C. Busletta

Redox mechanisms switch on hypoxia-dependent epithelial–mesenchymal transition in cancer cells

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

Epithelial–Mesenchymal Transition: From Molecular Mechanisms, Redox Regulation to Implications in Human Health and Disease

Epithelial to mesenchymal transition (EMT) is a fundamental process, paradigmatic of the concept of cell plasticity, that leads epithelial cells to lose their polarization and specialized junctional structures, to undergo cytoskeleton reorganization, and to acquire morphological and functional features of mesenchymal-like cells. Although EMT has been originally described in embryonic development, where cell migration and tissue remodeling have a primary role in regulating morphogenesis in multicellular organisms, recent literature has provided evidence suggesting that the EMT process is a more general biological process that is also involved in several pathophysiological conditions, including cancer progression and organ fibrosis. This review offers first a comprehensive introduction to describe major relevant features of EMT, followed by sections dedicated on those signaling mechanisms that are known to regulate or affect the process, including the recently proposed role for oxidative stress and reactive oxygen species (ROS). Current literature data involving EMT in both physiological conditions (i.e., embryogenesis) and major human diseases are then critically analyzed, with a special final focus on the emerging role of hypoxia as a relevant independent condition able to trigger EMT.

Intracellular reactive oxygen species are required for directional migration of resident and bone marrow-derived hepatic pro-fibrogenic cells

BACKGROUND & AIMS Liver fibrogenesis is sustained by myofibroblast-like cells originating from hepatic stellate cells (HSC/MFs), portal fibroblasts or bone marrow-derived cells, including mesenchymal stem cells (MSCs). Herein, we investigated the mechanistic role of intracellular generation of reactive oxygen species (ROS) and redox-sensitive signal transduction pathways in mediating chemotaxis, a critical profibrogenic response for human HSC/MFs and for MSC potentially engrafting chronically injured liver. METHODS Intracellular generation of ROS and signal transduction pathways were evaluated by integrating morphological and molecular biology techniques. Chemokinesis and chemotaxis were evaluated by wound healing assay and modified Boydens chamber assay, respectively. Additional in vivo evidence was obtained in human specimens from HCV-related cirrhosis. RESULTS Human MSCs and HSC/MFs migrate in response to a panel of polypeptide chemoattractants and extracellularly generated superoxide anion. All polypeptides induced a NADPH-oxidase-dependent intracellular rise in ROS, resulting in activation of ERK1/2 and JNK1/2. Moreover, menadione or 2,3-dimethoxy-1,4-naphthoquinone, which generate intracellular superoxide anion or hydrogen peroxide, respectively, induced ERK1/2 and JNK1/2 activation and migration. JNK1 activation was predominant for migration as shown by specific silencing. Finally, activation of ERK1/2 and JNK1/2 was found in extracts obtained from HSC/MFs during the course of an oxidative stress-mediated model of liver injury and phosphorylated JNK1/2 isoforms were detected in α-smooth muscle actin-positive myofibroblasts lining fibrotic septa in human cirrhotic livers. CONCLUSIONS Intracellular generation of ROS, through activation of specific signaling pathways, is a critical event for directional migration of HSC/MFs and MSCs.

The biphasic nature of hypoxia-induced directional migration of activated human hepatic stellate cells.

Liver fibrogenesis is sustained by pro‐fibrogenic myofibroblast‐like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia‐dependent angiogenesis. Human HSC/MFs were reported to express vascular‐endothelial growth factor (VEGF) and VEGF‐receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia‐induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non‐oriented and oriented migration were evaluated by using wound healing assay and the modified Boydens chamber assay, respectively. The data indicate that hypoxia‐induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria‐related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF‐1α‐dependent increase in VEGF expression (facilitated by ROS) and its progressive, time‐dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV‐related fibrotic/cirrhotic livers revealed HIF‐2α and haem‐oxygenase‐1 positivity in hepatocytes and α‐SMA‐positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia‐induced migration of HSC/MFs involves an early, mitochondrial‐dependent ROS‐mediated activation of ERK and JNK, followed by a delayed‐ and HIF‐1α‐dependent up‐regulation and release of VEGF. Copyright

Dissection of the Biphasic Nature of Hypoxia-Induced Motogenic Action in Bone Marrow-Derived Human Mesenchymal Stem Cells†‡§

