Elevated expression of mitogen-activated protein kinase phosphatase 3 in breast tumors: a mechanism of tamoxifen resistance

Abstract

Antiestrogen resistance is a major clinical problem in the treatment of breast cancer. Altered growth factor signaling with estrogen receptor (ER)-alpha is associated with the development of resistance. Gene expression profiling was used to identify mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP3) whose expression was correlated with response to the antiestrogen tamoxifen in both patients and in vitro-derived cell line models. Overexpression of MKP3 rendered ER-alpha-positive breast cancer cells resistant to the growth-inhibitory effects of tamoxifen and enhanced tamoxifen agonist activity in endometrial cells. MKP3 overexpression was associated with lower levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in the presence of estrogen but that estrogen deprivation and tamoxifen treatment decreased MKP3 phosphatase activity, leading to an up-regulation of pERK1/2 MAPK, phosphorylated Ser(118)-ER-alpha, and cyclin D1. The MAPK/ERK kinase inhibitor PD98059 blocked tamoxifen-resistant growth. Accumulation of reactive oxygen species was observed with tamoxifen treatment of MKP3-overexpressing cells, and antioxidant treatment increased MKP3 phosphatase activity, thereby blocking resistance. Furthermore, PD98059 increased the levels of phosphorylated c-Jun NH(2)-terminal kinase (JNK) in tamoxifen-treated MKP3-overexpressing cells, suggesting an interaction between MKP3 levels, activation of ERK1/2 MAPK, and JNK signaling in human breast cancer cells. MKP3 represents a novel mechanism of resistance, which may be a potential biomarker for the use of ERK1/2 and/or JNK inhibitors in combination with tamoxifen treatment.

Fig. 1

A. Scatter plot of MKP3 RNA expression in 9 tumors; 4 tumors were Tam sensitive and 5 were Tam-resistant. Circles show the individual RNA values in arbritary units. Parametric p-value = 0.006; permutation p-value adjusted for multiple comparisons = 0.09. B. Relative MKP3 mRNA levels were determined using qRT-PCR in various cell lines. Relative levels were normalized to the levels of 18S ribosomal RNA in each RNA sample. C. Relative MKP3 mRNA levels of MCF-7 cells treated with estrogen (E2, 10 nM) or Tam (Tam, 100 nM) for 24 hours. Relative MKP3 RNA levels were normalized to the levels of 18S ribosomal RNA in each RNA sample.

Fig. 1

A. Scatter plot of MKP3 RNA expression in 9 tumors; 4 tumors were Tam sensitive and 5 were Tam-resistant. Circles show the individual RNA values in arbritary units. Parametric p-value = 0.006; permutation p-value adjusted for multiple comparisons = 0.09. B. Relative MKP3 mRNA levels were determined using qRT-PCR in various cell lines. Relative levels were normalized to the levels of 18S ribosomal RNA in each RNA sample. C. Relative MKP3 mRNA levels of MCF-7 cells treated with estrogen (E2, 10 nM) or Tam (Tam, 100 nM) for 24 hours. Relative MKP3 RNA levels were normalized to the levels of 18S ribosomal RNA in each RNA sample.

Fig. 1

A. Scatter plot of MKP3 RNA expression in 9 tumors; 4 tumors were Tam sensitive and 5 were Tam-resistant. Circles show the individual RNA values in arbritary units. Parametric p-value = 0.006; permutation p-value adjusted for multiple comparisons = 0.09. B. Relative MKP3 mRNA levels were determined using qRT-PCR in various cell lines. Relative levels were normalized to the levels of 18S ribosomal RNA in each RNA sample. C. Relative MKP3 mRNA levels of MCF-7 cells treated with estrogen (E2, 10 nM) or Tam (Tam, 100 nM) for 24 hours. Relative MKP3 RNA levels were normalized to the levels of 18S ribosomal RNA in each RNA sample.

Fig. 2

A. Immunoblot (IB) analysis of two vector-alone, control transfected MCF-7 clones (Con 1 and 2), and two MKP-3 transfected clones (MKP3-1 and 2). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. B. Relative MKP3 levels in Con1 and MKP3.1 transfectants were determined using qRT-PCR. Levels were normalized to 18S ribosomal RNA in each sample. C. Anchorage-independent colony formation assay with the above transfectants in the presence of 1.0 nM estrogen (E2), 100 nM tamoxifen (Tam), 100 nM Tam + 10 nM ICI 182,780, or 100 nM Tam + 20 nM PD98059. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. D. Immunoblot analysis of vector-alone, control transfected Ishikawa cells (Con), and a MKP-3 transfected Ishikawa cell (MKP3) (upper panel). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. Anchorage-independent colony formation assay with the Ishikawa transfectants in the presence of ethanol (C), estrogen (E2) or Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. E. Knockdown of MKP3 in Ishikawa cells was performed using a siRNA to MKP3. Relative MKP3 levels in control siRNA transfected (Ctrl Si) and MKP3 siRNA transfectants were determined using qRT-PCR (upper panel). Colony formation assays were performed in the presence of ethanol vehicle (C), 1.0 nM E2 and 100 nM Tam (lower panel). ** significance level at p<0.05 using a two-taileld Student’s t-test. F. and G. MCF-7-vector control 1 and MCF-7-MKP3-2 cells were injected into athymic nude mice supplemented with E2 for 21 days, and then randomly assigned to either continue E2 treatment (F.), or to have their E2 pellets removed and were treated with Tam (G.). There were n=6 animals each for the E2-treated vector control, and MKP3-overexpressing cells; there were n=4 for Tam-treated vector control, and n=5 animals for the Tam-treated MKP3-overexpressing cells.

Fig. 2

A. Immunoblot (IB) analysis of two vector-alone, control transfected MCF-7 clones (Con 1 and 2), and two MKP-3 transfected clones (MKP3-1 and 2). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. B. Relative MKP3 levels in Con1 and MKP3.1 transfectants were determined using qRT-PCR. Levels were normalized to 18S ribosomal RNA in each sample. C. Anchorage-independent colony formation assay with the above transfectants in the presence of 1.0 nM estrogen (E2), 100 nM tamoxifen (Tam), 100 nM Tam + 10 nM ICI 182,780, or 100 nM Tam + 20 nM PD98059. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. D. Immunoblot analysis of vector-alone, control transfected Ishikawa cells (Con), and a MKP-3 transfected Ishikawa cell (MKP3) (upper panel). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. Anchorage-independent colony formation assay with the Ishikawa transfectants in the presence of ethanol (C), estrogen (E2) or Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. E. Knockdown of MKP3 in Ishikawa cells was performed using a siRNA to MKP3. Relative MKP3 levels in control siRNA transfected (Ctrl Si) and MKP3 siRNA transfectants were determined using qRT-PCR (upper panel). Colony formation assays were performed in the presence of ethanol vehicle (C), 1.0 nM E2 and 100 nM Tam (lower panel). ** significance level at p<0.05 using a two-taileld Student’s t-test. F. and G. MCF-7-vector control 1 and MCF-7-MKP3-2 cells were injected into athymic nude mice supplemented with E2 for 21 days, and then randomly assigned to either continue E2 treatment (F.), or to have their E2 pellets removed and were treated with Tam (G.). There were n=6 animals each for the E2-treated vector control, and MKP3-overexpressing cells; there were n=4 for Tam-treated vector control, and n=5 animals for the Tam-treated MKP3-overexpressing cells.

