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Cited by 126Ferroptosis and cancer prognosisCell death mechanisms and regulationIron Metabolism and Disorders

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CREB stimulates GPX4 transcription to inhibit ferroptosis in lung adenocarcinoma

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Wang, Zhixian, Zhang, Xiao, Tian, Xiaoting, Yang, Yueyue, Ma, Lifang, Wang, Jiayi, Yu, Yongchun·2021·Oncology Reports
DOI 10.3892/or.2021.8039PMC PMC8042667
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Abstract

Ferroptosis is a new form of regulated cell death and closely related to cancer. However, the mechanism underlying the regulation of ferroptosis in lung adenocarcinoma (LUAD) remains unclear. IB, IHC and ELISA were performed to analyze protein expression. RT‑qPCR was used to analyze mRNA expression. Cell viability, 3D cell growth, MDA, the generation of lipid ROS and the Fe2+ concentration were measured to evaluate the responses to the induction of ferroptosis. Measurement of luciferase activity and ChIP were used to analyze the promoter activity regulated by the transcriptional regulator. Co‑IP assays were performed to identify protein‑protein interactions. In the present study, it was revealed that cAMP response element‑binding protein (CREB) was highly expressed in LUAD, and knockdown of CREB inhibited cell viability and growth by promoting apoptosis‑ and ferroptosis‑like cell death, concurrently. It was observed that CREB suppressed lipid peroxidation by binding the promoter region of glutathione peroxidase 4 (GPX4), and this binding could be enhanced by E1A binding protein P300 (EP300). The bZIP domain in CREB and the CBP/p300‑HAT domain in EP300 were essential for CREB‑EP300 binding in LUAD cells. Finally, it was revealed that CREB, GPX4, EP300 and 4‑HNE were closely related to tumor size and stage, and tumors with a higher degree of malignancy were more likely to have a low degree of lipid peroxidation. Therefore, targeting this CREB/EP300/GPX4 axis may provide new strategies for treating LUAD.

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Figures

Figure 1. CREB is upregulated in LUAD. (A) UALCAN database was used to analyze TCGA data of CREB expression between LUAD (n=515) and normal lung (n=59) tissues. (B) CREB expression in lung fibroblast, LUSC and LUAD cell lines, as measured by IB and IHC. (C and D) CREB expression in LUAD and adjacent normal tissues was measured by ELISA, as presented in a (C) line plot and (D) scatter plot. (E) CREB expression in 10 pairs of LUAD tissues with the most significant increase of CREB according to the ELISA data of C and D, as measured by IB and qPCR. (F) CREB expression in 3 pairs of LUAD tissues, as measured by IHC (scale bar, 800 µm). The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from A were analyzed using an independent-sample Student's t-test, D and E were analyzed using paired Student's t-test. CREB, cAMP response element-binding protein; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; LUSC, lung squamous cell carcinoma; IB, immunoblotting; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorbent assay.
Figure 1. CREB is upregulated in LUAD. (A) UALCAN database was used to analyze TCGA data of CREB expression between LUAD (n=515) and normal lung (n=59) tissues. (B) CREB expression in lung fibroblast, LUSC and LUAD cell lines, as measured by IB and IHC. (C and D) CREB expression in LUAD and adjacent normal tissues was measured by ELISA, as presented in a (C) line plot and (D) scatter plot. (E) CREB expression in 10 pairs of LUAD tissues with the most significant increase of CREB according to the ELISA data of C and D, as measured by IB and qPCR. (F) CREB expression in 3 pairs of LUAD tissues, as measured by IHC (scale bar, 800 µm). The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from A were analyzed using an independent-sample Student's t-test, D and E were analyzed using paired Student's t-test. CREB, cAMP response element-binding protein; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; LUSC, lung squamous cell carcinoma; IB, immunoblotting; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorbent assay.CC BY-NC-ND · PubMed Central
Figure 2. CREB knockdown inhibits cell growth via stimulating apoptosis- and ferroptosis-like cell death concurrently. (A) Cell viability, 3D cell with SYTOX Green staining and MDA were measured in CREB-knockdown cells with additional treatment using Nec-1 (20 µM), ZVAD-FMK (20 µM) or Fer-1 (1 µM) (scale bar, 50 µm). (B) Cell viability, 3D cell with SYTOX Green staining and MDA were measured in cells treated with erastin (10 µM) or apoptozole (10 µM) with or without ectopically expressed CREB (scale bar, 50 µm). (C) Fe2+ and lipid ROS generation were measured in CREB-knockdown cells with additional treatment using Nec-1 (20 µM), ZVAD-FMK (20 µM) or Fer-1 (1 µM) or cells treated with erastin (10 µM) or apoptozole (10 µM) with or without ectopically expressed CREB. The data are presented as the mean ± SD from three biological replicates. *P<0.05, **P<0.01 indicates statistical significance. Data from A, B and C were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. CREB, cAMP response element-binding protein; MDA, malondialdehyde; NEC-1, necrostatin-1; Fer-1, ferrostatin-1; ROS, reactive oxygen species; sh, short hairpin.
Figure 2. CREB knockdown inhibits cell growth via stimulating apoptosis- and ferroptosis-like cell death concurrently. (A) Cell viability, 3D cell with SYTOX Green staining and MDA were measured in CREB-knockdown cells with additional treatment using Nec-1 (20 µM), ZVAD-FMK (20 µM) or Fer-1 (1 µM) (scale bar, 50 µm). (B) Cell viability, 3D cell with SYTOX Green staining and MDA were measured in cells treated with erastin (10 µM) or apoptozole (10 µM) with or without ectopically expressed CREB (scale bar, 50 µm). (C) Fe2+ and lipid ROS generation were measured in CREB-knockdown cells with additional treatment using Nec-1 (20 µM), ZVAD-FMK (20 µM) or Fer-1 (1 µM) or cells treated with erastin (10 µM) or apoptozole (10 µM) with or without ectopically expressed CREB. The data are presented as the mean ± SD from three biological replicates. *P<0.05, **P<0.01 indicates statistical significance. Data from A, B and C were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. CREB, cAMP response element-binding protein; MDA, malondialdehyde; NEC-1, necrostatin-1; Fer-1, ferrostatin-1; ROS, reactive oxygen species; sh, short hairpin.CC BY-NC-ND · PubMed Central
Figure 3. CREB positively regulates GPX4. (A and B) Indicated mRNA levels were measured in (A) A549 and (B) H1299 cells with or without CREB overexpression or knockdown. (C) Indicated protein expression levels were measured in A549 or H1299 cells with or without CREB overexpression or knockdown. (D) CREB, GPX4 and SAT1 mRNA levels were measured in 10 paired of LUAD tissues. (E) Correlation between GPX4 and CREB mRNA as well as SAT1 and CREB mRNA was calculated. (F) CREB, GPX4 and SAT1 protein expressions were measured in 10 paired of LUAD tissues. (G) Correlation between GPX4 and CREB protein level as well as SAT1 and CREB protein level was calculated. The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from A and B were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. Data from D were analyzed using a paired Student's t-test. Data from E and G were analyzed using Spearman's rank correlation coefficient. CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4, SAT1, spermidine/spermine N1-acetyltransferase 1; LUAD, lung adenocarcinoma; IB, immunoblotting; CARS, cysteinyl-tRNA synthetase; NRF2, nuclear factor, erythroid 2 like 2; HSPB1, heat shock protein family B small member 1; ACSL4, acyl-CoA synthetase long chain family member 4; sh, short hairpin; N, normal; T, tumor.
Figure 3. CREB positively regulates GPX4. (A and B) Indicated mRNA levels were measured in (A) A549 and (B) H1299 cells with or without CREB overexpression or knockdown. (C) Indicated protein expression levels were measured in A549 or H1299 cells with or without CREB overexpression or knockdown. (D) CREB, GPX4 and SAT1 mRNA levels were measured in 10 paired of LUAD tissues. (E) Correlation between GPX4 and CREB mRNA as well as SAT1 and CREB mRNA was calculated. (F) CREB, GPX4 and SAT1 protein expressions were measured in 10 paired of LUAD tissues. (G) Correlation between GPX4 and CREB protein level as well as SAT1 and CREB protein level was calculated. The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from A and B were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. Data from D were analyzed using a paired Student's t-test. Data from E and G were analyzed using Spearman's rank correlation coefficient. CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4, SAT1, spermidine/spermine N1-acetyltransferase 1; LUAD, lung adenocarcinoma; IB, immunoblotting; CARS, cysteinyl-tRNA synthetase; NRF2, nuclear factor, erythroid 2 like 2; HSPB1, heat shock protein family B small member 1; ACSL4, acyl-CoA synthetase long chain family member 4; sh, short hairpin; N, normal; T, tumor.CC BY-NC-ND · PubMed Central
Figure 4. CREB directly binds the promoter region of GPX4. (A) CREB motif (searched in the JASPAR database) location in GPX4 promoter. (B and C) WT- or Mut-GPX4-Promoter Luc was measured in (B) A549 and (C) H1299 cells with CREB overexpression or knockdown. (D-F) The enrichment of CREB in (D) A549, (E) H1299 cells and (F) 10 paired LUAD tissues at indicated regions of GPX4 promoter was calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies. Anti-IgG and anti-HNF4A were used as the parallel controls. The data are presented as the mean ± SD from three biological replicates. **P<0.01 indicates statistical significance. Data from B-E were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. Data from F were analyzed using a paired Student's t-test. CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4; WT, wild-type; MUT, mutant; LUAD, lung adenocarcinoma; ChIP, chromatin immunoprecipitation; HNF4A, hepatocyte nuclear factor 4α; sh, short hairpin.
