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P97/VCP inhibition radiosensitises cancer cells through excessive MRE11-dependent DNA end resection

ABSTRACT: Cancer cells are under constant proteotoxic stress and have an increased genomic instability. They therefore rely on upregulation of protein homeostasis and the DNA damage response (DDR) for survival. The ubiquitin-dependent ATPase p97 is a central component of the ubiquitin-proteasome degradation system (UPS), involved in both protein homeostasis and DDR. p97 is overexpressed in many tumours and its overexpression correlates with poor prognosis in patients. To explore the concept of cancer cell killing by combined targeting of DDR and proteostasis, we induced DNA damage by ionising radiation (IR) and inhibited proteostasis by CB-5083, a specific small molecule inhibitor of p97, in bladder cancer cell lines and a mouse xenograft model. Combined therapy induced radiosensitivity and supressed xenograft tumour growth. Mechanistically, inactivation of the p97 ATPase activity results in proteotoxic accumulation of the nuclease MRE11 at the sites of IR-induced DNA lesions. This leads to excessive formation of single-stranded DNA (ssDNA), inhibition of HR, reliance on SSA for DSB repair and synthetic lethality after IR exposure in non-homologous end-joining-defective tumour cells such as those in muscle-invasive bladder carcinoma patients

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Urinary Bladder Cancer

SUBMITTER: iolanda Vendrell

LAB HEAD: Anne E Kiltie

PROVIDER: PXD016279 | Pride | 2021-05-18

REPOSITORIES: Pride

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			Action	DRS
	OTE0365_JudeNicholson_1_MRE11-HA_F162229.xml	Xml
	OTE0365_JudithNicholson_20150126_MRE_HA_1.mgf	Mgf
	OTE0365_JudithNicholson_20150126_MRE_HA_1.raw	Raw

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Project description:DNA damage response (DDR) is a complex process, essential for cell survival. Especially deleterious type of DNA damage are DNA double-strand breaks (DSB), which can lead to genomic instability and malignant transformation if not repaired correctly. The central player in DSB detection and repair is the ATM kinase which orchestrates the action of several downstream factors. Despite substantial knowledge of DNA repair processes, still several aspects of DNA damage detection and signaling are not fully understood. Recent studies have suggested that long non-coding RNAs (lncRNAs) are involved in DDR. Here, we aimed to identify lncRNAs induced upon DNA damage in an ATM-dependent manner. 7 mRNAs and 10 lncRNAs were significantly induced 1h after ionizing radiation (IR) in healthy donors, whereas none in AT patients. 447 mRNAs and 149 lncRNAs were induced 8h after IR in the control group, while only 100 mRNAs and 3 lncRNAs in AT patients. Among IR-induced mRNAs, we found several genes with well-known functions in DDR. Gene Set Enrichment Analysis and Gene Ontology revealed delayed induction of key DDR pathways in AT patients compared to controls. The induction and dynamics of selected 9 lncRNAs were confirmed by RT-qPCR at several time-points after IR on a larger cohort. Moreover, inhibition of ATM with a specific inhibitor (KU-60019) proved that those lncRNAs are dependent on ATM. Some of the detected lncRNAs are localized next to protein-coding genes involved in DDR. We observed that induction of lncRNAs after IR preceded changes in expression of adjacent genes. This indicates that IR-induced lncRNAs may regulate the transcription of nearby genes. Subcellular fractionation into chromatin, nuclear, and cytoplasmic fractions revealed that the majority of studied lncRNAs are localized in chromatin, which further suggests their role in regulation of gene expression. In summary, our study revealed several lncRNAs induced by IR in an ATM-dependent manner. Their co-localization and co-expression with genes involved in DDR suggest that those lncRNAs may be important players in cellular response to DNA damage.

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P97/VCP inhibition radiosensitises cancer cells through excessive MRE11-dependent DNA end resection

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