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Tyrosine kinase inhibitors protect the salivary gland from radiation damage by increasing DNA double-strand break repair.

ABSTRACT: We have previously shown that the tyrosine kinase inhibitors (TKIs) dasatinib and imatinib can protect salivary glands from irradiation (IR) damage without impacting tumor therapy. However, how they induce this protection is unknown. Here we show that TKIs mediate radioprotection by increasing the repair of DNA double-stranded breaks. DNA repair in IR-treated parotid cells, but not oral cancer cells, occurs more rapidly following pretreatment with imatinib or dasatinib and is accompanied by faster formation of DNA damage-induced foci. Similar results were observed in the parotid glands of mice pretreated with imatinib prior to IR, suggesting that TKIs "prime" cells for DNA repair. Mechanistically, we observed that TKIs increased IR-induced activation of DNA-PK, but not ATM. Pretreatment of parotid cells with the DNA-PK inhibitor NU7441 reversed the increase in DNA repair induced by TKIs. Reporter assays specific for homologous recombination (HR) or nonhomologous end joining (NHEJ) verified regulatation of both DNA repair pathways by imatinib. Moreover, TKIs also increased basal and IR-induced expression of genes associated with NHEJ (DNA ligase 4, Artemis, XLF) and HR (Rad50, Rad51 and BRCA1); depletion of DNA ligase 4 or BRCA1 reversed the increase in DNA repair mediated by TKIs. In addition, TKIs increased activation of the ERK survival pathway in parotid cells, and ERK was required for the increased survival of TKI-treated cells. Our studies demonstrate a dual mechanism by which TKIs provide radioprotection of the salivary gland tissues and support exploration of TKIs clinically in head and neck cancer patients undergoing IR therapy.

SUBMITTER: Affandi T

PROVIDER: S-EPMC7973138 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background Triple-negative breast cancer (TNBC) is associated with aggressiveness and a poor prognosis. Besides surgery, radiotherapy serves as the major treatment modality for TNBC. However, response to radiotherapy is limited in many patients, most likely because of DNA damage response (DDR) signaling mediated radioresistance. Y-box binding protein-1 (YB-1) is a multifunctional protein that regulates the cancer hallmarks among them resisting to radiotherapy-induced cell death. Fisetin, is a plant flavonol of the flavonoid family of plant polyphenols that has anticancer properties, partially through inhibition of p90 ribosomal S6 kinase (RSK)-mediated YB-1 phosphorylation. The combination of fisetin with radiotherapy has not yet been investigated. Methods Activation status of the RSK signaling pathway in total cell lysate and in the subcellular fractions was analyzed by Western blotting. Standard clonogenic assay was applied to test post-irradiation cell survival. γH2AX foci assay and 3 color fluorescence in situ hybridization analyses were performed to study frequency of double-strand breaks (DSB) and chromosomal aberrations, respectively. The underlying repair pathways targeted by fisetin were studied in cells expressing genomically integrated reporter constructs for the DSB repair pathways via quantifying the expression of green fluorescence protein by flow cytometry. Flow cytometric quantification of sub-G1 cells and the protein expression of LC3-II were employed to measure apoptosis and autophagy, respectively. Kinase array and phosphoproteomics were performed to study the effect of fisetin on DDR response signaling. Results We showed that the effect of fisetin on YB-1 phosphorylation in TNBC cells is comparable to the effect of the RSK pharmacological inhibitors. Similar to ionizing radiation (IR), fisetin induces DSB. Additionally, fisetin impairs repair of IR-induced DSB through suppressing the classical non-homologous end-joining and homologous recombination repair pathways, leading to chromosomal aberration as tested by metaphase analysis. Effect of fisetin on DSB repair was partially dependent on YB-1 expression. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. Conclusion Fisetin acts as a DSB-inducing agent and simultaneously inhibits repair of IR-induced DSB. Thus, fisetin may serve as an effective therapeutic strategy to improve TNBC radiotherapy outcome. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-022-02442-x.

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Tyrosine kinase inhibitors protect the salivary gland from radiation damage by increasing DNA double-strand break repair.

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