Immunogenicity of neuroblastoma tumors is controlled by impaired activity of NF-kB and IRF1 transcription factors.

La maggior parte dei tumori riescono ad evadere il sistema immunitario inibendo l’espressione di antigeni tumorali associati alle molecole del complesso maggiore di istocompatibilità di classe I (MHC I) sulla superficie cellulare. La mancata espressione di questi complessi è spesso dovuta alla presenza di difetti strutturali dei geni codificanti le molecole MHC I, oppure all’aberrante espressione delle molecole responsabili del processamento degli antigeni legati alle molecole MHC I. Il neuroblastoma (NB), il tumore extracraniale solido più comune dell’infanzia, non è un’eccezione. Sia la maggior parte delle linee cellulari di NB, che i tumori primari esprimono bassi, se non nulli, livelli di MHC I che possono essere aumentati trattando le cellule con l’interferone-gamma (IFN-γ). Questo fenotipo è compatibile con la presenza di difetti nella regolazione trascrizionale delle molecole coinvolte nel processamento e nella presentazione dell’antigene. Il presente studio ha lo scopo di indagare il meccanismo molecolare che determina la mancata o ridotta espressione delle molecole MHC I, e delle due aminopeptidasi del reticolo endoplasmatico ERAP1 ed ERAP2. Le forme più aggressive di NB sono caratterizzate dall’amplificazione dell’oncogene MYCN. Sebbene una correlazione inversa tra l’espressione di MYCN le molecole MHC I nelle linee cellulari umane di NB sia stata riportata, un coinvolgimento diretto di MYCN nella regolazione di MHC I non è stato dimostrato. I nostri risultati dimostrano che MYCN non è responsabile dei bassi livelli di MHC I, ERAP1 ed ERAP2 nelle cellule di NB analizzate, infatti la loro espressione non è influenzata nè dalla forzata espressione ne dall’inibizione di MYCN. Abbiamo invece identificato due fattori di trascrizione, NF-kB e IRF1, che sono direttamente coinvolti nella regolazione delle proteine MHC I ed ERAPs. Mediante il saggio di immunoprecipitazione della cromatina abbiamo dimostrato che il reclutamento di p65 (una subunità di NF-kB) sui promotori di MHC I, ERAP1 ed ERAP2 è direttamente proporzionale all’espressione di questi geni. Inoltre, il fenotipo negativo per MHC I, ERAP1 ed ERAP2, caratteristico delle forme più aggressive di NB, coincide con una bassa attività nucleare di NF-kB ed IRF1. L’overespressione dei due fattori trascrizionali da soli è in grado di recuperare solo parzialmente l’espressione di MHC I, ERAP1 ed ERAP2, inoltre il risultato dipende dalla linea cellulare trasfettata. Comunque, la trasfezione contemporanea di NF-kB ed IRF1 produce un incremento sinergico dei geni target in tutte le linee trasfettate. Degno di nota è il fatto che l’espressione di p65 nei tumori primari di neuroblastoma è simile a quella osservata nelle linee cellulari. Infatti, solo le cellule gangliari, ovvero le cellule più differenziate presenti nel tessuto tumorale, esprimono sia l’MHC I che il p65 nucleare. Quindi, questo studio mette in luce il meccanismo molecolare responsabile della mancata espressione delle molecole MHC I, ERAP1 ed ERAP2 nei tumori di neuroblastoma più aggressivi fornendo un importante punto di partenza per lo sviluppo di protocolli immunoterapeutici più efficaci basati sull’utilizzo delle cellule T.Low expression of major histocompatibility complex class I (MHC I) molecules on the cell surface allows tumors to evade the host T cell-based immune response. These abnormalities are often related to either genetic defects of MHC I genes or aberrant expression of antigen processing machinery (APM) components. Neuroblastoma (NB), the most common solid extracranial cancer of childhood, is not an exception. MHC I surface expression is virtually undetectable in the most NB cell lines and primary tumors, and upregulated by gamma-interferon (IFN-γ). This phenotype is compatible with defects in the regulation of antigen processing and presentation components. In this study, the molecular mechanism underlying low immunogenicity in neuroblastoma was investigated. Amplification of the MYCN oncogene characterizes the most aggressive forms of NB and is believe to downregulate expression of MHC class I molecules. Although an inverse correlation between MYCN and MHC I has been reported in human NB cell lines, a direct demonstration of the MYCN-mediated down-regulation of MHC I expression has been questioned. Herein, we demonstrate that MYCN is not responsible for low MHC I, ERAP1 and ERAP2 protein levels in human NB cell lines, since their expression is not affected by neither transfection-mediated overexpression nor siRNA suppression of MYCN. Instead, we identified NF-kB and IRF1 as the main factors involved in the transcriptional regulation of MHC I and ERAPs proteins. By chromatin immunoprecipitation assay, we show a recruitment of p65 NF-kB to the MHC I, ERAP1 and ERAP2 promoters that is proportional with the expression of these genes. Moreover, low nuclear activity of both NF-kB and IRF1 factors correlated with the MHC I, ERAP1 and ERAP2-low phenotype of the most aggressive NB cell lines. Overexpression of either the transcription factors alone rescued the MHC I, ERAP1 and ERAP2-low phenotype, but only partially and in a cell-type depending manner. Important, the co-transfection of both NF-kB and IRF1 cooperated to strongly enhance the transactivation of MHC I, ERAP1 and ERAP2 in any cell lines. Notheworthy, NF-kB and IRF1 acted in a synergistic manner. We found an intriguing parallel in primary NB tumors, in fact, nuclear p65 was detected in the maturing neuroblastic cells (i.e. ganglionic cells) which express higher levels of MHC I molecules in human NB specimens. These findings provide molecular insight into defective MHC I expression in NB tumors and indicate that activating NF-kB and IRF1 in MHC I-low, aggressive NB cells could be instrumental for successful application of T cell-based immunotherapy

