VC-resist glioblastoma cell state: vessel co-option as a key driver of chemoradiation resistance.

Glioblastoma (GBM) is a highly lethal type of cancer. GBM recurrence following chemoradiation is typically attributed to the regrowth of invasive and resistant cells. Therefore, there is a pressing need to gain a deeper understanding of the mechanisms underlying GBM resistance to chemoradiation and its ability to infiltrate. Using a combination of transcriptomic, proteomic, and phosphoproteomic analyses, longitudinal imaging, organotypic cultures, functional assays, animal studies, and clinical data analyses, we demonstrate that chemoradiation and brain vasculature induce cell transition to a functional state named VC-Resist (vessel co-opting and resistant cell state). This cell state is midway along the transcriptomic axis between proneural and mesenchymal GBM cells and is closer to the AC/MES1-like state. VC-Resist GBM cells are highly vessel co-opting, allowing significant infiltration into the surrounding brain tissue and homing to the perivascular niche, which in turn induces even more VC-Resist transition. The molecular and functional characteristics of this FGFR1-YAP1-dependent GBM cell state, including resistance to DNA damage, enrichment in the G2M phase, and induction of senescence/stemness pathways, contribute to its enhanced resistance to chemoradiation. These findings demonstrate how vessel co-option, perivascular niche, and GBM cell plasticity jointly drive resistance to therapy during GBM recurrence.

The P2X4 purinergic receptor impacts liver regeneration after partial hepatectomy in mice through the regulation of biliary homeostasis.

UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH), to initiate growth, protect liver cells, and sustain remnant liver functions. Extracellular adenosine triphosphate rises in blood and bile after PH and contributes to liver regeneration, although purinergic receptors and mechanisms remain to be precisely explored. In this work we analyzed during regeneration after PH the involvement of P2X4 purinergic receptors, highly expressed in the liver. P2X4 receptor expression in the liver, liver histology, hepatocyte proliferation, plasma bile acid concentration, bile flow and composition, and lysosome distribution in hepatocytes were studied in wild-type and P2X4 knockout (KO) mice, before and after PH. P2X4 receptors were expressed in hepatocytes and Kupffer cells; in hepatocytes, P2X4 was concentrated in subcanalicular areas closely costained with lysosomal markers. After PH, delayed regeneration, hepatocyte necrosis, and cholestasis were observed in P2X4-KO mice. In P2X4-KO mice, post-PH biliary adaptation was impaired with a smaller increase in bile flow and HCO3 (-) biliary output, as well as altered biliary composition with reduced adenosine triphosphate and lysosomal enzyme release. In line with these data, lysosome distribution and biogenesis were altered in P2X4-KO compared with wild-type mice. CONCLUSION: During liver regeneration after PH, P2X4 contributes to the complex control of biliary homeostasis through mechanisms involving pericanalicular lysosomes, with a resulting impact on hepatocyte protection and proliferation. (Hepatology 2016;64:941-953).

The four and a half LIM-only protein 2 regulates liver homeostasis and contributes to carcinogenesis.

BACKGROUND & AIMS: The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2). METHODS: We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice. RESULTS: Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/ß-catenin signaling in Apc(lox/lox) animals. CONCLUSIONS: Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.

Cytosolic calcium regulates liver regeneration in the rat.

UNLABELLED: Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES-expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES-expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells. CONCLUSION: Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle.

ATP release after partial hepatectomy regulates liver regeneration in the rat.

BACKGROUND & AIMS: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study. METHODS: Hepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic acid), PPADS), and liver regeneration after hepatectomy was analyzed. RESULTS: A robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction. CONCLUSION: Extracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.

The CB(2) cannabinoid receptor controls myeloid progenitor trafficking: involvement in the pathogenesis of an animal model of multiple sclerosis.