Fig. 2

A. Immunoblot (IB) analysis of two vector-alone, control transfected MCF-7 clones (Con 1 and 2), and two MKP-3 transfected clones (MKP3-1 and 2). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. B. Relative MKP3 levels in Con1 and MKP3.1 transfectants were determined using qRT-PCR. Levels were normalized to 18S ribosomal RNA in each sample. C. Anchorage-independent colony formation assay with the above transfectants in the presence of 1.0 nM estrogen (E2), 100 nM tamoxifen (Tam), 100 nM Tam + 10 nM ICI 182,780, or 100 nM Tam + 20 nM PD98059. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. D. Immunoblot analysis of vector-alone, control transfected Ishikawa cells (Con), and a MKP-3 transfected Ishikawa cell (MKP3) (upper panel). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. Anchorage-independent colony formation assay with the Ishikawa transfectants in the presence of ethanol (C), estrogen (E2) or Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. E. Knockdown of MKP3 in Ishikawa cells was performed using a siRNA to MKP3. Relative MKP3 levels in control siRNA transfected (Ctrl Si) and MKP3 siRNA transfectants were determined using qRT-PCR (upper panel). Colony formation assays were performed in the presence of ethanol vehicle (C), 1.0 nM E2 and 100 nM Tam (lower panel). ** significance level at p<0.05 using a two-taileld Student’s t-test. F. and G. MCF-7-vector control 1 and MCF-7-MKP3-2 cells were injected into athymic nude mice supplemented with E2 for 21 days, and then randomly assigned to either continue E2 treatment (F.), or to have their E2 pellets removed and were treated with Tam (G.). There were n=6 animals each for the E2-treated vector control, and MKP3-overexpressing cells; there were n=4 for Tam-treated vector control, and n=5 animals for the Tam-treated MKP3-overexpressing cells.

Fig. 2

A. Immunoblot (IB) analysis of two vector-alone, control transfected MCF-7 clones (Con 1 and 2), and two MKP-3 transfected clones (MKP3-1 and 2). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. B. Relative MKP3 levels in Con1 and MKP3.1 transfectants were determined using qRT-PCR. Levels were normalized to 18S ribosomal RNA in each sample. C. Anchorage-independent colony formation assay with the above transfectants in the presence of 1.0 nM estrogen (E2), 100 nM tamoxifen (Tam), 100 nM Tam + 10 nM ICI 182,780, or 100 nM Tam + 20 nM PD98059. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. D. Immunoblot analysis of vector-alone, control transfected Ishikawa cells (Con), and a MKP-3 transfected Ishikawa cell (MKP3) (upper panel). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. Anchorage-independent colony formation assay with the Ishikawa transfectants in the presence of ethanol (C), estrogen (E2) or Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. E. Knockdown of MKP3 in Ishikawa cells was performed using a siRNA to MKP3. Relative MKP3 levels in control siRNA transfected (Ctrl Si) and MKP3 siRNA transfectants were determined using qRT-PCR (upper panel). Colony formation assays were performed in the presence of ethanol vehicle (C), 1.0 nM E2 and 100 nM Tam (lower panel). ** significance level at p<0.05 using a two-taileld Student’s t-test. F. and G. MCF-7-vector control 1 and MCF-7-MKP3-2 cells were injected into athymic nude mice supplemented with E2 for 21 days, and then randomly assigned to either continue E2 treatment (F.), or to have their E2 pellets removed and were treated with Tam (G.). There were n=6 animals each for the E2-treated vector control, and MKP3-overexpressing cells; there were n=4 for Tam-treated vector control, and n=5 animals for the Tam-treated MKP3-overexpressing cells.

Fig. 2

A. Immunoblot (IB) analysis of two vector-alone, control transfected MCF-7 clones (Con 1 and 2), and two MKP-3 transfected clones (MKP3-1 and 2). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. B. Relative MKP3 levels in Con1 and MKP3.1 transfectants were determined using qRT-PCR. Levels were normalized to 18S ribosomal RNA in each sample. C. Anchorage-independent colony formation assay with the above transfectants in the presence of 1.0 nM estrogen (E2), 100 nM tamoxifen (Tam), 100 nM Tam + 10 nM ICI 182,780, or 100 nM Tam + 20 nM PD98059. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. D. Immunoblot analysis of vector-alone, control transfected Ishikawa cells (Con), and a MKP-3 transfected Ishikawa cell (MKP3) (upper panel). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. Anchorage-independent colony formation assay with the Ishikawa transfectants in the presence of ethanol (C), estrogen (E2) or Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. E. Knockdown of MKP3 in Ishikawa cells was performed using a siRNA to MKP3. Relative MKP3 levels in control siRNA transfected (Ctrl Si) and MKP3 siRNA transfectants were determined using qRT-PCR (upper panel). Colony formation assays were performed in the presence of ethanol vehicle (C), 1.0 nM E2 and 100 nM Tam (lower panel). ** significance level at p<0.05 using a two-taileld Student’s t-test. F. and G. MCF-7-vector control 1 and MCF-7-MKP3-2 cells were injected into athymic nude mice supplemented with E2 for 21 days, and then randomly assigned to either continue E2 treatment (F.), or to have their E2 pellets removed and were treated with Tam (G.). There were n=6 animals each for the E2-treated vector control, and MKP3-overexpressing cells; there were n=4 for Tam-treated vector control, and n=5 animals for the Tam-treated MKP3-overexpressing cells.