A minimal promoter for TFIIIC-dependent in vitro transcription of snoRNA and tRNA genes by RNA polymerase III.

The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA(Asn)(GTT) gene family, but not for two tRNA(Pro)(AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA(Asn) genes, but only in the presence of TFIIIB reconstituted with a crude B'' fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA(Asn) genes was strongly influenced by their 5'-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIIC-interacting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription

Oncogenic EGFR Represses the TET1 DNA Demethylase to Induce Silencing of Tumor Suppressors in Cancer Cells

SummaryOncogene-induced DNA methylation-mediated transcriptional silencing of tumor suppressors frequently occurs in cancer, but the mechanism and functional role of this silencing in oncogenesis are not fully understood. Here, we show that oncogenic epidermal growth factor receptor (EGFR) induces silencing of multiple unrelated tumor suppressors in lung adenocarcinomas and glioblastomas by inhibiting the DNA demethylase TET oncogene family member 1 (TET1) via the C/EBPα transcription factor. After oncogenic EGFR inhibition, TET1 binds to tumor suppressor promoters and induces their re-expression through active DNA demethylation. Ectopic expression of TET1 potently inhibits lung and glioblastoma tumor growth, and TET1 knockdown confers resistance to EGFR inhibitors in lung cancer cells. Lung cancer samples exhibited reduced TET1 expression or TET1 cytoplasmic localization in the majority of cases. Collectively, these results identify a conserved pathway of oncogenic EGFR-induced DNA methylation-mediated transcriptional silencing of tumor suppressors that may have therapeutic benefits for oncogenic EGFR-mediated lung cancers and glioblastomas

Immunopurification of Pathological Prion Protein Aggregates

Background: Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP molecules. A great deal of effort has been devoted to developing protocols for purifying PrP for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases. Principal Findings: We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP and mutant PrP aggregates at electrophoretic homogeneity. PrP purified from prion-infected mice was able to seed misfolding of PrP in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons. Significance: The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates

Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma