Project description:Aims: Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi receptor tyrosine kinase inhibitor (TKI), has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored. Methods and Results: We performed lncRNA expression profiling to identify potential lncRNAs that are dysregulated in human induced pluripotent stem cells-derived ECs (iPSC-ECs) treated with sunitinib. We show that the lncRNA hyaluronan synthase 2 antisense 1 (HAS2-AS1) is significantly diminished in sunitinib-treated iPSC-ECs. Sunitinib was found to downregulate HAS2-AS1 by an epigenetic mechanism involving hypermethylation. Depletion of HAS2-AS1 recapitulated sunitinib-induced detrimental effects on iPSC-ECs, whereas CRISPR-mediated activation of HAS2-AS1 reversed sunitinib-induced dysfunction. We confirm that HAS2-AS1 stabilizes the expression of its sense gene HAS2 via an RNA/mRNA heteroduplex formation. Knockdown of HAS2-AS1 led to reduced synthesis of hyaluronic acid (HA) and upregulation of ADAMTS5, an enzyme involved in extracellular matrix degradation, resulting in disruption of the endothelial glycocalyx which is critical for ECs. In vivo, sunitinib-treated mice showed reduced coronary flow reserve, accompanied by a reduction in has2os and degradation of the endothelial glycocalyx. Finally, we identify that treatment with high molecular-weight HA can prevent the deleterious effects of sunitinib both in vitro and in vivo by preserving the endothelial glycocalyx. Conclusions: Our findings highlight the importance of lncRNA-mediated regulation of the endothelial glycocalyx as an important determinant of sunitinib-induced vascular toxicity and reveal potential novel therapeutic avenues to attenuate sunitinib-induced vascular dysfunction.

Project description:Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.

			Action	DRS
	SRR22025512_1.fastq.gz	Fastqsanger.gz
	SRR22025512_2.fastq.gz	Fastqsanger.gz
	SRR22025513_1.fastq.gz	Fastqsanger.gz
	SRR22025513_2.fastq.gz	Fastqsanger.gz
	SRR22025514_1.fastq.gz	Fastqsanger.gz

Dataset Information

Modulation of lncRNA links endothelial glycocalyx to vascular dysfunction of tyrosine kinase inhibitors.

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets