ErbB2-mediated Src and signal transducer and activator of transcription 3 activation leads to transcriptional up-regulation of p21Cip1 and chemoresistance in breast cancer cells

Abstract

Overexpression of the ErbB2 receptor tyrosine kinase is prevalent in approximately 30% of human breast cancers and confers Taxol resistance. Our previous work has shown that ErbB2 inhibits Taxol-induced apoptosis in breast cancer cells by transcriptionally up-regulating p21(Cip1). However, the mechanism of ErbB2-mediated p21(Cip1) up-regulation is unclear. Here, we show that ErbB2 up-regulates p21(Cip1) transcription through increased Src activity in ErbB2-overexpressing cells. Src activation further activated signal transducer and activator of transcription 3 (STAT3) that recognizes a SIE binding site on the p21(Cip1) promoter required for ErbB2-mediated p21(Cip1) transcriptional up-regulation. Both Src and STAT3 inhibitors restored Taxol sensitivity in resistant ErbB2-overexpressing breast cancer cells. Our data suggest that ErbB2 overexpression can activate STAT3 through Src leading to transcriptional up-regulation of p21(Cip1) that confers Taxol resistance of breast cancer cells. Our study suggests a potential clinical application of Src and STAT3 inhibitors in Taxol sensitization of ErbB2-overexpressing breast cancers.

FIGURE 1

Constitutive activation of STAT3 in ErbB2 overexpressing cells results in p21^Cip1 transcriptional upregulation. A. left, schematic diagram of the 5’ promoter region of the p21 gene. White boxes represent STAT SIE location on promoter; black box represents mutated SIE. right, Luciferase activity of extracts prepared from 435.Vec and 435.ErbB2 cells transfected with the indicated p21^Cip1 promoter reporter constructs. Bars indicate luciferase activity standardized to vector control for each construct. Error bars represent standard deviation (S.D.) B. Lysates from SKBR3, along with MDA-MB--435 and MDA-MB-231 breast cancer cells stably transfected with either control vector or wild-type ErbB2 were analyzed by Western blotting with the indicated antibodies. C. Nuclear extracts were collected from ErbB2 stable transfectants and vector control cells and SKBR3 cells for Electrophoretic Mobility Shift Assay (EMSA) analysis. Arrow indicates STAT protein:DNA complexes. Asterisk indicates addition of non-radiolabeled competitor probe. D. Chromatin Immunoprecipitation (ChIP assay) for SIE p21^Cip1 promoter sequence using antibodies against histone and STAT3 in ErbB2 overexpressing cells. Histone and IgG immunoprecipitation served as positive and negative controls, respectively. Input represents 10% of the total.

FIGURE 2

p21^Cip1 transcriptional activation by ErbB2 is dependant on STAT3 protein. A. 435.ErbB2 and 231.ErbB2 cells treated with 300 nM of Mismatch (MM) or STAT3 Antisense (AS) oligonucleotides. Cells were harvested 5 days post-transfection and analyzed by Western blot with the indicated antibodies. B. Standardized firefly luciferase activity of the wild type p21 promoter (pGL3-p21−2400) reporter plasmid in 435.ErbB2 and 231.ErbB2 cells treated with either STAT3 MM or AS oligonucleotides. Fold reduction was determined by standardizing to Mismatch control treated sample for each cell line. Error bars represent S.D.

FIGURE 3

ErbB2, Src, and STAT3 form a complex in ErbB2 overexpressing cells. Whole cell lysates from A. 435.ErbB2 or B. SKBR3 cells were immunoprecipitated (IP) with the indicated antibodies followed by immunoblot analysis for ErbB2, STAT3, and Src. Input lanes represent 10% of total protein immunoprecipitated. Asterisk indicates IgG heavy chain.

FIGURE 4

Src is required for ErbB2 mediated STAT3 activation and p21^Cip1 upregulation. A. ErbB2 overexpressing cells were treated with 5 μM control (PP3) or Src inhibitor (PP2). Cell lysates were collected and analyzed by Western blotting. B. 435.ErbB2 and SKBR3 cells were transfected with the p21^Cip1 reporter plasmids wild type (pGL3-p21−2400) or SIE mutant (pGL3-p21−2400mSIE) and treated with 5 μM PP3 or PP2 for 24 hours. Cell lysates were collected and luciferase activities were measured and standardized by transfection efficiency using renilla values. Error bars represent S.D. C. Indicated cells were transfected with either vector control (Vec) or dominant negative Src mutant (pSRC-DN) for 24 hours. Lysates were analyzed by Western blotting. D. Cells were co-transfected with vector control or pSrc-DN plasmids and the pGL3-p21−2400 reporter plasmid. Luciferase activities were measured as in (C).

FIGURE 5

Inhibition of STAT3 sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 and SKBR3 cells were treated with 50 μM of STAT3 inhibitory peptide or control peptide for 24 hours. Nuclear extracts were then subjected to EMSA. Band shifts indicated a STAT3 protein:DNA complex. Oct-1 protein:DNA binding serves as an loading control. B. 435.ErbB2 and SKBR3 cells were treated with control (CP) or STAT3 inhibitor peptide (SP) (100 μM for 435.ErbB2 and 50 μM for SKBR3) for 12 hours. Cells were then treated with different concentrations of Taxol for 24 hours. Cell growth inhibition was determined by MTS assay. Error bars represent S.D. Inset: Representative Western blot for p21^Cip1 and β-actin (loading control) from cells treated with either control (CP) or STAT3 inhibitor peptide (SP) treatment (** p = 0.004; * p = 0.016; t-test).

FIGURE 6

Inhibition of Src kinase sensitized ErbB2 overexpressing breast cancer cells to Taxol. A. 435.ErbB2 cells were treated as indicated for 18 hours. Cell lysates were collected and analyzed by western blot analysis for the indicated antibodies. Numbers represent relative intensity of each band compared to the non-treated (NT) control. Total STAT3 was used as the loading control for both P-STAT3-Y705 and p21. B. 435.ErbB2 cells were treated with DMSO (control) or AZD0530 for 18 hours. Chromatin Immunoprecipitation (ChIP assay) for SIE p21^Cip1 promoter sequence was performed using antibodies against histone and STAT3. IgG and Histone immunoprecipitation served as negative and positive controls, respectively. Input represents 10% of the total. C. 435.ErbB2 and SKBR3 cells were treated with DMSO only or AZD0530 (2 μM) for 12 hours. After 12 hours, cell media was replaced with medium containing either Taxol (50 nM) only, AZD0530 only or combination of Taxol and AZD0530. Cell growth inhibition was determined by MTS assay at 36 hours. Results were normalized to DMSO controls (** p = 0.002; * p = 0.013; t-test).