Fig. 2

A. Immunoblot (IB) analysis of two vector-alone, control transfected MCF-7 clones (Con 1 and 2), and two MKP-3 transfected clones (MKP3-1 and 2). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. B. Relative MKP3 levels in Con1 and MKP3.1 transfectants were determined using qRT-PCR. Levels were normalized to 18S ribosomal RNA in each sample. C. Anchorage-independent colony formation assay with the above transfectants in the presence of 1.0 nM estrogen (E2), 100 nM tamoxifen (Tam), 100 nM Tam + 10 nM ICI 182,780, or 100 nM Tam + 20 nM PD98059. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. D. Immunoblot analysis of vector-alone, control transfected Ishikawa cells (Con), and a MKP-3 transfected Ishikawa cell (MKP3) (upper panel). A V5 epitope tag was included in the expression vector and was used for visualization of MKP3 levels. Anchorage-independent colony formation assay with the Ishikawa transfectants in the presence of ethanol (C), estrogen (E2) or Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. E. Knockdown of MKP3 in Ishikawa cells was performed using a siRNA to MKP3. Relative MKP3 levels in control siRNA transfected (Ctrl Si) and MKP3 siRNA transfectants were determined using qRT-PCR (upper panel). Colony formation assays were performed in the presence of ethanol vehicle (C), 1.0 nM E2 and 100 nM Tam (lower panel). ** significance level at p<0.05 using a two-taileld Student’s t-test. F. and G. MCF-7-vector control 1 and MCF-7-MKP3-2 cells were injected into athymic nude mice supplemented with E2 for 21 days, and then randomly assigned to either continue E2 treatment (F.), or to have their E2 pellets removed and were treated with Tam (G.). There were n=6 animals each for the E2-treated vector control, and MKP3-overexpressing cells; there were n=4 for Tam-treated vector control, and n=5 animals for the Tam-treated MKP3-overexpressing cells.

Fig. 3

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to V5 to demonstrate MKP3 levels, or to pMAPK, total MAPK, ERα S118, total ERα antibodies, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of pMAPK normalized to Rho GDI levels. C. Densitometric scan of the immunoblot in panel A showing levels of pS118 normalized to Rho GDI levels. D. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of 20 nM PD98059. Immunoblots were stained with antibodies to V5, phospho-pMAPK and S118 ERα, or total MAPK and ERα. E. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of PD98059. Immunoblots were stained with antibodies to MKP1, phosphoJNK, and total JNK. F. Phosphatase assay using pNPP as a substrate using extracts prepared from MKP3 vector 1 and MKP3-2 cells treated for 2 hours with vehicle, E2, or Tam. The nonenzymatic hydrolysis of the substrate was corrected (absorbance at 405 nm) by subtracting the control vector transfected immunoprecipitates, from MKP3 levels and expressed as MKP3-Vector 405 nM. Phosphatase assays were performed in triplicate, n=3 separate experiments shown. G. MKP3/MAPK binding assay was performed with MKP3 Con 1 and MKP3-2 transfectants treated for 2 hours with ethanol vehicle (C), E2 (E), or Tam (T). Pre- and Post-V5 immunoprecipitated extracts (Pre-IP and Post-IP) were immunoblotted with antibodies to ERK2 and V5 to demonstrate levels of MAPK and MKP3, arrows respectively. Immunoglobulin heavy chain (HC) and light chain (LC) are shown. The specificity of the V5 and ERK2 antibodies are shown in the previous figures.

Fig. 3

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to V5 to demonstrate MKP3 levels, or to pMAPK, total MAPK, ERα S118, total ERα antibodies, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of pMAPK normalized to Rho GDI levels. C. Densitometric scan of the immunoblot in panel A showing levels of pS118 normalized to Rho GDI levels. D. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of 20 nM PD98059. Immunoblots were stained with antibodies to V5, phospho-pMAPK and S118 ERα, or total MAPK and ERα. E. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of PD98059. Immunoblots were stained with antibodies to MKP1, phosphoJNK, and total JNK. F. Phosphatase assay using pNPP as a substrate using extracts prepared from MKP3 vector 1 and MKP3-2 cells treated for 2 hours with vehicle, E2, or Tam. The nonenzymatic hydrolysis of the substrate was corrected (absorbance at 405 nm) by subtracting the control vector transfected immunoprecipitates, from MKP3 levels and expressed as MKP3-Vector 405 nM. Phosphatase assays were performed in triplicate, n=3 separate experiments shown. G. MKP3/MAPK binding assay was performed with MKP3 Con 1 and MKP3-2 transfectants treated for 2 hours with ethanol vehicle (C), E2 (E), or Tam (T). Pre- and Post-V5 immunoprecipitated extracts (Pre-IP and Post-IP) were immunoblotted with antibodies to ERK2 and V5 to demonstrate levels of MAPK and MKP3, arrows respectively. Immunoglobulin heavy chain (HC) and light chain (LC) are shown. The specificity of the V5 and ERK2 antibodies are shown in the previous figures.

Fig. 3

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to V5 to demonstrate MKP3 levels, or to pMAPK, total MAPK, ERα S118, total ERα antibodies, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of pMAPK normalized to Rho GDI levels. C. Densitometric scan of the immunoblot in panel A showing levels of pS118 normalized to Rho GDI levels. D. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of 20 nM PD98059. Immunoblots were stained with antibodies to V5, phospho-pMAPK and S118 ERα, or total MAPK and ERα. E. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of PD98059. Immunoblots were stained with antibodies to MKP1, phosphoJNK, and total JNK. F. Phosphatase assay using pNPP as a substrate using extracts prepared from MKP3 vector 1 and MKP3-2 cells treated for 2 hours with vehicle, E2, or Tam. The nonenzymatic hydrolysis of the substrate was corrected (absorbance at 405 nm) by subtracting the control vector transfected immunoprecipitates, from MKP3 levels and expressed as MKP3-Vector 405 nM. Phosphatase assays were performed in triplicate, n=3 separate experiments shown. G. MKP3/MAPK binding assay was performed with MKP3 Con 1 and MKP3-2 transfectants treated for 2 hours with ethanol vehicle (C), E2 (E), or Tam (T). Pre- and Post-V5 immunoprecipitated extracts (Pre-IP and Post-IP) were immunoblotted with antibodies to ERK2 and V5 to demonstrate levels of MAPK and MKP3, arrows respectively. Immunoglobulin heavy chain (HC) and light chain (LC) are shown. The specificity of the V5 and ERK2 antibodies are shown in the previous figures.

Fig. 3

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to V5 to demonstrate MKP3 levels, or to pMAPK, total MAPK, ERα S118, total ERα antibodies, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of pMAPK normalized to Rho GDI levels. C. Densitometric scan of the immunoblot in panel A showing levels of pS118 normalized to Rho GDI levels. D. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of 20 nM PD98059. Immunoblots were stained with antibodies to V5, phospho-pMAPK and S118 ERα, or total MAPK and ERα. E. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of PD98059. Immunoblots were stained with antibodies to MKP1, phosphoJNK, and total JNK. F. Phosphatase assay using pNPP as a substrate using extracts prepared from MKP3 vector 1 and MKP3-2 cells treated for 2 hours with vehicle, E2, or Tam. The nonenzymatic hydrolysis of the substrate was corrected (absorbance at 405 nm) by subtracting the control vector transfected immunoprecipitates, from MKP3 levels and expressed as MKP3-Vector 405 nM. Phosphatase assays were performed in triplicate, n=3 separate experiments shown. G. MKP3/MAPK binding assay was performed with MKP3 Con 1 and MKP3-2 transfectants treated for 2 hours with ethanol vehicle (C), E2 (E), or Tam (T). Pre- and Post-V5 immunoprecipitated extracts (Pre-IP and Post-IP) were immunoblotted with antibodies to ERK2 and V5 to demonstrate levels of MAPK and MKP3, arrows respectively. Immunoglobulin heavy chain (HC) and light chain (LC) are shown. The specificity of the V5 and ERK2 antibodies are shown in the previous figures.

