S-nitrosylation of EGFR and Src activates an oncogenic signaling network in human basal-like breast cancer

Abstract

Increased inducible nitric oxide synthase (NOS2) expression in breast tumors is associated with decreased survival of estrogen receptor negative (ER-) breast cancer patients. We recently communicated the preliminary observation that nitric oxide (NO) signaling results in epidermal growth factor receptor (EGFR) tyrosine phosphorylation. To further define the role of NO in the pathogenesis of ER- breast cancer, we examined the mechanism of NO-induced EGFR activation in human ER- breast cancer. NO was found to activate EGFR and Src by a mechanism that includes S-nitrosylation. NO, at physiologically relevant concentrations, induced an EGFR/Src-mediated activation of oncogenic signal transduction pathways (including c-Myc, Akt, and β-catenin) and the loss of PP2A tumor suppressor activity. In addition, NO signaling increased cellular EMT, expression and activity of COX-2, and chemoresistance to adriamycin and paclitaxel. When connected into a network, these concerted events link NO to the development of a stem cell-like phenotype, resulting in the upregulation of CD44 and STAT3 phosphorylation. Our observations are also consistent with the finding that NOS2 is associated with a basal-like transcription pattern in human breast tumors. These results indicate that the inhibition of NOS2 activity or NO signaling networks may have beneficial effects in treating basal-like breast cancer patients.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. NOS2 activity and NO-donor increases EGFR tyrosine phosphorylation

(A) Relative NOS2 expression in RAW 264.7 murine macrophages pretreated with or without IFN-γ/LPS. (B) Western blot showing EGFR tyrosine 1173 phosphorylation in MDA-MB-468 cells co-cultured with IFN-γ/LPS activated RAW cells in increasing ratios and in media containing or lacking the NOS2 inhibitor aminoguanidine (AG). Co-cultured samples are compared to monocultured MDA-MB-468 cells treated with AG or EGF. (C) Total nitrate and nitrite (NOx−) concentrations in conditioned media from co-culture experiments as an aggregate indicator of NOS2 activity. (D) Western blot of EGFR tyrosine 1045 phosphorylation in serum-starved MDA-MB-468 cells treated with either EGF (10 ng/ml) or DETANO for 24 hours. (E) Densitometric analyses of EGFR tyrosine 1173 phosphorylation indicating that EGF and 0.3 and 0.5 mM DETANO resulted in significant increases in EGFR activation compared to untreated controls (*P < 0.05).

Figure 2. NO activates EGFR via S-nitrosation post-translational modification

(A) Steady-state NO concentration in DETANO-supplemented media as determined by chemiluminescence. Mean [NO] (± sem) values are shown. (B) Nitrosative ability of DETANO as determined by DAN fluorescence assay. Mean DAN-triazole fluorescence (RFU) values (± sd) are shown and linear regression analysis (± 95% CI) indicates a linear response between DETANO concentration and nitrosative capacity. (C) Total cellular protein S-nitrosation from MDA-MB-468 cells treated with DETANO for 24 hours compared to total cellular protein content. (D) Total protein, immunoprecipitated EGFR and EGFR-SNO from MDA-MB-468 cells treated with DETANO for 24 hours. (E) Western blot of relative EGFR tyrosine 1173 phosphorylation and EGFR-SNO modification from MDA-MB-468 cells treated with EGF (10 ng/mL) or DETANO and chemical inhibitors of S-nitrosation.

Figure 3. NO activates EGFR and Src kinase signaling pathways in ER− breast cancer cells

