Raquel Urtasun

Epidermal Growth Factor Receptor Signaling in Hepatocellular Carcinoma: Inflammatory Activation and a New Intracellular Regulatory Mechanism

Background/Aims: Hepatocellular carcinoma (HCC) is a chemoresistant tumor strongly associated with chronic hepatitis. Identification of molecular links connecting inflammation with cell growth/survival, and characterization of pro-tumorigenic intracellular pathways is therefore of therapeutic interest. The epidermal growth factor receptor (EGFR) signaling system stands at a crossroad between inflammatory signals and intracellular pathways associated with hepatocarcinogenesis. We investigated the regulation and activity of different components of the EGFR system, including the EGFR ligand amphiregulin (AR) and its sheddase ADAM17, and the modulation of intracellular EGFR signaling by a novel mechanism involving protein methylation. Methods: ADAM17 protein expression was examined in models of liver injury and carcinogenesis. Crosstalk between tumor necrosis factor (TNF)-α, AR and EGFR signaling was evaluated in human HCC cells and mouse hepatocytes. Modulation of EGFR signaling and biological responses by methylation reactions was evaluated in AML12 mouse hepatocytes. Results: ADAM17 was upregulated in liver injury and hepatocarcinogenesis. TNF-α triggered AR shedding and EGFR transactivation in HCC cells. AR was necessary for TNF-α activation of ERK1/2 and Akt signaling in hepatocytes. Inhibition of methylation reactions increased the ERK1/2 signal amplitude triggered by AR/EGFR and reduced DNA synthesis in AML12 cells. Conclusions: Increased ADAM17 in pre-neoplastic liver injury further supports its implication in hepatocarcinogenesis. AR release and EGFR transactivation by TNF-α constitutes a novel link between inflammatory signals and pro-tumorigenic mechanisms in liver cells. Finally, the identification of a new mechanism controlling growth factor signaling, and biological responses, involving methylation reactions within the RAS/RAF/MEK/ERK pathway, exposes a new target for antineoplastic intervention.

Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment.

Matrix metalloproteinase‐10 expression is induced during hepatic injury and plays a fundamental role in liver tissue repair

Upon tissue injury, the liver mounts a potent reparative and regenerative response. A role for proteases, including serine and matrix metalloproteinases (MMPs), in this process is increasingly recognized. We have evaluated the expression and function of MMP10 (stromelysin‐2) in liver wound healing and regeneration.

Matrix metalloproteinase 10 contributes to hepatocarcinogenesis in a novel crosstalk with the stromal derived factor 1/C‐X‐C chemokine receptor 4 axis

Matrix metalloproteinases (MMPs) participate in tissue repair after acute injury, but also participate in cancer by promoting a protumorigenic microenvironment. Previously, we reported on a key role for MMP10 in mouse liver regeneration. Herein, we investigated MMP10 expression and function in human hepatocellular carcinoma (HCC) and diethylnitrosamine (DEN)‐induced mouse hepatocarcinogenesis. MMP10 was induced in human and murine HCC tissues and cells. MMP10‐deficient mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic, C‐X‐C chemokine receptor‐4 (CXCR4), was reduced in DEN‐induced MMP10‐deficient mice livers. Human HCC cells stably expressing MMP10 had increased CXCR4 expression and migratory capacity. Pharmacological inhibition of CXCR4 significantly reduced MMP10‐stimulated HCC cell migration. Furthermore, MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand, stromal‐derived factor‐1 (SDF1), through the extracellular signal‐regulated kinase 1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter. Conclusion: MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis. To our knowledge, this is the first report addressing the role of a MMP in hepatocarcinogenesis in the corresponding genetic mouse model. (Hepatology 2015;62:166‐178)

Regulation of amphiregulin gene expression by β-catenin signaling in human hepatocellular carcinoma cells: a novel crosstalk between FGF19 and the EGFR system.