Cannabinoids are potential agents for the development of therapeutic strategies against multiple sclerosis. Here we analyzed the role of the peripheral CB(2) cannabinoid receptor in the control of myeloid progenitor cell trafficking toward the inflamed spinal cord and their contribution to microglial activation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE). CB(2) receptor knock-out mice showed an exacerbated clinical score of the disease when compared with their wild-type littermates, and this occurred in concert with extended axonal loss, T-lymphocyte (CD4(+)) infiltration, and microglial (CD11b(+)) activation. Immature bone marrow-derived CD34(+) myeloid progenitor cells, which play a role in neuroinflammatory pathologies, were shown to express CB(2) receptors and to be abundantly recruited toward the spinal cords of CB(2) knock-out EAE mice. Bone marrow-derived cell transfer experiments further evidenced the increased contribution of these cells to microglial replenishment in the spinal cords of CB(2)-deficient animals. In line with these observations, selective pharmacological CB(2) activation markedly reduced EAE symptoms, axonal loss, and microglial activation. CB(2) receptor manipulation altered the expression pattern of different chemokines (CCL2, CCL3, CCL5) and their receptors (CCR1, CCR2), thus providing a mechanistic explanation for its role in myeloid progenitor recruitment during neuroinflammation. These findings demonstrate the protective role of CB(2) receptors in EAE pathology; provide evidence for a new site of CB(2) receptor action, namely the targeting of myeloid progenitor trafficking and its contribution to microglial activation; and support the potential use of non-psychoactive CB(2) agonists in therapeutic strategies for multiple sclerosis and other neuroinflammatory disorders.

The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK1 and 2). We previously showed that S1P receptors (S1P1, S1P2, and S1P3) are expressed in hepatic myofibroblasts (hMF), a population of cells that triggers matrix remodeling during liver injury. Here we investigated the function of these receptors in the wound healing response to acute liver injury elicited by carbon tetrachloride, a process that associates hepatocyte proliferation and matrix remodeling. Acute liver injury was associated with the induction of S1P2, S1P3, SphK1, and SphK2 mRNAs and increased SphK activity, with no change in S1P1 expression. Necrosis, inflammation, and hepatocyte regeneration were similar in S1P2-/- and wild-type (WT) mice. However, compared with WT mice, S1P2-/- mice displayed reduced accumulation of hMF, as shown by lower induction of smooth muscle alpha-actin mRNA and lower induction of TIMP-1, TGF-beta1, and PDGF-BB mRNAs, overall reflecting reduced activation of remodeling in response to liver injury. The wound healing response was similar in S1P3-/- and WT mice. In vitro, S1P enhanced proliferation of cultured WT hMF, and PDGF-BB further enhanced the mitogenic effect of S1P. In keeping with these findings, PDGF-BB up-regulated S1P2 and SphK1 mRNAs, increased SphK activity, and S1P2 induced PDGF-BB mRNA. These effects were blunted in S1P2-/- cells, and S1P2-/- hMF exhibited reduced mitogenic and comitogenic responses to S1P. These results unravel a novel major role of S1P2 in the wound healing response to acute liver injury by a mechanism involving enhanced proliferation of hMF.

Cannabinoids induce glioma stem-like cell differentiation and inhibit gliomagenesis.

Glioma stem-like cells constitute one of the potential origins of gliomas, and therefore, their elimination is an essential factor for the development of efficient therapeutic strategies. Cannabinoids are known to exert an antitumoral action on gliomas that relies on at least two mechanisms: induction of apoptosis of transformed cells and inhibition of tumor angiogenesis. However, whether cannabinoids target human glioma stem cells and their potential impact in gliomagenesis are unknown. Here, we show that glioma stem-like cells derived from glioblastoma multiforme biopsies and the glioma cell lines U87MG and U373MG express cannabinoid type 1 (CB(1)) and type 2 (CB(2)) receptors and other elements of the endocannabinoid system. In gene array experiments, CB receptor activation altered the expression of genes involved in the regulation of stem cell proliferation and differentiation. The cannabinoid agonists HU-210 and JWH-133 promoted glial differentiation in a CB receptor-dependent manner as shown by the increased number of S-100beta- and glial fibrillary acidic protein-expressing cells. In parallel, cannabinoids decreased the cell population expressing the neuroepithelial progenitor marker nestin. Moreover, cannabinoid challenge decreased the efficiency of glioma stem-like cells to initiate glioma formation in vivo, a finding that correlated with decreased neurosphere formation and cell proliferation in secondary xenografts. Gliomas derived from cannabinoid-treated cancer stem-like cells were characterized with a panel of neural markers and evidenced a more differentiated phenotype and a concomitant decrease in nestin expression. Overall, our results demonstrate that cannabinoids target glioma stem-like cells, promote their differentiation, and inhibit gliomagenesis, thus giving further support to their potential use in the management of malignant gliomas.

