CLPTM1L induces estrogen receptor β signaling-mediated radioresistance in non-small cell lung cancer cells

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: CLPTM1L; ERβ; Non-small cell lung cancer; Radioresistance; Radiotherapy.

Fig. 1

CLPTM1L induces the radioresistance of NSCLC cells. a The cell viabilities of different NSCLC cell lines exposed to 4 Gy of γ-ray irradiation (IR) were examined by colony formation assay after 12 days of IR and expressed as percent change compared with the control group (group without IR). b The expression levels of CLPTM1L in different NSCLC cell lines exposed to 4 Gy of IR were determined by western blot and real-time PCR analysis and expressed as fold change compared with the H460 group. c Heatmap showing the relationship between the expression level of CLPTM1L and the viability of NSCLC cells exposed to IR. d–e The expression levels of CLPTM1L in A549 and H460 cells exposed to various doses of γ-ray IR were detected by real-time PCR and western blot analysis. f The effect of CLPTM1L siRNA and/or 4 Gy of γ-ray IR on cell proliferation was measured by EdU assay in A549 cells. g The effect of CLPTM1L overexpression and/or 4 Gy of IR on cell proliferation was tested by EdU assay in H460 cells. h–i Cells (A549 and H460) pretreated with CLPTM1L siRNA or pcDNA-CLPTM1L (24 h) were exposed to IR (0, 2, 4, and 8 Gy). After 12 days, colonies were counted for quantification. Data are representative of three independent experiments; Student’s t test; * P < 0.05; ** P < 0.01; *** P < 0.001

Fig. 2

Identification of candidate targets of CLPTM1L by iTRAQ-based analysis. a Expression levels of CLPTM1L in lung adenocarcinoma compared with normal lung tissues obtained from public databases (Selamat dataset). Student’s t test; ** P < 0.01. b Proteins upregulated or downregulated by CLPTM1L in A549 cells exposed to IR were identified by iTRAQ-based analysis based on the P-value (P < 0.05). c–d Distribution of mass or isoelectric point of the 1237 proteins identified. e–f Analysis of parallelism between CLPTM1L overexpressing groups or control groups from iTRAQ-based analysis. g Volcano plot showing 233 CLPTM1L-modulated genes among the 1237 genes identified based on fold changes in expression [log₁₀(FC) < − 0.14 or log₁₀(FC) > 0.14]

Fig. 3

CLPTM1L expression is positively associated with the ERβ-induced genes in NSCLC. a Venn diagram showing the overlapping target genes of CLPTM1L, ERβ, and IR. b Real-time PCR analysis of the expression of 16 candidate target genes in A549 cells treated with CLPTM1L siRNAs/control siRNA and IR. c CLPTM1L binding to the promoters of CDC25A, c-Jun, and BCL2 was examined by ChIP-qPCR assay in A549 cells exposed to IR. d The expression levels of CLPTM1L, CDC25A, c-Jun, and BCL2 were detected by IHC staining in 110 cases of NSCLC tissue samples using tissue microarray. Representative images were taken from the same sample in 110 cases of the tissue microarray (see the No.55 case in Additional file 1: Table S11). e–g Real-time PCR analysis of the correlation between CLPTM1L and CDC25A (or c-Jun and BCL2) mRNA levels in seven NSCLC cell lines exposed to IR (CDC25A, P < 0.01, R² = 0.8876; c-Jun, P < 0.01, R² = 0.8302; BCL2, P < 0.01, R² = 0.7850; Pearson’s correlation). All experiments were repeated at least three times. Statistically significant differences are indicated. Student’s t test; ** P < 0.01

Fig. 4

CLPTM1L upregulates the levels of CDC25A, c-Jun, and BCL2 through ERβ in NSCLC cells. a Real-time PCR analysis of the mRNA levels of CLPTM1L, CDC25A, c-Jun, and BCL2 in A549 cells exposed to different doses of γ-ray IR. b Western blot analysis of the protein levels of these genes in A549 cells. c Western blot analysis of the protein levels of these genes in H460 cells exposed to 4 Gy IR. d–e Real-time PCR and western blot analysis of the mRNA and protein levels of CLPTM1L, ERβ, CDC25A, c-Jun, and BCL2 in A549 cells treated with IR and siRNAs. f–g Real-time PCR and western blot analysis of the expression of these genes in H460 cells treated with the indicated plasmids/siRNAs and 4 Gy IR. All experiments were repeated at least three times. Statistically significant differences are indicated. Student’s t test; * P < 0.05; ** P < 0.01

