Antagonism between wild-type and mutant ß-catenin controls hepatoblastoma differentiation via fascin-1.

Background & Aims: ß-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of ß-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated ß-catenins in tumour cells. We investigated the interplay between WT and mutated ß-catenins in liver tumour cells, and searched for new actors of the ß-catenin pathway. Methods: Using an RNAi strategy in ß-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of ß-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of ß-catenin in hepatocytes (APCKO and ß-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples. Results: We highlighted an antagonistic role of WT and mutated ß-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated ß-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs. Conclusions: Fascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with ß-catenin pathway alteration in the liver, and as a new potential target in HB. Impact and implications: The FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.

Silencing of RND3/RHOE inhibits the growth of human hepatocellular carcinoma and is associated with reversible senescence.

Rnd3/RhoE is an atypical Rho GTPase family member, known to be deregulated in many types of cancer. Previously, we showed that RND3 expression is downregulated in hepatocellular carcinoma (HCC) cell lines and tissues. In cancer cells, Rnd3 is involved in the regulation of cell proliferation and cell invasion. The implication of Rnd3 in HCC invasion was importantly studied whereas its role in cell growth needs further investigation. Thus, in this work, we aimed to better understand the impact of Rnd3 on tumor hepatocyte proliferation. Our results indicate that the silencing of RND3 induces a cell growth arrest both in vitro in 2D and 3D culture conditions and in vivo in tumor xenografts. The growth alteration after RND3 silencing in HCC cells is not due to an increase of cell death but to the induction of senescence. This RND3 knockdown-mediated phenomenon is dependent on the decrease of hTERT expression. Interestingly, after re-expression of RND3, these cells are able to bypass senescence and regain the ability to proliferate, with a re-expression of hTERT. Given that a low expression of Rnd3 is linked to the presence of satellite nodules in HCC, the transient senescence state observed might play a role in cancer progression.

Discoidin Domain Receptor 2 orchestrates melanoma resistance combining phenotype switching and proliferation.

Combined therapy with anti-BRAF plus anti-MEK is currently used as first-line treatment of patients with metastatic melanomas harboring the somatic BRAF V600E mutation. However, the main issue with targeted therapy is the acquisition of tumor cell resistance. In a majority of resistant melanoma cells, the resistant process consists in epithelial-to-mesenchymal transition (EMT). This process called phenotype switching makes melanoma cells more invasive. Its signature is characterized by MITF low, AXL high, and actin cytoskeleton reorganization through RhoA activation. In parallel of this phenotype switching phase, the resistant cells exhibit an anarchic cell proliferation due to hyper-activation of the MAP kinase pathway. We show that a majority of human melanoma overexpress discoidin domain receptor 2 (DDR2) after treatment. The same result was found in resistant cell lines presenting phenotype switching compared to the corresponding sensitive cell lines. We demonstrate that DDR2 inhibition induces a decrease in AXL expression and reduces stress fiber formation in resistant melanoma cell lines. In this phenotype switching context, we report that DDR2 control cell and tumor proliferation through the MAP kinase pathway in resistant cells in vitro and in vivo. Therefore, inhibition of DDR2 could be a new and promising strategy for countering this resistance mechanism.

Preferences for Conditional Economic Incentives to Improve Pre-exposure Prophylaxis Adherence: A Discrete Choice Experiment Among Male Sex Workers in Mexico.

Pre-exposure prophylaxis (PrEP) is highly effective in preventing HIV but requires sustained adherence. Conditional economic incentives (CEIs) can improve medication-taking behaviors, yet preferences for programs that employ CEIs to increase PrEP use among male sex workers (MSWs) have not been investigated. We conducted a discrete choice experiment in Mexico City to elicit stated preferences for a CEI-based PrEP adherence program among MSWs. Respondents expressed their preferences for different program characteristics: incentive amount; incentive format; incentive type; and adherence-verification method. We used a random utility logit model to estimate the relative importance of each attribute and estimated willingness-to-pay. MSWs preferred a higher, fixed incentive, with PrEP adherence measured via hair sampling. MSWs were willing to forego up to 21% of their potential maximum CEI amount to ensure receipt of a fixed payment. MSWs are highly willing to accept a CEI-based intervention for PrEP adherence, if offered along with fixed payments.

RESUMEN: La profilaxis previa a la exposición (PrEP) es muy eficaz para prevenir el VIH, pero requiere una adherencia sostenida. Los incentivos económicos condicionales (IEC) pueden mejorar los comportamientos de toma de medicamentos, sin embargo, no se han investigado las preferencias por los programas que emplean IEC para aumentar el uso de PrEP entre los trabajadores sexuales masculinos (TSM). Realizamos un experimento de elección discreta con TSM en la Ciudad de México para obtener preferencias declaradas para un programa de adherencia a la PrEP basado en IEC. Los participantes expresaron sus preferencias en cuanto a diferentes características: monto del incentivo; formato del incentivo; tipo de incentivo; y método de verificación de la adherencia. Utilizamos un modelo logit de utilidad aleatoria para estimar la importancia relativa de cada atributo y la disposición a pagar estimada (DAP). Los TSM prefirieron un incentivo fijo más alto, con la adherencia a la PrEP medida a través de muestras de cabello. Los TSM estaban dispuestos a renunciar hasta el 21% de su monto máximo potencial de IEC para garantizar la recepción de un pago fijo. Los TSM están muy dispuestos a aceptar una intervención basada en IEC para la adherencia a la PrEP, si se ofrece junto con pagos fijos.

Reptin/RUVBL2 is required for hepatocyte proliferation in vivo, liver regeneration and homeostasis.

Previous studies have shown that Reptin is overexpressed in hepatocellular carcinoma and that it is necessary for in vitro proliferation and cell survival. However, its pathophysiological role in vivo remains unknown. We aimed to study the role of Reptin in hepatocyte proliferation after regeneration using a liver Reptin knock-out model (ReptinLKO ). Interestingly, hepatocyte proliferation is strongly impaired in ReptinLKO mice 36 h after partial hepatectomy, associated with a decrease of cyclin-A expression and mTORC1 and MAPK signalling, leading to an impaired liver regeneration. Moreover, in the ReptinLKO model, we have observed a progressive loss of Reptin invalidation associated with an atypical liver regeneration. Hypertrophic and proliferative hepatocytes gradually replace ReptinKO hypotrophic hepatocytes. To conclude, our results show that Reptin is required for hepatocyte proliferation in vivo and liver regeneration and that it plays a crucial role in hepatocyte survival and liver homeostasis.

Punto Seguro: A Randomized Controlled Pilot Using Conditional Economic Incentives to Reduce Sexually Transmitted Infection Risks in Mexico.

Randomized controlled pilot evaluated effect of conditional economic incentives (CEIs) on number of sex partners, condom use, and incident sexually transmitted infections (STIs) among male sex workers in Mexico City. Incentives were contingent on testing free of new curable STIs and/or clinic attendance. We assessed outcomes for n = 227 participants at 6 and 12 months (during active phase with incentives), and then at 18 months (with incentives removed). We used intention-to-treat and inverse probability weighting for the analysis. During active phase, CEIs increased clinic visits (10-13 percentage points) and increased condom use (10-15 percentage points) for CEI groups relative to controls. The effect on condom use was not sustained once CEIs were removed. CEIs did not have an effect on number of partners or incident STIs. Conditional incentives for male sex workers can increase linkage to care and retention and reduce some HIV/STI risks such as condomless sex, while incentives are in place.

First identification of small-molecule inhibitors of Pontin by combining virtual screening and enzymatic assay.

The human protein Pontin, which belongs to the AAA+ (ATPases associated with various cellular activities) family, is overexpressed in several cancers and its silencing in vitro leads to tumour cell growth arrest and apoptosis, making it a good target for cancer therapy. In particular, high levels of expression were found in hepatic tumours for which the therapeutic arsenal is rather limited. The three-dimensional structure of Pontin has been resolved previously, revealing a hexameric assembly with one ADP molecule co-crystallized in each subunit. Using Vina, DrugScore and Xscore, structure-based virtual screening of 2200 commercial molecules was conducted into the ATP-binding site formed by a dimer of Pontin in order to prioritize the best candidates. Complementary to the in silico screening, a versatile and sensitive colorimetric assay was set up to measure the disruption of the ATPase activity of Pontin. This assay allowed the determination of inhibition curves for more than 20 top-scoring compounds, resulting in the identification of four ligands presenting an inhibition constant in the micromolar concentration range. Three of them inhibited tumour cell proliferation. The association of virtual screening and experimental assay thus proved successful for the discovery of the first small-molecule inhibitors of Pontin.

Combined targeting of interleukin-6 and vascular endothelial growth factor potently inhibits glioma growth and invasiveness.

Interleukin-6 (IL6) and vascular endothelial growth factor (VEGFA) are abundantly produced by glioma cells and contribute to malignancy by promoting angiogenesis, cell proliferation and resistance to apoptosis. We compared the effect of inhibiting IL6 and VEGF on U87-derived experimental glioma grown on the chick chorio-allantoic membrane (CAM) or in the brain of xenografted mice. Tumor growth was monitored by biomicroscopy and immunohistology. In vitro, IL6 knockdown had no effect on proliferation but substantially enhanced invasion. In the CAM experimental glioma, IL6 or VEGF knockdown reduced growth and vascularization of the tumors with a comparable efficiency, but increased invasion of residual tumor cells. In contrast, combined IL6/VEGF knockdown not only showed enhanced reduction of tumor growth and angiogenesis but also significantly prevented invasion of residual tumor cells. In mice, combining IL6 knockdown and Avastin treatment completely abrogated tumor development and infiltration. Molecular response of tumor cells to single or combined treatment was studied by transcriptomic profiling. Many cell cycle promoting genes and chromatin components were silenced in the double knockdown. In addition, specific migratory signatures detected in tumors under single IL6 or VEGF knockdown were partially erased in combined IL6/VEGF knockdown tumors. Our results show that treatment with a combination of IL6 and VEGF inhibitors brings synergistic antitumoral benefit and reduces global activity of major pathways of cell survival, proliferation and invasiveness in remaining tumor cells that may be induced by using VEGF or IL6 inhibitors alone.

FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients.

BACKGROUND: In the quest for novel molecular mediators of glioma progression, we studied the regulation of FBXW7 (hCDC4/hAGO/SEL10), its association with survival of patients with glioblastoma and its potential role as a tumor suppressor gene in glioma cells. The F-box protein Fbxw7 is a component of SCFFbxw7, a Skp1-Cul1-F-box E3 ubiquitin ligase complex that tags specific proteins for proteasome degradation. FBXW7 is mutated in several human cancers and functions as a haploinsufficient tumor suppressor in mice. Any of the identified targets, Cyclin E, c-Myc, c-Jun, Notch1/4 and Aurora-A may have oncogenic properties when accumulated in tumors with FBXW7 loss. RESULTS: We tested the expression of FBXW7 in human glioma biopsies by quantitative PCR and compared the transcript levels of grade IV glioma (glioblastoma, G-IV) with those of grade II tumors (G-II). In more than 80% G-IV, expression of FBXW7 was significantly reduced. In addition, levels of FBXW7 were correlated with survival indicating a possible implication in tumor aggressiveness. Locus 4q31.3 which carries FBXW7 was investigated by in situ hybridization on biopsy touchprints. This excluded allelic loss as the principal cause for low expression of FBXW7 in G-IV tumors. Two targets of Fbxw7, Aurora-A and Notch4 were preferentially immunodetected in G-IV biopsies. Next, we investigated the effects of FBXW7 misregulation in glioma cells. U87 cells overexpressing nuclear isoforms of Fbxw7 lose the expression of the proliferation markers PCNA and Ki-67, and get counterselected in vitro. This observation fits well with the hypothesis that Fbxw7 functions as a tumor suppressor in astroglial cells. Finally, FBXW7 knockdown in U87 cells leads to defects in mitosis that may promote aneuploidy in progressing glioma. CONCLUSION: Our results show that FBXW7 expression is a prognostic marker for patients with glioblastoma. We suggest that loss of FBXW7 plays an important role in glioma malignancy by allowing the accumulation of multiple oncoproteins and that interfering with Fbxw7 or its downstream targets would constitute a new therapeutic advance.

