Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Early targets of miR-34a in neuroblastoma.

Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3'-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3'-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-ß, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL.

TMA in Kidney Transplantation.

Thrombotic microangiopathy (TMA) is a rare and devastating complication of kidney transplantation, which often leads to graft failure. Posttransplant TMA (PT-TMA) may occur either de novo or as a recurrence of the disease. De novo TMA can be triggered by immunosuppressant drugs, antibody-mediated rejection, viral infections, and ischemia/reperfusion injury in patients with no evidence of the disease before transplantation. Recurrent TMA may occur in the kidney grafts of patients with a history of atypical hemolytic uremic syndrome (aHUS) in the native kidneys. Studies have shown that some patients with aHUS carry genetic abnormalities that affect genes that code for complement regulators (CFH, MCP, CFI) and components (C3 and CFB), whereas in 10% of patients (mostly children), anti-FH autoantibodies have been reported. The incidence of aHUS recurrence is determined by the underlying genetic or acquired complement abnormality. Although treatment of the causative agents is usually the first line of treatment for de novo PT-TMA, this approach might be insufficient. Plasma exchange typically resolves hematologic abnormalities but does not improve kidney function. Targeted complement inhibition is an effective treatment for recurrent TMA and may be effective in de novo PT-TMA as well, but it is necessary to establish which patients can benefit from different therapeutic options and when and how these can be applied.

An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome.

Introduction: Comprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants. Methods: To address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives). Results: Sera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent. Discussion: In conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.

A GWAS in the pandemic epicenter highlights the severe COVID-19 risk locus introgressed by Neanderthals.

Large GWAS indicated that genetic factors influence the response to SARS-CoV-2. However, sex, age, concomitant diseases, differences in ancestry, and uneven exposure to the virus impacted the interpretation of data. We aimed to perform a GWAS of COVID-19 outcome in a homogeneous population who experienced a high exposure to the virus and with a known infection status. We recruited inhabitants of Bergamo province-that in spring 2020 was the epicenter of the SARS-Cov-2 pandemic in Europe-via an online questionnaire followed by personal interviews. Cases and controls were matched by age, sex and risk factors. We genotyped 1195 individuals and replicated the association at the 3p21.31 locus with severity, but with a stronger effect size that further increased in gravely ill patients. Transcriptome-wide association study highlighted eQTLs for LZTFL1 and CCR9. We also identified 17 loci not previously reported, suggestive for an association with either COVID-19 severity or susceptibility.

Impact of SARS-CoV-2 Omicron and Delta variants in patients requiring intensive care unit (ICU) admission for COVID-19, Northern Italy, December 2021 to January 2022.

This multicenter observational study included 171 COVID-19 adult patients hospitalized in the ICUs of nine hospitals in Lombardy (Northern Italy) from December, 1st 2021, to February, 9th 2022. During the study period, the Delta/Omicron variant ratio of cases decreased with a delay of two weeks in ICU patients compared to that in the community; a higher proportion of COVID-19 unvaccinated patients was infected by Delta than by Omicron whereas a higher rate of COVID-19 boosted patients was Omicron-infected. A higher number of comorbidities and a higher comorbidity score in ICU critically COVID-19 inpatients was positively associated with the Omicron infection as well in vaccinated individuals. Although people infected by Omicron have a lower risk of severe disease than those infected by Delta variant, the outcome, including the risk of ICU admission and the need for mechanical ventilation due to infection by Omicron versus Delta, remains uncertain. The continuous monitoring of the circulating SARS-CoV-2 variants remains a milestone to counteract this pandemic.

Case Report: Tackling Complement Hyperactivation With Eculizumab in Atypical Hemolytic Uremic Syndrome Triggered by COVID-19.

Hemolytic uremic syndrome (HUS) is a rare life-threatening disease of unrestrained complement system dysregulation, microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure in genetically predisposed individuals. In this report, we describe two cases of SARS-CoV-2-associated HUS treated with eculizumab, a C5-blocking monoclonal antibody reported to be remarkably effective in the treatment of HUS. Detailed biochemical and genetic complement system analysis is reported, and the prompt clinical response after C5 pharmacological blockade is documented. Our report provides the rationale and supports the use of terminal complement pathway inhibition for the treatment of SARS-CoV-2-associated HUS.

Mycoplasma pneumoniae Infection Associated with Anti-Factor H Autoantibodies in Atypical Hemolytic Uremic Syndrome.

Hemolytic uremic syndrome (HUS) is a rare disease characterized by hemolytic anemia, thrombocytopenia, and renal impairment mostly triggered by strains of Shiga-like toxin-producing Escherichia coli (STEC-HUS). A rarer form of HUS, defined as atypical HUS (aHUS), is associated with genetic or acquired dysregulation of the alternative pathway of the complement system and presents a poorer prognosis than STEC-HUS. Factor H autoantibodies (anti-FHs) have been reported in aHUS in 5-11% of cases and are strongly associated with the homozygous deletion of CFHR3-CFHR1 genes. In the large majority of patients, anti-FH-associated aHUS is commonly preceded by gastrointestinal or respiratory tract infections. Here, we described the clinical case of a 3-year-old boy who was hospitalized for aHUS preceded by Mycoplasma pneumoniae (MP) infection. He resulted positive for anti-FHs and carried the homozygous deletion of CFHR3-CFHR1. Of relevance, he also showed a variant of unknown significance in the C5 gene. The patient was successfully treated with eculizumab and achieved hematological and renal remission. The anti-FH titer decreased, became negative after 6 months of mycophenolate mofetil (MMF) treatment, and remained negative for 21-month follow-up indicating that immunosuppression was effective and could prevent the reappearance of anti-FHs. We hypothesized that MP, likely through an evasion strategy of immunosurveillance based on binding of pathogen to FH, triggers anti-FH antibody generation and aHUS in a subject genetically predisposed. In conclusion, to the best of our knowledge, here, we reported the first case of anti-FH-mediated aHUS after an MP infection who benefited from eculizumab and immunosuppressive therapy based on MMF. Hence, monitoring of anti-FHs in patients with post-MP infection glomerulonephritis could be recommended, especially in those with low C3 plasma levels.

C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19.

Unrestrained activation of the complement system till the terminal products, C5a and C5b-9, plays a pathogenetic role in acute and chronic inflammatory diseases. In endothelial cells, complement hyperactivation may translate into cell dysfunction, favoring thrombus formation. The aim of this study was to investigate the role of the C5a/C5aR1 axis as opposed to C5b-9 in inducing endothelial dysfunction and loss of antithrombogenic properties. In vitro and ex vivo assays with serum from patients with atypical hemolytic uremic syndrome (aHUS), a prototype rare disease of complement-mediated microvascular thrombosis due to genetically determined alternative pathway dysregulation, and cultured microvascular endothelial cells, demonstrated that the C5a/C5aR1 axis is a key player in endothelial thromboresistance loss. C5a added to normal human serum fully recapitulated the prothrombotic effects of aHUS serum. Mechanistic studies showed that C5a caused RalA-mediated exocytosis of von Willebrand factor (vWF) and P-selectin from Weibel-Palade bodies, which favored further vWF binding on the endothelium and platelet adhesion and aggregation. In patients with severe COVID-19 who suffered from acute activation of complement triggered by severe acute respiratory syndrome coronavirus 2 infection, we found the same C5a-dependent pathogenic mechanisms. These results highlight C5a/C5aR1 as a common prothrombogenic effector spanning from genetic rare diseases to viral infections, and it may have clinical implications. Selective C5a/C5aR1 blockade could have advantages over C5 inhibition because the former preserves the formation of C5b-9, which is critical for controlling bacterial infections that often develop as comorbidities in severely ill patients. The ACCESS trial registered at www.clinicaltrials.gov as #NCT02464891 accounts for the results related to aHUS patients treated with CCX168.

CFH and CFHR structural variants in atypical Hemolytic Uremic Syndrome: Prevalence, genomic characterization and impact on outcome.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that manifests with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is associated with dysregulation of the alternative complement pathway. The chromosomal region including CFH and CFHR1-5 is rich in repeated sequences, favoring genomic rearrangements that have been reported in several patients with aHUS. However, there are limited data on the prevalence of uncommon CFH-CFHR genomic rearrangements in aHUS and their impact on disease onset and outcomes. Methods: In this study, we report the results of CFH-CFHR Copy Number Variation (CNV) analysis and the characterization of resulting structural variants (SVs) in a large cohort of patients, including 258 patients with primary aHUS and 92 with secondary forms. Results: We found uncommon SVs in 8% of patients with primary aHUS: 70% carried rearrangements involving CFH alone or CFH and CFHR (group A; n=14), while 30% exhibited rearrangements including only CFHRs (group B; n=6). In group A, 6 patients presented CFH::CFHR1 hybrid genes, 7 patients carried duplications in the CFH-CFHR region that resulted either in the substitution of the last CFHR1 exon(s) with those of CFH (CFHR1::CFH reverse hybrid gene) or in an internal CFH duplication. In group A, the large majority of aHUS acute episodes not treated with eculizumab (12/13) resulted in chronic ESRD; in contrast, anti-complement therapy induced remission in 4/4 acute episodes. aHUS relapse occurred in 6/7 grafts without eculizumab prophylaxis and in 0/3 grafts with eculizumab prophylaxis. In group B, 5 subjects had the CFHR31-5::CFHR410 hybrid gene and one had 4 copies of CFHR1 and CFHR4. Compared with group A, patients in group B exhibited a higher prevalence of additional complement abnormalities and earlier disease onset. However, 4/6 patients in this group underwent complete remission without eculizumab treatment. In secondary forms we identified uncommon SVs in 2 out of 92 patients: the CFHR31-5::CFHR410 hybrid and a new internal duplication of CFH. Discussion: In conclusion, these data highlight that uncommon CFH-CFHR SVs are frequent in primary aHUS and quite rare in secondary forms. Notably, genomic rearrangements involving the CFH are associated with a poor prognosis but carriers respond to anti-complement therapy.

Electrophilic Aromatic Alkylation by Hydroperoxides. Competition between Ionic and Radical Mechanisms with Phenols.

Tertiary hydroperoxides have been utilized for the electrophilic alkylation of activated aromatic substrates, particularly phenols and phenol ethers. Cumyl (1) and tert-butyl (2) hydroperoxides have shown a greatly different behavior as concerns the catalysis and the regioselectivity. The best catalyst for 1 is TiCl(4), which is completely inactive with 2. With the latter an effective catalyst is FeCl(3), which, however, can give rise to a combination of electrophilic and radical reactions with alkyl phenols. 2,2'-Dihydroxy-3,3'-di-tert-butyl-5,5'-dimethyldiphenyl is obtained in high yields from p-cresol.

New Methods of Free-Radical Perfluoroalkylation of Aromatics and Alkenes. Absolute Rate Constants and Partial Rate Factors for the Homolytic Aromatic Substitution by n-Perfluorobutyl Radical.

New methods of free-radical perfluoroalkylation of aromatics and alkenes are reported. n-C(4)F(9)I has been utilized as source of C(4)F(9)(*) radical through iodine abstraction by phenyl or methyl radical. The reaction with alkenes, carried out in the presence of catalytic amount of Cu(OAc)(2), leads to substitution by a mechanism substantially identical to the aromatic substitution and not to the usual chain addition of perfluoroalkyl group and iodine atom to the double bond. This has allowed to measure for the first time the absolute rate constants and the partial rate factors for the homolytic aromatic perfluoroalkylation by competition kinetics. The C(4)F(9)(*) radical shows a clear-cut electrophilic character in the aromatic substitution, as already reported for the addition to alkenes, but the low regio- and chemoselectivities suggest that the polar effect is not the main factor in determining the high reactivity of perfluoroalkyl radicals toward aromatics (10(5)-10(6) M(-1) s(-1), 2-3 orders of magnitude more reactive than alkyl radicals). The enthalpic factor, related to the involved bond energies, appears to be the major cause of the increased reactivity. The polar effect is considered as related more to the polarizability than to the polarity of a radical (the sigma-perfluoroalkyl radicals are considered less polarizable and hence less sensitive to polar effects than pi-radicals).