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony‐forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia‐dependent migration of bone marrow‐derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boydens chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia‐dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c‐Jun N‐terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor‐1 (HIF‐1)–dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia‐dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF‐1‐dependent expression and release of VEGF. STEM CELLS 2011;29:952–963

Hypoxia, hypoxia-inducible factors and fibrogenesis in chronic liver diseases

Fibrogenic progression of chronic liver diseases (CLDs) towards the end-point of cirrhosis is currently regarded, whatever the aetiology, as a dynamic and highly integrated cellular response to chronic liver injury. Liver fibrogenesis (i.e., the process) is sustained by hepatic populations of highly proliferative, pro-fibrogenic and contractile myofibroblast-like cells (MFs) that mainly originate from hepatic stellate cells (HSC) or, to a less extent, from portal fibroblasts or bone marrow-derived cells. As is well known, liver fibrosis (i.e., the result) is accompanied by perpetuation of liver injury, chronic hepatitis and persisting activation of tissue repair mechanisms, leading eventually to excess deposition of extracellular matrix (ECM) components. In this scenario, hypoxic areas represent a very common and major feature of fibrotic and cirrhotic liver during the progression of CLDs. Cells exposed to hypoxia respond by means of heterodimeric hypoxia-inducible factors (HIFs) that translocate into the nucleus and binds to a specific core sequence defined hypoxia-responsive element (HRE), present in the promoter on several genes which are considered as hypoxia-regulated target genes. HIFs transcription factors can activate a complex genetic program designed to sustain several changes necessary to efficiently counteract the decrease in oxygen tension. Accordingly, hypoxia, through up-regulation of angiogenesis, is currently believed to significantly contribute to fibrogenic progression of CLDs, mostly by affecting the pro-fibrogenic and pro-angiogenic behaviour of hepatic MFs. In addition, experimental and clinical evidence generated in the last decade also indicates that angiogenesis and fibrogenesis in CLDs may also be sustained by HIF-dependent but hypoxia-independent mediators.

SerpinB3 promotes pro-fibrogenic responses in activated hepatic stellate cells

SerpinB3 is a hypoxia- and hypoxia-inducible factor-2α-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-β1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. “In vitro” experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and αSMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.

Human-induced pluripotent stem cells as a source of hepatocyte-like cells: new kids on the block

One of the most exciting recent discoveries in the field of biology was the demonstration that both mouse and human somatic cells engineered via epigenetic reprogramming for the expression of combinations of few defined transcription factors (including Oct4, KLF4, Sox2, NANOG, LIN28, and c-Myc) can become pluripotent [1–3]. These cells closely resemble embryonic stem (ES) cells for their pluripotency and have been defined as induced pluripotent stem (iPS) cells. These cells have rapidly emerged as very promising tools for the achievement of significant advancement in different fields without the controversies and ethical concerns associated with the use of human ES cells [4]. In particular, the possibility to get iPS cells from readily obtainable somatic cells theoretically opened the way to a number of perspectives and practical applications, including (1) to design and test patient-customized (i.e., autologous) cell therapy without the need for immune suppression and (2) to increase our knowledge on mechanisms of inherited diseases with a realistic opportunity, as recently shown for human iPS-derived hepatocytes [5–7], for modeling inherited metabolic human diseases, understanding disease pathogenesis, and for drug discovery and testing. It should be anticipated, however, that the latter objective (i.e., the use of human iPS cell-derived hepatocytes for drug discovery and testing) can currently be envisaged as the most useful and safe application. Indeed, a number of relevant limitations have progressively emerged in relation to the clinical use of iPS cells and different laboratories have developed strategies in order to at least partially overcome such limitations. The original methods used in order to derive iPS cells from either human or mouse somatic cells employed viral vectors, a strategy leading to the integration of both desired transgenes and vector backbone into the host cell genome [1–3]. Of relevance, the use of these vectors is at risk to produce insertional mutations and genetic alterations able to interfere with normal functions of cells derived from iPS cells and to favor reactivation of reprogramming factors at later stages. Indeed, residual transgene expression has been reported to affect differentiation into specific lineages [2] or even result in tumorigenesis [8–10]. At present, the oncogenic potential of iPS cells represents a major concern for clinical application [9] since these cells, much as ES cells, can readily form teratomas when injected into immunodeficient mice. This may rely, for both iPS and ES cells, on the presence of residual diploid pluripotent cells that have not undergone differentiation in the population of transplanted human ES or iPS cells [9]. Moreover, tumor formation in iPS cell chimeric mice has been attributed to the expression of c-Myc in iPS cell-derived somatic cells, irrespective of the type of the original somatic cell employed [9–13]. However, as well reviewed [9], several other reasons are likely to underlie iPS cell tumorigenicity. In particular, although iPS cells may behave similarly to ES cells, differences between these two kinds of cells indeed exist and this is particularly significant for human cells, with human iPS cells being more tumorigenic than human ES cells because of genetic and epigenetic causes [9]. Along these lines, human iPS cells have been reported to acquire chromosomal alterations and genetic changes (including aneuploidy, gain or loss of small chromosomes, and point mutations) even more readily than ES cells C. Busletta E. Novo M. Parola (&) Department of Experimental Medicine and Oncology, InterUniversity Center for Hepatic Pathophysiology, University of Torino, Corso Raffaello 30, 10125 Turin, Italy e-mail: maurizio.parola@unito.it

