Current view of liver cancer cell-of-origin and proposed mechanisms precluding its proper determination.

Hepatocellular carcinoma and intrahepatic cholangiocarcinoma are devastating primary liver cancers with increasing prevalence in many parts of the world. Despite intense investigation, many aspects of their biology are still largely obscure. For example, numerous studies have tackled the question of the cell-of-origin of primary liver cancers using different experimental approaches; they have not, however, provided a clear and undisputed answer. Here, we will review the evidence from animal models supporting the role of all major types of liver epithelial cells: hepatocytes, cholangiocytes, and their common progenitor as liver cancer cell-of-origin. Moreover, we will also propose mechanisms that promote liver cancer cell plasticity (dedifferentiation, transdifferentiation, and epithelial-to-mesenchymal transition) which may contribute to misinterpretation of the results and which make the issue of liver cancer cell-of-origin particularly complex.

What makes oncogenes mutually exclusive?

Cancer is driven by mutations in genes whose products participate in major signaling pathways that fuel cell proliferation and survival. It is easy to assume that the more of these so-called driver mutations a tumor accumulates, the faster it progresses. However, this does not appear to be the case: Data from large-scale genome sequencing studies indicate that mutations in driver oncogenes often are mutually exclusive. The mechanisms underlying the mutual exclusivity of oncogenes are not completely understood, but recent reports suggest that the mechanisms may depend on the tumor type, and the nature of interacting oncogenes. Here we discuss our recent findings that the oncogenes KRASG12D and BRAFV600E are mutually exclusive in lung cancer in mouse models because their coexpression leads to oncogene-induced senescence.

Targeting protease-activated receptor-1 with cell-penetrating pepducins in lung cancer.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer.

Overexpression of biliverdin reductase enhances resistance to chemotherapeutics.

Biliverdin reductase (BVR) converts biliverdin to bilirubin. Additionally, acting as a transcription factor and possessing a capacity of a serine/threonine kinase, it may modulate signaling pathways. In order to gain better understanding of BVR functions, we used genetically modified line of mouse fibroblasts with reversible overexpression of BVR. Current study revealed that enhanced activity of BVR may protect cells in stressful conditions arising from anti-cancer drugs, cisplatin and doxorubicin, the effect most probably related to PKC α/ß activity, as its inhibition reversed BVR action. Therefore activity of BVR may be of significance in tumors and may influence the effectiveness of therapies.

15d-PGJ2 upregulates synthesis of IL-8 in endothelial cells through induction of oxidative stress.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a cyclopentenone prostaglandin regarded as antiinflammatory mediator, which can act through peroxisome proliferator-activated receptor-gamma (PPARgamma) or through G protein-coupled surface receptors. It has been demonstrated that 15d-PGJ(2) potently increases the generation of interleukin-8 (IL-8) in human microvascular endothelial cells (HMEC-1s); however, the mechanism of this induction is not known. The aim of the study was to find the pathway involved in 15d-PGJ(2)-mediated IL-8 stimulation. Our data confirmed that the effect of 15d-PGJ(2) is independent of PPARgamma. For the first time, we excluded the activation of G proteins and the contribution of G protein-coupled surface receptors in endothelial cells treated with 15d-PGJ(2). Instead, we demonstrated that stimulation of IL-8 involved induction of oxidative stress, activation of p38 kinases, and increase in stability of IL-8 mRNA. Upregulation of IL-8 promoter, although measurable, seemed to play a less-pronounced role. Additionally, our results indicate the involvement of cAMP elevation and may suggest a role for ATF2 transcription factor. Concomitant induction of heme oxygenase-1 in HMEC-1s did not influence the synthesis of IL-8. In summary, we showed that 15d-PGJ(2), acting through oxidative stress, may exert proinflammatory effects. The upregulation of IL-8 is mostly associated with p38-mediated stabilization of mRNA.

Heme oxygenase-1 and the vascular bed: from molecular mechanisms to therapeutic opportunities.

Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.

Effect of heme and heme oxygenase-1 on vascular endothelial growth factor synthesis and angiogenic potency of human keratinocytes.

BACKGROUND: Skin injury leads to the release of heme, a potent prooxidant which is degraded by heme oxygenase-1 (HO-1) to carbon monoxide, iron, and biliverdin, subsequently reduced to bilirubin. Recently the involvement of HO-1 in angiogenesis has been shown; however, the role of heme and HO-1 in wound healing angiogenesis has not been yet investigated. RESULTS: Treatment of HaCaT keratinocytes with hemin (heme chloride) induced HO-1 expression and activity. The effect of heme on vascular endothelial growth factor (VEGF) synthesis is variable: induction is significant after a short, 6 h treatment with heme, while longer stimulation may attenuate its production. The involvement of HO-1 in VEGF synthesis was confirmed by inhibition of VEGF expression by SnPPIX, a blocker of HO activity and by attenuation of HO-1 mRNA expression with specific siRNA. Importantly, induction of HO-1 by hemin was able to overcome the inhibitory effect of high glucose on VEGF synthesis. Moreover, HO-1 expression was also induced in keratinocytes cultured in hypoxia, with concomitant augmentation of VEGF production, which was further potentiated by hemin stimulation. Accordingly, conditioned media from keratinocytes overexpressing HO-1 enhanced endothelial cell proliferation and augmented formation of capillaries in angiogenic assay in vitro. CONCLUSIONS: HO-1 is involved in hemin-induced VEGF expression in HaCaT and may play a role in hypoxic regulation of this protein. HO-1 overexpression may be beneficial in restoring the proper synthesis of VEGF disturbed in diabetic conditions.

Role of heme oxygenase-1 in hydrogen peroxide-induced VEGF synthesis: effect of HO-1 knockout.

Hydrogen peroxide is an important mediator of intracellular signaling, which potently enhances the expression of heme oxygenase-1 (HO-1) and upregulates synthesis of vascular endothelial growth factor (VEGF). The purpose of the present study was to explore the involvement of HO-1 in regulation of H(2)O(2)-mediated induction of VEGF synthesis. We provide genetic evidence that basal and H(2)O(2)-induced VEGF synthesis is partially dependent on HO-1. Inhibition of HO-1 activity by tin protoporphyrin (SnPPIX) resulted in downregulation of VEGF synthesis in murine fibroblasts and human keratinocytes. The relationship between HO-1 and VEGF was corroborated by using cells derived from HO-1 knockout mice, which demonstrated lower basal and H(2)O(2)-induced production of VEGF. Additionally, knock out of HO-1 gene impaired induction of VEGF by hemin, lysophosphatidylcholine, and prostaglandin-J(2). Our results provide confirmation for the involvement of HO-1 in regulation of angiogenesis.

Gene transfer of CuZn superoxide dismutase enhances the synthesis of vascular endothelial growth factor.

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H2O2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H2O2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1.

Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells.

OBJECTIVES: HMG-CoA reductase inhibitors (statins) can modulate the formation of new blood vessels, but the reports on their contribution to angiogenesis are contradictory. Therefore, we investigated whether the effect of statins is dependent either on the concentration of the drug or on the cell type. METHODS AND RESULTS: Under basal conditions human vascular smooth muscle cells (HVSMC) and microvascular endothelial cells (HMEC-1) constitutively generate and release vascular endothelial growth factor (VEGF). In contrast, primary macrovascular endothelial cells (HUVEC) produce minute amounts of VEGF. Different statins (atorvastatin, simvastatin and lovastatin, 1-10 micromol/l) significantly reduced basal and cytokine-, nitric oxide- or lysophosphatidylcholine (LPC)-induced VEGF synthesis in HMEC-1 and HVSMC. Interestingly, at the same concentrations statins upregulated VEGF generation in HUVEC. Furthermore, statins exerted dual, concentration-dependent influence on angiogenic activities of HUVEC as determined by tube formation assay. At low concentrations (0.03-1 micromol/l) the pro-angiogenic activity of statins is prevalent, whereas at higher concentrations statins inhibit angiogenesis, despite increasing VEGF synthesis. CONCLUSION: Our data show that statins exert concentration- and cell type-dependent effects on angiogenic activity of endothelial cells and on VEGF synthesis. The data are of relevance for elucidating the differential activity of statins on angiogenesis in cardiovascular diseases and cancer.

Prostaglandin-J(2) upregulates expression of matrix metalloproteinase-1 independently of activation of peroxisome proliferator-activated receptor-gamma.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-inducible nuclear receptor that functions as a transcription factor involved in lipid metabolism, inflammatory response and angiogenesis. The most potent endogenous PPARgamma activator is 15-deoxy-Delta(12,14)prostaglandin-J(2) (15d-PGJ(2)), whereas synthetic ligands include the oral antidiabetic drugs thiazolidinediones (TZDs). Activation of PPARgamma was reported to decrease the synthesis of matrix metalloproteinases (MMPs) in vascular smooth muscle cells and macrophages. We aimed to investigate the effect of PPARgamma ligands on expression of MMP-1 and urokinase plasminogen activator (uPA) in human microvascular endothelial cells (HMEC-1). We found that treatment of HMEC-1 with 15d-PGJ(2) increased the synthesis of MMP-1 protein up to 168% comparing to untreated cells. TZDs (ciglitazone and troglitazone), more potent activators of PPARgamma in HMEC-1, did not influence MMP-1 production, arguing against the involvement of PPARgamma in this process. Importantly, the stimulatory effect of 15d-PGJ(2) was reversed by the antioxidant N-acetyl-cysteine (NAC), suggesting a contribution of oxidative stress. We demonstrated also that 15d-PGJ(2) did not change the activity of MMP-1 promoter, but increased the stability of MMP-1 mRNA. In contrast, 15d-PGJ(2) very potently inhibited the synthesis of uPA. This effect was in part mimicked by ciglitazone and troglitazone implying an involvement of PPARgamma. Accordingly, NAC did not modify the inhibitory effect of 15d-PGJ(2) on uPA expression. In conclusion, we postulate that 15d-PGJ(2) may differently regulate the synthesis of proteases involved in angiogenesis: it upregulates MMP-1 expression in HMEC-1 through induction of oxidative stress, and inhibits uPA synthesis partly by activation of PPARgamma.

IL-1-mediated inhibition of IL-6-induced STAT3 activation is modulated by IL-4, MAP kinase inhibitors and redox state of HepG2 cells.

It is known that during acute phase reaction IL-6 activates STAT3 in hepatoma cells and IL-1 downregulates this response. We found that the inhibitory properties of IL-1 on STAT signalling cascade in human hepatoma HepG2 cells are considerably decreased not only in the presence of MAP kinase inhibitors SB203580 and PD98059 but also by some antioxidants (N-acetyl cysteine and pyrrolidine dithiocarbamate) and by anti-inflammatory cytokine IL-4. It appears that cytokine crosstalk between IL-6 and IL-1 includes a direct pathway sensitive to antioxidants and MAP kinase inhibitors, whereas the indirect prolonged response depends probably on synthesis of suppressors of cytokine signalling (SOCS).