Unknown,Transcriptomics,Genomics,Proteomics

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RNAseq of the two isogenic variants of the CAL27 head-and-neck cancer cell line with wild-type or mutated CYP1B1 genotype

ABSTRACT: In order to understand the role of CYP1B1 and of its common polymorphism V432L, we characterized head-and-neck squamous cell carcinoma (HNSCC) cell lines that do not express this cytochrome, and we generated isogenic derivatives of these lines expressing similar levels of the two variant forms of CYP1B1. Our study reveals that the variant (L432) CYP1B1 form is a strong enhancer of cell proliferation, both in vitro and in vivo, and of migration and invasion capacity in vitro. It is also associated to resistance to DNA-damaging agents in vitro and in vivo. Transcriptome analysis by RNA-seq as well as protein blots showed that the cells expressing the variant CYP1B1 genotype presented an accentuated epithelial character as compared to those expressing the wild-type form, in agreement with in silico data extracted from cell line collections. The variant CYP1B1 cell cultures are enriched in ALDH-positive cells as compared to wild-type CYP1B1 cell cultures, which could be related the phenotypic differences detected. Finally, in order to identify associations between germline CYP1B1 polymorphism and patients’ survival), a clinical study was done, including in advanced HNSCC patients treated with chemotherapy and cetuximab. This prospective study revealed that the variant CYP1B1 genotype was associated with poor prognosis and was confirmed in a retrospective validation study.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Jacques ROBERT

PROVIDER: E-MTAB-8512 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.

Dataset Information

RNAseq of the two isogenic variants of the CAL27 head-and-neck cancer cell line with wild-type or mutated CYP1B1 genotype

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