Co-Expression Network Analysis Unveiled lncRNA-mRNA Links Correlated to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance and/or Intermediate Epithelial-to-Mesenchymal Transition Phenotypes in a Human Non-Small Cell Lung Cancer Cellular Model System.

We investigated mRNA-lncRNA co-expression patterns in a cellular model system of non-small cell lung cancer (NSCLC) sensitive and resistant to the epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) erlotinib/gefitinib. The aim of this study was to unveil insights into the complex mechanisms of NSCLC targeted therapy resistance and epithelial-to-mesenchymal transition (EMT). Genome-wide RNA expression was quantified for weighted gene co-expression network analysis (WGCNA) to correlate the expression levels of mRNAs and lncRNAs. Functional enrichment analysis and identification of lncRNAs were conducted on modules associated with the EGFR-TKI response and/or intermediate EMT phenotypes. We constructed lncRNA-mRNA co-expression networks and identified key modules and their enriched biological functions. Processes enriched in the selected modules included RHO (A, B, C) GTPase and regulatory signaling pathways, apoptosis, inflammatory and interleukin signaling pathways, cell adhesion, cell migration, cell and extracellular matrix organization, metabolism, and lipid metabolism. Interestingly, several lncRNAs, already shown to be dysregulated in cancer, are connected to a small number of mRNAs, and several lncRNAs are interlinked with each other in the co-expression network.

Effects of structurally distinct human HDAC6 and HDAC6/HDAC8 inhibitors against S. mansoni larval and adult worm stages.

Schistosomiasis is a major neglected parasitic disease that affects more than 240 million people worldwide caused by Platyhelminthes of the genus Schistosoma. The treatment of schistosomiasis relies on the long-term application of a single safe drug, praziquantel (PZQ). Unfortunately, PZQ is very effective on adult parasites and poorly on larval stage and immature juvenile worms; this can partially explain the re-infection in endemic areas where patients are likely to host parasites at different developmental stages concurrently. Moreover, the risk of development of drug resistance because of the widespread use of a single drug in a large population is nowadays a serious threat. Hence, research aimed at identifying novel drugs to be used alone or in combination with PZQ is needed. Schistosomes display morphologically distinct stages during their life cycle and epigenetic mechanisms are known to play important roles in parasite growth, survival, and development. Histone deacetylase (HDAC) enzymes, particularly HDAC8, are considered valuable for therapeutic intervention for the treatment of schistosomiasis. Herein, we report the phenotypic screening on both larvae and adult Schistosoma mansoni stages of structurally different HDAC inhibitors selected from the in-house Siena library. All molecules have previously shown inhibition profiles on human HDAC6 and/or HDAC8 enzymes. Among them we identified a quinolone-based HDAC inhibitor, NF2839, that impacts larval and adult parasites as well as egg viability and maturation in vitro. Importantly, this quinolone-based compound also increases histone and tubulin acetylation in S. mansoni parasites, thus representing a leading candidate for the development of new generation anti-Schistosoma chemotherapeutics.

The activity of a PI3K δ-sparing inhibitor, MEN1611, in non-small cell lung cancer cells with constitutive activation of the PI3K/AKT/mTOR pathway.

Background: Lung cancer remains the leading cause of cancer-related death worldwide. Targeted therapies with tyrosine kinase inhibitors (TKIs) result in improvement in survival for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR). Unfortunately, most patients who initially respond to EGFR-TKI ultimately develop resistance to therapy, resulting in cancer progression and relapse. Combination therapy is today a common strategy for the treatment of tumors to increase the success rate, improve the outcome and survival of patients, and avoid the selection of resistant cancer cells through the activation of compensatory pathways. In NSCLC, the phosphoinositide-3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway has been heavily implicated in both tumorigenesis and the progression of disease. Objectives: In this study, we investigated the efficacy of a PI3K δ-sparing inhibitor, MEN1611, in models of NSCLC sensitive and resistant to EGFR inhibitors (erlotinib and gefitinib) with a wild-type PIK3CA gene. Methods: We performed functional, biochemical, and immunohistochemistry studies. Results: We demonstrated good efficacy of MEN1611 in NSCLC devoid of PIK3CA gene mutations but with constitutive activation of the PI3K/AKT pathway and its synergistic effect with gefitinib both in vitro and in vivo. Conclusions: Overall, this preclinical study indicates that the inhibitor could be a candidate for the treatment of NSCLC with an erlotinib/gefitinib-resistant phenotype and constitutive activation of the PI3K/AKT pathway, a phenotype mimicked by our model system.

Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.

Azetidin-2-one-based small molecules as dual hHDAC6/HDAC8 inhibitors: Investigation of their mechanism of action and impact of dual inhibition profile on cell viability.

The search of new therapeutic tools for the treatment of cancer is being a challenge for medicinal chemists. Due to their role in different pathological conditions, histone deacetylase (HDAC) enzymes are considered valuable therapeutic targets. HDAC6 is a well-investigated HDAC-class IIb enzyme mainly characterized by a cytoplasmic localization; HDAC8 is an epigenetic eraser, unique HDAC-class I member that displays some aminoacidic similarity to HDAC6. New polypharmacological agents for cancer treatment, based on a dual hHDAC6/hHDAC8 inhibition profile were developed. The dual inhibitor design investigated the diphenyl-azetidin-2-one scaffold, typified in three different structural families, that, combined to a slender benzyl linker (6c, 6i, and 6j), displays nanomolar inhibition potency against hHDAC6 and hHDAC8 isoforms. Notably, their selective action was also corroborated by measuring their low inhibitory potency towards hHDAC1 and hHDAC10. Selectivity of these compounds was further demonstrated in human cell-based western blots experiments, by testing the acetylation of the non-histone substrates alpha-tubulin and SMC3. Furthermore, the compounds reduced the proliferation of colorectal HCT116 and leukemia U937 cells, after 48 h of treatment. The toxicity of the compounds was evaluated in rat perfused heart and in zebrafish embryos. In this latter model we also validated the efficacy of the dual hHDAC6/hHDAC8 inhibitors against their common target acetylated-alpha tubulin. Finally, the metabolic stability was verified in rat, mouse, and human liver microsomes.

In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines.

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Comparative metabolic profiling by 1H-NMR spectroscopy analysis reveals the adaptation of S. mansoni from its host to in vitro culture conditions: a pilot study with ex vivo and GSH-supplemented medium-cultured parasites.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms (blood fluke) of the genus Schistosoma. Parasites acquire most nutrients for their development and sustainment within the definitive host either by ingestion into the gut or across the body surface. Over the years, the best conditions for long-term maintenance of parasites in vitro have been thoroughly established. In our hands, 1H-NMR spectroscopy represents a powerful tool to characterize the metabolic changes in S. mansoni in response to culturing condition perturbations. In order to compare the metabolic fingerprint of ex vivo and parasites cultured in vitro with or without the supplement of reduced glutathione, we conducted a pilot study applying the 1H-NMR spectroscopy-based metabolomics. We obtained new insight into specific metabolic pathways modulated under these different experimental conditions.

Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novel hHDAC6 inhibitors, having low inhibitory potency over hHDAC1 and hHDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with low in vitro and in vivo toxicity. Structural analysis of 6h and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-ß1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.

Novel quinolone-based potent and selective HDAC6 inhibitors: Synthesis, molecular modeling studies and biological investigation.

In this work we describe the synthesis of potent and selective quinolone-based histone deacetylase 6 (HDAC6) inhibitors. The quinolone moiety has been exploited as an innovative bioactive cap-group for HDAC6 inhibition; its synthesis was achieved by applying a multicomponent reaction. The optimization of potency and selectivity of these products was performed by employing computational studies which led to the discovery of the diethylaminomethyl derivatives 7g and 7k as the most promising hit molecules. These compounds were investigated in cellular studies to evaluate their anticancer effect against colon (HCT-116) and histiocytic lymphoma (U9347) cancer cells, showing good to excellent potency, leading to tumor cell death by apoptosis induction. The small molecules 7a, 7g and 7k were able to strongly inhibit the cytoplasmic and slightly the nuclear HDAC enzymes, increasing the acetylation of tubulin and of the lysine 9 and 14 of histone 3, respectively. Compound 7g was also able to increase Hsp90 acetylation levels in HCT-116 cells, thus further supporting its HDAC6 inhibitory profile. Cytotoxicity and mutagenicity assays of these molecules showed a safe profile; moreover, the HPLC analysis of compound 7k revealed good solubility and stability profile.