Fig. 5

CLPTM1L activates the promoter of ERβ-induced genes by stimulating ERβ in NSCLC cells. a–i Cells were transfected with relative plasmids and siRNAs, exposed to 4 Gy IR, followed by luciferase reporter gene assays. a ERE-LUC activity was examined using luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-CLPTM1L plasmids (E2, also called 17β-estradiol, was used as the positive control). b ERE-LUC activity was examined using luciferase reporter gene assays in A549 cells transfected with pcDNA-CLPTM1L plasmids or CLPTM1L siRNA. c ERE-LUC activity was determined using luciferase reporter gene assays in H460 cells treated with various doses of pcDNA-CLPTM1L plasmids. d The mutation of the ERE is shown in the ERE-LUC construct. e Luciferase reporter gene assays in A549 and H460 cells transfected with ERE-LUC constructs with wild-type or mutant ERβ-binding sites (E2 was used as the positive control). f ERE-LUC activity was detected by luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-CLPTM1L and ERβ siRNAs. g The effect of CLPTM1L on ERE-LUC activity was tested by luciferase reporter gene assays in ER-negative cells (MDA-MB-231) transfected with or without pcDNA-ERβ/pcDNA-ERα. NS, not significant. h ERE-LUC activity was detected by luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-ERβ and CLPTM1L siRNAs. i ERE-LUC activity was examined by luciferase reporter gene assays in A549 and H460 cells transfected with pcDNA-CLPTM1L and/or pcDNA-ERβ plasmids. All experiments were repeated at least three times. Student’s t test; * P < 0.05; ** P < 0.01; *** P < 0.001

Fig. 6

CLPTM1L directly interacts with and coactivates ERβ. a Model showing the effect of mutation of the LXXLL motif on abolishing the interaction between CLPTM1L and ERβ. b Confocal microscopy images of the localization of Flag-CLPTM1L (wt or mut) and ERβ in A549 and H460 cells exposed to 4 Gy IR. c The interaction of CLPTM1L (wt or mut) with ERβ was detected by Co-IP assays in A549 cells exposed to 4 Gy IR. d The direct interaction of recombinant GST-CLPTM1L (wt or mut) with His-ERβ was detected by GST pull-down assays and western blot analysis. e–f CLPTM1L binding to the promoters of CDC25A, c-Jun, and BCL2 was examined by ChIP-qPCR in A549 cells exposed to 4 Gy IR. g Luciferase activities of Gal4-ERβ were measured by luciferase reporter gene assays in A549 cells exposed to 4 Gy IR. NS, not significant. Each experiment was repeated at least three times. Student’s t test; * P < 0.05; ** P < 0.01

Fig. 7

CLPTM1L induces radioresistance of NSCLC cells by coactivating ERβ. a Effect of CLPTM1L and ERβ on A549 and H460 cell proliferation after exposure to 0 or 4 Gy of IR as determined by the EdU assay. b–c MTT assays of the effect of ERβ/pcDNA-CLPTM1L-m (CLPTM1L containing a mutated LXXLL motif) on CLPTM1L-modulated radiosensitivity in A549 and H460 cells were performed after 3 days of IR. d–e A549/H460 cells pretreated with CLPTM1L siRNA/pcDNA-CLPTM1L and pcDNA-ERβ/ERβ siRNA/pcDNA-CLPTM1L-m were exposed to IR (0, 2, 4, and 8 Gy) and analyzed by clonogenic cell survival assay after 12 days of IR. The data presented are from three independent experiments; Student’s t test; * P < 0.05; ** P < 0.01; NS, not significant

Fig. 8

Silencing of CLPTM1L sensitizes xenograft NSCLC tumors to IR in an animal model. a Mice (n = 40) bearing NSCLC xenograft tumors developed from H460 or A549 cells transfected with control or CLPTM1L shRNA were treated with or without local IR, as illustrated in the diagram. b Growth of tumors generated by transplantation of NSCLC cells. c Images of dissected tumors from nude mice (n = 5/group). d Weight of resected xenograft tumors in nude mice. e Representative images (400× magnification) of H460 and A549 xenografts stained for CLPTM1L and Ki-67. f Real-time PCR analysis of the mRNA levels of CDC25A, c-Jun, and BCL2 in H460 and A549 xenografts. Statistically significant differences are indicated. NS, not significant. Student’s t test; * P < 0.05; ** P < 0.01. g The model shows that IR can increase the expression of CLPTM1L, and CLPTM1L upregulates the expression of ERβ-induced genes by coactivating ERβ via the LXXLL motif, leading to the radioresistance of NSCLC cells