Limits...
CCAAT/enhancer binding protein β is dispensable for development of lung adenocarcinoma.

Cai Y, Hirata A, Nakayama S, VanderLaan PA, Levantini E, Yamamoto M, Hirai H, Wong KK, Costa DB, Watanabe H, Kobayashi SS - PLoS ONE (2015)

Bottom Line: We found that overall lung architecture was maintained in Cebpb knockout mice.Neither overexpression of nuclear C/EBPβ nor suppression of CEBPB expression had significant effects on cell proliferation.Taken together, our data suggest that C/EBPβ is dispensable for development of lung adenocarcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Lung cancer is the leading cause of cancer death worldwide. Although disruption of normal proliferation and differentiation is a vital component of tumorigenesis, the mechanisms of this process in lung cancer are still unclear. A transcription factor, C/EBPβ is a critical regulator of proliferation and/or differentiation in multiple tissues. In lung, C/EBPβ is expressed in alveolar pneumocytes and bronchial epithelial cells; however, its roles on normal lung homeostasis and lung cancer development have not been well described. Here we investigated whether C/EBPβ is required for normal lung development and whether its aberrant expression and/or activity contribute to lung tumorigenesis. We showed that C/EBPβ was expressed in both human normal pneumocytes and lung adenocarcinoma cell lines. We found that overall lung architecture was maintained in Cebpb knockout mice. Neither overexpression of nuclear C/EBPβ nor suppression of CEBPB expression had significant effects on cell proliferation. C/EBPβ expression and activity remained unchanged upon EGF stimulation. Furthermore, deletion of Cebpb had no impact on lung tumor burden in a lung specific, conditional mutant EGFR lung cancer mouse model. Analyses of data from The Cancer Genome Atlas (TCGA) revealed that expression, promoter methylation, or copy number of CEBPB was not significantly altered in human lung adenocarcinoma. Taken together, our data suggest that C/EBPβ is dispensable for development of lung adenocarcinoma.

No MeSH data available.


Related in: MedlinePlus

C/EBPβ fails to contribute to cell proliferation in vitro.(A) Fluorescent images of NCI-H358 cells stably expressing C/EBPβ-ER. Cells were treated with 1 μM β-estradiol or 0.1% ethanol (vehicle) for 24 hours and stained with anti ER antibody and visualized by Alexa Fluor 488. Scale Bar = 10 μm. (B) Luciferase assay to determine C/EBPβ transcriptional activity in NCI-H358 cells stably expressing C/EBPβ-ER and control cells. Cells were transfected with C/ebpb0.3-luc reporter and pCMV-Renilla control plasmid were assayed after 24 hours of treatment with 1 μM β-estradiol or vehicle control. Data represents mean ± standard deviation from three independent experiments. ** denotes p ≤ 0.01. (C) Cell viability determined by MTS assay. NCI-H358 cells expressing C/EBPβ-ER cells or control cells were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from eight independent experiments. * denotes p ≤ 0.05. (D) Immunoblots of C/EBPβ in NCI-H358 cells transduced with shRNA against CEBPB. Three clones each for control and shRNA against CEBPB are shown. (E) Cell viability determined by MTS assay. NCI-H358 cells transduced with shRNA against CEBPB or control shRNA described in (D) were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from three independent experiments. N.S. denotes not significant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4358974&req=5

pone.0120647.g003: C/EBPβ fails to contribute to cell proliferation in vitro.(A) Fluorescent images of NCI-H358 cells stably expressing C/EBPβ-ER. Cells were treated with 1 μM β-estradiol or 0.1% ethanol (vehicle) for 24 hours and stained with anti ER antibody and visualized by Alexa Fluor 488. Scale Bar = 10 μm. (B) Luciferase assay to determine C/EBPβ transcriptional activity in NCI-H358 cells stably expressing C/EBPβ-ER and control cells. Cells were transfected with C/ebpb0.3-luc reporter and pCMV-Renilla control plasmid were assayed after 24 hours of treatment with 1 μM β-estradiol or vehicle control. Data represents mean ± standard deviation from three independent experiments. ** denotes p ≤ 0.01. (C) Cell viability determined by MTS assay. NCI-H358 cells expressing C/EBPβ-ER cells or control cells were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from eight independent experiments. * denotes p ≤ 0.05. (D) Immunoblots of C/EBPβ in NCI-H358 cells transduced with shRNA against CEBPB. Three clones each for control and shRNA against CEBPB are shown. (E) Cell viability determined by MTS assay. NCI-H358 cells transduced with shRNA against CEBPB or control shRNA described in (D) were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from three independent experiments. N.S. denotes not significant.

Mentions: Expression of C/EBPβ in lung adenocarcinoma cell lines (Fig. 1B) and its reported proliferative role in epithelial cancer development [32] prompted us to hypothesize that C/EBPβ may be important for lung cancer progression. To examine proliferative effects of C/EBPβ, we generated stable NCI-H358 cell lines expressing an estrogen receptor hormone-binding domain fused to C/EBPβ (C/EBPβ-ER) or expressing the estrogen receptor hormone-binding domain alone [25, 26]. Upon stimulation with β-estradiol, C/EBPβ-ER is translocated to the nucleus (Fig. 3A) and activated(Fig. 3B)Fig. Unexpectedly, nuclear translocation of C/EBPβ-ER to the nucleus led to suppression of cell proliferation in NCI-H358 cells (Fig. 3C). Next, to examine requirement of C/EBPβ expression in lung adenocarcinoma cells, we transduced NCI-H358 cells with retrovirus containing shRNA constructs against C/EBPβ and isolated three clones. Despite greater than 90% reduction in C/EBPβ expression achieved in all clones (Fig. 3D), we observed no significant difference in cell proliferation upon C/EBPβ suppression in NCI-H358 cells (Fig. 3E). Similar results were obtained in NCI-H1975 cells (data not shown). These results suggest that C/EBPβ is not essential for and does not promote lung cancer cell growth.


CCAAT/enhancer binding protein β is dispensable for development of lung adenocarcinoma.

Cai Y, Hirata A, Nakayama S, VanderLaan PA, Levantini E, Yamamoto M, Hirai H, Wong KK, Costa DB, Watanabe H, Kobayashi SS - PLoS ONE (2015)

C/EBPβ fails to contribute to cell proliferation in vitro.(A) Fluorescent images of NCI-H358 cells stably expressing C/EBPβ-ER. Cells were treated with 1 μM β-estradiol or 0.1% ethanol (vehicle) for 24 hours and stained with anti ER antibody and visualized by Alexa Fluor 488. Scale Bar = 10 μm. (B) Luciferase assay to determine C/EBPβ transcriptional activity in NCI-H358 cells stably expressing C/EBPβ-ER and control cells. Cells were transfected with C/ebpb0.3-luc reporter and pCMV-Renilla control plasmid were assayed after 24 hours of treatment with 1 μM β-estradiol or vehicle control. Data represents mean ± standard deviation from three independent experiments. ** denotes p ≤ 0.01. (C) Cell viability determined by MTS assay. NCI-H358 cells expressing C/EBPβ-ER cells or control cells were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from eight independent experiments. * denotes p ≤ 0.05. (D) Immunoblots of C/EBPβ in NCI-H358 cells transduced with shRNA against CEBPB. Three clones each for control and shRNA against CEBPB are shown. (E) Cell viability determined by MTS assay. NCI-H358 cells transduced with shRNA against CEBPB or control shRNA described in (D) were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from three independent experiments. N.S. denotes not significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358974&req=5

