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DAL-1 attenuates epithelial-to mesenchymal transition in lung cancer.

Chen X, Guan X, Zhang H, Xie X, Wang H, Long J, Cai T, Li S, Liu Z, Zhang Y - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: DAL-1 levels were strongly reduced even lost in lymph node metastasis and advanced pathological stage of human lung carcinomas.Overexpression of DAL-1 altered the expression of numerous EMT markers, such as E-cadherin, β-catenin Vimentin and N-cadherin expression, meanwhile changed the morphological shape of lung cancer cells, and whereas silencing DAL-1 had an opposite effect.Strikingly, HSPA5 was found as DAL-1 direct binding protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou 510180, Guangdong, People's Republic of China. yajie.zhang@163.com.

ABSTRACT

Background: Epithelial-to mesenchymal transition (EMT) involves in metastasis, causing loss of epithelial polarity. Metastasis is the major cause of carcinoma-induced death, but mechanisms are poorly understood. Here we identify differentially expressed in adenocarcinoma of the lung-1 (DAL-1), a protein belongs to the membrane-associated cytoskeleton protein 4.1 family, as an efficient suppressor of EMT in lung cancer.

Methods: The relationship between DAL-1 and EMT markers were analyzed by using immunohistochemistry in the clinical lung cancer tissues. Quantitative real-time PCR and western blot were used to characterize the expression of the EMT indicator mRNAs and proteins in DAL-1 overexpressed or knockdown cells. DAL-1 combined proteins were assessed by co-immunoprecipitation.

Results: DAL-1 levels were strongly reduced even lost in lymph node metastasis and advanced pathological stage of human lung carcinomas. Overexpression of DAL-1 altered the expression of numerous EMT markers, such as E-cadherin, β-catenin Vimentin and N-cadherin expression, meanwhile changed the morphological shape of lung cancer cells, and whereas silencing DAL-1 had an opposite effect. DAL-1 directly combined with E-cadherin promoter and regulated its expression that could be the reason for impairing EMT and decreasing cell migration and invasion. Strikingly, HSPA5 was found as DAL-1 direct binding protein.

Conclusions: These results suggest that tumor suppressor DAL-1 could also attenuate EMT and be important for tumor metastasis in the early transformation process in lung cancer.

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Related in: MedlinePlus

Effect of DAL-1 overexpression on morphology and EMT markers in A549 cells. Cells were stably transfected with pcDNA3-DAL-1 or pcDNA3 plasmid respectively. A, Morphological changes in DAL-1 overexpression cells, from spindle-like mesenchymal morphology into epithelial morphology, by manifesting an increased cell-to-cell adhesion; B, Q-PCR analysis of the levels of EMT related markers after DAL-1stably transfected with pcDNA3-DAL-1 plasmid; The levels of E-cadherin and β-catenin mRNAs were higher in the cells treated with the pcDNA3-DAL-1 plasmid than in the pcDNA3 treated cells (mock) or A549 cells (blank), but the level of Vimentin and N-cadherin mRNAs were lower. The data are presented as the mean ± SD. n = 3, *P <0.05,#P <0.01. C, Western blot analysis of DAL-1 and EMT marker protein expression levels in A549 cells. When the cells were transfected with the pcDNA3-DAL-1, the protein levels of E-cadherin and β-catenin increased while protein levels of Vimentin N-cadherin were down-regulated.
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Fig2: Effect of DAL-1 overexpression on morphology and EMT markers in A549 cells. Cells were stably transfected with pcDNA3-DAL-1 or pcDNA3 plasmid respectively. A, Morphological changes in DAL-1 overexpression cells, from spindle-like mesenchymal morphology into epithelial morphology, by manifesting an increased cell-to-cell adhesion; B, Q-PCR analysis of the levels of EMT related markers after DAL-1stably transfected with pcDNA3-DAL-1 plasmid; The levels of E-cadherin and β-catenin mRNAs were higher in the cells treated with the pcDNA3-DAL-1 plasmid than in the pcDNA3 treated cells (mock) or A549 cells (blank), but the level of Vimentin and N-cadherin mRNAs were lower. The data are presented as the mean ± SD. n = 3, *P <0.05,#P <0.01. C, Western blot analysis of DAL-1 and EMT marker protein expression levels in A549 cells. When the cells were transfected with the pcDNA3-DAL-1, the protein levels of E-cadherin and β-catenin increased while protein levels of Vimentin N-cadherin were down-regulated.

Mentions: To verify whether DAL-1 is a crucial mediator of lung cancer, we established A549 cell line stably overexpressing DAL-1. We then analyzed DAL-1/A549 cells and the control cells through cell morphological observation. In the initial characterization of these cells, we observed a distinct morphological change in the cells overexpressing DAL-1. The A549 cells expressing DAL-1 underwent a phenotypic change and appear planar epithelial morphological shape, while the control A549 cells (Vector/A549) appear fibroblast-like mesenchymal cell morphological shape (Figure 2A).Figure 2


DAL-1 attenuates epithelial-to mesenchymal transition in lung cancer.

