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Targeted Profiling of Epitranscriptomic Reader, Writer and Eraser Proteins in Radioresistant Breast Cancer Cells

ABSTRACT: Epitransciptomic reader, writer and eraser (RWE) proteins recognize, install, and remove modified nucleosides in RNA, which are known to play essential roles in RNA processing, splicing, and stability. Here, we established a liquid chromatography–parallel reaction monitoring (LC-PRM) method for high-throughput profiling of a total of 152 epitransciptomic RWE proteins, including 68 human epitranscriptomic RWE proteins deposited in the Modomics database. To our knowledge, this is the first report of quantifying a comprehensive list of epitranscriptomic RWE proteins using LC-PRM. To access the roles of epitranscriptomic RWE proteins in breast cancer radioresistance, we applied the LC-PRM method, in conjunction with stable isotope labelling by amino acids in cell culture (SILAC), to quantify these proteins in two pairs of matched parental/radioresistant breast cancer cells (i.e., MDA-MB-231 and MCF-7 cells, and their radioresistant counterparts, i.e., C5 and C6 cells). We found that eight epitranscriptomic RWE proteins were commonly altered by over 1.5-fold in the two pairs of matched breast cancer cells, from which TRMT1 (an m2,2G writer) may play a role in promoting breast cancer radioresistance due to its clinical relevance and correlation with DNA repair gene sets. Furthermore, we envision that the LC-PRM method is applicable for studying the roles of epitranscriptomic RWE proteins in the metastatic transformation of cancer and therapeutic resistance of other types of cancer.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tianyu Qi

LAB HEAD: Yinsheng Wang

PROVIDER: PXD030387 | Pride | 2022-08-15

REPOSITORIES: Pride

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Project description:Withaferin A isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to concentrations of Withaferin A (WA) which can be detected systemically in in vivo experiments. Whereas WA revealed attenuation of multiple cancer hallmarks the withanolide analogue Withanone (WN), did not exert any of the described effects in these cells at comparable concentrations. Pathway enrichment analysis identified that WA targeted relevant cancer programs related to cell death, cell cycle and proliferation, which could functionally be validated flow cytometrically and by real-time proliferation xCELLigence assay. With respect to cell motility, invasion and metastasis processes, WA was found to strongly decrease MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This is further supported by gene expression changes which demonstrate increased expression of a validated breast cancer metastasis suppressor gene (BRMS1) and concomitant decreased expression of extracellular matrix-degrading proteases (PLAU, PLAT, ADAM8), cell adhesion molecules (integrins, laminins) as well as pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R). Subsequent in silico network analysis listed key node transcription factors regulating these processes (p53, E2F1, CDKN2A, NRF2) and revealed the role of chromatin modifying enzymes (p300, JARID1B) as central master switches, linking multiple anticancer activities of WA. Furthermore, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking in account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy resistant triple negative breast cancer, WA based therapeutic strategies hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer. Total RNA from 2 different cell lines treated with WA, are compared to their untreated states

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	C5_F_a-inj2-upd.raw	Raw
	C5_F_a-inj3-upd.raw	Raw
	C5_F_b-inj1-upd.raw	Raw
	C5_F_b-inj2-upd.raw	Raw

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Targeted Profiling of Epitranscriptomic Reader, Writer and Eraser Proteins in Radioresistant Breast Cancer Cells

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