Radioresistance is a major challenge in lung cancer radiotherapy, and new radiosensitizers are urgently needed. Estrogen receptor β (ERβ) is involved in the progression of non-small cell lung cancer (NSCLC), however, the role of ERβ in the response to radiotherapy in lung cancer remains elusive. In the present study, we investigated the mechanism underlying ERβ-mediated transcriptional activation and radioresistance of NSCLC cells. Quantitative real-time PCR, western blot and immunohistochemistry were used to detect the expression of CLPTM1L, ERβ and other target genes. The mechanism of CLPTM1L in modulation of radiosensitivity was investigated by chromatin immunoprecipitation assay, luciferase reporter gene assay, immunofluorescence staining, confocal microscopy, coimmunoprecipitation and GST pull-down assays. The functional role of CLPTM1L was detected by function assays in vitro and in vivo. CLPTM1L expression was negatively correlated with the radiosensitivity of NSCLC cell lines, and irradiation upregulated CLPTM1L in radioresistant (A549) but not in radiosensitive (H460) NSCLC cells. Meanwhile, IR induced the translocation of CLPTM1L from the cytoplasm into the nucleus in NSCLC cells. Moreover, CLPTM1L induced radioresistance in NSCLC cells. iTRAQ-based analysis and cDNA microarray identified irradiation-related genes commonly targeted by CLPTM1L and ERβ, and CLPTM1L upregulated ERβ-induced genes CDC25A, c-Jun, and BCL2. Mechanistically, CLPTM1L coactivated ERβ by directly interacting with ERβ through the LXXLL NR (nuclear receptor)-binding motif. Functionally, ERβ silencing was sufficient to block CLPTM1L-enhanced radioresistance of NSCLC cells in vitro. CLPTM1L shRNA treatment in combination with irradiation significantly inhibited cancer cell growth in NSCLC xenograft tumors in vivo. The present results indicate that CLPTM1L acts as a critical coactivator of ERβ to promote the transcription of its target genes and induce radioresistance of NSCLC cells, suggesting a new target for radiosensitization in NSCLC therapy.

中文翻译：

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CLPTM1L 在非小细胞肺癌细胞中诱导雌激素受体 β 信号介导的放射抗性。

放射抗性是肺癌放疗的一大挑战，急需新型放射增敏剂。雌激素受体β（ERβ）参与非小细胞肺癌（NSCLC）的进展，然而，ERβ在肺癌放疗反应中的作用仍然难以捉摸。在本研究中，我们研究了 ERβ 介导的 NSCLC 细胞转录激活和放射抗性的机制。采用定量实时PCR、蛋白质印迹和免疫组化检测CLPTM1L、ERβ等靶基因的表达。通过染色质免疫沉淀试验、荧光素酶报告基因试验、免疫荧光染色、共聚焦显微镜、共免疫沉淀和 GST 下拉试验研究了 CLPTM1L 调节放射敏感性的机制。CLPTM1L 的功能作用是通过体外和体内的功能测定来检测的。CLPTM1L 表达与 NSCLC 细胞系的放射敏感性呈负相关，辐射上调了放射抗性 (A549) 中的 CLPTM1L，但在放射敏感性 (H460) NSCLC 细胞中没有。同时，IR诱导CLPTM1L从细胞质转移到NSCLC细胞的细胞核中。此外，CLPTM1L 在 NSCLC 细胞中诱导放射抗性。基于 iTRAQ 的分析和 cDNA 微阵列确定了 CLPTM1L 和 ERβ 通常靶向的辐射相关基因，并且 CLPTM1L 上调了 ERβ 诱导的基因 CDC25A、c-Jun 和 BCL2。从机制上讲，CLPTM1L 通过 LXXLL NR（核受体）结合基序与 ERβ 直接相互作用来共激活 ERβ。在功能上，ERβ 沉默足以在体外阻断 CLPTM1L 增强的 NSCLC 细胞的放射抗性。CLPTM1L shRNA 治疗与辐射相结合显着抑制了体内 NSCLC 异种移植肿瘤中的癌细胞生长。目前的结果表明，CLPTM1L 作为 ERβ 的关键共激活剂，促进其靶基因的转录并诱导 NSCLC 细胞的放射抗性，为 NSCLC 治疗中的放射增敏提供了新的靶点。