pone.0120647.g003: C/EBPβ fails to contribute to cell proliferation in vitro.(A) Fluorescent images of NCI-H358 cells stably expressing C/EBPβ-ER. Cells were treated with 1 μM β-estradiol or 0.1% ethanol (vehicle) for 24 hours and stained with anti ER antibody and visualized by Alexa Fluor 488. Scale Bar = 10 μm. (B) Luciferase assay to determine C/EBPβ transcriptional activity in NCI-H358 cells stably expressing C/EBPβ-ER and control cells. Cells were transfected with C/ebpb0.3-luc reporter and pCMV-Renilla control plasmid were assayed after 24 hours of treatment with 1 μM β-estradiol or vehicle control. Data represents mean ± standard deviation from three independent experiments. ** denotes p ≤ 0.01. (C) Cell viability determined by MTS assay. NCI-H358 cells expressing C/EBPβ-ER cells or control cells were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from eight independent experiments. * denotes p ≤ 0.05. (D) Immunoblots of C/EBPβ in NCI-H358 cells transduced with shRNA against CEBPB. Three clones each for control and shRNA against CEBPB are shown. (E) Cell viability determined by MTS assay. NCI-H358 cells transduced with shRNA against CEBPB or control shRNA described in (D) were plated in 96 well plates at 2,000 cells/well and assayed after 72 hours of incubation. Data represents mean ± standard deviation from three independent experiments. N.S. denotes not significant.
Mentions: Expression of C/EBPβ in lung adenocarcinoma cell lines (Fig. 1B) and its reported proliferative role in epithelial cancer development [32] prompted us to hypothesize that C/EBPβ may be important for lung cancer progression. To examine proliferative effects of C/EBPβ, we generated stable NCI-H358 cell lines expressing an estrogen receptor hormone-binding domain fused to C/EBPβ (C/EBPβ-ER) or expressing the estrogen receptor hormone-binding domain alone [25, 26]. Upon stimulation with β-estradiol, C/EBPβ-ER is translocated to the nucleus (Fig. 3A) and activated(Fig. 3B)Fig. Unexpectedly, nuclear translocation of C/EBPβ-ER to the nucleus led to suppression of cell proliferation in NCI-H358 cells (Fig. 3C). Next, to examine requirement of C/EBPβ expression in lung adenocarcinoma cells, we transduced NCI-H358 cells with retrovirus containing shRNA constructs against C/EBPβ and isolated three clones. Despite greater than 90% reduction in C/EBPβ expression achieved in all clones (Fig. 3D), we observed no significant difference in cell proliferation upon C/EBPβ suppression in NCI-H358 cells (Fig. 3E). Similar results were obtained in NCI-H1975 cells (data not shown). These results suggest that C/EBPβ is not essential for and does not promote lung cancer cell growth.

Bottom Line: We found that overall lung architecture was maintained in Cebpb knockout mice.Neither overexpression of nuclear C/EBPβ nor suppression of CEBPB expression had significant effects on cell proliferation.Taken together, our data suggest that C/EBPβ is dispensable for development of lung adenocarcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Lung cancer is the leading cause of cancer death worldwide. Although disruption of normal proliferation and differentiation is a vital component of tumorigenesis, the mechanisms of this process in lung cancer are still unclear. A transcription factor, C/EBPβ is a critical regulator of proliferation and/or differentiation in multiple tissues. In lung, C/EBPβ is expressed in alveolar pneumocytes and bronchial epithelial cells; however, its roles on normal lung homeostasis and lung cancer development have not been well described. Here we investigated whether C/EBPβ is required for normal lung development and whether its aberrant expression and/or activity contribute to lung tumorigenesis. We showed that C/EBPβ was expressed in both human normal pneumocytes and lung adenocarcinoma cell lines. We found that overall lung architecture was maintained in Cebpb knockout mice. Neither overexpression of nuclear C/EBPβ nor suppression of CEBPB expression had significant effects on cell proliferation. C/EBPβ expression and activity remained unchanged upon EGF stimulation. Furthermore, deletion of Cebpb had no impact on lung tumor burden in a lung specific, conditional mutant EGFR lung cancer mouse model. Analyses of data from The Cancer Genome Atlas (TCGA) revealed that expression, promoter methylation, or copy number of CEBPB was not significantly altered in human lung adenocarcinoma. Taken together, our data suggest that C/EBPβ is dispensable for development of lung adenocarcinoma.

No MeSH data available.


Related in: MedlinePlus