Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, 238 Bai-Di Road, Tianjin, 300192, P.R. China.
Department of Radiation Oncology, Affiliated Hospital of Jiangnan University, 200 Hui-He Road, Wuxi, 214062, Jiangsu, P.R. China.
Cell Commun Signal. 2020 Sep 17;18(1):152. doi: 10.1186/s12964-020-00571-4.
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. Video Abstract.
肺癌放射治疗的主要挑战是放射抵抗，因此迫切需要新的放射增敏剂。雌激素受体β（ERβ）参与非小细胞肺癌（NSCLC）的进展，然而，ERβ在肺癌对放射治疗的反应中的作用仍不清楚。在本研究中，我们研究了 ERβ介导的 NSCLC 细胞转录激活和放射抵抗的机制。
使用定量实时 PCR、western blot 和免疫组织化学检测 CLPTM1L、ERβ和其他靶基因的表达。通过染色质免疫沉淀分析、荧光素酶报告基因分析、免疫荧光染色、共聚焦显微镜、免疫共沉淀和 GST 下拉实验研究 CLPTM1L 调节放射敏感性的机制。体外和体内功能实验检测 CLPTM1L 的功能作用。
CLPTM1L 的表达与 NSCLC 细胞系的放射敏感性呈负相关，照射可上调放射抵抗（A549）而非放射敏感（H460）的 NSCLC 细胞中的 CLPTM1L。同时，IR 诱导 CLPTM1L 在 NSCLC 细胞中从细胞质转位到细胞核。此外，CLPTM1L 可诱导 NSCLC 细胞产生放射抵抗。iTRAQ 分析和 cDNA 微阵列鉴定出 CLPTM1L 和 ERβ共同靶向的照射相关基因，CLPTM1L 上调了 ERβ 诱导的基因 CDC25A、c-Jun 和 BCL2。机制上，CLPTM1L 通过 LXXLL NR（核受体）结合基序直接与 ERβ相互作用，从而共同激活 ERβ。功能上，ERβ 沉默足以阻断 CLPTM1L 在体外增强 NSCLC 细胞的放射抵抗。CLPTM1L shRNA 处理联合照射显著抑制 NSCLC 异种移植肿瘤的癌细胞生长。
本研究结果表明，CLPTM1L 作为 ERβ 的关键共激活因子，可促进其靶基因的转录，诱导 NSCLC 细胞的放射抵抗，为 NSCLC 治疗中的放射增敏提供了新的靶点。视频摘要。
