ABSTRACT
Transforming growth factor ß (TGF-ß) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-ß role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-ß inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-ß effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-ß-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-ß actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.
Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Proliferation , Hepatocytes/metabolism , Liver Diseases/genetics , Liver Neoplasms/genetics , Smad7 Protein/genetics , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cells, Cultured , Female , Gene Expression Regulation, Neoplastic/drug effects , Hep G2 Cells , Humans , Liver Diseases/metabolism , Liver Diseases/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice, 129 Strain , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Middle Aged , Multivariate Analysis , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Smad7 Protein/metabolism , Survival Analysis , Transforming Growth Factor beta/pharmacology , Y-Box-Binding Protein 1/genetics , Y-Box-Binding Protein 1/metabolismABSTRACT
Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-ß is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-ß effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-ß inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-ß and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-ß signatures correlated with better outcome in HCC patients. TGF-ß dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-ß and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-ß signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-ß signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-ß expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-ß sensitivity. Cell lines responsive to the TGF-ß cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-ß/Smad3 signaling are insensitive to TGF-ß dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-ß functions for the design of efficient TGF-ß directed therapy in liver cancer.
Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Cycle , Liver Neoplasms/pathology , Signal Transduction , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism , Base Sequence , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , DNA Primers , Humans , Liver Neoplasms/metabolism , RNA Interference , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/biosynthesisABSTRACT
Microarrays are widely used in biomedical research. However, researchers conducting the biomedical assays are often not skilled to perform the necessary biostatistical preprocessing of the resulting data. As a result, researchers with different backgrounds contribute to the analysis, but often without documenting how the data were transformed. For a biomedical research network on liver cancer, we implemented a prototype that has two major aims: First, it should guide biomedical researchers through the analysis of microarray data by providing a limited amount of appropriate choices for the biostatistical procedures to be applied. Second, it should help to ensure data quality by documenting all transformations applied to the data set.
