BackgroundEpidermal growth factor receptor (EGFR) mutations enable constitutive active downstream signaling of PI3K/AKT, KRAS/ERK and JAK/STAT pathways, and promote tumor progression by inducing uncontrolled proliferation, evasion of apoptosis and migration of non-small cell lung cancer (NSCLC). In addition, such EGFR mutations increase the susceptibility of patients with NSCLC to tyrosine kinase inhibitor (TKI) therapy, but treated patients will invariably relapse with resistant disease. A global understanding of underlying molecular mechanisms of EGFR signaling may improve the management of NSCLC patients.Methodsmicroarray analysis was performed to identify PI3K/AKT-regulated miRNAs. Phosphoproteomic analysis and cell based assays were performed using NSCLC cell lines lentivirally transduced with anti-miR or miR overexpressing constructs.ResultsHere, we show that 17 miRNAs including members of the miR-17~ 92 cluster are dysregulated following PI3K/AKT inhibition of EGFR mutant NSCLC cells. Bioinformatics analysis revealed that dysregulated miRNAs act in a concerted manner to enhance the activity of the EGFR signaling pathway. These findings were closely mirrored by attenuation of miR-17~ 92 family member miR-19b in NSCLC cell lines which resulted in reduced phosphorylation of ERK, AKT and STAT and effector proteins in EGFR mutant NSCLC cells. Consistent with this finding, cell cycle progression, clonogenic growth and migration were reduced and apoptosis was enhanced. Co-treatment of NSCLC cells with the tyrosine kinase inhibitor (TKI) gefitinib and anti-miR-19b construct reduced migration and clonogenic growth in a synergistic manner suggesting that EGFR and miR-19b act together to control oncogenic processes. Serine/threonine phosphatase PP2A subunit PPP2R5E and BCL2L11 encoding BIM were identified as major targets of miR-19b by target validation assays. Consistent with this finding, PP2A activity was strongly enhanced in NSCLC transduced with anti-miR-19b construct, but not in cells co-transduced with anti-miR-19b and shPPP2R5E, suggesting that PPP2R5E is a major constituent of the PP2A complex. Accordingly, enhanced proliferation by miR-19b was due to targeting PPP2R5E. In contrast, apoptosis resistance was mainly due to targeting BCL2L11.ConclusionOur results provide insight into the importance of targeting PPP2R5E and BCL2L11 by miR-19b in oncogenic processes of NSCLC. Attenuation of miR-19b expression could potentially be exploited in adjuvant therapy of EGFR mutant NSCLC.Electronic supplementary materialThe online version of this article (10.1186/s12943-018-0781-5) contains supplementary material, which is available to authorized users.
Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.
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