Fibroblast growth factor 15 (FGF15), FGF19 in humans, is a gut-derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF15 also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell growth. Here we addressed for the first time the role of endogenous Fgf15 in hepatocarcinogenesis. Fgf15(+/) (+) and Fgf15(-/-) mice were subjected to a clinically relevant model of liver inflammation and fibrosis-associated carcinogenesis. Fgf15(-/-) mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf15(+/) (+) animals. Importantly, ileal Fgf15 mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF15 protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf15(-/-) mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP). Interestingly, lack of FGF15 resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF15/FGF19. Conversely we demonstrate that FGF15/FGF19 induces the expression of the pro-fibrogenic and pro-tumorigenic connective tissue growth factor (CTGF) in hepatocytes. These findings suggest the existence of an FGF15-triggered CTGF-mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF15 via CTGF production. In summary, our observations indicate that ileal FGF15 may contribute to HCC development in a context of chronic liver injury and fibrosis.
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 4? (Hnf4?), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.
Radioembolization may rarely induce liver disease resulting in a syndrome that is similar to veno-occlusive disease complicating bone marrow transplantation where inflammation, endothelial cell activation and thrombosis are likely involved. We hypothesized that similar mechanisms could be implicated in radioembolization-induced liver disease (REILD). Moreover, lobar radioembolization may induce hypertrophy of the non-treated hemiliver most probably by inducing liver regeneration.
Amphiregulin (AREG) is a ligand of the epidermal growth factor receptor (EGFR), a widely expressed transmembrane tyrosine kinase. AREG is synthesized as a membrane-anchored precursor protein that can engage in juxtacrine signaling on adjacent cells. Alternatively, after proteolytic processing by cell membrane proteases, mainly TACE/ADAM17, AREG is secreted and behaves as an autocrine or paracrine factor. AREG gene expression and release is induced by a plethora of stimuli including inflammatory lipids, cytokines, hormones, growth factors and xenobiotics. Through EGFR binding AREG activates major intracellular signaling cascades governing cell survival, proliferation and motility. Physiologically, AREG plays an important role in the development and maturation of mammary glands, bone tissue and oocytes. Chronic elevation of AREG expression is increasingly associated with different pathological conditions, mostly of inflammatory and/or neoplastic nature. Here we review the essential aspects of AREG structure, function and regulation, discuss the basis for its differential role within the EGFR family of ligands, and identify emerging aspects in AREG research with translational potential.
The liver displays an outstanding wound healing and regenerative capacity unmatched by any other organ. This reparative response is governed by a complex network of inflammatory mediators, growth factors and metabolites that are set in motion in response to hepatocellular injury. However, when liver injury is chronic, these regenerative mechanisms become dysregulated, facilitating the accumulation of genetic alterations leading to unrestrained cell proliferation and the development of hepatocellular carcinoma (HCC). The epidermal growth factor receptor (EGFR or ErbB1) signaling system has been identified as a key player in all stages of the liver response to injury, from early inflammation and hepatocellular proliferation to fibrogenesis and neoplastic transformation. The EGFR system engages in extensive crosstalk with other signaling pathways, acting as a true signaling hub for other growth factors, cytokines and inflammatory mediators. Here, we briefly review essential aspects of the biology of the EGFR, the other ErbB receptors, and their ligands in liver injury, regeneration and HCC development. Some aspects of the preclinical and clinical experience with EGFR therapeutic targeting in HCC are also discussed.
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.
Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. Fibroblast growth factor 15 (Fgf15, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf15 in the preservation of BA homeostasis after PH and its potential role in the regenerative process.
The identification of molecular mechanisms involved in the maintenance of the transformed phenotype of hepatocellular carcinoma (HCC) cells is essential for the elucidation of therapeutic strategies. Here, we show that human HCC cells display an autocrine loop mediated by connective tissue growth factor (CTGF) that promotes DNA synthesis and cell survival. Expression of CTGF was stimulated by epidermal growth factor receptor (EGFR) ligands and was dependent on the expression of the transcriptional coactivator, Yes-associated protein (YAP). We identified elements in the CTGF gene proximal promoter that bound YAP-enclosing complexes and were responsible for basal and EGFR-stimulated CTGF expression. We also demonstrate that YAP expression can be up-regulated through EGFR activation not only in HCC cells, but also in primary human hepatocytes. CTGF contributed to HCC cell dedifferentiation, expression of inflammation-related genes involved in carcinogenesis, resistance toward doxorubicin, and in vivo HCC cell growth. Importantly, CTGF down-regulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 expression and was involved in the reduced sensitivity of these cells toward TRAIL-mediated apoptosis. Conclusion: We have identified autocrine CTGF as a novel determinant of HCC cells neoplastic behavior. Expression of CTGF can be stimulated through the EGFR-signaling system in HCC cells in a novel cross-talk with the oncoprotein YAP. Moreover, to our knowledge, this is the first study that identifies a signaling mechanism triggering YAP gene expression in healthy and transformed liver parenchymal cells.
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.
Bile acids (BA) are increasingly recognized as important modulators of liver regeneration. Increased enterohepatic BA flux has been proposed to generate specific signals that activate hepatocyte proliferation after partial hepatectomy (PH). We have investigated the role of the BA membrane transporter Mrp3 (Abcc3), which is expressed in the liver and gut, in the hepatic growth response elicited by BA and in liver regeneration after PH.
Cell therapy has been used to attenuate liver injury. Here we evaluated whether genetic engineering of either bone marrow-derived mononuclear cells (MNC) or endothelial progenitor cells (EPC) many enhance their hepatoprotective properties.
