An Atypical System for Studying Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma

Abstract

Intrahepatic and extrahepatic metastases are frequently detected in hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is believed to drive metastasis. There are not many well-established model systems to study EMT in HCC. Here we identified an atypical EMT while characterizing a population of mesenchymal cells in Huh7.5 hepatoma cell cultures. Cells with distinct morphology appeared during geneticin treatment of Huh7.5 cultures. Molecular characterization of geneticin resistant Huh7.5M cells confirmed EMT. Huh7.5M cells expressed cancer stem cell markers. p38MAPK and ERK1/2 were substantially activated in Huh7.5M cells. Their Inhibition elevated E-Cadherin expression with concerted suppression of Vimentin and anchorage independent growth in Huh7.5M cells. TGFβ could not induce EMT in Huh7.5 cultures, but enriched mesenchymal populations, similar to geneticin. Huh7.5M cells formed more aggressive solid tumors, primarily comprising cells with epithelial morphology, in nude mice. Canonical EMT-TFs did not participate in this atypical EMT, indicating that the established canonical EMT-TFs do not drive every EMT and there is a dire need to identify additional factors. The system that we characterized is a unique model to study EMT, MET and biphasic TGFβ signaling in HCC and offers considerable potential to facilitate more insightful studies on deeper questions in tumor metastasis.

Unconventional MAPK-GSK-3β Pathway Behind Atypical Epithelial-Mesenchymal Transition In Hepatocellular Carcinoma

“…Out of this, 476 genes showed activation in Huh7.5M cells. This phenomenal regulation is in sync with the activation of HDAC 1 , 6 and 8 in Huh7.5M that we reported previously 24 .…”

“…We previously reported that β-Catenin may not be a critical regulator of this EMT based on the absence of a credible change in β-Catenin levels between the two cells 24 . Contradicting this hypothesis, IPA of the transcription profiles flagged a highly active Wnt/β-Catenin signaling in Huh7.5M cells based on the upregulation of majority of its receptors and ligands (Fig.…”

“…We had previously demonstrated that TGFβ was unable to induce EMT in Huh7.5 cells 24 . Upon TGFβ treatment, E-Cadherin levels were augmented in Huh7.5 cells 24 . Interestingly, SMAD 2 and 3 phosphorylations remained elevated in Huh7.5 cells than the mesenchymal Huh7.5M cells.…”

“…We previously reported an atypical EMT in Huh7.5 cells that resulted in generation of Huh7.5M mesenchymal cells through geneticin resistance 24 . The atypicalness was attributed to two key features: (i) inability of canonical EMT-TFs to induce EMT in Huh7.5 and (ii) concurrent expression of epithelial markers and EMT-TFs in Huh7.5.…”

“…We recently characterized an atypical EMT in HCC cultures 24 . Huh7.5M cells with mesenchymal characteristics were generated from the epithelial Huh7.5 cells.…”