B-RAF mutation in non-small cell lung cancer: the sleeping beauty is waking up.
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ABSTRACT:
SUBMITTER: Rolfo C
PROVIDER: S-EPMC5009081 | biostudies-literature | 2016 Aug
REPOSITORIES: biostudies-literature
Publications
B-RAF mutation in non-small cell lung cancer: the sleeping beauty is waking up.
Rolfo Christian C Caparica Rafael R
Translational lung cancer research 20160801 4
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Project description:Lung cancer is the leading cause of cancer-related death globally, with non-small-cell lung cancer (NSCLC) being the predominant subtype. Overall survival remains low for NSCLC patients, and novel targets are needed to improve outcome. Raf-1 is a key component of the Ras/Raf/MEK signalling pathway, but its role and downstream targets in NSCLC are not completely understood. Our previous study indicated a possible correlation between Raf-1 levels and ribosomal protein S6 kinase (p70S6K) function. In this study, we aimed to investigate whether p70S6K is a downstream target of Raf-1 in NSCLC. Raf-1 was silenced in NSCLC cell lines by using small hairpin RNA, and Raf-1 and p70S6K protein levels were measured via Western blot. p70S6K was then overexpressed following Raf-1 knock-down; then, cell proliferation, apoptosis and the cell cycle in NSCLC cell lines were examined. Tumour xenografts with NSCLC cells were then transplanted for in vivo study. Tumours were measured and weighed, and Raf-1 and p70S6K expression, cell proliferation and apoptosis were examined in tumour tissues by Western blot, Ki-67 staining and TUNEL staining, respectively. When Raf-1 was silenced, p70S6K protein levels were markedly decreased in the A549 and H1299 NSCLC cell lines. A significant decrease in NSCLC cell proliferation, a profound increase in apoptosis and cell cycle arrest were observed in vitro following Raf-1 knock-down. Overexpression of p70S6K after Raf-1 depletion effectively reversed these effects. Xenograft studies confirmed these results in vivo. In conclusion, Raf-1 targets p70S6K as its downstream effector to regulate NSCLC tumorigenicity, making Raf-1/p70S6K signalling a promising target for NSCLC treatment.
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Project description:BackgroundSleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells. It is widely thought that SB exclusively integrates into TA dinucleotides. However, this strict TA-preference has not been rigorously tested in large numbers of insertion sites that now can be detected with next generation sequencing. Li et al. found 71 SB insertions in non-TA dinucleotides in 2013, suggesting that TA dinucleotides are not the only sites of SB integration, yet further studies on this topic have not been carried out.ResultsIn this study, we re-analyzed 600 million pairs of Illumina sequence reads from a high-throughput SB mutagenesis screen and identified 28 thousand SB insertions in non-TA sites. We recovered some of these non-TA sites using PCR and confirmed that at least a subset of the insertions at non-TA sites are real integrations. The consensus sequence of these non-TA sites shows an asymmetric pattern distinct from the symmetric pattern of the canonical TA sites. Perfect similarity between the downstream flanking sequence and SB transposon ends indicates there may be interaction between the transposon DNA binding domain of transposase and the target DNA.ConclusionThe TA-preference of SB transposon is not as strict as what people had thought. And the SB integrations at non-TA sites might be guided by the interaction between the transposon DNA binding domain of SB transposase and the target DNA.
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Project description:We have investigated the role of individual members of the Raf/Mek/Erk cascade in the onset of K-Ras oncogene-driven non-small cell lung carcinoma (NSCLC). Ablation of Erk1 or Erk2 in K-Ras oncogene-expressing lung cells had no significant effect due to compensatory activities. Yet, elimination of both Erk kinases completely blocked tumor development. Similar results were obtained with Mek kinases. Ablation of B-Raf had no significant effect on tumor development. However, c-Raf expression was absolutely essential for the onset of NSCLC. Interestingly, concomitant elimination of c-Raf and B-Raf in adult mice had no deleterious consequences for normal homeostasis. These results indicate that c-Raf plays a unique role in mediating K-Ras signaling and makes it a suitable target for therapeutic intervention.
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Project description:Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1-derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).
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2016-07-03 | E-GEOD-43263 | biostudies-arrayexpress
Project description:We extracted RNA of 39 mouse tissue of various genotypes and performed expression microarrays. Subsequently a screen was conducted using the Sleeping Beauty (SB) transposon to identify breast cancer candidate genes.
2016-01-01 | GSE43263 | GEO
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Project description:For appropriate treatment selection, the updated NCCN Guidelines for Non-Small Cell Lung Cancer (NSCLC) recommend broad molecular profiling for all patients with nonsquamous disease. Three different tyrosine kinase inhibitors (TKIs) are recommended as first-line treatment of EGFR mutation-positive NSCLC: gefitinib, erlotinib, and afatinib. Most patients whose disease responds will still experience progression, and the type of disease progression drives management. Systemic progression requires switching TKI treatment, whereas patients with oligoprogression and central nervous system progression may have their new lesions treated but continue on their TKI. A new third-generation TKI has been approved and others are currently under development, and new combinations of these drugs with a VEGFR inhibitor offer promise to improve outcomes.
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Project description:The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR V? and TCR V? repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).
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