Daily cannabis smoking as a risk factor for progression of fibrosis in chronic hepatitis C.

Cannabinoids present in Cannabis sativa (marijuana) exert biological effects via cannabinoid receptors CB1 and CB2. We recently demonstrated that CB1 and CB2 receptors regulate progression of experimental liver fibrosis. We therefore investigated the impact of cannabis smoking on fibrosis progression rate in patients with chronic hepatitis C (CHC). Two hundred seventy consecutive untreated patients with CHC of known duration undergoing liver biopsy were studied. Demographic, epidemiological, metabolic, and virological data were recorded, and detailed histories of cannabis, alcohol, and tobacco use over the span of hepatitis C virus infection were obtained. Fibrosis stage, steatosis, and activity grades were scored according to Metavir system. Patients were categorized as noncannabis users (52.2%), occasional users (14.8%), or daily users (33.0%), and the relationship between cannabis use and fibrosis progression rate (FPR) or fibrosis stage was assessed. On multivariate analysis, six factors were independently related to a FPR greater than 0.074 (median value of the cohort): daily cannabis use (OR = 3.4 [1.5-7.4]), Metavir activity grade A2 or higher (OR = 5.4 [2.9-10.3]), age at contamination of more than 40 years (OR = 10.5 [3.0-37.1]), genotype 3 (OR = 3.4 [1.5-7.7]), excessive alcohol intake (OR = 2.2 [1.1-4.5]), and steatosis (OR = 2.0 [1.0-4.1]). Daily cannabis use was also an independent predictor of a rapid FPR (>0.15) (OR = 3.6 [1.5-7.5]). Finally, severe fibrosis (> or =F3) was also predicted by daily cannabis use (OR = 2.5 [1.1-5.6]; P = .034), independently of Metavir activity grade, excessive alcohol intake, age at liver biopsy, steatosis, and tobacco smoking. In conclusion, daily cannabis smoking is significantly associated with fibrosis progression during CHC. Patients with ongoing CHC should be advised to refrain from regular cannabis use.

Antifibrogenic role of the cannabinoid receptor CB2 in the liver.

BACKGROUND & AIMS: Hepatic myofibroblasts are central for the development of liver fibrosis associated with chronic liver diseases, and blocking their accumulation may prevent fibrogenesis. Cannabinoids are the active components of marijuana and act via 2 G-protein-coupled receptors, CB1 and CB2. Here, we investigated whether liver fibrogenic cells are a target of cannabinoids. METHODS: CB2 receptors were characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultures of hepatic stellate cells and hepatic myofibroblasts by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and GTPgammaS assays. Functional studies were performed in cultured hepatic myofibroblasts and activated hepatic stellate cells. Carbon tetrachloride-induced liver fibrosis was studied in mice invalidated for CB2 receptors. RESULTS: In liver biopsy specimens from patients with active cirrhosis of various etiologies, CB2 receptors were expressed in nonparenchymal cells located within and at the edge of fibrous septa in smooth muscle alpha-actin-positive cells. In contrast, CB2 receptors were not detected in normal human liver. CB2 receptors were also detected in cultured hepatic myofibroblasts and in activated hepatic stellate cells. Their activation triggered potent antifibrogenic effects, namely, growth inhibition and apoptosis. Growth inhibition involved cyclooxygenase-2, and apoptosis resulted from oxidative stress. Finally, mice invalidated for CB2 receptors developed enhanced liver fibrosis following chronic carbon tetrachloride treatment as compared with wild-type mice. CONCLUSIONS: These data constitute the first demonstration that CB2 receptors are highly up-regulated in the cirrhotic liver, predominantly in hepatic fibrogenic cells. Moreover, this study also highlights the antifibrogenic role of CB2 receptors during chronic liver injury.