Hepatic Myofibroblasts in Liver Fibrogenesis

On a worldwide perspective chronic liver diseases (CLDs) are very common pathologic conditions which are characterized by reiteration of hepatocyte injury that is mainly induced by chronic infection by hepatitis B and C viruses (HBV and HCV), autoimmune injury and metabolic and/or toxic/drug – induced causes, with chronic alcohol consumption being predominant particularly in western countries. Chronic liver injury is reported to result in the chronic activation of both inflammatory and wound healing response that, in association with other major pathogenic mechanisms (oxidative stress, derangement of epithelial-mesenchymal interactions and possibly epithelial to mesenchymal transition, see later in section 3), can sustain persistent liver fibrogenesis (i.e., the process) and represent the prominent driving force for liver fibrosis (i.e., the result) (Parola and Robino, 2001; Friedman 2003; Bataller and Brenner, 2005; Friedman 2008b; Novo and Parola, 2008; Parola et al., 2008).

T-5 Oncostatin M, overexpressed in hepatocellular carcinoma, up-regulates SERPIN-B3 expression in hepatic cancer cells

Background: In the western world HCV is the leading etiologic factor of HCC. Overall HCV impact on HCC-related mortality is increasing, but recent data from Italy suggest an initial drop. Aims: To evaluate epidemiology, clinical features and survival of HCV-related HCC in a wide time range in Italy. Methods: Multicenter retrospective study including 2769 patients prospectively recruited by the ITA.LI.CA group. The patients were classified in 3 groups (Group A [G.A.], HCV associated or not with other etiologies, Group B [G.B.], pure HCV, and Group C [G.C.], alternative etiologies) and sub-grouped in 5 years time cohorts (1986-90, 91-95, 96-2000, 2001-2005) but for the last one (2006-2008). Age, gender, Child-Pugh status, diagnosis for surveillance, stage, thrombosis and metastases, treatment and survival (Kaplan-Meier, Log-Rank) were analyzed. Results: 1780 patients were included in G.A., 1430 in G.B., 989 in G.C. The number of G.A. and G.B. patients reached a peak in the 1996-2000 to then gradually drop, lowest values being observed in the last three years (p=0.0001 G.A., 0.01 G.B.). Mean age at diagnosis progressively increased in G.A. (p<0.0001) and G.B. (p<0.001), but not in G.C, as did the percentage of cases diagnosed under surveillance (G.A. p=0.019, G.B. p=0.0038) and the share of patients in Child-Pugh A stage (G.A. p=0.01, G.B. p=0.007). Tumor size decreased in all subgroups, stage improved significantly only in G.B. Median survival significantly increased in all groups but more significantly in G.A. and G.B., with a significant difference among G.A. and G.B. vs G.C. in the last 3 time periods (p=0.02). Conclusions: The incidence of HCC in HCV-related liver disease is decreasing in Italy since 2001. HCV-related HCC patients are older, more frequently diagnosed under surveillance, more frequently characterized by conserved liver function and smaller tumors, with an earlier tumor stage when HCV is the only etiologic factor. Finally their survival dramatically improved in the last 15 years, more than in patients with different etiology. We therefore expect a further drop in incidence and mortality for the disease in Italy in the years to come.