We recently characterized an atypical EMT in HCC cultures24. Huh7.5M cells with mesenchymal characteristics were generated from the epithelial Huh7.5 cells. Huh7.5:Huh7.5M EMT system was non-responsive to canonical EMT-TFs. Ectopic expression of EMT-TFs in Huh7.5 cells did neither drive EMT, nor did Slug depletion revert EMT phenotype in Huh7.5M cells. Whole genome transcription profiling is a powerful tool to gain valuable details on critical molecules and pathways reprogrammed during EMT. Various studies have reported transcriptional reprogramming behind EMT in cultured cells or from clinical samples25,26,27. Metadata generated from various such curated datasets identified a core gene set in EMT programs28. We performed microarray based transcriptome analysis of Huh7.5 and Huh7.5M cells in order to gain insight into transcriptional reprogramming behind this atypical EMT and to identify novel molecules involved in it. We identify in this study that β-Catenin signaling is phenomenally dysregulated during EMT. GSK-3β was critical in the maintenance of mesenchymal markers in Huh7.5M cells as its inhibition reverted EMT features. We also demonstrate that GSK-3β is in turn regulated by p38MAPK and ERK1/2 through a novel regulation. In addition to this pathway, BMP and NF-κB pathways were also highly dysregulated. Another important finding from our study is the loss of liver characteristics in Huh7.5M cells upon EMT. We also identify a large set of novel transcription factors, cell surface receptors, cytoskeletal molecules and regulators of various metabolic pathways in this study.
Information generated over the past decade reiterates that cancer EMT is a vast and diverse process with significant variation observed in the induction mechanism and the extent of EMT. During EMT, the cell undergoes dramatic changes. On either end of the transcriptional reprogramming, the cell experiences changes at signaling and metabolic levels. To understand the EMT process in a holistic way one should be able to connect all these major changes to one another. Despite the complexity in the presentation of EMT, roles of the canonical EMT-TFs have been undoubtedly proven. Intriguingly, Snail and Zeb1 were detected at robust levels in the epithelial Huh7.5 and their over-expression had no change on the cells24, indicating that additional factors were required to promote EMT. At this outset, we profiled transcriptomes of Huh7.5 and Huh7.5M cells and also studied the major pathways that drive EMT. Our analysis identified a large-scale transcriptional reprogramming and several factors with potential to influence EMT. However, the novel factors regulating this atypical EMT remain elusive.
What is atypical about Huh7.5:Huh7.5M system and what do we learn from it To the best of our understanding a few points justify our claim of it being atypical. Firstly, expression of EMT-TFs Snail and Zeb in Huh7.5 cells with concomitant expression of epithelial markers is strange. Secondly, detection of other mesenchymal markers such as N-Cadherin, Fibronectin and β-Catenin co-residing with epithelial markers is unique. The dispensability of these factors in the perceivably epithelial cells needs further interrogation. Thirdly, MAPK-GSK-3β signaling behaved in total contradiction to its conventional role in EMT. Even with activation of Wnt and BMP pathways in Huh7.5M, their regulation was anything but straightforward. Since such mechanisms do exist in cultured cells, their existence in vivo cannot be ruled out. So, these results not only highlight the dynamic and diverse nature of mechanisms that drive EMT in different biological systems including numerous cancer EMT models, but underline the necessity of scrutiny that should be exercised while extrapolating results from them in the search for therapeutic targets. 59ce067264