Abstract 1465: Metastatic potential of hepatocellular carcinoma determined by dynamic modeling of EMT network and Snail1 expression
Authors: Ding Wei , You Hanning , Dang Hien , Steinway Steve , Albert Reka , Rountree Carl B. 
Published: June 2012

Intro: Epithelial-to-mesenchymal transition (EMT) is drives metastasis in hepatocellular carcinoma (HCC). The micro-RNA 200 family blocks EMT by targeting Zeb1, a suppressors of E-cadherin. We investigated a dynamic network model of EMT to predict metastatic potential of HCC cells and confirmed metastasis in-vivo using inducible expression of miR-200 in mesenchymal cells.

Methods: We constructed an EMT signaling network by integrating the signaling pathways involved in EMT during liver development and hepatocarcinogenesis. This network was translated into a predictive, dynamic computer model. We utilized published murine epithelial and mesenchymal HCC lines. miR-200 family members were assessed using qPCR. A tet-on inducible system expressing the miR-200b was developed using pmRi-ZsGreen1 miRNA vector, which co-express miRNA and green fluorescent protein (GFP). Functional analyses included gene and protein expression, cell proliferation, and migration assays. 3×105 tumor cells stably expressing either empty vector (EV) or miR-200b were orthotopically transplanted into the liver of nude mice.

Results: Dynamic network modeling using Boolean algorithms initially predicted miR200b would block Zeb1 expression and induce an epithelial phenotype. Compared to epithelial cells, mesenchymal cells express extremely low levels of miR200a and miR200b (400 and 1400 fold decreased, p&lt;0.01). The mesenchymal tumor cells with low miR-200 expression have aggressive growth in-vivo (1000 mm3/week vs. 150 mm3/week high miR-200, p&lt;0.01) with significant tissue invasion and metastasis. After doxycycline treatment using inducible miR-200b system, the elongated spindle-like mesenchymal cells underwent a morphology change to epithelial-like cells with a cobblestone pattern, demonstrated significant reduction of Zeb1 and increase in E-cadherin. However, expression of miR-200b in orthotopically transplanted mesenchymal tumor cells was unable to suppress liver cancer metastasis to the lungs compared to empty vector (miR-200b 3/8 mets vs. EV 2/9 mets) or reduce the number of circulating tumor cells, identified by as GFP+ nucleated blood cells (miR-200b 0.15% vs. EV 0.12%). Other miR-200 species were also reduced with miR-200b over-expression. Review of dynamic model indicates that over-expression of miR-200b is predicted to reduce Zeb1 but have no effect on Snail1. Stable Snail1 was confirmed in-vitro and in-vivo as miR-200b induction had no significant effect on Snail1 expression.

Conclusion: Stable/inducible expression of miR-200b targeted Zeb1 and partially reversed EMT by increasing expression of E-cadherin. This incomplete reversal of EMT by over-expression of miR-200 is not sufficient to suppress metastasis to the lungs, and dynamic network modeling predicts Snail1 as the primary node which maintains metastatic phenotype.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1465. doi:10.1158/1538-7445.AM2011-1465