IQGAP1 protein binds human epidermal growth factor receptor 2 (HER2) and modulates trastuzumab resistance

Abstract

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast cancers. Increased HER2 expression is an adverse prognostic factor and correlates with decreased patient survival. HER2-positive (HER2(+)) breast cancer is treated with trastuzumab. Unfortunately, some patients are intrinsically refractory to therapy, and many who do respond initially become resistant within 1 year. Understanding the molecular mechanisms underlying HER2 signaling and trastuzumab resistance is essential to reduce breast cancer mortality. IQGAP1 is a ubiquitously expressed scaffold protein that contains multiple protein interaction domains. By regulating its binding partners IQGAP1 integrates signaling pathways, several of which contribute to breast tumorigenesis. We show here that IQGAP1 is overexpressed in HER2(+) breast cancer tissue and binds directly to HER2. Knockdown of IQGAP1 decreases HER2 expression, phosphorylation, signaling, and HER2-stimulated cell proliferation, effects that are all reversed by reconstituting cells with IQGAP1. Reducing IQGAP1 up-regulates p27, and blocking this increase attenuates the growth inhibitory effects of IQGAP1 knockdown. Importantly, IQGAP1 is overexpressed in trastuzumab-resistant breast epithelial cells, and reducing IQGAP1 both augments the inhibitory effects of trastuzumab and restores trastuzumab sensitivity to trastuzumab-resistant SkBR3 cells. These data suggest that inhibiting IQGAP1 function may represent a rational strategy for treating HER2(+) breast carcinoma.

FIGURE 1.

IQGAP1 is overexpressed in HER2(+) breast cancer. A and B, IQGAP1 protein expression was evaluated by immunohistochemistry in normal breast specimens (Nl, upper panels) and HER2(+) (3+ by immunohistochemistry and/or amplified by FISH) invasive breast carcinomas (Ca, lower panels). Representative images are shown. The final magnification is 100× (A) and 400× (B). H&E, hematoxylin and eosin. C, IQGAP1 protein expression was evaluated by immunohistochemistry in hormone receptor-positive (HR(+); upper panel) and triple-negative (TNBC; lower panel) invasive breast carcinomas. Representative images are shown. The final magnification is 100×.

FIGURE 2.

IQGAP1 binds HER2. A, GST alone, GST-HER2 (HER2), or calmodulin-Sepharose (CaM) was incubated with equal amounts of purified IQGAP1 (upper panel) or equal amounts of protein from SkBR3 cell lysates (lower panel). Complexes were isolated and washed as described under “Experimental Procedures.” The samples were resolved by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and probed with anti-IQGAP1 antibodies. An aliquot of each sample (equivalent to 2% of the amount in each pulldown) was also processed by Western blotting (Input, top panel; Lysate, bottom panel). The data are representative of five independent experiments. B, equivalent amounts of GST alone, GST-HER2 (HER2), or calmodulin-Sepharose (CaM) to those used in the pulldown assays in A were resolved by SDS-PAGE. The gel was stained with Coomassie Blue. The data are representative of five independent experiments.

FIGURE 3.

IQGAP1 and HER2 co-immunoprecipitate. Equal amounts of protein from SkBR3 cell lysates were immunoprecipitated (IP) with anti-IQGAP1 antibodies (upper panel) or anti-HER2 antibody (lower panel). Non-immune rabbit serum (NIRS) was used as a negative control. Both unfractionated lysates (Lysate) and complexes (IP) were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1 and anti-HER2 antibodies. The data are representative of five independent experiments.

FIGURE 4.

The IQ region of IQGAP1 is both necessary and sufficient for HER2 binding. A, shown is a schematic representation of IQGAP1 constructs depicting full-length IQGAP1, truncated IQGAP1 fragments, and a deletion mutant. The specific amino acids absent from each construct are indicated. FL, full-length IQGAP1 (amino acids 2–1657); N, N-terminal half of IQGAP1 (amino acids 2–863); IQ, IQ region of IQGAP1 (amino acids 717–916); C, C-terminal half of IQGAP1 (amino acids 864–1657); ΔIQ, full length IQGAP1 from which the IQ region has been deleted. B, [³⁵S]methionine-labeled full length IQGAP1 (FL), IQGAP1-N (N), IQGAP1-IQ (IQ), IQGAP1-C (C), and IQGAP1ΔIQ (ΔIQ) were incubated with equal amounts of GST-HER2 (HER2, top right panel), GST-Cdc42V12 (Cdc42, bottom left panel), or GST alone (GST, bottom right panel). Complexes were isolated and washed as described under “Experimental Procedures.” The samples were resolved by SDS-PAGE, and the gels were dried and processed by autoradiography. An aliquot of each [³⁵S]methionine-labeled TNT product (equivalent to 2% of the amount in each pulldown) was also resolved by SDS-PAGE and processed by autoradiography (Input, top left panel). The data are representative of five independent experiments.

FIGURE 5.

Knockdown of IQGAP1 reduces HER2 expression, phosphorylation, and signaling and enhances the inhibitory effects of trastuzumab. A, SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-pHER2, anti-HER2, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, and anti-β-tubulin antibodies. The data are representative of five independent experiments. B, the amounts of IQGAP1 and HER2 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The amounts of pHER2 and pAKT were quantified by densitometry and corrected for the amounts of total HER2 and total AKT, respectively, in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and treated with vehicle represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05). C, SkBR3 cells were transiently transfected with siRNA against renilla luciferase and vector (siRen and Vector), siRNA against IQGAP1 and vector (siIQ12 and Vector), or siRNA against IQGAP1 and siRNA-resistant IQGAP1 (siIQ12 and IQGAP1). 36 h after transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-pHER2, anti-HER2, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, and anti-β-tubulin antibodies. The data are representative of three independent experiments. D, the amounts of IQGAP1 and HER2 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The amounts of pHER2 and pAKT were quantified by densitometry and corrected for the amounts of total HER2 and total AKT, respectively, in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and vector and treated with vehicle, represent the mean ± S.E. (n = 3). *, significantly different from control cells transfected with siRen and vector and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05); #, significantly different from cells transfected with siIQ12 and vector (p < 0.05).

FIGURE 6.

Knockdown of IQGAP1 inhibits SkBR3 cell growth and increases the sensitivity of SkBR3 cells to trastuzumab. A, SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection cells were serum-starved in the presence of vehicle (black bars) or 21 μg/ml trastuzumab (white bars) for 48 h. Cell growth was measured using sulforhodamine B staining. The data, expressed relative to the number of cells in wells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05). B, SkBR3 cells were transiently transfected with siRNA against renilla luciferase and vector (siRen and vector), siRNA against IQGAP1 and vector (siIQ12 and vector), or siRNA against IQGAP1 and siRNA-resistant IQGAP1 (siIQ12 and IQGAP1). 36 h after transfection, cells were serum-starved in the presence of vehicle (black bars) or 21 μg/ml trastuzumab (white bars) for 48 h. Cell growth was measured using sulforhodamine B staining. The data, expressed relative to the number of cells in wells transfected with siRen and vector and treated with vehicle, represent the mean ± S.E. (n = 3). *, significantly different from control cells transfected with siRen and vector and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05); #, significantly different from cells transfected with siIQ12 and vector (p < 0.05).

FIGURE 7.

Knockdown of IQGAP1 increases p27 expression and augments trastuzumab-induced p27 up-regulation. A, SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-p27, and anti-β-tubulin antibodies. The data are representative of five independent experiments. B, the amounts of IQGAP1 and p27 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05). C, SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen), siRNA against IQGAP1 (siIQ12), or siRNAs against IQGAP1 and p27 (siIQ12 and sip27). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-p27, and anti-β-tubulin antibodies. The data are representative of three independent experiments. D, the amounts of IQGAP1 and p27 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 3). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05); #, significantly different from cells transfected with siIQ12 (p < 0.05). E, SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen), siRNA against IQGAP1 (siIQ12), or siRNAs against IQGAP1 and p27 (siIQ12 and sip27). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (black bars) or 21 μg/ml trastuzumab (white bars) for 48 h. Cell growth was measured using sulforhodamine B staining. The data, expressed relative to the number of cells in wells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 3). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05); #, significantly different from cells transfected with siIQ12 (p < 0.05).

