Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jun 26.
Published in final edited form as: Cancer Res. 2012 Mar 29;72(10):2672–2682. doi: 10.1158/0008-5472.CAN-11-3594

HER3 Is Required for HER2-Induced Preneoplastic Changes to the Breast Epithelium and Tumor Formation

David B Vaught 1, Jamie C Stanford 1, Christian Young 2, Donna J Hicks 1, Frank Wheeler 1, Cammie Rinehart 2, Violeta Sánchez 2, John Koland 4, William J Muller 5, Carlos L Arteaga 1,2,3, Rebecca S Cook 1,3
PMCID: PMC3693553  NIHMSID: NIHMS407226  PMID: 22461506

Abstract

Increasing evidence suggests that HER2-amplified breast cancer cells use HER3/ErbB3 to drive therapeutic resistance to HER2 inhibitors. However, the role of ErbB3 in the earliest events of breast epithelial transformation remains unknown. Using mouse mammary specific models of Cre-mediated ErbB3 ablation, we show that ErbB3 loss prevents the progressive transformation of HER2-overexpressing mammary epithelium. Decreased proliferation and increased apoptosis were seen in MMTV-HER2 and MMTV-Neu mammary glands lacking ErbB3, thus inhibiting premalignant HER2-induced hyperplasia. Using a transgenic model in which HER2 and Cre are expressed from a single polycistronic transcript, we showed that palpable tumor penetrance decreased from 93.3% to 6.7% upon ErbB3 ablation. Penetrance of ductal carcinomas in situ was also decreased. In addition, loss of ErbB3 impaired Akt and p44/42 phosphorylation in preneoplastic HER2-overexpressing mammary glands and in tumors, decreased growth of preexisting HER2-overexpressing tumors, and improved tumor response to the HER2 tyrosine kinase inhibitor lapatinib. These events were rescued by reexpression of ErbB3, but were only partially rescued by ErbB36F, an ErbB3 mutant harboring six tyrosine-to-phenylalanine mutations that block its interaction with phosphatidyl inositol 3-kinase. Taken together, our findings suggest that ErbB3 promotes HER2-induced changes in the breast epithelium before, during, and after tumor formation. These results may have important translational implications for the treatment and prevention of HER2-amplified breast tumors through ErbB3 inhibition.

Introduction

Aberrant regulation of ErbB receptor tyrosine kinases is common in human cancers (1). This family of 4 related members, EGFR (epidermal growth factor receptor), HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4 (2) exhibit dimerization-induced tyrosine phosphorylation and catalytic activation, with the exception of ErbB3, which has only weak intrinsic kinase activity, and therefore relies on heterodimeric transphosphorylation for signal transduction (3). Nonetheless, ErbB2/ErbB3 heterodimers are powerful oncogenic units, in part, because P-ErbB3 amplifies signaling through phosphatidyl inositol 3-kinase (PI3K; refs. 4–6). The p85 regulatory subunit of PI3K interacts directly with P-ErbB3 at 6 consensus P-Tyr p85-binding motifs within ErbB3 (3). In contrast, ErbB2 cannot directly engage p85. Therefore, it is thought that ErbB3 functions primarily to drive ErbB2-mediated PI3K signaling (7, 8). This is of critical therapeutic importance, as currently approved ErbB2 inhibitors (e.g., trastuzumab and the dual EGFR/ErbB2 TK inhibitor lapatinib) decrease P-ErbB3/PI3K signaling (9–11). However, many metastatic ERBB2/HER2-amplified breast cancers do not respond to, or eventually escape, trastuzumab and lapatinib, often with recovery of P-ErbB3/PI3K signaling (12). These studies would predict that therapeutic combinations targeting ErbB2 and ErbB3 would improve outcome in ERBB2/HER2-amplified breast cancers.

It is unclear how ErbB3 contributes to earliest events in ErbB2-mediated transformation of the breast epithelium, although ErbB3 is expressed in ErbB2-overexpressing hyperplasias in mice (13, 14). Tumorigenesis is a multistep process proceeding first through hyperplasia, to noninvasive ductal carcinoma in situ (DCIS), invasive adenocarcinoma, and finally to the formation of distant metastases. Although the expression of ErbB3 in DCIS lesions has not been reported, DCIS frequently overexpress ErbB2 (15, 16). It is important to understand what supports the transition of ErbB2+ DCIS to invasive, metastatic breast cancers.

To study the role of ErbB3 in all stages of ErbB2-mediated breast transformation ranging from premalignant hyperplasia to therapeutic response, we used genetically engineered mouse models to impair ErbB3 expression within ErbB2+ mammary epithelial cells (MEC; ref. 17). These studies revealed that the formation of ErbB2-mediated hyperplasias, DCIS, adenocarcinomas, and distant metastatic lesions were substantially decreased upon ErbB3 ablation. Inducible ErbB3 ablation in preexisting ErbB2-overexpressing tumors improved lapatinib-mediated growth inhibition. Genetic ablation of ErbB3 reduced cell proliferation and survival, and interfered with PI3K-Akt signaling. These signaling events were rescued by ErbB3 reexpression, but only partially rescued by ErbB36F, a mutant ErbB3 isoform incapable of interacting with p85 (4). These findings support a role for ErbB3 throughout the continuum of ErbB2-mediated tumorigenesis. Strategies targeting ErbB3 expression may prove useful in combination with ErbB2 inhibitors to improve the clinical outcome of ErbB2/HER2-amplified breast cancer patients. Furthermore, these studies suggest that ErbB3 targeting may be a novel prevention strategy to limit premalignant ErbB2-induced changes to the breast epithelium.

Materials and Methods

Histologic analysis

Mammary glands were whole mounted on glass slides and stained with hematoxylin as previously described (18). Paraffin-embedded tissue sections (5 μm) were stained with hematoxylin and eosin (H&E), with the ApopTag kit (Millipore), or by immunohistochemistry (IHC) using the following antibodies as described previously: ErbB3 (C-17), ErbB2 (F-11), and Ki67 (Santa Cruz Biotechnology, Inc.), S473 P-Akt (Cell Signaling Technology, