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Legend for Supplementary Figures S1-S5.

TGFβR inhibition suppresses mesenchymal features in human HCC cell lines (S1); AXIN2 and GLI are induced by Wnt and SHH signals in the epithelial-to-mesenchymal transition network model (S2); All stable motifs (feedback loops) stabilizing the TGFβ-driven EMT phenotype (S3); The ratio between the timescale of the slow and fast processes does not matter as long as it is significantly larger than one (S4); Exploration of alternative rules to describe nuclear localization of β-catenin (S5).

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue, and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is TGFβ, which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We have constructed an EMT network of 70 nodes and 135 edges by integrating the signaling pathways involved in developmental EMT and known dysregulations in invasive HCC. We then used discrete dynamic modeling to understand the dynamics of the EMT network driven by TGFβ. Our network model recapitulates known dysregulations during the induction of EMT and predicts the activation of the Wnt and Sonic hedgehog (SHH) signaling pathways during this process. We show, across multiple murine (P2E and P2M) and human HCC cell lines (Huh7, PLC/PRF/5, HLE, and HLF), that the TGFβ signaling axis is a conserved driver of mesenchymal phenotype HCC and confirm that Wnt and SHH signaling are induced in these cell lines. Furthermore, we identify by network analysis eight regulatory feedback motifs that stabilize the EMT process and show that these motifs involve cross-talk among multiple major pathways. Our model will be useful in identifying potential therapeutic targets for the suppression of EMT, invasion, and metastasis in HCC. Cancer Res; 74(21); 5963–77. ©2014 AACR.

c-Met, a high affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion and metastasis. Hepatocellular carcinoma (HCC) patients with active HGF/c-Met signaling have a significantly worse prognosis. CD44, the receptor for hyaluronic acid, is utilized as a surface marker to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers. CD44v6 (spice variant 6) has been shown to promote HGF activation of c-Met. In this work, we examine the role of CD44s as a deregulator and activator of c-Met signaling in HCC. In a transcriptome profile analysis of 149 human HCC cases, we identified a significant positive correlation (R2=0.8993) between up-regulated CD44 and c-Met expression. The human HCC cell lines MHCC97-H and Huh7 were further utilized to investigate the direct relationship between c-Met and CD44s. Metastatic MHCC97-H cells are 95% CD44+ and demonstrate ligand-independent c-Met activation. CD44+/c-Met+ MHCC97-H cells demonstrate a mesenchymal phenotype with low expression of E-cadherin, high level of tumorsphere formation, increased expression of fibronectin and Zeb2 compared to Huh7 cells, which have are CD44neg/c-Metneg. In MHCC97-H cells, a direct interaction between CD44s and c-Met was demonstrated by immunoprecipitation/immuno-blot analysis. Transient and stable inhibition of c-Met in MHCC97-H cells results in increased E-cadherin, decreased CD44s, and reduced tumor-initiating characteristics and loss of tumorsphere formation. Likewise, inhibition of CD44s demonstrates similar phenotype with increased E-cadherin, decreased tumorsphere formation, and decreased c-Met activation and loss of downstream Akt and Erk phosphorylation. CD44s knockdown recovered c-Met to a regulated HGF-dependent state. Selective inhibition of either PI3K (Ly294002) or MAPK (PD98059) pathways in MHCC97-H cells demonstrate that PI3K/Akt activation signals drive CD44s expression in a feed forward fashion. Conversely, the over-expression of CD44s in Huh7 cells demonstrates a mesenchymal phenotype with fibroblastic-like cell morphology, decreased E-cadherin, increased Zeb1, increased PI3K/Akt signaling, and increased tumorsphere formation compared to control Huh7 cells. Selective inhibition of PI3K/Akt signaling in CD44+ Huh7 cells resulted in reduced CD44s expression and decreased tumorsphere formation. In conclusion, CD44s induces a mesenchymal phenotype and tumor initiating characteristics in HCC through the deregulation of c-Met and activation of a positive feedback loop through PI3K/Akt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2408. doi:1538-7445.AM2012-2408