Figure 4. CREB directly binds the promoter region of GPX4. (A) CREB motif (searched in the JASPAR database) location in GPX4 promoter. (B and C) WT- or Mut-GPX4-Promoter Luc was measured in (B) A549 and (C) H1299 cells with CREB overexpression or knockdown. (D-F) The enrichment of CREB in (D) A549, (E) H1299 cells and (F) 10 paired LUAD tissues at indicated regions of GPX4 promoter was calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies. Anti-IgG and anti-HNF4A were used as the parallel controls. The data are presented as the mean ± SD from three biological replicates. **P<0.01 indicates statistical significance. Data from B-E were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. Data from F were analyzed using a paired Student's t-test. CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4; WT, wild-type; MUT, mutant; LUAD, lung adenocarcinoma; ChIP, chromatin immunoprecipitation; HNF4A, hepatocyte nuclear factor 4α; sh, short hairpin.CC BY-NC-ND · PubMed Central
Figure 5. EP300 stimulates CREB-dependent GPX4 transcription. (A) STRING analysis revealed CREB interacted with methyltransferase and acyltransferase (confidence=0.980). (B) Co-immunoprecipitation experiments performed in A549 cells using indicated antibodies, and further analysis of CREB, EP300 and SETDB2 expression by IB. (C) Schematic diagram shows the domains in CREB and EP300 protein. (D and E) Co-immunoprecipitation experiments performed using anti-HA or anti-CREB in A549 and H1299 cells with indicated CREB or EP300 plasmids overexpressed, and further analysis of Myc and HA levels by IB. (F and G) The enrichments of H3K27Ac, EP300 and CREB at −2 k, CREB motif or 2 k regions of GPX4 promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in (F) A549 and (G) H1299 cells with CREB or EP300 overexpressed or knocked down. Anti-IgG was used as the parallel control. (H-K) The enrichments of H3K27Ac, EP300 and CREB at −2 k, CREB motif or 2 k regions of GPX4 promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in (H and J) A549 and H1299 (I and K) cells with WT or mutant (H and I) CREB or (J and K) EP300 overexpressed. Anti-IgG was used as the parallel control. (L and M) Cell viability and MDA, respectively, were measured in A549 and H1299 cells treated with erastin (10 µM) with or without ectopically expressed CREB with or without EP300 knockdown. The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from F-M were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. EP300, E1A binding protein P300; CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4; SETDB2, SET domain bifurcated histone lysine methyltransferase 2; IB, immunoblotting; ChIP, chromatin immunoprecipitation; WT, wild-type; MDA, MDA, malondialdehyde; sh, short hairpin; Ctrl, control.
Figure 5. EP300 stimulates CREB-dependent GPX4 transcription. (A) STRING analysis revealed CREB interacted with methyltransferase and acyltransferase (confidence=0.980). (B) Co-immunoprecipitation experiments performed in A549 cells using indicated antibodies, and further analysis of CREB, EP300 and SETDB2 expression by IB. (C) Schematic diagram shows the domains in CREB and EP300 protein. (D and E) Co-immunoprecipitation experiments performed using anti-HA or anti-CREB in A549 and H1299 cells with indicated CREB or EP300 plasmids overexpressed, and further analysis of Myc and HA levels by IB. (F and G) The enrichments of H3K27Ac, EP300 and CREB at −2 k, CREB motif or 2 k regions of GPX4 promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in (F) A549 and (G) H1299 cells with CREB or EP300 overexpressed or knocked down. Anti-IgG was used as the parallel control. (H-K) The enrichments of H3K27Ac, EP300 and CREB at −2 k, CREB motif or 2 k regions of GPX4 promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in (H and J) A549 and H1299 (I and K) cells with WT or mutant (H and I) CREB or (J and K) EP300 overexpressed. Anti-IgG was used as the parallel control. (L and M) Cell viability and MDA, respectively, were measured in A549 and H1299 cells treated with erastin (10 µM) with or without ectopically expressed CREB with or without EP300 knockdown. The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from F-M were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. EP300, E1A binding protein P300; CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4; SETDB2, SET domain bifurcated histone lysine methyltransferase 2; IB, immunoblotting; ChIP, chromatin immunoprecipitation; WT, wild-type; MDA, MDA, malondialdehyde; sh, short hairpin; Ctrl, control.CC BY-NC-ND · PubMed Central
Figure 6. Clinical significance of CREB, EP300, GPX4 and 4-HNE. (A-D) Relative (A) CREB, (B) GPX4, (C) EP300 mRNA levels and (D) 4-HNE concentration in 36 paired LUAD tissues. (E-G) Correlations between 4-HNE concentration between (E) CREB, (F) GPX4 and (G) EP300 mRNA levels in 36 paired LUAD tissues. (H-J) Correlations of mRNA levels between (H) GPX4 and CREB, (I) EP300 and CREB, as well as (J) EP300 and GPX4. (K) TCGA data of CREB and EP300 co-expression in 515 LUAD specimens from UALCAN database. (L) Survival in LUAD patients with CREB high (n=26) or low (n=26) expression. (M and N) Correlations between 4-HNE levels and (M) tumor stage and (N) tumor diameter. The data are presented as the mean ± SD from three biological replicates. **P<0.01 indicates statistical significance. Data from A-D were analyzed using a paired Student's t-test. Data from E-K were analyzed using Spearman's rank correlation coefficient. Data from L were analyzed using the log rank analysis. Data from M were analyzed using Fisher's exact test. Data from N were analyzed using χ2 test. CREB, cAMP response element-binding protein; EP300, E1A binding protein P300; GPX4, glutathione peroxidase 4; 4-HNE, 4-hydroxynonenal; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas.
