Doris Schneller

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.

Hepatic tumor-stroma crosstalk guides epithelial to mesenchymal transition at the tumor edge.

The tumor–stroma crosstalk is a dynamic process fundamental in tumor development. In hepatocellular carcinoma (HCC), the progression of malignant hepatocytes frequently depends on transforming growth factor (TGF)-β provided by stromal cells. TGF-β induces an epithelial to mesenchymal transition (EMT) of oncogenic Ras-transformed hepatocytes and an upregulation of platelet-derived growth factor (PDGF) signaling. To analyse the influence of the hepatic tumor–stroma crosstalk onto tumor growth and progression, we co-injected malignant hepatocytes and myofibroblasts (MFBs). For this, we either used in vitro-activated p19ARF MFBs or in vivo-activated MFBs derived from physiologically inflamed livers of Mdr2/p19ARF double-null mice. We show that co-transplantation of MFBs with Ras-transformed hepatocytes strongly enhances tumor growth. Genetic interference with the PDGF signaling decreases tumor cell growth and maintains plasma membrane-located E-cadherin and β-catenin at the tumor–host border, indicating a blockade of hepatocellular EMT. We further generated a collagen gel-based three dimensional HCC model in vitro to monitor the MFB-induced invasion of micro-organoid HCC spheroids. This invasion was diminished after inhibition of TGF-β or PDGF signaling. These data suggest that the TGF-β/PDGF axis is crucial during hepatic tumor–stroma crosstalk, regulating both tumor growth and cancer progression.

Nuclear β-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.

ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression.

In human hepatocellular carcinoma (HCC), epithelial to mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. We employed a model of EMT based on immortalized p19ARF null hepatocytes (MIM), which display tumor growth upon expression of oncogenic Ras and undergo EMT through the synergism of Ras and transforming growth factor (TGF)-β. Here, we show that the interleukin-related protein interleukin-like EMT inducer (ILEI), a novel EMT-, tumor- and metastasis-inducing protein, cooperates with oncogenic Ras to cause TGF-β-independent EMT. Ras-transformed MIM hepatocytes overexpressing ILEI showed cytoplasmic E-cadherin, loss of ZO-1 and induction of α-smooth muscle actin as well as platelet-derived growth factor (PDGF)/PDGF-R isoforms. As shown by dominant-negative PDGF-R expression in these cells, ILEI-induced PDGF signaling was required for enhanced cell migration, nuclear accumulation of β-catenin, nuclear pY-Stat3 and accelerated growth of lung metastases. In MIM hepatocytes expressing the Ras mutant V12-C40, ILEI collaborated with PI3K signaling resulting in tumor formation without EMT. Clinically, human HCC samples showed granular or cytoplasmic localization of ILEI correlating with well and poorly differentiated tumors, respectively. In conclusion, these data indicate that ILEI requires cooperation with oncogenic Ras to govern hepatocellular EMT through mechanisms involving PDGF-R/β-catenin and PDGF-R/Stat3 signaling.

Disruption of the Growth Hormone—Signal Transducer and Activator of Transcription 5—Insulinlike Growth Factor 1 Axis Severely Aggravates Liver Fibrosis in a Mouse Model of Cholestasis

Growth hormone (GH) resistance and low serum levels of insulinlike growth factor 1 (IGF‐1) are common features in human liver fibrosis and cirrhosis. Signal transducer and activator of transcription 5 (STAT5) controls several vital functions in the liver, including GH‐mediated transcription of IGF‐1. To investigate the role of STAT5 in liver fibrogenesis, we specifically deleted the Stat5a/b locus both in hepatocytes and cholangiocytes in the multidrug resistance gene 2 knockout (Mdr2−/−) mouse model of cholestasis. Double knockout mice develop an early and severe liver fibrosis phenotype, accompanied by perturbed expression of key regulators of bile acid homeostasis. Deletion of Stat5 resulted in GH resistance, and IGF‐1 levels in serum were undetectable. We could observe reduced expression of important hepatoprotective genes, such as epidermal growth factor receptor (Egfr), hepatocyte nuclear factor 6 (Hnf6), prolactin receptor (Prlr), and leukemia inhibitory factor receptor (Lifr) as well as increased numbers of apoptotic hepatocytes. Conclusion: Our data suggest that loss of STAT5 sensitizes hepatocytes to bile acid–induced damage and apoptosis caused by disruption of GH‐induced transcription of Igf‐1 and down‐regulation of hepatoprotective genes. These findings could contribute to the understanding of liver fibrosis and future treatment strategies for liver fibrosis. (HEPATOLOGY 2010.)

Signal Transducer and Activator of Transcription 3 Protects From Liver Injury and Fibrosis in a Mouse Model of Sclerosing Cholangitis

BACKGROUND & AIMS Signal transducer and activator of transcription 3 (Stat3) is the main mediator of interleukin-6-type cytokine signaling required for hepatocyte proliferation and hepatoprotection, but its role in sclerosing cholangitis and other cholestatic liver diseases remains unresolved. METHODS We investigated the role of Stat3 in inflammation-induced cholestatic liver injury and used mice lacking the multidrug resistance gene 2 (mdr2(-/-)) as a model for SC. RESULTS We show that conditional inactivation of Stat3 in hepatocytes and cholangiocytes (stat3(Deltahc)) of mdr2(-/-) mice strongly aggravated bile acid-induced liver injury and fibrosis. A similar phenotype was observed in mdr2(-/-) mice lacking interleukin-6 production. Biochemical and molecular characterization suggested that Stat3 exerts hepatoprotective functions in both hepatocytes and cholangiocytes. Loss of Stat3 led to increased expression of tumor necrosis factor alpha, which might reduce the barrier function of bile ducts. Moreover, Stat3-deficient hepatocytes displayed up-regulation of bile acid biosynthesis genes and down-regulation of hepatoprotective epidermal growth factor receptor and insulin-like growth factor 1 signaling pathways. Consistently, stat3(Deltahc) mice were more sensitive to cholic acid-induced liver damage than control mice. CONCLUSIONS Our data suggest that Stat3 prevents cholestasis and liver damage in sclerosing cholangitis via regulation of pivotal functions in hepatocytes and cholangiocytes.

p19ARF/p14ARF controls oncogenic functions of signal transducer and activator of transcription 3 in hepatocellular carcinoma

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 p19ARF/p14ARF. We show that constitutive active (ca) Stat3 is tumor suppressive in Ras‐transformed p19ARF−/− hepatocytes, whereas the expression of Stat3 lacking Tyr705 phosphorylation (U‐Stat3) enhances tumor formation. Accordingly, Ras‐transformed Stat3Δhc/p19ARF−/− hepatocytes (lacking Stat3 and p19ARF) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor‐suppressor activity of Stat3 in cells lacking p19ARF. Notably, endogenous expression of p19ARF 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 p14ARF (the human homolog of p19ARF) 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 p14ARF levels, indicating that p14ARF controls the oncogenic function of pY‐Stat3 downstream of Jak. Conclusion: These data show evidence that p19ARF/p14ARF determines the pro‐ or anti‐oncogenic activity of U‐Stat3 and pY‐Stat3 in Ras‐dependent HCC progression. (HEPATOLOGY 2011;)

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.