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The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

Li CF, Wu WJ, Wu WR, Liao YJ, Chen LR, Huang CN, Li CC, Li WM, Huang HY, Chen YL, Liang SS, Chow NH, Shiue YL - Oncotarget (2015)

Bottom Line: Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival.Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models.Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.

ABSTRACT
In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding protein 1 (CREB1) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2, in vitro and in vivo. Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

No MeSH data available.


Related in: MedlinePlus

Stable knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes enhanced tumor growth in NOD/SCID xenograft models(A) The EMP2 mRNA and protein levels were downregulated after stable transfection of shEMP#1 and cotransfection of shCREB1#3 and shEMP2# plasmids into RT4 cells. Knockdown cells (5 × 106) were mixed with Matrigel and injected into flank sites of mice (n = 6 for each group). (B) Stable knockdown of EMP2 gene (shEMP2#1) increased tumor growth, compared to the control group, shLuc (*, p < 0.05). Double knockdown of CREB1 and EMP2 genes (shCREB1#3 & shEMP2#1) further enhanced tumor growth, compared to the shEMP2#1 group (#, p < 0.05). (C) After sacrifice, tumors were dissected from animals and tumors from each group are shown. (D) Immunohistochemistry on xenograft tissues displayed that knockdown of EMP2 gene notably downregulated EMP2 protein level, however, double knockdown of CREB1 and EMP2 genes markedly downregulated both CREB1 and EMP2 protein levels.
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Figure 5: Stable knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes enhanced tumor growth in NOD/SCID xenograft models(A) The EMP2 mRNA and protein levels were downregulated after stable transfection of shEMP#1 and cotransfection of shCREB1#3 and shEMP2# plasmids into RT4 cells. Knockdown cells (5 × 106) were mixed with Matrigel and injected into flank sites of mice (n = 6 for each group). (B) Stable knockdown of EMP2 gene (shEMP2#1) increased tumor growth, compared to the control group, shLuc (*, p < 0.05). Double knockdown of CREB1 and EMP2 genes (shCREB1#3 & shEMP2#1) further enhanced tumor growth, compared to the shEMP2#1 group (#, p < 0.05). (C) After sacrifice, tumors were dissected from animals and tumors from each group are shown. (D) Immunohistochemistry on xenograft tissues displayed that knockdown of EMP2 gene notably downregulated EMP2 protein level, however, double knockdown of CREB1 and EMP2 genes markedly downregulated both CREB1 and EMP2 protein levels.

Mentions: The mouse xenograft model was also used to evaluate whether knockdown of EMP2 and double knockdown of CREB1 and EMP2 affected tumor growth in vivo. In RT4 cells, both stable knockdown of EMP2 gene (shEMP2#1), and double knockdown of CREB1 and EMP2 genes (shCREB1#3 & shEMP2#1) inhibited EMP2 mRNA (p < 0.05; p < 0.01) and protein levels, compared to the control group (Figure 5A). In NOD/SCID mice, xenografts with EMP2 stable knocked down RT4 cells showed larger tumors, compared to the control group (*, p < 0.05). Double knockdown of CREB1 and EMP2 genes exhibited larger tumors, compared to the EMP2 knockdown group (#, p < 0.05; Figure 5B, 5C). Immunohistochemistry further demonstrated that stable knockdown of EMP2 gene suppressed EMP2 protein levels, compared to the controls (shLuc). Double knockdown of CREB1 and EMP2 genes downregulated CREB1 and EMP2 immunointensities, compared to knockdown of EMP2 gene alone (Figure 5D).


The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

Li CF, Wu WJ, Wu WR, Liao YJ, Chen LR, Huang CN, Li CC, Li WM, Huang HY, Chen YL, Liang SS, Chow NH, Shiue YL - Oncotarget (2015)

Stable knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes enhanced tumor growth in NOD/SCID xenograft models(A) The EMP2 mRNA and protein levels were downregulated after stable transfection of shEMP#1 and cotransfection of shCREB1#3 and shEMP2# plasmids into RT4 cells. Knockdown cells (5 × 106) were mixed with Matrigel and injected into flank sites of mice (n = 6 for each group). (B) Stable knockdown of EMP2 gene (shEMP2#1) increased tumor growth, compared to the control group, shLuc (*, p < 0.05). Double knockdown of CREB1 and EMP2 genes (shCREB1#3 & shEMP2#1) further enhanced tumor growth, compared to the shEMP2#1 group (#, p < 0.05). (C) After sacrifice, tumors were dissected from animals and tumors from each group are shown. (D) Immunohistochemistry on xenograft tissues displayed that knockdown of EMP2 gene notably downregulated EMP2 protein level, however, double knockdown of CREB1 and EMP2 genes markedly downregulated both CREB1 and EMP2 protein levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496213&req=5

Figure 5: Stable knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes enhanced tumor growth in NOD/SCID xenograft models(A) The EMP2 mRNA and protein levels were downregulated after stable transfection of shEMP#1 and cotransfection of shCREB1#3 and shEMP2# plasmids into RT4 cells. Knockdown cells (5 × 106) were mixed with Matrigel and injected into flank sites of mice (n = 6 for each group). (B) Stable knockdown of EMP2 gene (shEMP2#1) increased tumor growth, compared to the control group, shLuc (*, p < 0.05). Double knockdown of CREB1 and EMP2 genes (shCREB1#3 & shEMP2#1) further enhanced tumor growth, compared to the shEMP2#1 group (#, p < 0.05). (C) After sacrifice, tumors were dissected from animals and tumors from each group are shown. (D) Immunohistochemistry on xenograft tissues displayed that knockdown of EMP2 gene notably downregulated EMP2 protein level, however, double knockdown of CREB1 and EMP2 genes markedly downregulated both CREB1 and EMP2 protein levels.
Mentions: The mouse xenograft model was also used to evaluate whether knockdown of EMP2 and double knockdown of CREB1 and EMP2 affected tumor growth in vivo. In RT4 cells, both stable knockdown of EMP2 gene (shEMP2#1), and double knockdown of CREB1 and EMP2 genes (shCREB1#3 & shEMP2#1) inhibited EMP2 mRNA (p < 0.05; p < 0.01) and protein levels, compared to the control group (Figure 5A). In NOD/SCID mice, xenografts with EMP2 stable knocked down RT4 cells showed larger tumors, compared to the control group (*, p < 0.05). Double knockdown of CREB1 and EMP2 genes exhibited larger tumors, compared to the EMP2 knockdown group (#, p < 0.05; Figure 5B, 5C). Immunohistochemistry further demonstrated that stable knockdown of EMP2 gene suppressed EMP2 protein levels, compared to the controls (shLuc). Double knockdown of CREB1 and EMP2 genes downregulated CREB1 and EMP2 immunointensities, compared to knockdown of EMP2 gene alone (Figure 5D).

Bottom Line: Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival.Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models.Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.

ABSTRACT
In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding protein 1 (CREB1) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2, in vitro and in vivo. Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

No MeSH data available.


Related in: MedlinePlus