H-Prune through GSK-3ß interaction sustains canonical WNT/ß-catenin signaling enhancing cancer progression in NSCLC.

H-Prune hydrolyzes short-chain polyphosphates (PPase activity) together with an hitherto cAMP-phosphodiesterase (PDE), the latest influencing different human cancers by its overexpression. H-Prune promotes cell migration in cooperation with glycogen synthase kinase-3 (Gsk-3ß). Gsk-3ß is a negative regulator of canonical WNT/ß-catenin signaling. Here, we investigate the role of Gsk-3ß/h-Prune complex in the regulation of WNT/ß-catenin signaling, demonstrating the h-Prune capability to activate WNT signaling also in a paracrine manner, through Wnt3a secretion. In vivo study demonstrates that h-Prune silencing inhibits lung metastasis formation, increasing mouse survival. We assessed h-Prune levels in peripheral blood of lung cancer patients using ELISA assay, showing that h-Prune is an early diagnostic marker for lung cancer. Our study dissects out the mechanism of action of h-Prune in tumorigenic cells and also sheds light on the identification of a new therapeutic target in non-small-cell lung cancer.

MicroRNA 199b-5p delivery through stable nucleic acid lipid particles (SNALPs) in tumorigenic cell lines.

MicroRNA (miR)-199b-5p has been shown to regulate Hes-1, a downstream effector of the canonical Notch and noncanonical SHH pathways, whereby it impairs medulloblastoma (MB) cancer stem cells (CSCs) through a decrease in the CD133+/CD15+ cell population. Here, we have developed stable nucleic acid lipid particles (SNALPs) that encapsulate miR-199b-5p. The efficacy of the miR-199b-5p delivery by these SNALPs is demonstrated by significant impairment of Hes-1 levels and CSC markers in a range of different tumorigenic cell lines: colon (HT-29, CaCo-2, and SW480), breast (MDA-MB231T and MCF-7), prostate (PC-3), glioblastoma (U-87), and MB (Daoy, ONS-76, and UW-228). After treatment with SNALP miR-199b-5p, there is also impairment of cell proliferation and no signs of apoptosis, as measured by caspases 3/7 activity and annexin V fluorescence cell sorter analyses. These data strengthen the importance of such carriers for miRNA delivery, which show no cytotoxic effects and provide optimal uptake into cells. Thus, efficient target downregulation in different tumorigenic cell lines will be the basis for future preclinical studies.

Extraction, isolation, and purification of analytes from samples of marine origin--a multivariate task.

The development of a multivariate study for a quantitative analysis of six different polybrominated diphenyl ethers (PBDEs) in tissue of Atlantic Salmo salar L. is reported. An extraction, isolation, and purification process based on an accelerated solvent extraction system was designed, investigated, and optimized by means of statistical experimental design and multivariate data analysis and regression. An accompanying gas chromatography-mass spectrometry analytical method was developed for the identification and quantification of the analytes, BDE 28, BDE 47, BDE 99, BDE 100, BDE 153, and BDE 154. These PBDEs have been used in commercial blends that were used as flame-retardants for a variety of materials, including electronic devices, synthetic polymers and textiles. The present study revealed that an extracting solvent mixture composed of hexane and CH2Cl2 (10:90) provided excellent recoveries of all of the six PBDEs studied herein. A somewhat lower polarity in the extracting solvent, hexane and CH2Cl2 (40:60) decreased the analyte %-recoveries, which still remain acceptable and satisfactory. The study demonstrates the necessity to perform an intimately investigation of the extraction and purification process in order to achieve quantitative isolation of the analytes from the specific matrix.

Targeting monocyte chemotactic protein-1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models.

Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-ß and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor-stroma niche microenvironment.

The micro-RNA 199b-5p regulatory circuit involves Hes1, CD15, and epigenetic modifications in medulloblastoma.