Chen X, Guan X, Zhang H, Xie X, Wang H, Long J, Cai T, Li S, Liu Z, Zhang Y - J. Exp. Clin. Cancer Res. (2015)

Effect of DAL-1 overexpression on morphology and EMT markers in A549 cells. Cells were stably transfected with pcDNA3-DAL-1 or pcDNA3 plasmid respectively. A, Morphological changes in DAL-1 overexpression cells, from spindle-like mesenchymal morphology into epithelial morphology, by manifesting an increased cell-to-cell adhesion; B, Q-PCR analysis of the levels of EMT related markers after DAL-1stably transfected with pcDNA3-DAL-1 plasmid; The levels of E-cadherin and β-catenin mRNAs were higher in the cells treated with the pcDNA3-DAL-1 plasmid than in the pcDNA3 treated cells (mock) or A549 cells (blank), but the level of Vimentin and N-cadherin mRNAs were lower. The data are presented as the mean ± SD. n = 3, *P <0.05,#P <0.01. C, Western blot analysis of DAL-1 and EMT marker protein expression levels in A549 cells. When the cells were transfected with the pcDNA3-DAL-1, the protein levels of E-cadherin and β-catenin increased while protein levels of Vimentin N-cadherin were down-regulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4307741&req=5

Fig2: Effect of DAL-1 overexpression on morphology and EMT markers in A549 cells. Cells were stably transfected with pcDNA3-DAL-1 or pcDNA3 plasmid respectively. A, Morphological changes in DAL-1 overexpression cells, from spindle-like mesenchymal morphology into epithelial morphology, by manifesting an increased cell-to-cell adhesion; B, Q-PCR analysis of the levels of EMT related markers after DAL-1stably transfected with pcDNA3-DAL-1 plasmid; The levels of E-cadherin and β-catenin mRNAs were higher in the cells treated with the pcDNA3-DAL-1 plasmid than in the pcDNA3 treated cells (mock) or A549 cells (blank), but the level of Vimentin and N-cadherin mRNAs were lower. The data are presented as the mean ± SD. n = 3, *P <0.05,#P <0.01. C, Western blot analysis of DAL-1 and EMT marker protein expression levels in A549 cells. When the cells were transfected with the pcDNA3-DAL-1, the protein levels of E-cadherin and β-catenin increased while protein levels of Vimentin N-cadherin were down-regulated.
Mentions: To verify whether DAL-1 is a crucial mediator of lung cancer, we established A549 cell line stably overexpressing DAL-1. We then analyzed DAL-1/A549 cells and the control cells through cell morphological observation. In the initial characterization of these cells, we observed a distinct morphological change in the cells overexpressing DAL-1. The A549 cells expressing DAL-1 underwent a phenotypic change and appear planar epithelial morphological shape, while the control A549 cells (Vector/A549) appear fibroblast-like mesenchymal cell morphological shape (Figure 2A).Figure 2

Bottom Line: DAL-1 levels were strongly reduced even lost in lymph node metastasis and advanced pathological stage of human lung carcinomas.Overexpression of DAL-1 altered the expression of numerous EMT markers, such as E-cadherin, β-catenin Vimentin and N-cadherin expression, meanwhile changed the morphological shape of lung cancer cells, and whereas silencing DAL-1 had an opposite effect.Strikingly, HSPA5 was found as DAL-1 direct binding protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Basic Medical Science, Guangzhou Medical University, 195# Dongfeng West Road, Guangzhou 510180, Guangdong, People's Republic of China. yajie.zhang@163.com.

ABSTRACT

Background: Epithelial-to mesenchymal transition (EMT) involves in metastasis, causing loss of epithelial polarity. Metastasis is the major cause of carcinoma-induced death, but mechanisms are poorly understood. Here we identify differentially expressed in adenocarcinoma of the lung-1 (DAL-1), a protein belongs to the membrane-associated cytoskeleton protein 4.1 family, as an efficient suppressor of EMT in lung cancer.

Methods: The relationship between DAL-1 and EMT markers were analyzed by using immunohistochemistry in the clinical lung cancer tissues. Quantitative real-time PCR and western blot were used to characterize the expression of the EMT indicator mRNAs and proteins in DAL-1 overexpressed or knockdown cells. DAL-1 combined proteins were assessed by co-immunoprecipitation.

Results: DAL-1 levels were strongly reduced even lost in lymph node metastasis and advanced pathological stage of human lung carcinomas. Overexpression of DAL-1 altered the expression of numerous EMT markers, such as E-cadherin, β-catenin Vimentin and N-cadherin expression, meanwhile changed the morphological shape of lung cancer cells, and whereas silencing DAL-1 had an opposite effect. DAL-1 directly combined with E-cadherin promoter and regulated its expression that could be the reason for impairing EMT and decreasing cell migration and invasion. Strikingly, HSPA5 was found as DAL-1 direct binding protein.

Conclusions: These results suggest that tumor suppressor DAL-1 could also attenuate EMT and be important for tumor metastasis in the early transformation process in lung cancer.

Show MeSH
Related in: MedlinePlus