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. 2010 Sep;37(3):541-50.
doi: 10.3892/ijo_00000703.

Regulation of ERalpha-mediated transcription of Bcl-2 by PI3K-AKT crosstalk: implications for breast cancer cell survival

Melyssa R Bratton  1 , Bich N Duong, Steven Elliott, Christopher B Weldon, Barbara S Beckman, John A McLachlan, Matthew E Burow
Affiliations

Regulation of ERalpha-mediated transcription of Bcl-2 by PI3K-AKT crosstalk: implications for breast cancer cell survival

Melyssa R Bratton et al. Int J Oncol. 2010 Sep.

Abstract

Both estrogen, through the estrogen receptor (ER), and growth factors, through the phosphatidylinositol-3-kinase (PI3K)-AKT pathway, have been shown to independently promote cell survival. Here, we investigated the role of ER/PI3K-AKT crosstalk in the regulation of cell survival in MCF-7 breast carcinoma cells. The ER inhibitor ICI 182,780 was used to determine the requirement of the ER for estrogen in the suppression of tumor necrosis factor-alpha (TNFalpha) induced apoptosis. Gene reporter assays and Western blot analyses were used to determine the involvement of the pro-survival factor Bcl-2 and the coactivator GRIP1 in this survival crosstalk. We demonstrated that an intact ER signaling pathway was required for estrogen to suppress apoptosis induced by TNFalpha. Our gene reporter assays revealed that ERalpha, not ERbeta, was targeted by AKT, resulting in transcriptional potentiation of the full-length Bcl-2 promoter, ultimately leading to increased Bcl-2 protein levels. AKT targeted both activation function (AF) domains of the ERalpha for maximal induction of Bcl-2 reporter activity, although the AF-II domain was predominately targeted. In addition, AKT also caused an upregulation of GRIP1 protein levels. Finally, AKT and GRIP1 cooperated to increase Bcl-2 protein expression to a greater level than either factor alone. Collectively, our study suggests a role for ER/PI3K-AKT crosstalk in cell survival and documents the ability of AKT to regulate Bcl-2 expression via differential activation of ERalpha and ERbeta as well as regulation of GRIP1.

