The genomic landscape of hepatoblastoma and their progenies with HCC-like features.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most common childhood liver cancer and occasionally presents with histological and clinical features reminiscent of hepatocellular carcinoma (HCC). Identification of molecular mechanisms that drive the neoplastic continuation towards more aggressive HCC phenotypes may help to guide the new stage of targeted therapies. METHODS: We performed comprehensive studies on genetic and chromosomal alterations as well as candidate gene function and their clinical relevance. RESULTS: Whole-exome sequencing identified HB as a genetically very simple tumour (2.9 mutations per tumour) with recurrent mutations in ß-catenin (CTNNB1) (12/15 cases) and the transcription factor NFE2L2 (2/15 cases). Their HCC-like progenies share the common CTNNB1 mutation, but additionally exhibit a significantly increased mutation number and chromosomal instability due to deletions of the genome guardians RAD17 and TP53, accompanied by telomerase reverse-transcriptase (TERT) promoter mutations. Targeted genotyping of 33 primary tumours and cell lines revealed CTNNB1, NFE2L2, and TERT mutations in 72.5%, 9.8%, and 5.9% of cases, respectively. All NFE2L2 mutations affected residues of the NFE2L2 protein that are recognized by the KEAP1/CUL3 complex for proteasomal degradation. Consequently, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signalling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumours was significantly associated with metastasis, vascular invasion, the adverse prognostic C2 gene signature, as well as poor outcome. CONCLUSIONS: Our study demonstrates the importance of CTNNB1 mutations and NFE2L2-KEAP1 pathway activation in HB development and defines loss of genomic stability and TERT promoter mutations as prominent characteristics of aggressive HB with HCC features.

Epigallocatechin-3-gallate inhibits hepatoblastoma growth by reactivating the Wnt inhibitor SFRP1.

Activation of Wnt signaling plays a central role in the formation of hepatoblastoma (HB), the most common pediatric liver cancer. Blocking this pathway with specific inhibitors is currently the target of various research endeavours. This study provides evidence that the naturally occurring flavonoid epigallocatechin-3-gallate (EGCG) is highly effective against HB growth through inhibition of Wnt signaling. We demonstrate that EGCG has a strong cytotoxic effect on HB cells in a time- and dose-dependent manner by impinging on cell viability, while leaving normal fibroblasts unaffected. Apoptotic features, including morphological changes, caspase 3 activity, and proteolytic cleavage of poly(ADP-ribose) polymerase, were frequently found in EGCG-treated HB cells, thereby suggesting involvement of the mitochondrial intrinsic apoptotic pathway. We furthermore show that EGCG effectively inhibits Wnt signaling, as evidenced by down-regulation of Wnt-responsive reporter gene activity and expression of the Wnt target genes MYC and CCND1. Interestingly, EGCG induced reexpression of the tumor suppressor gene SFRP1, which is transcriptionally silenced in HB cells and known to down-regulate Wnt signaling. Considering the lack of toxic effects on normal cells, EGCG should be preclinically validated as an adjuvant therapy in vivo with the ultimate goal of determining its efficacy in human trials.

IGFBP3 impedes aggressive growth of pediatric liver cancer and is epigenetically silenced in vascular invasive and metastatic tumors.

BACKGROUND: Hepatoblastoma (HB) is an embryonal liver neoplasm of early childhood with a poor prognosis for patients with distant metastases and vascular invasion. We and others have previously shown that the overexpression of insulin-like growth factor 2 (IGF2), loss of imprinting at the IGF2/H19 locus, and amplification of pleomorphic adenoma gene 1 (PLAG1) are common features in HB, suggesting a critical role of the IGF axis in hepatoblastomagenesis. In this study, we investigated the role of the insulin-like growth factor binding protein 3 (IGFBP3), a known competitor of the IGF axis, in pediatric liver cancers. RESULTS: The IGFBP3 gene was highly expressed in normal pediatric livers but was heavily downregulated in four HB cell lines and the majority of HB primary tumors (26/36). Detailed methylation analysis of CpG sites in the IGFBP3 promoter region by bisulfite sequencing revealed a high degree of DNA methylation, which is causatively associated with the suppression of IGFBP3 in HB cell lines. Consequently, the treatment of HB cell lines with 5-aza-2'-deoxycytidine resulted in DNA demethylation and reactivation of the epigenetically silenced IGFBP3 expression. Interestingly, IGFBP3 promoter methylation predominantly occurred in metastatic HB with vascular invasion. Restoring IGFBP3 expression in HB cells resulted in reduced colony formation, migration, and invasion. CONCLUSION: This study provides the first direct evidence that the reactivation of IGFBP3 decreases aggressive properties of pediatric liver cancer cells and that IGFBP3 promoter methylation might be used as an indicator for vessel-invasive tumor growth in HB patients.

Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1).

Ca(2+) signaling includes store-operated Ca(2+) entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca(2+) stores. On store depletion, the ER Ca(2+) sensor STIM1 activates Orai1, the pore-forming unit of Ca(2+)-release-activated Ca(2+) (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, I(CRAC), and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active (S422D)SGK1 (by+81, +378, and+136%, respectively) but not by inactive (K127N)SGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by -48%) and I(CRAC) (by -60%), an effect reversed by expression of (S422D)SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1(-/-)) mice (by -37% and -52%, respectively) than in mast cells from wild-type (sgk1(+/+)) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase-inhibitor thapsigargin (2 µM) stimulated migration, an effect significantly higher (by +306%) in (S422D)SGK1-expressing than in (K127N)SGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1(+/+) than in sgk1(-/-) mast cells. SGK1 is thus a novel key player in the regulation of SOCE.

SGK1-dependent stimulation of intestinal SGLT1 activity by vitamin D.

The serum- and glucocorticoid-inducible kinase SGK1 has previously been shown to mediate the glucocorticoid-dependent stimulation of several intestinal transport systems including the electrogenic glucose transporter SGLT1. In squamous carcinoma cells, SGK1 expression is stimulated by 1,25(OH)2D3, the biologically active metabolite of vitamin D. The present study explored whether vitamin D influences the intestinal SGLT1 activity. Jejunal SGLT1 activity was determined by Ussing chamber experiments. Under a normal diet, the electrogenic glucose transport was similar in SGK1 knockout (sgk1 ( -/- )) and wild type mice (sgk1 ( +/+ )). Following a vitamin D-rich diet (14 days 10,000 I.U. vitamin D), the SGK1 transcript levels as well as the SGLT1 protein abundance were increased in sgk1(+/+) mice. Moreover, SGLT1 activity was increased in sgk1(+/+) mice but not in sgk1(-/-) mice following a vitamin D-rich diet. Furthermore, an oral glucose load was followed by an increase in the plasma glucose concentration to significantly higher values in sgk1(+/+) mice treated with a vitamin D-rich diet than in untreated sgk1(+/+) mice. In conclusion, vitamin D treatment upregulates the expression of SGK1, which in turn enhances SGLT1 activity.

Hypothyroidism of gene-targeted mice lacking Kcnq1.

Thyroid hormones T3/T4 participate in the fine tuning of development and performance. The formation of thyroid hormones requires the accumulation of I(-) by the electrogenic Na(+)/I(-) symporter, which depends on the electrochemical gradient across the cell membrane and thus on K(+) channel activity. The present paper explored whether Kcnq1, a widely expressed voltage-gated K(+) channel, participates in the regulation of thyroid function. To this end, Kcnq1 expression was determined by RT-PCR, confocal microscopy, and thyroid function analyzed in Kcnq1 deficient mice (Kcnq1 ( -/- )) and their wild-type littermates (Kcnq1 ( +/+ )). Moreover, Kcnq1 abundance and current were determined in the thyroid FRTL-5 cell line. Furthermore, mRNA encoding KCNQ1 and the subunits KCNE1-5 were discovered in human thyroid tissue. According to patch-clamp TSH (10 mUnits/ml) induced a voltage-gated K(+) current in FRTL-5 cells, which was inhibited by the Kcnq inhibitor chromanol (10 µM). Despite a tendency of TSH plasma concentrations to be higher in Kcnq1 ( -/- ) than in Kcnq1 ( +/+ ) mice, the T3 and T4 plasma concentrations were significantly smaller in Kcnq1 ( -/- ) than in Kcnq1 ( +/+ ) mice. Moreover, body temperature was significantly lower in Kcnq1 ( -/- ) than in Kcnq1 ( +/+ ) mice. In conclusion, Kcnq1 is required for proper function of thyroid glands.

Downregulation of Klotho expression by dehydration.

Klotho, a transmembrane protein, protease, and hormone mainly expressed in renal tissue counteracts aging. Overexpression of Klotho substantially prolongs the life span. Klotho deficiency leads to excessive formation of 1,25(OH)(2)D(3), growth deficit, accelerated aging, and early death. Aging is frequently paralleled by dehydration, which is considered to accelerate the development of age-related disorders. The present study explored the possibility that dehydration influences Klotho expression. Klotho transcript levels were determined by RT-PCR, and Klotho protein abundance was detected by Western blotting in renal tissue from hydrated and 36-h-dehydrated mice as well as in human embryonic kidney (HEK293) cells. Dehydration was followed by a significant decline of renal Klotho transcript levels and protein abundance, accompanied by an increase in plasma osmolarity as well as plasma ADH, aldosterone, and 1,25(OH)(2)D(3) levels. Antidiuretic hormone (ADH; 50 nM) and aldosterone (1 µM) significantly decreased Klotho transcription and protein expression in HEK293 cells. In conclusion, the present observations disclose a powerful effect of dehydration on Klotho expression, an effect at least partially mediated by enhanced release of ADH and aldosterone.

Influence of dexamethasone on na+/h+ exchanger activity in dendritic cells.

Glucocorticoids regulate the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. Glucocorticoids influence the function of other cell types by modulating the activity of the Na(+)/H(+)exchanger (NHE), a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether dexamethasone influences Na(+)/H(+) exchanger activity in DCs. The DCs were isolated from mouse bone marrow, cell volume was estimated from forward scatter in FACS analysis, cytosolic pH (pH(i)) utilizing BCECF fluorescence and Na(+)/H(+) exchanger activity from the Na(+) dependent realkalinization after an ammonium pulse. Treatment with the glucocorticoid dexamethasone (100 nM; 1, 4, 16 and 24h) significantly decreased pH(i) (≥4 h) and gradually increased Na(+)/H(+) exchanger activity (=16 h). The stimulation of Na(+)/H(+) exchanger activity by dexamethasone was virtually abrogated by glucocorticoid receptor blocker mefiprestone (1 µM) and NHE3 inhibitor dimethyl amiloride (5 µM), but not prevented by NHE1 inhibitor cariporide (10 µM). Dexamethasone treatment significantly increased SGK1 mRNA levels. Stimulation of Na(+)/H(+) exchanger activity by dexamethasone was blunted in DCs lacking SGK1. Dexamethasone treatment did not significantly alter ROS formation but significantly decreased the forward scatter. Exposure of DCs to lipopolysacharide (LPS, 1 µg/ml) led to a transient increase followed by a decline of Na(+)/H(+) exchanger activity and to enhanced forward scatter as well as ROS formation, all effects significantly blunted in the presence of dexamethasone (100 nM). In conclusion, glucocorticoid treatment decreased pH(i) and cell volume, effects paralleled by upregulation of Na(+)/H(+) exchanger activity in DCs. Moreover, glucocorticoids blunted the stimulation of Na(+)/H(+) exchanger activity, cell swelling and ROS formation following LPS treatment.

Ca2+ activated K+ channel Kca3.1 as a determinant of gastric acid secretion.

The Ca(2+) activated K(+) channel K(ca)3.1 is expressed in a variety of tissues. In the gastric gland it is expressed in the basolateral cell membrane. To determine the functional significance of K(ca)3.1 activity for gastric acid secretion, gastric acid secretion was determined in isolated glands from gene targeted mice lacking functional K(ca)3.1 (K(ca)3.1(-/-)) and from their wild type littermates (K(ca)3.1(+/+)). According to BCECF-fluorescence cytosolic pH in isolated gastric glands was similar in K(ca)3.1(-/-) and K(ca)3.1(+/+) mice. Na(ca)-independent pH recovery (ΔpH/min) following an ammonium pulse, a measure of H(ca)/K(ca) ATPase activity, was, however, significantly faster in K(ca)3.1(-/-) than in K(ca)3.1(+/+) mice. Accordingly, the luminal pH was significantly lower and the acid content significantly higher in K(ca)3.1(-/-) than in K(ca)3.1(+/+) mice. The abundance of mRNA encoding H(ca)/K(ca) ATPase and KCNQ1 was similar in both genotypes. Increase of extracellular K(ca) concentrations to 35 mM (replacing Na(ca)/NMDG) and treatment with histamine (100 µM) significantly increased ΔpH/min to a larger extent in K(ca)3.1(+/+) than in K(ca)3.1(-/-) mice and dissipated the differences between the genotypes. Carbachol (100 µM) increased ΔpH/min in both genotypes but did not abolish the difference between K(ca)3.1(-/-) and K(ca)3.1(+/+) mice. In K(ca)3.1(+/+) mice the K(ca)3.1 opener DCEBIO (100 µM) did not significantly alter basal ΔpH/min but significantly blunted ΔpH/min in the presence of carbachol. In conclusion, K(ca)3.1 activity suppresses carbachol stimulated gastric acid secretion.

Downregulation of NaPi-IIa and NaPi-IIb Na-coupled phosphate transporters by coexpression of Klotho.

Klotho, a transmembrane protein, protease and hormone has been shown to exert a profound effect on phosphate metabolism. Klotho overexpression lowers and Klotho deficiency increases the plasma phosphate concentration, effects in part attributed to an inhibitory effect of Klotho on the formation of 1,25-dihydroxycholecalciferol (1,25(OH) (2)D(3)), the active form of Vitamin D. Beyond that Klotho has been shown to decrease renal tubular phosphate transport more directly. The influence of Klotho on the plasma phosphate concentration contributes to the profound effect of Klotho on ageing and life span. The present study explored whether Klotho influences the major renal tubular (NaPi-IIa) and the major intestinal (NaPi-IIb) phosphate transporters. For functional analysis NaPi-IIa or NaPi-IIb were expressed in Xenopus oocytes both, without or with additional coexpression of Klotho and electrogenic phosphate transport was estimated from the phosphate-induced current (Ip). According to RT-PCR Klotho is expressed in the murine kidney and intestine. Coexpression of Klotho decreased Ip in both NaPi-IIa- and NaPi-IIb-expressing oocytes. Klotho decreased the maximal Ip without appreciably affecting the concentration required for halfmaximal Ip. Treatment of NaPi-IIa- or NaPi-IIb-expressing oocytes with Klotho protein similarly decreased Ip. In conclusion, Klotho down regulates both, renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters.

Hydration-sensitive gene expression in brain.

Dehydration has a profound influence on neuroexcitability. The mechanisms remained, however, incompletely understood. The present study addressed the effect of water deprivation on gene expression in the brain. To this end, animals were exposed to a 24 hours deprivation of drinking water and neuronal gene expression was determined by microarray technology with subsequent confirmation by RT-PCR. As a result, water deprivation was followed by significant upregulation of clathrin (light polypeptide Lcb), serum/glucocorticoid-regulated kinase (SGK) 1, and protein kinase A (PRKA) anchor protein 8-like. Water deprivation led to downregulation of janus kinase and microtubule interacting protein 1, neuronal PAS domain protein 4, thrombomodulin, purinergic receptor P2Y - G-protein coupled 13 gene, gap junction protein beta 1, neurotrophin 3, hyaluronan and proteoglycan link protein 1, G protein-coupled receptor 19, CD93 antigen, forkhead box P1, suppressor of cytokine signaling 3, apelin, immunity-related GTPase family M, serine (or cysteine) peptidase inhibitor clade B member 1a, serine (or cysteine) peptidase inhibitor clade H member 1, glutathion peroxidase 8 (putative), discs large (Drosophila) homolog-associated protein 1, zinc finger and BTB domain containing 3, and H2A histone family member V. Western blotting revealed the downregulation of forkhead box P1, serine (or cysteine) peptidase inhibitor clade H member 1, and gap junction protein beta 1 protein abundance paralleling the respective alterations of transcript levels. In conclusion, water deprivation influences the transcription of a wide variety of genes in the brain, which may participate in the orchestration of brain responses to water deprivation.

Phosphoinositide 3-kinase-dependent regulation of Na+/H+ exchanger in dendritic cells.

Dendritic cells (DCs), antigen-presenting cells that are able to initiate primary immune responses and to establish immunological memory, are activated by exposure to bacterial lipopolysaccharides (LPS), which leads to cell swelling, triggering ROS formation and stimulating migration. The function of DCs is regulated by the phosphoinositide 3 (PI3) kinase pathway. On the other hand, PI3 kinase is an important regulator of diverse transporters including the Na(+)/H(+) exchanger (NHE). The present study was performed to elucidate the role of PI3 kinase in NHE activity, cell volume, ROS formation, and migration. To this end, DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization after an ammonium pulse, cell volume from forward scatter in fluorescence-activated cell sorter analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate fluorescence, and migration utilizing transwell migration assays. Exposure of DCs to LPS led within 4 h to a gradual cytosolic acidification paralleled by a transient time- and dose-dependent increase of Na(+)/H(+) exchanger activity, cell swelling, enhanced ROS production, and stimulation of migration. The PI3K inhibitors Wortmannin (1 µM) or LY294002 (10 µM) significantly blunted the effects of LPS on NHE activity, cell volume, ROS production, and migration. The present observations disclose a critical role of PI3K signaling in the regulation of DC function following exposure to LPS.

Effect of bacterial lipopolysaccharide on Na(+)/H(+) exchanger activity in dendritic cells.

The function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, is stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS). In macrophages, ROS formation is paralleled by activation of the Na(+)/H(+) exchanger, a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether LPS influence Na(+)/H(+) exchanger activity in DCs. The DCs were isolated from murine bone marrow, cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, apoptosis from annexin V binding, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Na(+)/H(+) exchanger activity from the Na(+) dependent realkalinization following an ammonium pulse. Exposure of DCs to LPS (1 µg/ml) led to a transient increase of Na(+)/H(+) exchanger activity. Moreover, LPS increased forward scatter and ROS formation and decreased apoptosis. The NHE1 inhibitor cariporide (10 µM) virtually abrogated Na(+)/H(+) exchanger activity, inhibited LPS-induced cell swelling, blunted LPS-induced ROS formation and reversed the antiapoptotic effect of LPS. Na(+)/H(+) exchanger activity was stimulated by oxidative stress and LPS induced stimulation of NHE activity was abolished in presence of ROS chelators (Tempol, Tiron and Vitamin C). In conclusion, LPS treatment transiently upregulates the Na(+)/H(+) exchanger in DCs, an effect required for the effects of LPS on DC survival, cell volume and ROS formation.

Phosphoinositide-dependent kinase PDK1 in the regulation of Ca2+ entry into mast cells.

The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKCδ. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKCδ inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 µM) increased the K(+) channel conductance to similar values in both genotypes. ß-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKCδ inhibitor rottlerin increased ß-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKCδ and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.

Regulation of gastric acid secretion by PKB/Akt2.

