Generation and characterization of hepatocellular carcinoma cell lines with enhanced cancer stem cell potential.

Hepatocellular carcinoma (HCC) is one of the most common causes for cancer-related death worldwide with rapidly increasing incidence and mortality rates. As for other types of cancers, also in HCC cancer stem cells (CSCs) are thought to be responsible for tumour initiation, progression and therapy failure. However, as rare subpopulations of tumour tissue, CSCs are difficult to isolate, thus making the development of suitable and reliable model systems necessary. In our study, we generated HepG2 subclones with enriched CSC potential by application of the spheroid formation method and subsequent single-cell cloning. Analyses in several 2D and 3D cell culture systems as well as a panel of functional assays both in vitro and in vivo revealed that the generated subclones displayed characteristic and sustained features of tumour initiating cells as well as highly aggressive properties related to tumour progression and metastasis. These characteristics could clearly be correlated with the expression of CSC markers that might have prognostic value in the clinical HCC setting. Therefore, we conclude that our CSC enriched HepG2 clones certainly represent suitable model systems to study the role of CSCs during HCC initiation, progression and drug resistance.

A Gene Signature Derived from the Loss of CDKN1A (p21) Is Associated with CMS4 Colorectal Cancer.

The epithelial-mesenchymal transition (EMT) is associated with tumor aggressiveness and increased invasion, migration, metastasis, angiogenesis, and drug resistance. Although the HCT116 p21-/- cell line is well known for its EMT-associated phenotype, with high Vimentin and low E-cadherin protein levels, the gene signature of this rather intermediate EMT-like cell line has not been determined so far. In this work, we present a robust molecular and bioinformatics analysis, to reveal the associated gene expression profile and its correlation with different types of colorectal cancer tumors. We compared the quantitative signature obtained with the NanoString platform with the expression profiles of colorectal cancer (CRC) Consensus Molecular Subtypes (CMS) as identified, and validated the results in a large independent cohort of human tumor samples. The expression signature derived from the p21-/- cells showed consistent and reliable numbers of upregulated and downregulated genes, as evaluated with two machine learning methods against the four CRC subtypes (i.e., CMS1, 2, 3, and 4). High concordance was found between the upregulated gene signature of HCT116 p21-/- cells and the signature of the CMS4 mesenchymal subtype. At the same time, the upregulated gene signature of the native HCT116 cells was similar to that of CMS1. Using a multivariate Cox regression model to analyze the survival data in the CRC tumor cohort, we selected genes that have a predictive risk power (with a significant gene risk incidence score). A set of genes of the mesenchymal signature was proven to be significantly associated with poor survival, specifically in the CMS4 CRC human cohort. We suggest that the gene signature of HCT116 p21-/- cells could be a suitable metric for mechanistic studies regarding the CMS4 signature and its functional consequences in CRC. Moreover, this model could help to discover the molecular mechanisms of intermediate EMT, which is known to be associated with extraordinarily high stemness and drug resistance.

EMT transcription factor ZEB1 alters the epigenetic landscape of colorectal cancer cells.

Epigenetic deregulation remarkably triggers mechanisms associated with tumor aggressiveness like epithelial-mesenchymal transition (EMT). Since EMT is a highly complex, but also reversible event, epigenetic processes such as DNA methylation or chromatin alterations must be involved in its regulation. It was recently described that loss of the cell cycle regulator p21 was associated with a gain in EMT characteristics and an upregulation of the master EMT transcription factor ZEB1. In this study, in silico analysis was performed in combination with different in vitro and in vivo techniques to identify and verify novel epigenetic targets of ZEB1, and to proof the direct transcriptional regulation of SETD1B by ZEB1. The chorioallantoic-membrane assay served as an in vivo model to analyze the ZEB1/SETD1B interaction. Bioinformatical analysis of CRC patient data was used to examine the ZEB1/SETD1B network under clinical conditions and the ZEB1/SETD1B network was modeled under physiological and pathological conditions. Thus, we identified a self-reinforcing loop for ZEB1 expression and found that the SETD1B associated active chromatin mark H3K4me3 was enriched at the ZEB1 promoter in EMT cells. Moreover, clinical evaluation of CRC patient data showed that the simultaneous high expression of ZEB1 and SETD1B was correlated with the worst prognosis. Here we report that the expression of chromatin modifiers is remarkably dysregulated in EMT cells. SETD1B was identified as a new ZEB1 target in vitro and in vivo. Our study demonstrates a novel example of an activator role of ZEB1 for the epigenetic landscape in colorectal tumor cells.