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Figures

Figure 1
Figure 1
The ER is required for cell survival mediated by signaling through AKT. (A) MCF-7 cells were treated with vehicle control (Con) or E2 (10 nM) in the presence or absence of ICI 182,780 (100 nM) for 24 h and then exposed to TNFα (10 ng/ml). Cells were harvested for CV viability assay 24 h later. Asterisk denotes statistical significance from control (p<0.05). (B) MCF-7 cells were transfected with 2 μg of empty vector (Vec) or constitutive active AKT (AKT-CA) along with pGL3-Luc (200 ng). Cells were treated with vehicle (Con) or ICI 182,780 (100 nM) followed by addition of TNFα (10 ng/ml) for 48 h and harvested for viability assay. Asterisk denotes statistical significance from vector control (p<0.05). All data are the means and standard errors of double treatments from a single experiment, and are representative of at least two independent experiments. (C) MCF-7 cells were transfected with 500 ng of empty vector (Vec) or constitutive active AKT (AKT-CA) along with a pEGFP expression vector (100 ng). Cells were treated with vehicle control (Con) or ICI 182,780 (100 nM), followed by the addition of vehicle or TNF-α (10 ng/ml) and analyzed for cell death by fluorescence microscopy 24 h later.
Figure 2
Figure 2
The ER is required for potentiation of Bcl-2 expression by AKT. (A) MCF-7 cells were transfected with the pBcl-2-Luc reporter (200 ng) along with 300 ng empty vector (Vec), constitutive active AKT (AKT-CA), or dominant negative AKT (AKT-DN). Cells were treated with E2 (1 nM), ICI 182,780 (100 nM), or E2 + ICI and were harvested 24 h later and assayed for luciferase activity. Data are represented as fold change normalized to vehicle-treated Vec control, and the values are the means and standard errors of double treatments from a single experiment, and representative of at least two independent experiments. Asterisk denotes statistical significance from vector control (p<0.05). (B) MCF-7 cells were transfected with 5 μg empty vector (Vec) or constitutive active AKT (AKT-CA) and were treated with vehicle control (Con) or E2 (1 nM) with or without ICI 182,780 (100 nM), and harvested 24 h later. Fifty micrograms whole cell extracts were subjected to Western blot analysis using a Bcl-2 antibody. The blots were then stripped and re-probed with an actin antibody as an internal loading control. Data are represented as Bcl-2 protein level relative to actin (Bcl2/actin) and normalized to vehicle-treated Vec control. The blot shown is representative of at least three independent experiments. (C) Densitometry of Western blot analysis from 2B. Asterisk denotes statistical significance from vector control (p<0.05).
Figure 3
Figure 3
AKT differentially potentiates ERα and ERβ activity at the Bcl-2 promoter. (A) HEK 293 cells were transfected with the pBcl-2-Luc reporter (200 ng), along with 300 ng empty vector (Vec) or constitutive active AKT (AKT-CA), along with either 100 ng ERα or ERβ. Cells were treated with vehicle control (Veh) or E2 (1 nM) and were harvested after 24 h of treatment and assayed for luciferase activity. Data are represented as RLUs normalized to vehicle-treated Vec control, and the values are the means and standard errors of double treatments from a single experiment and are representative of at least two independent experiments. Asterisk denotes statistical significance from vector control (p<0.05). (B) HEK 293 cells were transfected as in (A) with the addition of ER domain mutants ERα-AF-I and ER-AF-II. The experiment was carried out as in (A). Asterisk denotes statistical significance from vector control of each ER construct (p<0.05). (C) HEK 293 cells were transfected with a total of 5 μg DNA of empty vector (Vec) or ERα, with or without constitutive active AKT (AKT-CA) and were treated with vehicle control (Con) or E2 (1 nM), and harvested 24 h later. Whole cell extracts (50 μg) were subjected to Western blot analysis using Bcl-2 antibody. The blots were then stripped and re-probed with actin antibody as an internal loading control. Data are represented as Bcl-2 protein level relative to actin (Bcl2/actin) and normalized to vehicle-treated Vec control. (D) Densitometry of the Western blot analysis from E. The blot shown is representative and the data are the results of at least three independent experiments. Asterisk denotes statistical significance from vector control (p<0.05).
Figure 4
Figure 4
AKT enhances GRIP1 protein expression. (A) MCF-7 cells were transfected with 5 μg empty vector (Vec) or constitutive active AKT (AKTCA) and were treated with either vehicle (con) or E2 (1 nM) for 24 h. Once harvested, 50 μg whole cell extracts were subjected to Western blot analysis using a GRIP1 antibody. The blots were then stripped and re-probed with an actin antibody as an internal loading control. (B) Densitometry of the Western blot analysis. Data are represented as GRIP1 protein level relative to actin (GRIP1/actin) and are normalized to vehicle-treated Vec control and are the results of three independent experiments. Asterisk denotes statistical significance from vector control (p<0.05).
Figure 5
Figure 5
AKT and GRIP1 cooperatively enhance ERα-mediated Bcl-2 promoter activation. (A) HEK 293 cells were transfected with a pBcl-2-Luc reporter, along with constitutive active AKT (AKT-CA) or GRIP1 and ERα (full length). Cells were treated with vehicle control (Con) or E2 (1 nM) and were harvested after 24 h of treatment for measurement of luciferase activity. Data are represented as fold change and are normalized to vehicle-treated Vec control. All the values are the means and standard errors of double treatments from a single experiment, and are representative of at least two independent experiments. Asterisk denotes statistical significance from vector control cells. Two asterisks denotes statistical significance from AKT-CA control cells. (B) Experimental design the same as in (A) except ER-AF-I domain mutant was used. (C) Experimental design the same as in (A) except ER-AF-II domain mutant was used.
Figure 6
Figure 6
AKT and GRIP1 cooperatively enhance Bcl-2 protein expression. (A) MCF-7 cells were transfected with 5 μg vector control (Vec), constitutive active AKT (AKT-CA), pSG5-GRIP1-HA (GRIP1), or 2.5 μg each AKT-CA and GRIP1. Cells were treated with E2 (1 nM) for 24 h prior to harvesting. Whole cell extracts (50 μg) were subjected to Western blot analysis using a Bcl-2 antibody. The blots were then stripped and re-probed with an actin antibody as an internal loading control. (B) Densitometry of the Western blot analysis. Data are represented as Bcl-2 protein level relative to actin (Bcl2/actin) and are normalized to vector control. Data are the results of two independent experiments. Asterisk denotes statistical significance from vector E2, and two asterisks denotes statistical significance from AKTCA E2 and GRIP-1 E2 (p<0.05).
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References

    1. Kumar R, Vadlamudi RK, Adam L. Apoptosis in mammary gland and cancer. Endocr Relat Cancer. 2000;7:257–269. - PubMed
    1. Green DR, Evan GI. A matter of life and death. Cancer Cell. 2002;1:19–30. - PubMed
    1. Imagawa W, Pedchenko VK, Helber J, Zhang H. Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis. J Steroid Biochem Mol Biol. 2002;80:213–230. - PubMed
    1. Feigelson HS, Henderson BE. Estrogens and breast cancer. Carcinogenesis. 1996;17:2279–2284. - PubMed
    1. Gross JM, Yee D. The type-1 insulin-like growth factor receptor tyrosine kinase and breast cancer: Biology and therapeutic relevance. Cancer Metastasis Rev. 2003;22:327–336. - PubMed

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