Pharmacological inhibition of phosphoinositol 3 kinase (PI3K) and partial deficiency of phosphoinositide dependent kinase PDK1 have previously been shown to enhance basal gastric acid secretion. PI3K/PDK1 dependent signaling involves activation of protein kinase B/Akt, which may thus be similarly involved in the regulation of gastric acid secretion. To test that hypothesis, gastric acid secretion was determined in isolated glands from gene targeted mice lacking functional Akt2 (akt2(-/-)) or from their wild type littermates (akt2(+/+)). According to BCECF-fluorescence cytosolic pH in isolated gastric glands was similar in akt2(-/-) and akt2(+/+) mice. Na(+)-independent pH recovery (DeltapH/min) following an ammonium pulse, a measure of H(+)/K(+) ATPase activity, was, however, significantly faster in akt2(-/-) than in akt2(+/+) mice. In both genotypes, DeltapH/min was virtually abolished by H(+)/K(+) ATPase inhibitor omeprazole (100 muM). Increase of extracellular K(+) concentrations to 35 mM (replacing Na(+)) increased DeltapH/min to a significantly larger extent in akt2(+/+) than in akt2(-/-) mice and dissipated the differences between the genotypes. Similarly, treatment with 5 muM forskolin enhanced DeltapH/min significantly only in akt2(+/+) mice and abolished the differences between the genotypes. Conversely, protein kinase A inhibitor H89 (50 nM) decreased DeltapH/min to similarly low values in both genotypes. In conclusion, Akt2 suppresses gastric acid secretion and contributes to or even accounts for the inhibition of gastric acid secretion by PI3K.

Upregulation of Na+/H+ exchanger by the AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O(2) deficiency glycolytic degradation of glucose leads to formation of lactate and H(+), thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H(+) export. The present study explored whether AMPK influences Na(+)/H(+) exchanger (NHE) activity and/or Na(+)-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH(i)) was estimated utilizing BCECF fluorescence and Na(+)/H(+) exchanger activity from the Na(+)-dependent re-alkalinization (DeltapH(i)) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH(i) and DeltapH(i) in those cells were significantly increased by treatment with AMPK stimulator AICAR (1mM) and significantly decreased by AMPK inhibitor compound C (10 microM). The effect of AICAR on pH(i) and DeltapH(i) was blunted in the presence of the Na(+)/H(+) exchanger inhibitor cariporide (10microM), but not by the H(+) ATPase inhibitor bafilomycin (10nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.

Blocking the hedgehog pathway inhibits hepatoblastoma growth.

UNLABELLED: Recent evidence has indicated that Hedgehog (Hh) signaling significantly contributes to liver development and regeneration and that activation of the pathway may contribute to growth of hepatocellular carcinoma (HCC) in adults. However, the role of Hh signaling in pediatric liver tumors remains to be elucidated. In this study, we show that Hh signaling is activated in hepatoblastoma (HB), the most common liver tumor in childhood, with most occurrences before the age of 3 years. The Hh target genes glioma-associated oncogene homolog 1 (GLI1) and Patched (PTCH1) showed increased transcript levels in 65% and 30% of HB samples, respectively, compared with normal liver tissues. Most interestingly, the gene encoding the hedgehog interacting protein (HHIP) is transcriptionally silenced by cytosine-phospho-guanosine (CpG) island promoter hypermethylation in 26% of HB cases and treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine partially restored HHIP expression. Blocking Hh signaling with the antagonist cyclopamine had a strong inhibitory effect on cell proliferation of HB cell lines with an activated pathway. We further demonstrate that this decrease in cell viability is caused by a massive induction of apoptosis, as shown by morphological changes and phosphatidylserine membrane asymmetry. In cyclopamine-exposed HB cells, caspase 3 and poly(adenosine diphosphate-ribose) polymerase proteins were specifically activated by their proteolytic cleavage. CONCLUSION: This study demonstrates, for the first time, the frequent occurrence of GLI1 and PTCH1 overexpression and HHIP promoter methylation in early childhood HB, thus indicating a key role for Hh signaling activation in the malignant transformation of embryonal liver cells.

