Transforming Growth Factor-ß Drives the Transendothelial Migration of Hepatocellular Carcinoma Cells.

The entry of malignant hepatocytes into blood vessels is a key step in the dissemination and metastasis of hepatocellular carcinoma (HCC). The identification of molecular mechanisms involved in the transmigration of malignant hepatocytes through the endothelial barrier is of high relevance for therapeutic intervention and metastasis prevention. In this study, we employed a model of hepatocellular transmigration that mimics vascular invasion using hepatic sinusoidal endothelial cells and malignant hepatocytes evincing a mesenchymal-like, invasive phenotype by transforming growth factor (TGF)-ß. Labelling of respective cell populations with various stable isotopes and subsequent mass spectrometry analyses allowed the "real-time" detection of molecular changes in both transmigrating hepatocytes and endothelial cells. Interestingly, the proteome profiling revealed 36 and 559 regulated proteins in hepatocytes and endothelial cells, respectively, indicating significant changes during active transmigration that mostly depends on cell-cell interaction rather than on TGF-ß alone. Importantly, matching these in vitro findings with HCC patient data revealed a panel of common molecular alterations including peroxiredoxin-3, epoxide hydrolase, transgelin-2 and collectin 12 that are clinically relevant for the patient's survival. We conclude that hepatocellular plasticity induced by TGF-ß is crucially involved in blood vessel invasion of HCC cells.

Transforming growth factor-ß-induced plasticity causes a migratory stemness phenotype in hepatocellular carcinoma.

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-ß induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells. Whether EMT induces changes in tumor cell plasticity has not been fully explored yet. Here, we analyze the effects of TGF-ß on the EMT and stem-related properties of HCC cells and the potential correlation among those processes. The translational aim of the study was to propose a TGF-ß/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-ß therapy. Results indicate that when TGF-ß induces EMT in HCC cells, a switch in the expression of stem genes is observed and their stemness potential and migratory/invasive capacity are enhanced. However, TGF-ß may induce a partial EMT in some epithelial HCC cells, increasing the expression of mesenchymal genes and CD44, but maintaining epithelial gene expression. Epithelial cells show higher stemness potential than the mesenchymal ones, but respond to TGF-ß increasing their migratory and invasive capacity. In HCC patient samples, TGFB1 expression most frequently correlates with a partial EMT, increase in mesenchymal genes and CD44 expression, as well as maintenance or over-expression of epithelial-related genes.

Role of epithelial to mesenchymal transition in hepatocellular carcinoma.

The epithelial to mesenchymal transition (EMT) is a multistep biological process whereby epithelial cells change in plasticity by transient de-differentiation into a mesenchymal phenotype. EMT and its reversal, mesenchymal to epithelial transition (MET), essentially occur during embryogenetic morphogenesis and have been increasingly described in fibrosis and cancer during the last decade. In carcinoma progression, EMT plays a crucial role in early steps of metastasis when cells lose cell-cell contacts due to ablation of E-cadherin and acquire increased motility to spread into surrounding or distant tissues. Epithelial plasticity has become a hot issue in hepatocellular carcinoma (HCC), as strong inducers of EMT such as transforming growth factor-ß are able to orchestrate both fibrogenesis and carcinogenesis, showing rising cytokine levels in cirrhosis and late stage HCC. In this review, we consider the significance of EMT-MET in malignant hepatocytes as well as changes in the plasticity of hepatic stellate cells for cellular heterogeneity of HCC, and further aim at explaining the current limiting insights into EMT by snapshot analyses of HCC tissues. Recent advances in the identification of clinically relevant mechanisms that impinge on important EMT-transcription factors, as well as on miRNAs causing EMT signatures and HCC progression are highlighted. In addition, we draw particular attention to framing EMT in the context of potential clinical relevance for HCC patients. We conclude that some aspects of EMT are still elusive and further studies are required to better link the clinical management of HCC with biomarkers and targeted therapies related to EMT.

Liver Sinusoidal Endothelial Cells Escape Senescence by Loss of p19ARF.

Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.

Halogenation of N6-benzyladenosine decreases its cytotoxicity in human leukemia cells.

Cytotoxicity of two halogen derivatives of N6-benzyladenosine (BAPR), N6-(3-iodobenzyl)-adenosine (I-BAPR) and 2-chloro-N6-(3-iodobenzyl)-adenosine (Cl-I-BAPR), was tested in human leukemia U937 cell line. Our results revealed that their cytotoxicity was surprisingly low. I-BAPR and also Cl-I-BAPR induced cell death with morphological and biochemical hallmarks of apoptosis, although the number of apoptotic cells was significantly lower than that found for BAPR. Our data strongly suggested that the decreased cytotoxic effect of halogenated derivatives of N6-benzyladenosine was related to their reduced intracellular phosphorylation by adenosine kinase.