Christer Einvik

Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma

Background:MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation.Methods:A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3′-untranslated region (3′UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3′UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line.Results:The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells.Conclusion:The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.

Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma

BackgroundNeuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers.ResultsIn this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti-MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced.ConclusionWe have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.

Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment.

Two group I ribozymes with different functions in a nuclear rDNA intron.

DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self‐splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS‐1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self‐splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS‐1. Much of the P2 stem and all of the associated loop could be deleted without effect on self‐splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5′ splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1‐mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron‐encoded endonuclease I‐DirI.

Group I twintrons : Genetic elements in myxomycete and schizopyrenid amoeboflagellate ribosomal DNAs

Protists are unicellular eukaryotes which represent a significant fraction of the global biodiversity. The myxomycete Didymium and the schizopyrenid amoeboflagellate Naegleria are distantly related protists. However, we have noted several striking similarities in life cycle, cell morphology, and ribosomal DNA organization between these organisms. Both have multicopy nuclear extrachromosomal ribosomal DNAs. Here the small subunit ribosomal RNA genes are interrupted by an optional group I twintron, a novel category among the group I introns. Group I twintrons are mobile self-splicing introns of 1.3-1.4 kb in size, with a complex organization at the RNA level. A group I twintron consists of two distinct ribozymes (catalytic RNAs) with different functions in RNA processing, and an open reading frame encoding a functional homing endonuclease--all with prospects of application as molecular tools in biotechnology. Updated RNA secondary structure models of group I twintrons, as well as an example of in vitro ribozyme activity, are presented. We suggest that the group I twintrons have been independently established in myxomycetes and schizopyrenid amoeboflagellates by horizontal gene transfer due to a combination of the phagocytotic behavior in natural environments and the extrachromosomal multicopy nature of ribosomal DNA.

N-myc and Noncoding RNAs in Neuroblastoma

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Amplification and overexpression of the MYCN proto-oncogene occurs in approximately 20% of neuroblastomas and is associated with advanced stage disease, rapid tumor progression, and poor prognosis. MYCN encodes the transcriptional regulator N-myc, which has been shown to both up- and downregulate many target genes involved in cell cycle, DNA damage, differentiation, and apoptosis in neuroblastoma. During the last years, it has become clear that N-myc also modulates the expression of several classes of noncoding RNAs, in particular microRNAs. MicroRNAs are the most widely studied noncoding RNA molecules in neuroblastoma. They function as negative regulators of gene expression at the posttranscriptional level in diverse cellular processes. Aberrant regulation of miRNA expression has been implicated in the pathogenesis of neuroblastoma. While the N-myc protein is established as an important regulator of several miRNAs involved in neuroblastoma tumorigenesis, tumor suppressor miRNAs have also been documented to repress MYCN expression and inhibit cell proliferation of MYCN-amplified neuroblastoma cells. It is now becoming increasingly evident that N-myc also regulates the expression of long noncoding RNAs such as T-UCRs and ncRAN. This review summarizes the current knowledge about the interplay between N-myc and noncoding RNAs in neuroblastoma and how this contributes to neuroblastoma tumorigenesis. Mol Cancer Res; 10(10); 1243–53. ©2012 AACR.

Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines

Conditional expression of short hairpin RNAs (shRNAs) to knock down target genes is a powerful tool to study gene function. The most common inducible expression systems are based on tetracycline-regulated RNA polymerase III promoters. During the last years, several tetracycline-inducible U6 and H1 promoter variants have been reported in different experimental settings showing variable efficiencies. In this study, we compare the most common variants of these promoters in several mammalian cell lines. For all cell lines tested, we find that several inducible U6 and H1 promoters containing single tetracycline operator (tetO) sequences show high-transcriptional background in the non-induced state. Promoter variants containing two tetO sequences show tight suppression of transcription in the non-induced state, and high tet responsiveness and high gene knockdown efficiency upon induction in all cell lines tested. We report a variant of the H1 promoter containing two O2-type tetO sequences flanking the TATA box that shows little transcriptional background in the non-induced state and up to 90% target knockdown when the inducer molecule (dox–doxycycline) is added. This inducible system for RNAi-based gene silencing is a good candidate for use both in basic research on gene function and for potential therapeutic applications.

Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells

BACKGROUND Neuroblastoma is a malignant childhood tumour arising from precursor cells of the sympathetic nervous system. Genomic amplification of the MYCN oncogene is associated with dismal prognosis. For this group of high-risk tumours, the induction of tumour cell differentiation is part of current treatment protocols. MicroRNAs (miRNAs) are small non-coding RNA molecules that effectively reduce the translation of target mRNAs. MiRNAs play an important role in cell proliferation, apoptosis, differentiation and cancer. In this study, we investigated the role of N-myc on miRNA expression in MYCN-amplified neuroblastoma. We performed a miRNA profiling study on SK-N-BE (2) cells, and determined differentially expressed miRNAs during differentiation initiated by MYCN knockdown, using anti-MYCN short-hairpin RNA (shRNA) technology. RESULTS Microarray analyses revealed 23 miRNAs differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and down-regulated, respectively. Among the down-regulated miRNAs, we found several members of the mir-17 family of miRNAs. Mir-21, an established oncomir in a variety of cancer types, became strongly up-regulated upon MYCN knockdown and the subsequent differentiation. Neither overexpression of mir-21 in the high-MYCN neuroblastoma cells, nor repression of increased mir-21 levels during MYCN knockdown-mediated differentiation had any significant effects on cell differentiation or proliferation. CONCLUSIONS We describe a subset of miRNAs that were altered during the N-myc deprived differentiation of MYCN-amplified neuroblastoma cells. In this context, N-myc acts as both an activator and suppressor of miRNA expression. Mir-21 was up-regulated during cell differentiation, but inhibition of mir-21 did not prevent this process. We were unable to establish a role for this miRNA during differentiation and proliferation of the two neuroblastoma cell lines used in this study.

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes.

Twin‐ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre‐lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P.