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Keap1 inhibition sensitizes head and neck squamous cell carcinoma cells to ionizing radiation via impaired non-homologous end joining and induced autophagy.

ABSTRACT: The function of Keap1 (Kelch-like ECH-associated protein 1), a sensor of oxidative and electrophilic stress, in the radiosensitivity of cancer cells remains elusive. Here, we investigated the effects of pharmacological inhibition of Keap1 with ML344 on radiosensitivity, DNA double-strand break (DSB) repair and autophagy in head and neck squamous cell carcinoma (HNSCC) cell lines. Our data demonstrate that Keap1 inhibition enhances HNSCC cell radiosensitivity. Despite elevated, Nrf2-dependent activity of non-homologous end joining (NHEJ)-related DNA repair, Keap1 inhibition seems to impair DSB repair through delayed phosphorylation of DNA-PKcs. Moreover, Keap1 inhibition elicited autophagy and increased p62 levels when combined with X-ray irradiation. Our findings suggest HNSCC cell radiosensitivity, NHEJ-mediated DSB repair, and autophagy to be co-regulated by Keap1.

SUBMITTER: Deville SS

PROVIDER: S-EPMC7578798 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Keap1 inhibition sensitizes head and neck squamous cell carcinoma cells to ionizing radiation via impaired non-homologous end joining and induced autophagy.

Deville Sara Sofia SS Luft Susanne S Kaufmann Maria M Cordes Nils N

Cell death & disease 20201021 10

The function of Keap1 (Kelch-like ECH-associated protein 1), a sensor of oxidative and electrophilic stress, in the radiosensitivity of cancer cells remains elusive. Here, we investigated the effects of pharmacological inhibition of Keap1 with ML344 on radiosensitivity, DNA double-strand break (DSB) repair and autophagy in head and neck squamous cell carcinoma (HNSCC) cell lines. Our data demonstrate that Keap1 inhibition enhances HNSCC cell radiosensitivity. Despite elevated, Nrf2-dependent act ...[more]

PMID: 33087706

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Project description:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Experiment Overall Design: Number of plants pooled:20

Project description:Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor radiation response.

Project description:BackgroundDrug resistance limits the treatment effect of cisplatin-based chemotherapy in head and neck squamous cell carcinoma (HNSCC), and the underlying mechanism is not fully understood. The aim of this study was to explore the cause of cisplatin resistance in HNSCC.MethodsWe performed survival and gene set variation analyses based on HNSCC cohorts and identified the critical role of tumor necrosis factor alpha-induced protein 2 (TNFAIP2) in cisplatin-based chemotherapy resistance. Half-maximal inhibitory concentration (IC50) examination, colony formation assays and flow cytometry assays were conducted to examine the role of TNFAIP2 in vitro, while xenograft models in nude mice and 4-nitroquinoline N-oxide (4NQO)-induced HNSCC models in C57BL/6 mice were adopted to verify the effect of TNFAIP2 in vivo. Gene set enrichment analysis (GSEA) and coimmunoprecipitation coupled with mass spectrometry (Co-IP/MS) were performed to determine the mechanism by which TNFAIP2 promotes cisplatin resistance.ResultsHigh expression of TNFAIP2 is associated with a poor prognosis, cisplatin resistance, and low reactive oxygen species (ROS) levels in HNSCC. Specifically, it protects cancer cells from cisplatin-induced apoptosis by inhibiting ROS-mediated c-JUN N-terminal kinase (JNK) phosphorylation. Mechanistically, the DLG motif contained in TNFAIP2 competes with nuclear factor-erythroid 2-related factor 2 (NRF2) by directly binding to the Kelch domain of Kelch-like ECH-associated protein 1 (KEAP1), which prevents NRF2 from undergoing ubiquitin proteasome-mediated degradation. This results in the accumulation of NRF2 and confers cisplatin resistance. Positive correlations between TNFAIP2 protein levels and NRF2 as well as its downstream target genes were validated in HNSCC specimens. Moreover, the small interfering RNA (siRNA) targeting TNFAIP2 significantly enhanced the cisplatin treatment effect in a 4NQO-induced HNSCC mouse model.ConclusionsOur results reveal the antioxidant and cisplatin resistance-regulating roles of the TNFAIP2/KEAP1/NRF2/JNK axis in HNSCC, suggesting that TNFAIP2 might be a potential target in improving the cisplatin treatment effect, particularly for patients with cisplatin resistance.

Dataset Information

Keap1 inhibition sensitizes head and neck squamous cell carcinoma cells to ionizing radiation via impaired non-homologous end joining and induced autophagy.

Publications

Keap1 inhibition sensitizes head and neck squamous cell carcinoma cells to ionizing radiation via impaired non-homologous end joining and induced autophagy.

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