Downregulation of SFRP1 is a protumorigenic event in hepatoblastoma and correlates with beta-catenin mutations.

BACKGROUND: Hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are the most common malignant liver tumors in childhood. Both tumor types exhibit genetic and epigenetic alterations in the WNT/ß-catenin signaling pathway, which is a key regulator of liver progenitor cells in embryonic development. The tumors demonstrate a high rate of ß-catenin mutations and gene expression changes of several WNT antagonists. However, the role of the WNT inhibitory factor secreted frizzled-related protein 1 (SFRP1) has not been addressed in pediatric liver cancer so far. RESULTS: In our study, we investigated the gene expression level, DNA methylation status and functional relevance of SFRP1 in HB cell lines and in pediatric liver tumor patient samples. SFRP1 was downregulated due to DNA promoter methylation in all tested HB cell lines. Overexpression of SFRP1 in HB cell lines diminished tumor cell proliferation, colony formation and migration potential. In addition, the SFRP1-expressing HB cell lines showed reduced WNT/ß-catenin signaling pathway activity and decreased expression of WNT target genes. To evaluate the utility of SFRP1 as a biomarker in pediatric liver cancer, we determined the gene expression level and DNA methylation status of SFRP1 in 45 pediatric liver tumor patient samples. The correlation analysis of different clinical parameters and tumor characteristics revealed a significant correlation of reduced SFRP1 expression with the presence of mutant ß-catenin. The methylation status of SFRP1 was furthermore associated to a pediatric liver tumor type with HCC-like characteristics, TERT mutations and an older age at diagnosis. CONCLUSION: Altogether, our data demonstrate that the epigenetic suppression of the WNT/ß-catenin antagonist SFRP1 has an important impact on the malignant behavior of HB cells. Although SFRP1 methylation is a common event in HCC-like pediatric liver tumors, its potential as a prognostic or diagnostic biomarker needs to be further investigated.

HIV entry inhibition by the envelope 2 glycoprotein of GB virus C.

Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.

Hedgehog-independent overexpression of transforming growth factor-beta1 in rhabdomyosarcoma of Patched1 mutant mice.

The tumor suppressor gene PATCHED1 (PTCH1) is a member of the hedgehog signaling pathway and causatively associated with several human sporadic and familial cancers, including those of the skin, muscle and brain. Inactivation of one Ptch1 allele in the mouse results in the development of medulloblastoma and rhabdomyosarcoma (RMS), the latter being a malignant tumor of skeletal muscle origin. To identify genes involved in the pathogenesis of Ptch1-associated RMS, we have monitored the expression of 588 genes in RMS and normal skeletal muscle (SM) of heterozygous Ptch1neo67/+ mice using cDNA array technology. RMS displayed increased transcript levels of several genes such as transforming growth factor-beta1 (Tgfb1), insulin-like growth factor 2 (Igf2), villin 2 (Vil2), integrin beta1 (Itgb1), Sloan-Kettering viral oncogene homolog (Ski), and insulin-like growth factor binding protein 3 (Igfbp3), as well as numerous genes coding for structural components of myogenic cells such as myosin light polypeptide 4 (Myl4), myosin light polypeptide 6 (Myl6), and vimentin (Vim). Detailed promoter analysis revealed a putative Gli binding site in the second promoter region (P2) of the murine Tgfb1 gene. However, using reporter assay we show that the P2 promoter is not responsive to hedgehog signaling. We furthermore describe that Tgfb1 expression could not be activated in C2C12 myoblasts in the presence of murine Shh-N peptide and that Tgfb1 is equally expressed in both wild-type and Ptch1-deficient mouse embryos. In line with this, TGFB1 was strongly expressed in human RMS cell lines independently of the GLI1 expression status. In summary, our results suggest that aberrant expression of Tgfb1 may be involved in RMS development in a way that is independent of hedgehog signaling.