Essential role for Rac in heregulin beta1 mitogenic signaling: a mechanism that involves epidermal growth factor receptor and is independent of ErbB4

Abstract

Heregulins are a family of ligands for the ErbB3/ErbB4 receptors that play important roles in breast cancer cell proliferation and tumorigenesis. Limited information is available on the contribution of Rho GTPases to heregulin-mediated signaling. In breast cancer cells, heregulin beta1 (HRG) causes a strong activation of Rac; however, it does so with striking differences in kinetics compared to epidermal growth factor, which signals through ErbB1 (epidermal growth factor receptor [EGFR]). Using specific ErbB receptor inhibitors and depletion of receptors by RNA interference (RNAi), we established that, surprisingly, activation of Rac by HRG is mediated not only by ErbB3 and ErbB2 but also by transactivation of EGFR, and it is independent of ErbB4. Similar receptor requirements are observed for HRG-induced actin cytoskeleton reorganization and mitogenic activity via extracellular signal-regulated kinase (ERK). HRG-induced Rac activation was phosphatidylinositol 3-kinase dependent and Src independent. Furthermore, inactivation of Rac by expression of the Rac GTPase-activating protein beta2-chimerin inhibited HRG-induced ERK activation, mitogenicity, and migration in breast cancer cells. HRG mitogenic activity was also impaired by depletion of Rac1 using RNAi. Our studies established that Rac is a critical mediator of HRG mitogenic signaling in breast cancer cells and highlight additional levels of complexity for ErbB receptor coupling to downstream effectors that control aberrant proliferation and transformation.

FIG. 1.

HRG and EGF induce Rac/Cdc42/RhoA activation in MCF-7 and T-47D cells. (A) Dose-dependent activation of Rac by HRG. MCF-7 and T-47D cells were serum starved for 48 h and then stimulated with HRG (0 to 30 ng/ml) for 10 or 5 min, respectively. Rac GTP levels were determined using a PBD “pull-down” assay. (B) Densitometric analysis of Rac activation, normalized to the corresponding total Rac levels. Data are presented as means ± standard deviations (n = 3). (C) Time-dependent activation of Rac by HRG and EGF. Rac activation was determined in serum-starved cells after HRG (10 ng/ml) or EGF (100 ng/ml) treatment. (D and E) Densitometric analysis of time-dependent activation of Rac. Data are presented as means ± standard deviations (n = 3). (F) Cdc42 and RhoA activation by HRG (10 ng/ml) in MCF-7 and T-47D cells. Similar results were observed in three independent experiments.

FIG. 2.

Effect of siRNA knock-down individual ErbB receptors on Rac activation by HRG. (A) siRNA duplexes for each ErbB receptor were transfected into T-47D cells. Twenty-four hours later, cells were serum starved for 48 h, and Rac activation was determined after stimulation with HRG (10 ng/ml, 5 min). ErbB2 and EGFR phosphorylation after HRG (10 ng/ml, 10 min) were analyzed by Western blotting using specific anti-phospho-ErbB2-Tyr1248 or anti-phospho-EGFR-Tyr992 antibody. Similar results were observed in three independent experiments. (B and C) Densitometric analysis of the effect of individual ErbB receptor knock-down and its effect on HRG-induced Rac activation, respectively, shown as percentages of expression relative to that for control (nontransfected) cells. Data are presented as means ± standard deviations (n = 3). *, P < 0.05, compared to results with nontransfected HRG-stimulated cells.

FIG. 3.

Requirement of EGFR for HRG-induced Rac activation. (A) T-47D cells were serum starved for 48 h, incubated with different concentrations of AG1478 for 1 h, and then stimulated with HRG (10 ng/ml) for 5 min. Rac GTP levels were then assayed. (B) T-47D cells were serum starved for 48 h, pretreated with different concentrations of cetuximab for 1 h, and then stimulated with EGF (100 ng/ml for 1 min for the Rac GTP assay and 2 min for EGFR phosphorylation analysis) or HRG (10 ng/ml, 5 min for the Rac GTP assay and 10 min for EGFR phosphorylation analysis). Similar results were observed in three independent experiments. Panel C. After 48 h of serum starvation, T47D cells were treated with HRG (10 ng/ml) or EGF (100 ng/ml). Cell extracts were subjected to Western blot analysis using the indicated antibodies. ErbB4 phosphorylation was detected by IP. Inset. Densitometric analysis of ErbB receptor phosphorylation, expressed as a percentage of the maximum response in each case. Data are presented as means ± standard deviations (n = 3).

FIG. 4.

