Synergism of the receptor tyrosine kinase Axl with ErbB receptors mediates resistance to regorafenib in hepatocellular carcinoma.

Introduction: Hepatocellular carcinoma (HCC) patients at advanced stages receive immunotherapy or treatment with tyrosine kinase inhibitors (TKIs) such as Sorafenib (Sora) or Lenvatinib in frontline as well as Regorafenib (Rego) or Cabozantinib in second-line. A major hindrance of TKI therapies is the development of resistance, which renders drug treatment futile and results in HCC progression. Methods: In this study, we addressed the impact of the receptor tyrosine kinase Axl binding to its ligand Gas6 in acquiring refractoriness to TKIs. The initial responses of Axl-positive and Axl-negative cell lines to different TKIs were assessed. Upon inducing resistance, RNA-Seq, gain- and loss-of-function studies were applied to understand and intervene with the molecular basis of refractoriness. Secretome analysis was performed to identify potential biomarkers of resistance. Results: We show that HCC cells exhibiting a mesenchymal-like phenotype were less sensitive to drug treatment, linking TKI resistance to changes in epithelial plasticity. Gas6/Axl expression and activation were upregulated in Rego-resistant HCC cells together with the induction of ErbB receptors, whereas HCC cells lacking Axl failed to stimulate ErbBs. Treatment of Rego-insensitive HCC cells with the pan-ErbB family inhibitor Afatinib rather than with Erlotinib blocking ErbB1 reduced cell viability and clonogenicity. Genetic intervention with ErbB2-4 but not ErbB1 confirmed their crucial involvement in refractoriness to Rego. Furthermore, Rego-resistant HCC cells secreted basic fibroblast growth factor (bFGF) depending on Axl expression. HCC patients treated with Sora in first-line and with Rego in second-line displayed elevated serum levels of bFGF, emphasizing bFGF as a predictive biomarker of TKI treatment. Discussion: Together, these data suggest that the inhibition of ErbBs is synthetic lethal with Rego in Axl-expressing HCC cells, showing a novel vulnerability of HCC.

Gas6 in chronic liver disease-a novel blood-based biomarker for liver fibrosis.

The expression of the receptor tyrosine kinase Axl and its cleavage product soluble Axl (sAxl) is increased in liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In this multicenter study, we evaluated the diagnostic value of Gas6, the high-affinity ligand of Axl, in patients with chronic liver disease. Levels of sAxl and Gas6, and their albumin (alb) ratios were analyzed in serum samples of patients with biopsy-proven liver fibrosis, end-stage liver disease, HCC, and healthy controls, and were compared to Fibrosis-4 (FIB-4), enhanced liver fibrosis (ELF™) test, Child-Pugh score (CPS), model of end-stage liver disease (MELD) score, hepatic venous pressure gradient, and α-fetoprotein, respectively. A total of 1111 patients (median age 57.8 y, 67.3% male) was analyzed. Gas6/alb showed high diagnostic accuracy for the detection of significant (≥F2: AUC 0.805) to advanced fibrosis (≥F3: AUC 0.818), and was superior to Fib-4 for the detection of cirrhosis (F4: AUC 0.897 vs. 0.878). In addition, Gas6/alb was highly predictive of liver disease severity (Odds ratios for CPS B/C, MELD ≥ 15, and clinically significant portal hypertension (CSPH) were 16.534, 10.258, and 12.115), and was associated with transplant-free survival (Hazard ratio 1.031). Although Gas6 and Gas6/alb showed high diagnostic accuracy for the detection of HCC in comparison to chronic liver disease patients without cirrhosis (AUC 0.852, 0.868), they failed to discriminate between HCC in cirrhosis versus cirrhosis only. In conclusion, Gas6/alb shows a high accuracy to detect significant to advanced fibrosis and cirrhosis, and predicts severity of liver disease including CSPH.

PRAME Is a Novel Target of Tumor-Intrinsic Gas6/Axl Activation and Promotes Cancer Cell Invasion in Hepatocellular Carcinoma.

(1) Background: Activation of the receptor tyrosine kinase Axl by Gas6 fosters oncogenic effects in hepatocellular carcinoma (HCC), associating with increased mortality of patients. The impact of Gas6/Axl signaling on the induction of individual target genes in HCC and its consequences is an open issue. (2) Methods: RNA-seq analysis of Gas6-stimulated Axl-proficient or Axl-deficient HCC cells was used to identify Gas6/Axl targets. Gain- and loss-of-function studies as well as proteomics were employed to characterize the role of PRAME (preferentially expressed antigen in melanoma). Expression of Axl/PRAME was assessed in publicly available HCC patient datasets and in 133 HCC cases. (3) Results: Exploitation of well-characterized HCC models expressing Axl or devoid of Axl allowed the identification of target genes including PRAME. Intervention with Axl signaling or MAPK/ERK1/2 resulted in reduced PRAME expression. PRAME levels were associated with a mesenchymal-like phenotype augmenting 2D cell migration and 3D cell invasion. Interactions with pro-oncogenic proteins such as CCAR1 suggested further tumor-promoting functions of PRAME in HCC. Moreover, PRAME showed elevated expression in Axl-stratified HCC patients, which correlates with vascular invasion and lowered patient survival. (4) Conclusions: PRAME is a bona fide target of Gas6/Axl/ERK signaling linked to EMT and cancer cell invasion in HCC.

Immunological Aspects of AXL/GAS-6 in the Context of Human Liver Regeneration.

AXL and its corresponding ligand growth arrest-specific 6 (GAS-6) are critically involved in hepatic immunomodulation and regenerative processes. Pleiotropic inhibitory effects on innate inflammatory responses might essentially involve the shift of macrophage phenotype from a pro-inflammatory M1 to an anti-inflammatory M2. We aimed to assess the relevance of the AXL/GAS-6-pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Soluble AXL (sAXL) and GAS-6 levels were analyzed at preoperative and postoperative stages in 154 patients undergoing partial hepatectomy and correlated with clinical outcome. Perioperative dynamics of interleukin (IL)-6, soluble tyrosine-protein kinase MER (sMerTK), soluble CD163 (sCD163), and cytokeratin (CK) 18 were assessed to reflect pathophysiological processes. Preoperatively elevated sAXL and GAS-6 levels predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P < 0.005) and worse clinical outcome. These patients failed to respond with an immediate increase of sAXL and GAS-6 upon induction of liver regeneration. Abolished AXL pathway response resulted in a restricted increase of sCD163, suggesting a disrupted phenotypical switch to regeneratory M2 macrophages. No association with sMerTK was observed. Concomitantly, a distinct association of IL-6 levels with an absent increase of AXL/GAS-6 signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by CK18M65. sAXL and GAS-6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome but also AXL/GAS-6 signaling might be of critical relevance in human liver regeneration. Refractory AXL/GAS-6 signaling, due to chronic overactivation/stimulation in the context of underlying liver disease, appears to abolish their immediate release following induction of liver regeneration, causing overwhelming immune activation, presumably via intrahepatic immune regulation.

Transforming Growth Factor-ß and Axl Induce CXCL5 and Neutrophil Recruitment in Hepatocellular Carcinoma.

Transforming growth factor (TGF)-ß suppresses early hepatocellular carcinoma (HCC) development but triggers pro-oncogenic abilities at later stages. Recent data suggest that the receptor tyrosine kinase Axl causes a TGF-ß switch toward dedifferentiation and invasion of HCC cells. Here, we analyzed two human cellular HCC models with opposing phenotypes in response to TGF-ß. Both HCC models showed reduced proliferation and clonogenic growth behavior following TGF-ß stimulation, although they exhibited differences in chemosensitivity and migratory abilities, suggesting that HCC cells evade traits of anti-oncogenic TGF-ß. Transcriptome profiling revealed differential regulation of the chemokine CXCL5, which positively correlated with TGF-ß expression in HCC patients. The expression and secretion of CXCL5 was dependent on Axl expression, suggesting that CXCL5 is a TGF-ß target gene collaborating with Axl signaling. Loss of either TGF-ß or Axl signaling abrogated CXCL5-dependent attraction of neutrophils. In mice, tumor formation of transplanted HCC cells relied on CXCL5 expression. In HCC patients, high levels of Axl and CXCL5 correlated with advanced tumor stages, recruitment of neutrophils into HCC tissue, and reduced survival. Conclusion: The synergy of TGF-ß and Axl induces CXCL5 secretion, causing the infiltration of neutrophils into HCC tissue. Intervention with TGF-ß/Axl/CXCL5 signaling may be an effective therapeutic strategy to combat HCC progression in TGF-ß-positive patients.

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells.

Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl--1, HLF-Axl--2, SNU449-Axl--1, SNU449-Axl--2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl- clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region. However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl--1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl--1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

The non-invasive serum biomarker soluble Axl accurately detects advanced liver fibrosis and cirrhosis.

Soluble Axl (sAxl) was recently shown to be strongly released into the blood during liver fibrogenesis and hepatocellular carcinoma suggesting sAxl as a biomarker of liver diseases. In this study we are the first to evaluate sAxl in human serum in comparison to Enhanced Liver Fibrosis (ELF) test and transient elastography (TE; Fibroscan) for its value to detect significant (F≥2), advanced fibrosis (F≥3), and cirrhosis (F4) in different liver disease etiologies and healthy controls. To properly determine the diagnostic accuracy of sAxl, a test cohort as well as a validation cohort was employed using liver biopsy as a reference method. Most notably, sAxl was confirmed to be an accurate biomarker of liver fibrosis and cirrhosis. Its accuracy was increased, if total serum albumin was added to build a sAxl/albumin ratio. Thereby an AUC of 0.763, 0.776, 0.826, and 0.832 was achieved corresponding to histological fibrosis stages F≥2, F≥3, F4 with liver biopsy as a reference method, and cirrhosis according to imaging techniques, respectively. With a cut-off of 1.29, a sensitivity, specificity, PPV, and NPV of 78.5%, 80.1%, 44%, 94.9% for the detection of cirrhosis was achieved. In comparison, ELF test and TE showed an AUC of 0.910, and 0.934, respectively, for the detection of cirrhosis. However, performance of TE was not possible in 14.4% of patients and both, ELF™ test and TE bear the disadvantage of high costs. In conclusion, the sAxl/albumin ratio is suggested as an accurate biomarker of liver fibrosis and cirrhosis. Due to its easy applicability and low costs it is suitable as screening parameter for significant to advanced liver fibrosis and cirrhosis, especially if TE is not available or not applicable.

Soluble Axl is an accurate biomarker of cirrhosis and hepatocellular carcinoma development: results from a large scale multicenter analysis.

Patients with chronic liver disease (CLD) and cirrhosis are at high risk for hepatocellular carcinoma (HCC). Current diagnostic tools for HCC detection include imaging techniques and serum biomarkers such as α-fetoprotein (AFP). Yet, these methods are limited in sensitivity and specificity to accurately detect early HCC. Here we focused on the potential of soluble Axl (sAxl) as a biomarker in CLD patients by analyzing serum samples of 1067 patients and healthy controls from centers in Europe and Asia. We show that serum concentrations of sAxl were significantly increased at early (82.57 ng/mL) and later stages of HCC (114.50 ng/mL) as compared to healthy controls (40.15 ng/mL). Notably, no elevated sAxl levels were detected in patients with CLD including chronic viral hepatitis, autoimmune hepatitis, cholestatic liver disease, or non-alcoholic fatty liver disease versus healthy controls. Furthermore, sAxl did not rise in liver adenomas or cholangiocarcinoma (CCA). Yet, patients with advanced fibrosis (F3) or cirrhosis (F4) showed enhanced sAxl concentrations (F3: 54.67 ng/mL; F4: 94.74 ng/mL). Hepatic myofibroblasts exhibited an increased release of sAxl, suggesting that elevated sAxl levels arise from these cells during fibrosis. Receiver operating characteristic curve analysis of sAxl displayed a strongly increased sensitivity and specificity to detect both cirrhosis (80.8%/92.0%) and HCC (83.3%/86.7%) with an area under the curve of 0.935/0.903 as compared to AFP. In conclusion, sAxl shows high diagnostic accuracy at early stage HCC as well as cirrhosis, thereby outperforming AFP. Importantly, sAxl remains normal in most common CLDs, liver adenomas and CCA.

Accurate Determination of Soluble Axl by Enzyme-Linked Immunosorbent Assay.

Levels of soluble Axl (sAxl) are routinely assessed in human sera by sandwich enzyme-linked immunosorbent assay (ELISA). Although sAxl values are suggested to diagnose different types of disorders, no uniform ELISA method is available, allowing the reliable interassay comparison between results. Furthermore, little is known about the stability of sAxl under storage conditions, which is a relevant parameter for biomedical trials. The evaluation of sAxl stability under various stress conditions and the determination of proper conditions to use the sAxl ELISA for routine clinical applications are of great interest. In this study, serum samples were subjected to freeze-thaw cycles and incubation at different temperatures to analyze the stability of sAxl by ELISA. Dilution and spike-in experiments were carried out to examine the impact of serum and diluent components on the ELISA performance. Various diluents and media were employed to resolve masking effects of the serum. The assay components were further optimized for long-term usability by treatment with stabilizers and validation under temperature stress. Indeed, sAxl showed long-term stability in serum during freeze-thaw cycles and incubation under temperature stress conditions. The dilution experiments revealed that unknown components in the serum caused masking effects that can be reduced by proper dilutions. The assay performance was further increased by using a standardized buffer system to dilute serum samples. Stabilization of coated plates and of streptavidin-horseradish peroxidase allowed long-term storage for up to 6 months. In sum, our data demonstrate proper ELISA conditions, allowing the accurate analysis of sAxl levels in human serum.

Neuropilin-2 induced by transforming growth factor-ß augments migration of hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-ß (TGF-ß) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-ß. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-ß/Smad signaling. METHODS: NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-ß signaling were analyzed by Western blotting and confocal immunofluorescence microscopy. RESULTS: We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-ß/Smad signaling while NRP2 expression has no impact on TGF-ß signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-ß signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-ß signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens. CONCLUSIONS: These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-ß-dependent HCC progression.

Axl activates autocrine transforming growth factor-ß signaling in hepatocellular carcinoma.

UNLABELLED: In hepatocellular carcinoma (HCC), intrahepatic metastasis frequently correlates with epithelial to mesenchymal transition (EMT) of malignant hepatocytes. Several mechanisms have been identified to be essentially involved in hepatocellular EMT, among them transforming growth factor (TGF)-ß signaling. Here we show the up-regulation and activation of the receptor tyrosine kinase Axl in EMT-transformed hepatoma cells. Knockdown of Axl expression resulted in abrogation of invasive and transendothelial migratory abilities of mesenchymal HCC cells in vitro and Axl overexpression-induced metastatic colonization of epithelial hepatoma cells in vivo. Importantly, Axl knockdown severely impaired resistance to TGF-ß-mediated growth inhibition. Analysis of the Axl interactome revealed binding of Axl to 14-3-3ζ, which is essentially required for Axl-mediated cell invasion, transendothelial migration, and resistance against TGF-ß. Axl/14-3-3ζ signaling caused phosphorylation of Smad3 linker region (Smad3L) at Ser213, resulting in the up-regulation of tumor-progressive TGF-ß target genes such as PAI1, MMP9, and Snail as well as augmented TGF-ß1 secretion in mesenchymal HCC cells. Accordingly, high Axl expression in HCC patient samples correlated with elevated vessel invasion of HCC cells, higher risk of tumor recurrence after liver transplantation, strong phosphorylation of Smad3L, and lower survival. In addition, elevated expression of both Axl and 14-3-3ζ showed strongly reduced survival of HCC patients. CONCLUSION: Our data suggest that Axl/14-3-3ζ signaling is central for TGF-ß-mediated HCC progression and a promising target for HCC therapy.

NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2(-/-)/p19(ARF-/-), Stat3(Δhc)/Mdr2(-/-)) and a model of experimental induced fibrosis (CCl(4)) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-ß pathway activation. In vitro TGF-ß-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-ß is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-ß-induced epithelial-mesenchymal transition (EMT), but was required for TGF-ß-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-ß-induced death of hepatocytes.

PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition.

The extracellular matrix protein Laminin B1 (LamB1) regulates tumor cell migration and invasion. Carcinoma cells acquire invasive properties by epithelial to mesenchymal transition (EMT), which is a fundamental step in dissemination of metastatic cells from the primary tumor. Recently, we showed that enhanced translation of LamB1 upon EMT of malignant hepatocytes is mediated by an internal ribosome entry site (IRES). We demonstrated that the IRES transacting factor La binds the minimal IRES motif and positively modulates IRES activity of LamB1. Here, we show that platelet-derived growth factor (PDGF) enhances IRES activity of LamB1 by the increasing cytoplasmic localization of La during EMT. Accordingly, cells expressing dominant negative PDGF receptor display reduced cytoplasmic accumulation of La and show no elevation of IRES activity or endogenous LamB1 levels after stimulation with PDGF. Furthermore, La-mediated regulation of LamB1 IRES activity predominantly depends on MAPK/ERK signaling downstream of PDGF. Notably, LamB1 expression is not significantly downregulated by the impairment of the translation initiation factor eIF4E. In vivo, knockdown of La associated with decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT.

