TGF-β family members play a relevant role in tumorigenic processes, including hepatocellular carcinoma (HCC), but a specific implication of the Bone Morphogenetic Protein (BMP) subfamily is still unknown. Although originally isolated from fetal liver, little is known about BMP9, a BMP family member, and its role in liver physiology and pathology. Our results show that BMP9 promotes growth in HCC cells, but not in immortalized human hepatocytes. In the liver cancer cell line HepG2, BMP9 triggers Smad1,5,8 phosphorylation and inhibitor of DNA binding 1 (Id1) expression up- regulation. Importantly, by using chemical inhibitors, ligand trap and gene silencing approaches we demonstrate that HepG2 cells autocrinely produce BMP9 that supports their proliferation and anchorage independent growth. Additionally, our data reveal that in HepG2 cells BMP9 triggers cell cycle progression, and strikingly, completely abolishes the increase in the percentage of apoptotic cells induced by long-term incubation in low serum. Collectively, our data unveil a dual role for BMP9, both promoting a proliferative response and exerting a remarkable anti-apoptotic function in HepG2 cells, which result in a robust BMP9 effect on liver cancer cell growth. Finally, we show that BMP9 expression is increased in 40% of human HCC tissues compared with normal human liver as revealed by immunohistochemistry analysis, suggesting that BMP9 signaling may be relevant during hepatocarcinogenesis in vivo. Our findings provide new clues for a better understanding of BMPs contribution, and in particular BMP9, in HCC pathogenesis that may result in the development of effective and targeted therapeutic interventions.

Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (DEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, DEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-b pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-b through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in DEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, DEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because DEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. Conclusion: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (HEPATOLOGY 2016;63:604-619) See Editorial on Page 371 T he liver is a unique organ in displaying reparative response and regenerative capacity. However, prolonged liver regeneration as a consequence of Abbreviations: DEN, diethyl-nitrosamine; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HCC, hepatocellular carcinoma; HGF, hepatocyte growth factor; mRNA, messenger RNA; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PH, partial hepatectomy; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; TGF-a/-b, transforming growth factor (a/b); TNF-a, tumor necrosis factor-a; uPA, urokinase-type plasminogen activator; WT, wild type.From the

Oval cells constitute an interesting hepatic cell population. They contribute to sustain liver regeneration during chronic liver damage, but in doing this they can be target of malignant conversion and become tumor-initiating cells and drive hepatocarcinogenesis. The molecular mechanisms beneath either their pro-regenerative or pro-tumorigenic potential are still poorly understood. In this study, we have investigated the role of the HGF/c-Met pathway in regulation of oval cell migratory and invasive properties. Our results show that HGF induces c-Met-dependent oval cell migration both in normal culture conditions and after in vitro wounding. HGF-triggered migration involves F-actin cytoskeleton reorganization, which is also evidenced by activation of Rac1. Furthermore, HGF causes ZO-1 translocation from cell-cell contact sites to cytoplasm and its concomitant activation by phosphorylation. However, no loss of expression of cell-cell adhesion proteins, including E-cadherin, ZO-1 and Occludin-1, is observed. Additionally, migration does not lead to cell dispersal but to a characteristic organized pattern in rows, in turn associated with Golgi compaction, providing strong evidence of a morphogenic collective migration. Besides migration, HGF increases oval cell invasion through extracellular matrix, a process that requires PI3K activation and is at least partly mediated by expression and activation of metalloproteases. Altogether, our findings provide novel insights into the cellular and molecular mechanisms mediating the essential role of HGF/c-Met signaling during oval cell-mediated mouse liver regeneration.