DataSheet_1_Nuclear Receptor PXR Confers Irradiation Resistance by Promoting DNA Damage Response Through Stabilization of ATF3.docx

Low response rate to radiotherapy remains a problem for liver and colorectal cancer patients due to inappropriate DNA damage response in tumors. Here, we report that pregnane X receptor (PXR) contributes to irradiation (IR) resistance by promoting activating transcription factor 3 (ATF3)-mediated ataxia-telangiectasia-mutated protein (ATM) activation. PXR stabilized ATF3 protein by blocking its ubiquitination. PXR–ATF3 interaction is required for regulating ATF3, as one mutant of lysine (K) 42R of ATF3 lost binding with PXR and abolished PXR-reduced ubiquitination of ATF3. On the other hand, threonine (T) 432A of PXR lost binding with ATF3 and further compromised ATM activation. Moreover, the PXR–ATF3 interaction increases ATF3 stabilization through disrupting ATF3–murine double minute 2 (MDM2) interaction and negatively regulating MDM2 protein expression. PXR enhanced MDM2 auto-ubiquitination and shortened its half-life, therefore compromising the MDM2-mediated degradation of ATF3 protein. Structurally, both ATF3 and PXR bind to the RING domain of MDM2, and on the other hand, MDM2 binds with PXR on the DNA-binding domain (DBD), which contains zinc finger sequence. Zinc finger sequence is well known for nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) playing E3 ligase activity to degrade nuclear factor κB (NFκB)/p65. However, whether zinc-RING sequence grants E3 ligase activity to PXR remains elusive. Taken together, these results provide a novel mechanism that PXR contributes to IR resistance by promoting ATF3-mediated ATM activation through stabilization of ATF3. Our result suggests that targeting PXR may sensitize liver and colon cancer cells to IR therapy.

放射治疗应答率低下仍是肝癌与结直肠癌患者面临的难题，其根源在于肿瘤细胞存在不恰当的DNA损伤应答（DNA damage response）机制。本研究证实，孕烷X受体（pregnane X receptor, PXR）可通过激活转录因子3（activating transcription factor 3, ATF3）介导的共济失调毛细血管扩张症突变蛋白（ataxia-telangiectasia-mutated protein, ATM）激活，促进肿瘤细胞产生放射抵抗（IR）。PXR通过阻断ATF3的泛素化修饰，稳定其蛋白表达。调控ATF3的过程依赖于PXR与ATF3的相互作用：当ATF3的赖氨酸42位点突变为精氨酸（K42R）时，其与PXR的结合能力丧失，同时无法抵消PXR对ATF3泛素化的抑制作用。另一方面，当PXR的苏氨酸432位点突变为丙氨酸（T432A）时，其与ATF3的结合能力受损，进而削弱ATM的激活效果。此外，PXR与ATF3的相互作用可通过破坏ATF3与鼠双微体2（murine double minute 2, MDM2）的结合，并负向调控MDM2的蛋白表达，进一步增强ATF3的稳定性。PXR可促进MDM2的自身泛素化并缩短其半衰期，从而抑制MDM2介导的ATF3蛋白降解过程。从结构层面来看，ATF3与PXR均能结合MDM2的环指结构域（RING domain）；而MDM2则通过DNA结合结构域（DNA-binding domain, DBD）与PXR结合，该结构域包含锌指序列。众所周知，核受体过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor-γ, PPARγ）可通过其锌指序列发挥E3泛素连接酶活性，降解核因子κB（nuclear factor κB, NFκB）/p65蛋白。但锌指-环指序列是否赋予PXR E3连接酶活性，目前仍难以阐明。综上，本研究揭示了一种全新的机制：PXR通过稳定ATF3蛋白，促进ATF3介导的ATM激活，进而参与肿瘤细胞的放射抵抗过程。本研究结果提示，靶向PXR可增强肝癌与结直肠癌细胞对放射治疗的敏感性。