Spiroindoline-Capped Selective HDAC6 Inhibitors: Design, Synthesis, Structural Analysis, and Biological Evaluation.

Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Drug effects on metabolic profiles of Schistosoma mansoni adult male parasites detected by 1H-NMR spectroscopy.

Schistosomiasis is one of the most devastating neglected tropical parasitic diseases caused by trematodes of the genus Schistosoma. Praziquantel (PZQ) is today the only drug used in humans and animals for the treatment of schistosomiasis but unfortunately it is poorly effective on larval and juvenile stages of the parasite. Therefore, it is urgent the discovery of new drug targets and compounds. We have recently showed that the anti-anginal drug perhexiline maleate (PHX) is very active on multiple developmental stages of Schistosoma mansoni in vitro. It is well known that PHX impacts the lipid metabolism in mammals, but the final target on schistosomes still remains unknown. The aim of this study was to evaluate the ability of 1H nuclear magnetic resonance (NMR) spectroscopy in revealing metabolic perturbations due to PHX treatment of S. mansoni adult male worms. The effects of PHX were compared with the ones induced by vehicle and gambogic acid, in order to detect different metabolic profiles and specificity of the PHX action. Remarkably a list of metabolites associated to PHX-treatment was identified with enrichment in several connected metabolic pathways including also the Kennedy pathway mediating the glycerophospholipid metabolism. Our study represents the first 1H-NMR metabolomic approach to characterize the response of S. mansoni to drug treatment. The obtained "metabolic fingerprint" associated to PHX treatment could represent a strategy for displaying cellular metabolic changes for any given drug and to compare compounds targeting similar or distinct biochemical pathways.

(+)-(R)- and (-)-(S)-Perhexiline maleate: Enantioselective synthesis and functional studies on Schistosoma mansoni larval and adult stages.

Schistosomiasis is a neglected tropical disease mainly affecting the poorest tropical and subtropical areas of the world with the impressive number of roughly 200 million infections per year. Schistosomes are blood trematode flukes of the genus Schistosoma causing symptoms in humans and animals. Organ morbidity is caused by the accumulation of parasite eggs and subsequent development of fibrosis. If left untreated, schistosomiasis can result in substantial morbidity and even mortality. Praziquantel (PZQ) is the most effective and widely used compound for the treatment of the disease, in prevention and control programs in the last 30 years. Unfortunately, it has no effect on juvenile immature schistosomes and cannot prevent reinfection or interfere with the schistosome life cycle; moreover drug-resistance represents a serious threat. The search for an alternative or complementary treatment is urgent and drug repurposing could accelerate a solution. The anti-anginal drug perhexiline maleate (PHX) has been previously shown to be effective on larval, juvenile, and adult stages of S. mansoni and to impact egg production in vitro. Since PHX is a racemic mixture of R-(+)- and S-(-)-enantiomers, we designed and realized a stereoselective synthesis of both PHX enantiomers and developed an analytical procedure for the direct quantification of the enantiomeric excess also suitable for semipreparative separation of PHX enantiomers. We next investigated the impact of each enantiomer on viability of newly transformed schistosomula (NTS) and worm pairs of S. mansoni as well as on egg production and vitellarium morphology by in vitro studies. Our results indicate that the R-(+)-PHX is mainly driving the anti-schistosomal activity but that also the S-(-)-PHX possesses a significant activity towards S. mansoni in vitro.

Luminescence-Based, Low- and Medium-Throughput Assays for Drug Screening in Schistosoma mansoni Larval Stage.

Schistosomiasis is one of the major parasitic diseases with more than  200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.

