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1.
Nat Commun ; 11(1): 5060, 2020 10 08.
Article in English | MEDLINE | ID: mdl-33033246

ABSTRACT

Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.


Subject(s)
CRISPR-Cas Systems/genetics , Neoplasms/genetics , Oncogene Fusion/genetics , Animals , Base Sequence , Cell Line, Tumor , Cell Proliferation/genetics , Doxorubicin/therapeutic use , Fusion Proteins, bcr-abl/genetics , Gene Deletion , Genetic Loci , Genomic Instability , HEK293 Cells , Humans , Introns/genetics , Mice, Nude , Neoplasms/drug therapy , Neoplasms/pathology , Oncogene Proteins, Fusion/genetics , RNA, Guide, Kinetoplastida/metabolism , Reproducibility of Results , Xenograft Model Antitumor Assays
2.
Oncogene ; 30(7): 854-64, 2011 Feb 17.
Article in English | MEDLINE | ID: mdl-20956942

ABSTRACT

The thyroid hormone receptor (TR) is a suppressor of ras-mediated responses. To characterize the receptor domains involved in this function, we analyzed a panel of TRß1 mutants for their ability to interfere with ras-driven cyclin D1 activation, formation of transformation foci and tumor growth in nude mice. Our results show that the domains and mechanisms responsible for the anti-transforming and anti-tumorigenic actions of the receptor are divergent from those operating in classical T3-dependent transcriptional activation. TRß1 mutants that do not bind coactivators and do not transactivate retained the capacity of suppressing cellular transformation and tumor growth, whereas selective mutations in the hinge region affecting corepressors recruitment abolished these actions, while preserving ligand-dependent transcription. There was a strict parallelism between anti-transforming activity of the various mutants and their ability to antagonize cyclin D1 stimulation by ras, indicating that transrepression mechanisms may have an important function in suppression of the transforming effects of the oncogene by TRß1. The inhibitory action of T3 on transformation was further enhanced after over-expression of corepressors, while corepressor depletion by means of small-interference RNA reversed significantly hormonal action. This shows an important functional role of endogenous corepressors in suppression of ras-mediated transformation and tumorigenesis by TRß1.


Subject(s)
Cell Transformation, Neoplastic/genetics , Co-Repressor Proteins/metabolism , Genes, ras , Protein Interaction Domains and Motifs , Thyroid Hormone Receptors beta/metabolism , Animals , Co-Repressor Proteins/genetics , Cyclin D1/metabolism , Hep G2 Cells , Humans , Mice , Mice, Nude , Mutation , RNA, Small Interfering/metabolism , Thyroid Hormone Receptors beta/genetics , Triiodothyronine/metabolism
3.
J Biol Chem ; 275(2): 1307-14, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10625678

ABSTRACT

The thyroid hormone (T3) blocks proliferation and induces differentiation of neuroblastoma N2a-beta cells that express the thyroid hormone receptor (TR) beta1 isoform. c-Myc is required for cell cycle progression, and this study shows that T3-induced neuronal differentiation is preceded by a rapid decrease of c-myc gene expression. A negative T3 responsive element (TRE), arranged as an inverted palindrome spaced by three nucleotides, has been identified within the first exon between nucleotides +237 and +268. The TRE is adjacent to the binding site for the transcriptional repressor CCCTC binding factor and maps precisely within the region of RNA polymerase II pausing and release, suggesting a direct implication of TR on premature termination of transcription. Furthermore, the TRE confers repression by T3 to an heterologous promoter only when inserted downstream of the transcription initiation site. Binding of CCCTC binding factor and TR to their cognate sites in the region of transcriptional attenuation, as well as direct interactions between both factors, could facilitate the formation of a repressor complex and the inhibition of c-myc gene expression. These studies provide insight into mechanisms by which TR mediate transcriptional repression and contribute to the understanding of the important effects of thyroid hormones on growth and differentiation of neuronal cells.


Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Genes, myc , Promoter Regions, Genetic/drug effects , Receptors, Thyroid Hormone/physiology , Transcription, Genetic , Triiodothyronine/pharmacology , Animals , Base Sequence , Cell Differentiation/drug effects , Humans , Mice , Neuroblastoma , Neurons/cytology , RNA, Messenger/genetics , Recombinant Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Transcription, Genetic/drug effects , Transfection , Tumor Cells, Cultured
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