Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19–27 (MGBR2_ex19–27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2,174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage, and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19–27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations.
To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.
Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.
17It has been gradually established that the vast majority of human tumors are extraordinarily 18 heterogeneous at a genetic level. To accurately recapitulate this complexity, it is now evident that 19in vivo animal models of cancers will require to recreate not just a handful of simple genetic 20 alterations, but possibly dozens and increasingly intricate. Here, we have combined the 21 RCAS/TVA system with the CRISPR/Cas9 genome editing tools for precise modeling of human 22 tumors. We show that somatic deletion in neural stem cells (NSCs) of a variety of known tumor 23 suppressor genes (Trp53, Cdkn2a and Pten), in combination with the expression of an oncogene 24 driver, leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of 25 guide RNAs (gRNAs) we generated different gene fusions, either by chromosomal deletion 26 (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk), and we show that they have 27 transforming potential in vitro and in vivo. Lastly, using homology-directed-repair (HDR), we 28 also produced tumors carrying the Braf V600E mutation, frequently identified in a variety of 29 subtypes of gliomas. In summary, we have developed an extremely powerful and versatile mouse 30 model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer 31 models particularly appropriate for pre-clinical testing. 32 33 34All rights reserved. No reuse allowed without permission.
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