Cadmium induces caspase-independent apoptosis in liver Hep3B cells: role for calcium in signaling oxidative stress-related impairment of mitochondria and relocation of endonuclease G and apoptosis-inducing factor.

Cadmium-induced cellular toxicity has been related to necrosis and/or caspase-dependent apoptosis. In the present study, we show that, on cadmium exposure, the human hepatocarcinoma Hep3B cells undergo caspase-independent apoptosis associated with nuclear translocation of endonuclease G and apoptosis-inducing factor, two mitochondrial apoptogenic proteins. Release of these proteins is likely related to calcium-induced alteration of mitochondrial homeostasis. Indeed, it was first preceded by a rapid and sustained increase in cytoplasmic calcium and then by a coincident loss in mitochondrial membrane potential and production of reactive oxygen species. Bapta-AM (acetoxymethyl ester of 5, 5'-dimethyl-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), a calcium chelator, blocked all these events and prevented cadmium-induced apoptosis. Production of reactive oxygen species was inhibited by ruthenium red and rotenone, two mitochondrial inhibitors, and by diphenyleneiodonium, a flavoprotein inhibitor, which also prevented both loss in mitochondrial membrane potential and apoptosis. In addition, Bapta-AM and diphenyleneiodonium were found to almost totally block decreased expression of the mitochondrial anti-apoptotic nuclear factor-kappaB-regulated bcl-x(L) protein in cadmium-treated cells. Taken together, our results show that cadmium induces Hep3B cells apoptosis mainly by calcium- and oxidative stress-related impairment of mitochondria, which probably favors release of apoptosis-inducing factor and endonuclease G.

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection.

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.

Blockage of multidrug resistance-associated proteins potentiates the inhibitory effects of arsenic trioxide on CYP1A1 induction by polycyclic aromatic hydrocarbons.

Arsenic is a toxic metalloid known to interact with drug-metabolizing enzymes. In the present study, we investigated the effects of arsenic trioxide (As2O3), recently used as an anticancer drug, on the expression of human cytochrome P450 (P450) 1A1, which bioactivates polycyclic aromatic hydrocarbons into mutagenic metabolites. Clinically relevant concentrations (0.25-5 microM) of As2O3 were demonstrated to inhibit CYP1A activity in primary human hepatocytes and hepatoma Hep3B and HepG2 cells coexposed to 3-methylcholanthrene (3MC), benzo(a)pyrene, or dioxin and the metalloid for 24 h. Inhibition reached 50 and 90% in Hep3B cells treated with 1 and 5 microM As2O3, respectively, and was not due to direct interaction of the metalloid with CYP1A1. As2O3 (2.5-5 microM) was demonstrated to markedly reduce induction of CYP1A1 mRNA and apoprotein levels and gene promotor activity in 3MC-treated Hep3B cells, whereas lower concentrations (0.25-1 microM) were ineffective. These effects of As2O3 were abrogated by N-acetylcysteine. Surprisingly, this agent was found 1) to block cellular arsenic uptake when coincubated with the metalloid and 2) to increase arsenic efflux through multidrug resistance-associated proteins. In addition, blockade of these transporters was shown to enhance intracellular amounts of metalloid and to potentiate its effects on CYP1A1 gene. Finally, our results have demonstrated that As2O3, at low concentrations routinely reached in As2O3-treated patients, prevents induction of human CYP1A1 gene expression and that such an effect is increased by blocking multidrug resistance-associated proteins.