La enhances IRES-mediated translation of laminin B1 during malignant epithelial to mesenchymal transition.

The majority of transcripts that harbor an internal ribosome entry site (IRES) are involved in cancer development via corresponding proteins. A crucial event in tumor progression referred to as epithelial to mesenchymal transition (EMT) allows carcinoma cells to acquire invasive properties. The translational activation of the extracellular matrix component laminin B1 (LamB1) during EMT has been recently reported suggesting an IRES-mediated mechanism. In this study, the IRES activity of LamB1 was determined by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1. Furthermore, no other LamB1 mRNA species arising from alternative transcription start sites or polyadenylation signals were detected that account for its translational control. Mapping of the LamB1 5'-untranslated region (UTR) revealed the minimal LamB1 IRES motif between -293 and -1 upstream of the start codon. Notably, RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction and its regulation during EMT were confirmed by ribonucleoprotein immunoprecipitation. In addition, La was able to positively modulate LamB1 IRES translation. In summary, these data indicate that the LamB1 IRES is activated by binding to La which leads to translational upregulation during hepatocellular EMT.

p19(ARF) /p14(ARF) controls oncogenic functions of signal transducer and activator of transcription 3 in hepatocellular carcinoma.

UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.

Nuclear beta-catenin induces an early liver progenitor phenotype in hepatocellular carcinoma and promotes tumor recurrence.

Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.

Epithelial-mesenchymal transition in hepatocellular carcinoma.

The transition of epithelial cells to a mesenchymal phenotype is of paramount relevance for embryonic development and adult wound healing. During the past decade, the epithelial-mesenchymal transition (EMT) has been increasingly recognized to occur during the progression of various carcinomas such as hepatocellular carcinoma (HCC). Here, we focus on EMT in both experimental liver models and human HCC, emphasizing the underlying molecular mechanisms which show partial recurrence of embryonic programs such as TGF-beta and Wnt/ beta-catenin signaling, including collaboration with hepatitis viruses. We further discuss the differentiation repertoire of malignant hepatocytes with respect to the potential acquisition of stemness, and the involvement of the mesenchymal to epithelial transition, the reversal of EMT, in cancer dissemination and metastatic colonization. The strong evidence for EMT in HCC patients demands novel strategies in pathological assessments and therapeutic concepts to efficiently combat HCC progression.

The leader region of Laminin B1 mRNA confers cap-independent translation.

Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5'-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5'-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes.

TGF-beta dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells.

BACKGROUND: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-beta. We analyzed the oxidative stress induced by TGF-beta and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs. RESULTS: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-beta/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense. CONCLUSION: The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

Immortalized p19ARF null hepatocytes restore liver injury and generate hepatic progenitors after transplantation.

Primary hepatocytes are blocked in mitotic activity and well-defined culture conditions only allow the limited expansion of these cells. Various genetic modifications have therefore been employed to establish immortalized hepatic cell lines, but, unfortunately, proper hepatocyte cultures conducting a faithful hepatic gene expression program and lacking malignancy are hardly available. Here we report the immortalization of primary hepatocytes isolated from p19(ARF) null mice using the rationale that loss of p19(ARF) lowers growth-suppressive functions of p53 and bypasses cellular senescence without losing genetic stability. The established hepatocytes, called MIM, express liver-specific markers, show a nontumorigenic phenotype, and competence to undergo Fas-mediated apoptosis. Intrasplenic transplantation of GFP-expressing parental MIM cells into Fas-injured livers of SCID mice revealed liver-reconstituting activity. In the regenerated liver, MIM cells localized in small-sized clusters and showed presence in structures comparable to canals of Hering, the site of oval cells. Transplantation of MIM-Bcl-X(L) cells, which are protected against apoptosis, and successive Fas-induced liver damage, enhanced donor-derived liver repopulation by showing differentiation into cholangiocytes and cells expressing markers characteristic of both fetal hepatocytes and oval cells. In conclusion, these data indicate that long-term cultivated p19(ARF) null hepatocytes are capable of generating hepatic progenitor cells during liver restoration, and thus represent a highly valuable tool to study the differentiation repertoire of hepatocytes.