Investigating the Antiparasitic Potential of the Marine Sesquiterpene Avarone, Its Reduced form Avarol, and the Novel Semisynthetic Thiazinoquinone Analogue Thiazoavarone.

The chemical analysis of the sponge Dysidea avara afforded the known sesquiterpene quinone avarone, along with its reduced form avarol. To further explore the role of the thiazinoquinone scaffold as an antiplasmodial, antileishmanial and antischistosomal agent, we converted the quinone avarone into the thiazinoquinone derivative thiazoavarone. The semisynthetic compound, as well as the natural metabolites avarone and avarol, were pharmacologically investigated in order to assess their antiparasitic properties against sexual and asexual stages of Plasmodium falciparum, larval and adult developmental stages of Schistosoma mansoni (eggs included), and also against promastigotes and amastigotes of Leishmania infantum and Leishmania tropica. Furthermore, in depth computational studies including density functional theory (DFT) calculations were performed. A toxic semiquinone radical species which can be produced starting both from quinone- and hydroquinone-based compounds could mediate the anti-parasitic effects of the tested compounds.

Thiazinoquinones as New Promising Multistage Schistosomicidal Compounds Impacting Schistosoma mansoni and Egg Viability.

Schistosomiasis is the most significant neglected tropical parasitic disease caused by helminths in terms of morbidity and mortality caused by helminths. In this work, we present the antischistosomal activity against Schistosoma mansoni of a rationally selected small set of thiazinoquinone derivatives, some of which were previously found to be active against Plasmodium falciparum and others synthesized ad hoc. The effects on larvae, juvenile, and adult parasite viability as well as on egg production and development were investigated, resulting in the identification of new multistage antischistosomal hit compounds. The most promising compounds 6, 8, 13, and 14 with a LC50 value on schistosomula from ∼5 to ∼15 µM also induced complete death of juvenile (28 days old) and adult worm pairs (7 weeks old) and a detrimental effect on egg production and development in vitro. Structure-activity relationships (SARs) were analyzed by means of computational studies leading to the hypothesis of a redox-based mechanism of action with a one-electron reduction bioactivation step and the subsequent formation of a toxic semiquinone species, similarly to what was previously observed for the antiplasmodial activity. Our results also evidenced that the selective toxicity against mammalian cells or parasites as well as specific developmental stages of a parasite can be addressed by varying the nature of the introduced substituents.

Screening and Phenotypical Characterization of Schistosoma mansoni Histone Deacetylase 8 (SmHDAC8) Inhibitors as Multistage Antischistosomal Agents.

Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by platyhelminths of the genus Schistosoma. The disease is endemic in tropical and subtropical areas of the world where water is infested by the intermediate parasite host, the snail. More than 800 million people live in endemic areas and more than 200 million are infected and require treatment. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment and transmission control being safe and very effective against adult worms of all the clinically relevant Schistosoma species. Unfortunately, it is ineffective on immature, juvenile worms; therefore, it does not prevent reinfection. Moreover, the risk of development and spread of drug resistance because of the widespread use of a single drug in such a large population represents a serious threat. Therefore, research aimed at identifying novel drugs to be used alone or in combination with PZQ are needed. Schistosoma mansoni histone deacetylase 8 (SmHDAC8) is a class I zinc-dependent HDAC, which is abundantly expressed in all stages of its life cycle, thus representing an interesting target for drug discovery. Through virtual screening and phenotypical characterization of selected hits, we discovered two main chemical classes of compounds characterized by the presence of a hydroxamate-based metal binding group coupled to a spiroindoline or a tricyclic thieno[3,2-b]indole core as capping groups. Some of the compounds of both classes were deeply investigated and showed to impair viability of larval, juvenile, and adult schistosomes, to impact egg production in vitro and/or to induce morphological alterations of the adult schistosome reproductive systems. Noteworthy, all of them inhibit the recombinant form of SmHDAC8 enzyme in vitro. Overall, we identified very interesting scaffolds, paving the way to the development of effective antischistosomal agents.