(A) Densitometric analyses from receptor tyrosine kinase (RTK) Pathscan spot-ELISA experiment in MDA-MB-468 cells treated with EGF or DETANO compared to serum-starved controls. EGFR, Src and Akt phosphorylation was increased by EGF and 0.5 mM DETANO. (B) Total input protein and Src, immunoprecipitated and S-nitrosated (SNO) Src from MDA-MB-468 cells treated with DETANO. (C) Relative Akt, Src and STAT3 phosphorylation from MDA-MB-468 cells treated with either EGF or DETANO compared to untreated serum-starved controls. (D) Akt, Src, STAT3 phosphorylation and CD44 expression in the presence of chemical inhibitors of nitrosation. (E) Western blot showing the stabilization of c-Myc in MDA-MB-468 cells in response to DETANO concentration. (F) Relative c-Myc-DNA binding from MDA-MB-468 cells treated with 0.5 mM DETANO and/or PP2 (100 nM). Normalized mean c-Myc activity (± sd) is shown. Statistical significance was determined by *P < 0.05. (G) Representative western blots from Her2+ SKBR3 cells exposed to EGF or DETANO showing NO-induced increases in EGFR and STAT3 tyrosine phosphorylation and increased CD44 expression.

Figure 4. NO activates the β-catenin signaling pathway in basal-like breast cancer cells

(A) TCF-luciferase reporter activity of MDA-MB-468 cells alone or co-cultured with RAW 264.7 cells under the conditions shown. Data are normalized to untreated controls and represent the fold-increase of mean relative luminescence units (RLU) (± sd). (B) TCF-luciferase reporter activity from breast cancer cell lines (MDA-MB-468, MDA-MB-231 and M6) exposed to LiCl or DETANO for 20 hours. Data represent mean fold-increase RLU (± sd) compared to untreated controls. (C) Representative immunofluorescent micrographs of MDA-MB-231 β-catenin cellular localization in response to LiCl or DETANO exposure. The yellow pixels in merged images represent nuclear β-catenin localization and the scale bars represent 10 µm. (D) Representative western blot of MDA-MB-468 nuclear fractions showing an increase in nuclear β-catenin in response to DETANO compared to tata-binding protein (TBP) nuclear content.

Figure 5. S-nitrosation is required for NO to activate β-catenin signaling

(A) TCF-luciferase activity from MDA-MB-468 cells exposed to LiCl or 0.3 mM DETANO and inhibitors of nitrosation. (B) TCF-luciferase activity from MDA-MB-468 cells pretreated with NEM (black) or untreated (grey) followed by DETANO incubation. (C) TCF-luciferase activity from MDA-MB-468 cells exposed to 0.3 mM DETANO and kinase (Gefitinib, PP2, wortmannin and triciribine) and soluble guanylate cyclase (ODQ) inhibitors. Data represent fold-RLU relative to control (± sd). (D) Representative immunofluorescent images of β-catenin localization in MDA-MB-231 cells treated with 0.3 mM DETANO ± 100 nM PP2.

Figure 6. NO increases cellular EMT

(A) Representative microphotographs of MDA-MB-468 cells compared to cells exposed to 0.5 mM DETANO showing altered cellular morphology and cell-to-cell adhesion. (B) Representative western blot of E-cadherin and vimentin expression in MDA-MB-468 cells exposed to 0.5 mM DETANO alone or with GSH, N3− or PP2. (C) Cellular proliferation in serum-starved MDA-MB-468 or SKBR3 cells exposed to EGF or 0.5 mM DETANO. Significance from control was determined by one-way ANOVA with Dunnett’s post-test analysis (*P < 0.01). (D) Western blot of relative COX-2 expression in MDA-MB-468 cells treated with DETANO for 24 hours as compared to actin. (E) COX-2 activity as determined by PGE2 measurements in DETANO-treated MDA-MB-468 conditioned media. Data represent mean PGE2 concentrations (± sd). Significance from control was determined by one-way ANOVA with Dunnett’s post-test analysis (*P < 0.05).

Figure 7. NO increases chemoresistance in basal-like breast cancer cells

(A) Representative western blot of P-glycoprotein expression in MDA-MB-231 cells exposed to wither EGF or DETANO. (B) Densitometric analyses of P-glycoprotein expression in response to EGF or DETANO. Significance from control was determined by one-way ANOVA with Dunnett’s post-test analysis (*P < 0.05). (C) Survival of MDA-MB-231 cells pre-treated with or without 0.5 mM DETANO in response to adriamycin and paclitaxel exposure. Data is shown as percent proliferation compared to cells not treated with chemotherapeutic.