Fig. 3

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to V5 to demonstrate MKP3 levels, or to pMAPK, total MAPK, ERα S118, total ERα antibodies, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of pMAPK normalized to Rho GDI levels. C. Densitometric scan of the immunoblot in panel A showing levels of pS118 normalized to Rho GDI levels. D. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of 20 nM PD98059. Immunoblots were stained with antibodies to V5, phospho-pMAPK and S118 ERα, or total MAPK and ERα. E. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of PD98059. Immunoblots were stained with antibodies to MKP1, phosphoJNK, and total JNK. F. Phosphatase assay using pNPP as a substrate using extracts prepared from MKP3 vector 1 and MKP3-2 cells treated for 2 hours with vehicle, E2, or Tam. The nonenzymatic hydrolysis of the substrate was corrected (absorbance at 405 nm) by subtracting the control vector transfected immunoprecipitates, from MKP3 levels and expressed as MKP3-Vector 405 nM. Phosphatase assays were performed in triplicate, n=3 separate experiments shown. G. MKP3/MAPK binding assay was performed with MKP3 Con 1 and MKP3-2 transfectants treated for 2 hours with ethanol vehicle (C), E2 (E), or Tam (T). Pre- and Post-V5 immunoprecipitated extracts (Pre-IP and Post-IP) were immunoblotted with antibodies to ERK2 and V5 to demonstrate levels of MAPK and MKP3, arrows respectively. Immunoglobulin heavy chain (HC) and light chain (LC) are shown. The specificity of the V5 and ERK2 antibodies are shown in the previous figures.

Fig. 3

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to V5 to demonstrate MKP3 levels, or to pMAPK, total MAPK, ERα S118, total ERα antibodies, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of pMAPK normalized to Rho GDI levels. C. Densitometric scan of the immunoblot in panel A showing levels of pS118 normalized to Rho GDI levels. D. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of 20 nM PD98059. Immunoblots were stained with antibodies to V5, phospho-pMAPK and S118 ERα, or total MAPK and ERα. E. An immunoblot analysis of MKP3 Con 1 and MKP3-2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of PD98059. Immunoblots were stained with antibodies to MKP1, phosphoJNK, and total JNK. F. Phosphatase assay using pNPP as a substrate using extracts prepared from MKP3 vector 1 and MKP3-2 cells treated for 2 hours with vehicle, E2, or Tam. The nonenzymatic hydrolysis of the substrate was corrected (absorbance at 405 nm) by subtracting the control vector transfected immunoprecipitates, from MKP3 levels and expressed as MKP3-Vector 405 nM. Phosphatase assays were performed in triplicate, n=3 separate experiments shown. G. MKP3/MAPK binding assay was performed with MKP3 Con 1 and MKP3-2 transfectants treated for 2 hours with ethanol vehicle (C), E2 (E), or Tam (T). Pre- and Post-V5 immunoprecipitated extracts (Pre-IP and Post-IP) were immunoblotted with antibodies to ERK2 and V5 to demonstrate levels of MAPK and MKP3, arrows respectively. Immunoglobulin heavy chain (HC) and light chain (LC) are shown. The specificity of the V5 and ERK2 antibodies are shown in the previous figures.

Fig. 4

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to CCND1, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of CCND1 normalized to Rho GDI levels.

Fig. 4

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to CCND1, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of CCND1 normalized to Rho GDI levels.

Fig. 4

A. Immunoblot analysis of two vector control (Con 1 and 2), and two MKP3-overexpressing transfectants (MKP3-1 and 2) treated for 2 hours with ethanol vehicle (C), 100 nM E2 (E), or 100 nM Tam (T). Immunoblots were stained with antibodies to CCND1, and anti-Rho GDI as a loading control. B. Densitometric scan of the immunoblot in panel A showing levels of CCND1 normalized to Rho GDI levels.

