymal tumor cells in a feed-forward mechanism.