Figure 6. Clinical significance of CREB, EP300, GPX4 and 4-HNE. (A-D) Relative (A) CREB, (B) GPX4, (C) EP300 mRNA levels and (D) 4-HNE concentration in 36 paired LUAD tissues. (E-G) Correlations between 4-HNE concentration between (E) CREB, (F) GPX4 and (G) EP300 mRNA levels in 36 paired LUAD tissues. (H-J) Correlations of mRNA levels between (H) GPX4 and CREB, (I) EP300 and CREB, as well as (J) EP300 and GPX4. (K) TCGA data of CREB and EP300 co-expression in 515 LUAD specimens from UALCAN database. (L) Survival in LUAD patients with CREB high (n=26) or low (n=26) expression. (M and N) Correlations between 4-HNE levels and (M) tumor stage and (N) tumor diameter. The data are presented as the mean ± SD from three biological replicates. **P<0.01 indicates statistical significance. Data from A-D were analyzed using a paired Student's t-test. Data from E-K were analyzed using Spearman's rank correlation coefficient. Data from L were analyzed using the log rank analysis. Data from M were analyzed using Fisher's exact test. Data from N were analyzed using χ2 test. CREB, cAMP response element-binding protein; EP300, E1A binding protein P300; GPX4, glutathione peroxidase 4; 4-HNE, 4-hydroxynonenal; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Wang, Z., Zhang, X., Tian, X., Yang, Y., Ma, L., Wang, J., & Yu, Y.
Affiliations
  • Shanghai University of Traditional Chinese Medicine (CN)
  • Shanghai Jiao Tong University (CN)
  • Tongji University (CN)
Corresponding author
  • Wang, Jiayi
  • Yu, Yongchun
ORCID
Venue
Oncology Reports
Publisher
Spandidos Publications
Published
2021-04-02
Volume
45
Issue
6
ISSN
  • 1021-335X (ISSN-L)
  • 1791-2431
DOI
10.3892/or.2021.8039
PMC
PMC8042667
OpenAlex
W3146768338
Cited by
122
References
53
Citations by year
2026: 14 · 2025: 16 · 2024: 34 · 2023: 25 · 2022: 22 · 2021: 11
FWCI
15.75
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Oncology Reports (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (81871907, 81822029, 81902869, 81902315, 81774291, 81672332)
  • Shanghai Education Development Foundation (18CG16)
  • Shanghai Municipal Education Commission
  • Shanghai Chest Hospital (2018YNJCQ06)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Ferroptosis and cancer prognosis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
  • Health Sciences › Medicine › Hematology › Iron Metabolism and Disorders
Keywords
CREB, GPX4, ferroptosis, lung adenocarcinoma, EP300, lipid peroxidation, transcriptional regulation, 4-hydroxynonenal, apoptosis, bZIP domain, protein-protein interaction, tumor malignancy
MeSH terms
Adenocarcinoma of Lung, Ferroptosis, Phospholipid Hydroperoxide Glutathione Peroxidase, Humans, Lung, Lung Neoplasms, Promoter Regions, Genetic, Protein Binding, Lipid Peroxidation, Transcriptional Activation, Gene Expression Regulation, Neoplastic, Cyclic AMP Response Element-Binding Protein, Cell Line, Tumor, E1A-Associated p300 Protein, Gene Knockdown Techniques, HEK293 Cells, Datasets as Topic

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