Chemical Investigation of the Indonesian Tunicate Polycarpa aurata and Evaluation of the Effects Against Schistosoma mansoni of the Novel Alkaloids Polyaurines A and B.

A deep study of the metabolic content of the tunicate Polycarpa aurata, collected from Indonesian coast, afforded the isolation of two novel alkaloids, polyaurines A (1) and B (2), along with two new p-substituted benzoyl derivatives (3 and 4) and four known compounds (5-8). The structural elucidation of the new secondary metabolites was assigned by 1D, 2D NMR, and HRESIMS techniques. Computational studies resulted a useful tool to unambiguously determine in polyaurine B the presence of rarely found 1,2,4-thiadiazole ring. The effects of polyaurines A and B on mammalian cells growth and on the viability of different blood-dwelling Schistosoma mansoni (phylum: Platyhelminthes) stages, as well as egg production, were evaluated. Both compounds resulted not cytotoxic; interestingly some of the eggs produced by polyaurine A-treated adult pairs in vitro are smaller, deformed, and/or fragmented; therefore, polyaurine A could represent an interesting bioactive natural molecule to be further investigated.

Identification of novel multi-stage histone deacetylase (HDAC) inhibitors that impair Schistosoma mansoni viability and egg production.

BACKGROUND: Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. METHODS: We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. RESULTS: Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. CONCLUSIONS: We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.

CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion.

Inflammatory caspases, including human caspase-4 (CASP4), play key roles in innate immune responses to promote fusion of phagosomes harboring pathogenic bacteria with lysosomes, halt intracellular replication of pathogens, maturation and secretion of pro-inflammatory cytokines. The role of inflammatory caspases in cancer cells remains poorly investigated. Here, we explored the consequences of modulating CASP4 expression levels on the migratory behavior of epithelial cancer cell lines. By a gene silencing approach and in vitro and in vivo studies we show that down-regulation of CASP4 leads to impaired cell migration and cell-matrix adhesion. This phenotype is accompanied by an increased actin cytoskeleton polymerization, changes in the overall organization of adherens junctions (AJs) and number and size of focal adhesions. Interestingly, the cell migration deficit could be reversed by epithelial growth factor treatment, and depletion of calcium ions unveiled a role of CASP4 in the novo assembly of AJs, suggesting that the role of CASP4 is not cell-autonomous. Finally, CASP4-silenced A431 cells exhibited a severe reduction in their ability to invade lung tissue, when injected into nude mice. Overall, our data support the emerging evidence that inflammatory caspases can regulate cell migration through actin remodeling and uncover a novel role of CASP4 in cancer cell behavior.

Tis21-gene therapy inhibits medulloblastoma growth in a murine allograft model.

Medulloblastoma (MB), the tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. Based on gene profiling, four MB subgroups have been identified, i.e., Wnt or Sonic Hedgehog (Shh) types, and subgroup 3 or 4. The Shh-type MB has been shown to arise from the cerebellar precursors of granule neurons (GCPs), where a hyperactivation of the Shh pathway leads to their neoplastic transformation. We have previously shown that the gene Tis21 (PC3/Btg2) inhibits the proliferation and promotes the differentiation and migration of GCPs. Moreover, the overexpression or the deletion of Tis21 in Patched1 heterozygous mice, a model of spontaneous Shh-type MB, highly reduces or increases, respectively, the frequency of MB. Here we tested whether Tis21 can inhibit MB allografts. Athymic nude mice were subcutaneously grafted with MB cells explanted from Patched1 heterozygous mice. MB allografts were then injected with adeno-associated viruses either carrying Tis21 (AAV-Tis21) or empty (AAV-CBA). We observed that the treatment with AAV-Tis21 significantly inhibited the growth of tumor nodules, as judged by their volume, and reduced the number of proliferating tumor cells (labeled with Ki67 or BrdU), relative to AAV-CBA-treated control mice. In parallel, AAV-Tis21 increased significantly tumor cells labeled with early and late neural differentiation markers. Overall the results suggest that Tis21-gene therapy slows down MB tumor growth through inhibition of proliferation and enhancement of neural differentiation. These results validate Tis21 as a relevant target for MB therapy.