Fig. 5

A. ROS levels in MKP3.2 MCF-7 cells treated for 2 hours with ethanol vehicle (C), 100 nM E2, or 100 nM Tam. were measured using the fluorogenic DCF. B. MKP3 phosphatase activity was examined in MKP3.2 and vector Con 1 control cells in the presence of hormonal treatments and GSH. C. Immunoblot of exogenous MKP3 levels in cells from panel B using an antibody to the V5 tag. D. Anchorage-independent colony formation assay with the above transfectants in the presence of vechicle, 1.0 nM E2, 100 nM Tam +/− 100 μM GSH. The mean colony number was assayed after growth under the respective treatment conditions for 14 days. E. An immunoblot analysis of MKP3 Con 1 and MKP3.2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of GSH Immunoblots were stained with antibodies to pMAPK, pS118 ERα, total Erk1/2, total ERα, and V5 to detect exogenous MKP3.

Fig. 5

A. ROS levels in MKP3.2 MCF-7 cells treated for 2 hours with ethanol vehicle (C), 100 nM E2, or 100 nM Tam. were measured using the fluorogenic DCF. B. MKP3 phosphatase activity was examined in MKP3.2 and vector Con 1 control cells in the presence of hormonal treatments and GSH. C. Immunoblot of exogenous MKP3 levels in cells from panel B using an antibody to the V5 tag. D. Anchorage-independent colony formation assay with the above transfectants in the presence of vechicle, 1.0 nM E2, 100 nM Tam +/− 100 μM GSH. The mean colony number was assayed after growth under the respective treatment conditions for 14 days. E. An immunoblot analysis of MKP3 Con 1 and MKP3.2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of GSH Immunoblots were stained with antibodies to pMAPK, pS118 ERα, total Erk1/2, total ERα, and V5 to detect exogenous MKP3.

Fig. 5

A. ROS levels in MKP3.2 MCF-7 cells treated for 2 hours with ethanol vehicle (C), 100 nM E2, or 100 nM Tam. were measured using the fluorogenic DCF. B. MKP3 phosphatase activity was examined in MKP3.2 and vector Con 1 control cells in the presence of hormonal treatments and GSH. C. Immunoblot of exogenous MKP3 levels in cells from panel B using an antibody to the V5 tag. D. Anchorage-independent colony formation assay with the above transfectants in the presence of vechicle, 1.0 nM E2, 100 nM Tam +/− 100 μM GSH. The mean colony number was assayed after growth under the respective treatment conditions for 14 days. E. An immunoblot analysis of MKP3 Con 1 and MKP3.2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of GSH Immunoblots were stained with antibodies to pMAPK, pS118 ERα, total Erk1/2, total ERα, and V5 to detect exogenous MKP3.

Fig. 5

A. ROS levels in MKP3.2 MCF-7 cells treated for 2 hours with ethanol vehicle (C), 100 nM E2, or 100 nM Tam. were measured using the fluorogenic DCF. B. MKP3 phosphatase activity was examined in MKP3.2 and vector Con 1 control cells in the presence of hormonal treatments and GSH. C. Immunoblot of exogenous MKP3 levels in cells from panel B using an antibody to the V5 tag. D. Anchorage-independent colony formation assay with the above transfectants in the presence of vechicle, 1.0 nM E2, 100 nM Tam +/− 100 μM GSH. The mean colony number was assayed after growth under the respective treatment conditions for 14 days. E. An immunoblot analysis of MKP3 Con 1 and MKP3.2 transfectants treated with vehicle, E2, or Tam for 2 hours in the absence(-) or presence of GSH Immunoblots were stained with antibodies to pMAPK, pS118 ERα, total Erk1/2, total ERα, and V5 to detect exogenous MKP3.

Fig. 6

A. Immunoblot analysis of vector-alone, control siRNA transfected Ishikawa cells (Ctrl Si ), and two MKP1 siRNA transfected Ishikawa cell polls (Si615 and 1126). MKP1 and total Erk1/2 levels were measured. B. Anchorage-independent colony formation assay with the above Ishikawa transfectants in the presence of ethanol (C), 1.0 nM E2 or 100 nM Tam. The mean colony number was assayed after growth under the respective treatment conditions for 14 days and statistical significance was assessed using a two-tailed Student’s t-test; **significance level at p<0.05. C. Immunoblot analysis of Ctrl SiRNA and two MKP1 siRNAs after treatment with ethanol (C), 100 nM E2, or 100 nM Tam using antibodies to pMAPK and total Erk2.