Micro-RNA (miR) 199b-5p targets Hes1 in medulloblastoma, one of the downstream effectors of both the canonical Notch and noncanonical Sonic Hedgehog pathways. In medulloblastoma patients, expression of miR-199b-5p is significantly decreased in metastatic cases, thus suggesting a downregulation mechanism. We studied this mechanism, which is mediated mostly by Hes1 and epigenetic promoter modifications. The miR-199b-5p promoter region was characterized, which identified a Hes1 binding site, thus demonstrating a negative feedback loop of regulation. MiR-199b-5p was shown to be downregulated in several medulloblastoma cell lines and in tumors by epigenetic methylation of a cytosine-phosphate-guanine island upstream of the miR-199b-5p promoter. Furthermore, the cluster of differention (CD) carbohydrate antigen CD15, a marker of medulloblastoma tumor-propagating cells, is an additional direct target of miR-199b-5p. Most importantly, regulation of miR-199b-5p expression in these CD15+/CD133+ tumor-propagating cells was influenced by only Hes1 expression and not by any epigenetic mechanism of regulation. Moreover, reverse-phase protein array analysis showed both the Akt and extracellular-signal-regulated kinase pathways as being mainly negatively regulated by miR-199b-5p expression in several medulloblastoma cell lines and in primary cell cultures. We present here the finely tuned regulation of miR-199b-5p in medulloblastoma, underlining its crucial role by its additional targeting of CD15.

The metallophosphodiesterase Mpped2 impairs tumorigenesis in neuroblastoma.

Through microarray analyses, we identified the Mpped2 gene as differentially expressed in two neuroblastoma cell lines induced to differentiation with all-trans retinoic acid. Mpped2 codes for a new metallophosphodiesterase protein, the expression of which inhibits cell proliferation and soft agar colony formation in SH -SY5Y cells. This inhibition is concomitant to an increased proportion of the cells in G0/G1 phase and enhanced caspase 3 activation, effects not seen for the other phosphodiesterases. A Mpped2-null mutation (H67R) abrogates these functions, which indicates that the biochemical activity of Mpped2 is advantageous for cancer suppression. Expression analyses in the "Los Angeles" and "Essen" neuroblastoma gene-array data sets show that increased expression of Mpped2 is associated with good patient prognosis according to Kaplan-Meier analyses. Tumorigenic assays in mice show that overexpression of Mpped2 improves survival rate, substantially impairs tumor growth and induces neuronal differentiation. Altogether, these data show that Mpped2 expression impairs neuroblastoma tumorigenesis, and they establish a basis for future therapeutic applications.

Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue.

Nm23-H1 has been identified as a metastatic suppressor gene in murine melanoma cell lines. Several functions have been attributed to its activity in cancer, including a histidine kinase activity, DNA repair, and regulation of other proteins involved in metastatic formation. While in breast cancer, NM23-H1 overexpression indicates a benign status through impairing progression of disease, its function is opposite in other cancers; e.g., neuroblastoma. To further understand this dichotomy of function in cancer, we have analyzed its function in prostate cancer, in which the relationship between NM23-H1 expression and prognostic state is today controversial. In vitro, overexpression of NM23-H1 in PC3 cells inhibited their cell motility, while downregulation of NM23-H1 expression in these cells by RNA interference showed enhanced cell motility. Immunohistochemistry analysis performed on 346 prostate cancer tissue samples showed a relationship between high levels of NM23-H1 expression in the nuclei of these tumorigenic cells and elevated Gleason score, with high levels of NM23-H1 cytoplasmic staining related to metastatic stage. This retrospective survival study demonstrates that high levels of NM23-H1 expression in the cytoplasm determine recurrence of prostate-specific antigen levels only in those patients with metastatic disease. Our findings suggest a correlation between high levels of NM23-H1 protein in the cytoplasm of the cells and progression of prostate cancer to metastasis, thus definitively identifying NM23-H1 as a new negative prognostic marker in prostate cancer.

MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma.

BACKGROUND: Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+)/CD15(+) tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/-) p53(-/-)), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. CONCLUSIONS/SIGNIFICANCE: Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.