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.
Pre-mRNA splicing is an essential step in the process of gene expression in eukaryotes and consists of the removal of introns and the linking of exons to generate mature mRNAs. This is a highly regulated mechanism that allows the alternative usage of exons, the retention of intronic sequences and the generation of exonic sequences of variable length. Most human genes undergo splicing events, and disruptions of this process have been associated with a variety of diseases, including cancer. Hepatocellular carcinoma (HCC) is a molecularly heterogeneous type of tumor that usually develops in a cirrhotic liver. Alterations in pre-mRNA splicing of some genes have been observed in liver cancer, and although still scarce, the available data suggest that splicing defects may have a role in hepatocarcinogenesis. Here we briefly review the general mechanisms that regulate pre-mRNA splicing, and discuss some examples that illustrate how this process is impaired in liver tumorigenesis, and may contribute to HCC development. We believe that a more thorough examination of pre-mRNA splicing is still needed to accurately draw the molecular portrait of liver cancer. This will surely contribute to a better understanding of the disease and to the development of new effective therapies.
Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-stimulation of tumor-specific CD8(+) T cells. Whether antigen cross-presenting DC are required for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8beta(+) T-cell-dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross-presentation revealed that CD11c(+) cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-->C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction against OVA(257-264) epitope and in the antitumor efficacy induced by anti-CD137 mAb.
Epidemiological studies have established that many tumours occur in association with persistent inflammation. One clear example of inflammation-related cancer is hepatocellular carcinoma (HCC). HCC slowly unfolds on a background of chronic inflammation triggered by exposure to infectious agents (hepatotropic viruses), toxic compounds (ethanol), or metabolic impairment. The molecular links that connect inflammation and cancer are not completely known, but evidence gathered over the past few years is beginning to define the precise mechanisms. A central role for cytokines such as interleukin-6 (IL-6) and IL-1 (alpha and beta) in liver cancer has been established in experimental models. Besides these inflammatory mediators, mounting evidence points to the dysregulation of specific growth and survival-related pathways in HCC development. Among them is the pathway governed by the epidermal growth factor receptor (EGFR), which can be bound and activated by a broad family of ligands. Of special relevance is the fact that the EGFR engages in extensive crosstalk with other signaling pathways, serving as a "signaling hub" for an increasing list of growth factors, cytokines, and inflammatory mediators. In this review, we summarize the most recent evidences supporting a role for the EGFR system in inflammation-related cell signaling, with special emphasis in liver inflammation and HCC. The molecular dissection of the pathways connecting the inflammatory reaction and neoplasia will facilitate the development of novel and more effective antitumor strategies.
The modulation of the hepatic acute-phase reaction (APR) that occurs during inflammation and liver regeneration is important for allowing normal hepatocellular proliferation and the restoration of homeostasis. Activation of acute-phase protein (APP) gene expression by interleukin-6 (IL-6)-type cytokines is thought to be counteracted by growth factors released during hepatic inflammation and regeneration. The epidermal growth factor receptor (EGFR) ligand amphiregulin (AR) is readily induced by inflammatory signals and plays a nonredundant protective role during liver injury. In this paper, we investigated the role of AR as a modulator of liver APP gene expression.
The Wilms tumor 1 gene (WT1) encodes a transcription factor involved in cell growth and development. As we previously reported, WT1 expression is hardly detectable in normal hepatic tissue but is induced in liver cirrhosis. Although WT1 has been found to be overexpressed in a number of malignancies, the role of WT1 in hepatocarcinogenesis has not been clarified. We found that WT1 is expressed in several human hepatocellular carcinoma (HCC) cell lines, including PLC/PRF/5 and HepG2, and in HCC tumor tissue in 42% of patients. WT1 small interfering RNAs did not affect proliferation rate of HCC cells but abrogated their resistance to anoikis. Transcriptome analysis of PLC/PRF/5 cells after WT1 knockdown showed up-regulation of 251 genes and down-regulation of 321. Ninety percent of the former corresponded to metabolic genes, mostly those characterizing the mature hepatocyte phenotype. On the contrary, genes that decreased upon WT1 inhibition were mainly related to defense against apoptosis, cell cycle, and tumor progression. In agreement with these findings, WT1 expression increased the resistance of liver tumor cells to doxorubicin, a compound used to treat HCC. Interestingly, doxorubicin strongly enhanced WT1 expression in both HCC cells and normal human hepatocytes. Among different chemotherapeutics, induction of WT1 transcription was restricted to topoisomerase 2 inhibitors. When WT1 expression was prohibited, doxorubicin caused a marked increase in caspase-3 activation. In conclusion, WT1 is expressed in a substantial proportion of HCC contributing to tumor progression and resistance to chemotherapy, suggesting that WT1 may be an important target for HCC treatment.
Inactivation of the product of the tumor suppressor gene TP73 does not usually occur by mutation but rather through expression of truncated isoforms that have dominant-negative effects on p73 and p53. The truncated oncogenic isoform DeltaEx2p73 is expressed in hepatocellular carcinomas (HCC) and is produced through the alternative splicing of p73 pre-messenger RNA (pre-mRNA); however, the underlying mechanisms regulating this process are unknown.
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 ?-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of ?-catenin signaling, or expression of the T41A ?-catenin active mutant, led to the induction of AR expression involving three specific ?-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A ?-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 ?-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the ?-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 ?-catenin signaling, and suggest that combined targeting of FGF19 and AR/EGFR may enhance therapeutic efficacy.
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.
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