Hepatic fibrosis: molecular mechanisms and drug targets.

Liver fibrosis is the common response to chronic liver injury, ultimately leading to cirrhosis and its complications, portal hypertension, liver failure, and hepatocellular carcinoma. Efficient and well-tolerated antifibrotic drugs are currently lacking, and current treatment of hepatic fibrosis is limited to withdrawal of the noxious agent. Efforts over the past decade have mainly focused on fibrogenic cells generating the scarring response, although promising data on inhibition of parenchymal injury and/or reduction of liver inflammation have also been obtained. A large number of approaches have been validated in culture studies and in animal models, and several clinical trials are underway or anticipated for a growing number of molecules. This review highlights recent advances in the molecular mechanisms of liver fibrosis and discusses mechanistically based strategies that have recently emerged.

Molecular mechanisms regulating the antifibrogenic protein heme-oxygenase-1 in human hepatic myofibroblasts.

BACKGROUND/AIMS: Hepatic myofibroblasts are central in liver fibrogenesis associated with chronic liver diseases. We previously showed that heme-oxygenase-1 (HO-1) displays antifibrogenic properties in human hepatic myofibroblasts. Here, we further investigated the mechanisms regulating HO-1 expression. METHODS: Expression of HO-1 was assayed in cultured human hepatic myofibroblasts by Northern and Western blot. Functional studies were also performed in cultured human hepatic myofibroblasts. RESULTS: 15-Deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) elicited inhibition of proliferation and of alpha1(I) collagen mRNA expression. These effects were reproduced by the glutathione depletor diethyl maleate and blunted by the glutathione precursor N-acetyl cysteine, indicating the involvement of oxidative stress. Two consecutive events mediated inhibition of proliferation and of alpha1(I) collagen mRNA expression by 15-d-PGJ2: (i) mild oxidative stress characterized by a transient GSH decrease and (ii) activation of p38 MAPK, resulting in increased HO-1 mRNA stability. CONCLUSIONS: Our results provide new insights into the regulatory mechanisms governing HO-1 expression in human hepatic myofibroblasts and identify mild oxidative stress and p38 MAPK as two consecutive early signals promoting HO-1 induction that are crucial for its antifibrogenic properties, namely inhibition of growth and extracellular matrix gene expression.

Heme oxygenase-1 is an antifibrogenic protein in human hepatic myofibroblasts.

BACKGROUND & AIMS: Hepatic myofibroblasts play a key role in the development of liver fibrosis associated with chronic liver diseases. We have shown that oxidative stress is a messenger of 15-deoxy-delta-12,14-prostaglandin J2 (15-d-PGJ2) in human hepatic myofibroblasts. The aim of the present study was to investigate the role of a stress-inducible protein, heme oxygenase-1 (HO-1), in the action of 15-d-PGJ2. METHODS: Expression of HO-1 was characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultured human hepatic myofibroblasts by Northern and Western blot analysis. Functional studies also were performed in cultured human hepatic myofibroblasts. RESULTS: Immunohistochemistry showed that in biopsy specimens from normal livers, HO-1 protein expression was restricted to Kupffer cells. Biopsy specimens from cirrhotic patients displayed HO-1 protein both in macrophages and in myofibroblasts within fibrotic septa. HO-1 messenger RNA (mRNA) and protein also were detected in cultured human hepatic myofibroblasts and increased in response to 15-d-PGJ2 in a time- and dose-dependent manner. Induction of HO-1 in human hepatic myofibroblasts mediated 2 major antifibrogenic properties of 15-d-PGJ2, namely, inhibition of proliferation and of procollagen I mRNA expression. These effects were ascribed to bilirubin, one of the products of HO-1-mediated heme degradation. CONCLUSIONS: This study shows that HO-1 is expressed in human hepatic myofibroblasts and induced during chronic liver injury. Moreover, these data unravel HO-1 as a major antifibrogenic pathway.