Hepatocellular carcinoma (HCC) is the most prevalent liver tumor and a deadly disease with limited therapeutic options. Dysregulation of cell signaling pathways is a common denominator in tumorigenesis, including hepatocarcinogenesis. The epidermal growth factor receptor (EGFR) signaling system is commonly activated in HCC, and is currently being evaluated as a therapeutic target in combination therapies. We and others have identified a central role for the EGFR ligand amphiregulin (AR) in the proliferation, survival and drug resistance of HCC cells. AR expression is frequently up-regulated in HCC tissues and cells through mechanisms not completely known. Here we identify the β-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of β-catenin signaling, or expression of the T41A β-catenin active mutant, led to the induction of AR expression involving three specific β-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A β-catenin active mutant show enhanced proliferation that is dependent in part on AR expression and EGFR signaling. We also demonstrate here a novel cross-talk of the EGFR system with fibroblast growth factor 19 (FGF19). FGF19 is a recently identified driver gene in hepatocarcinogenesis and an activator of β-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the β-catenin pathway in human HCC cells. Importantly, AR up-regulation and EGFR signaling participated in the induction of cyclin D1 and cell proliferation elicited by FGF19. Finally, we demonstrate a positive correlation between FGF19 and AR expression in human HCC tissues, therefore supporting in clinical samples our experimental observations. These findings identify the AR/EGFR system as a key mediator of FGF19 responses in HCC cells involving β-catenin signaling, and suggest that combined targeting of FGF19 and AR/EGFR may enhance therapeutic efficacy.

Splicing regulator SLU7 is essential for maintaining liver homeostasis

A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.

Oral Methylthioadenosine Administration Attenuates Fibrosis and Chronic Liver Disease Progression in Mdr2−/− Mice

Background Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5′-methylthioadenosine (MTA) in Mdr2−/− mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. Methodology MTA was administered daily by gavage to wild type and Mdr2−/− mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2−/− mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). Principal Findings MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. Conclusions/Significance Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration

Objective Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. Design Fgf15−/− mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. Results Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15−/− mice. Hepatic expression of Pparγ2 was elevated in Fgf15−/− mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. Conclusions FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.

Fibroblast Growth Factor 15/19 in Hepatocarcinogenesis

Background: Advanced hepatocellular carcinoma (HCC) is a neoplastic disease with a very bad prognosis and increasing worldwide incidence. HCCs are resistant to conventional chemotherapy and the multikinase inhibitor sorafenib is the only agent that has shown some clinical efficacy. It is therefore important to identify key molecular mechanisms driving hepatocarcinogenesis for the development of more efficacious therapies. However, HCCs are heterogeneous tumors and different molecular subclasses have been characterized. This heterogeneity may underlie the poor performance of most of the targeted therapies so far tested in HCC patients. The fibroblast growth factor 15/19 (FGF15/19), FGF receptor 4 (FGFR4) and beta-Klotho (KLB) correceptor signaling system, a key regulator of bile acids (BA) synthesis and intermediary metabolism, is emerging as an important player in hepatocarcinogenesis. Key Messages: Aberrant signaling through the FGF15/19-FGFR4 pathway participates in the neoplastic behavior of HCC cells, promotes HCC development in mice and its overexpression has been characterized in a subset of HCC tumors from patients with poorer prognosis. Pharmacological interference with FGF15/19-FGFR4 signaling inhibits experimental hepatocarcinogenesis, and specific FGFR4 inhibitors are currently being tested in selected HCC patients with tumoral FGF19-FGFR4/KLB expression. Conclusions: Interference with FGF19-FGFR4 signaling represents a novel strategy in HCC therapy. Selection of candidate patients based on tumoral FGF19-FGFR4/KLB levels as biomarkers may result in increased efficacy of FGFR4-targeted drugs. Nevertheless, attention should be paid to the potential on target toxic effects of FGFR4 inhibitors due to the key role of this signaling system in BA metabolism.

Alterations in the expression and activity of pre-mRNA splicing factors in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is a molecularly complex tumor that is resistant to standard and targeted therapies, and thus a deadly disease. In this context, the identification of key alterations driving HCC development is therefore essential. The implementation of next-generation sequencing techniques has underscored earlier realizations of the marked dysregulation of pre-mRNA splicing in HCC. Impairments in alternative splicing may lead to the expression of protumorigenic protein isoforms and to the generation of unstable mRNA species. Mechanistically, mutations in key nucleotides are responsible for many of these alterations in different types of tumors. However, changes in the expression of factors involved in the regulation of the splicing machinery are also important determinants in the derangement of pre-mRNA splicing. Here we discuss recent reports on the alteration of splicing factors in HCC, the pathological significance of these changes, and the identification of cell signaling pathways leading to the missplicing of genes in hepatocarcinogenesis.