HRG-induced Rac activation is Src independent and PI3K dependent. (A and D) Time course of Src and Akt activation by HRG. T-47D cells were serum starved for 48 h and then treated with HRG (10 ng/ml). Cell extracts were subjected to Western blot analysis using specific anti-phospho-Src and anti-phospho-Akt antibodies. (B and C) After 48 h of serum starvation, T-47D cells were treated either with PP2 (0 to 5 μM) or wortmannin (0 to 5 μM) for 1 h and stimulated with HRG (10 ng/ml) for 5 min, and Rac GTP levels were then determined. (E) Effect of AG1478 and wortmannin (Wortm) pretreatment on the activation of EGFR, ErbB2, and Akt. After 48 h of serum starvation, T-47D cells were treated with either AG1478 or wortmannin for 1 h and stimulated with HRG (10 ng/ml) for 10 min. Cell extracts were subjected to Western blotting. Similar results were observed in three independent experiments.

FIG. 5.

Differential kinetics of Erk1/2 and JNK activation by HRG and EGF. (A) Cells were serum starved for 48 h and then treated with either HRG (10 ng/ml) or EGF (100 ng/ml). Cell extracts were subjected to Western blot analysis using the indicated antibodies. (B) Effect of AG1478 and wortmannin (Wortm) on HRG-induced Erk1/2 and JNK activation in T-47D cells. Cells were treated as described in the legend to Fig. 4E, and cell extracts were subjected to Western blot analysis. (C) T-47D cells were serum starved for 48 h, pretreated with different concentrations of cetuximab for 1 h, and then stimulated with EGF (100 ng/ml, 2 min) or HRG (10 ng/ml, 10 min). Cell extracts were subjected to Western blot analysis. Similar results were observed in three independent experiments.

FIG. 6.

Inhibition of β2-chimerin on HRG-induced Rac, Erk1/2, and JNK activation. (A) MCF-7 and HA-V12Rac1-MCF-7 cells were serum starved for 8 h and then infected with either HA-β2-chimerin-AdV (β2-chim) or LacZ-AdV (LacZ) for 16 h in serum-free DMEM. After extensive washing, cells were grown for 24 h in serum-free DMEM and then stimulated with HRG (10 ng/ml) for 10 min. Activation of Rac, Akt, Erk1/2, and JNK was then determined. Expression of HA-β2-chimerin and HA-V12Rac1 was examined by Western blotting using an anti-HA antibody. (B) MCF-7 cells were treated as described for panel A. Cdc42-GTP and RhoA-GTP levels were determined using pull-down assays.

FIG. 7.

β2-Chimerin inhibits HRG-induced MCF-7 cell migration. (A) Effects of inhibitors and blocking antibodies on EGF/HRG-induced ruffle formation. After 48 h of serum starvation, T-47D cells were treated with AG1478 (1 μM), Cetuximab (1 μg/ml), wortmannin (1 μM), PP2 (1 μM), ErbB3, or ErbB4 blocking antibody (10 μg/ml) for 1 h, stimulated with EGF (100 ng/ml, 5 min) or HRG (10 ng/ml, 10 min), and then stained with phalloidin. Ruffles are indicated by arrows. Similar results were observed in three independent experiments. (B) Effect of β2-chimerin on HRG-induced cell migration. Migration of MCF-7 or HA-V12Rac1-MCF-7 cells infected with HA-β2-chimerin-AdV or LacZ-AdV (see Fig. 6) was determined using a Boyden chamber. Data are presented as means ± standard deviations (n = 4). *, P < 0.05, compared to results for non-AdV-infected HRG-stimulated cells. (C) Effect of Rac1 depletion on HRG-induced cell migration. MCF-7 cells were transfected with siRNA duplexes for Rac1 (RNAi1 or RNAi2) or a control duplex (CONT), and cell migration was determined 72 h after transfection. Data are presented as means ± standard deviations (n = 4). *, P < 0.05, compared to results for nontransfected, HRG-stimulated cells. Rac expression is shown in a representative Western blot. HMF, high-magnification field.

FIG. 8.

Effect of β2-chimerin, MAPK inhibitors, and siRNA knock-down of Rac1 and individual ErbB receptors on MCF-7 and T-47D cell proliferation. (A) HA-β2-chimerin-AdV- or LacZ-AdV-infected MCF-7 or HA-V12Rac1-MCF-7 cells were stimulated with HRG (10 ng/ml) for 24 h, and then BrdU incorporation was determined. Data are presented as means ± standard deviations (n = 3). *, P < 0.05, compared to results for non-AdV-infected HRG-stimulated cells. (B, C, and F) siRNA duplexes for Rac1 or each ErbB receptor were transfected into T-47D cells. Twenty-four hours later, cells were serum starved for 48 h and stimulated with HRG (10 ng/ml). BrdU incorporation was determined 24 h later. Data are presented as means ± standard deviations (n = 3). *, P < 0.05, compared to results for nontransfected HRG-stimulated cells. Panel B shows a representative Western blot for Rac1 knock-down. (D) After 48 h of serum starvation, T-47D cells were incubated with U0126 (5 μM) or SP600125 (25 μM) for 1 h and then stimulated with HRG (10 ng/ml) in the presence of the inhibitors. BrdU incorporation was determined 48 h later. (E) Phospho-Erk and phospho-ATF2 were analyzed by Western blotting after treatment with HRG (10 ng/ml, 10 min) in the presence of UO126 and SP600125.