FIGURE 8.

IQGAP1 is overexpressed in trastuzumab-resistant SkBR3 cells. A, equal amounts of protein from trastuzumab-sensitive (SkBR3) and trastuzumab-resistant (SkBR3^TR) SkBR3 cells were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1 and anti-β-tubulin antibodies. The data are representative of three independent experiments. B, the amount of IQGAP1 was quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The data, expressed relative to the amount of IQGAP1 in SkBR3 cells, represent the mean ± S.E. (n = 3). *, significantly different from SkBR3 cells (p < 0.05).

FIGURE 9.

Knockdown of IQGAP1 abrogates resistance to the growth inhibitory effects of trastuzumab. Trastuzumab-sensitive (SkBR3) or trastuzumab-resistant (SkBR3^TR) SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (black bars) or 21 μg/ml trastuzumab (white bars) for 48 h. Cell growth was measured using sulforhodamine B staining. The data, expressed relative to the number of control cells in wells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05).

FIGURE 10.

Knockdown of IQGAP1 reduces HER2 expression, phosphorylation, and signaling and restores trastuzumab sensitivity to trastuzumab-resistant SkBR3 cells. A, trastuzumab-resistant SkBR3 cells were transiently transfected with siRNA against renilla luciferase (siRen) or siRNAs against IQGAP1 (siIQ12 and siIQ14). 36 h after siRNA transfection, cells were serum-starved in the presence of vehicle (−) or 21 μg/ml trastuzumab (+) for 48 h. Equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IQGAP1, anti-pHER2, anti-HER2, anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-p27, and anti-β-tubulin antibodies. The data are representative of five independent experiments. B, the amounts of IQGAP1, HER2, and p27 were quantified by densitometry and corrected for the amount of β-tubulin in the corresponding lysate. The amounts of pHER2 and pAKT were quantified by densitometry and corrected for the amounts of total HER2 and total AKT, respectively, in the corresponding lysate. Samples that were treated with vehicle are depicted by black bars, and those treated with trastuzumab are depicted by white bars. The data, expressed relative to the amount of each protein in control cells transfected with siRen and treated with vehicle, represent the mean ± S.E. (n = 5). *, significantly different from control cells transfected with siRen and treated with vehicle (p < 0.05); θ, significantly different from cells treated with vehicle (p < 0.05).

FIGURE 11.

Model of IQGAP1 involvement in HER2 function. A1, HER2 (blue), which is constitutively phosphorylated, signals (red arrows) primarily through the AKT (orange ovals) and MAPK (not shown) pathways. HER2 signaling degrades p27 (light purple decagon), a CDK inhibitor, and thereby stimulates cell growth. A2, HER2 is internalized (green arrows) followed by degradation in the lysosome. A3, some receptors, particularly those bound to IQGAP1 (red), escape degradation and are recycled back to the plasma membrane where they are biologically active. B, trastuzumab-sensitive cells are shown. B1, trastuzumab (dark purple) inhibits signaling by HER2. The reduced activation of AKT results in accumulation of p27, which arrests the cell cycle, reducing growth. B2, trastuzumab also stimulates (purple arrow) HER2 internalization and degradation, reducing the number of HER2 receptors in the cell. C, trastuzumab-resistant cells are shown. These cells contain increased amounts of IQGAP1. The excess IQGAP1 stabilizes HER2 protein expression by reducing HER2 degradation. The resultant increase in the number of HER2 receptors enhances HER2 signaling, inducing trastuzumab resistance and promoting cell growth. p, phosphate.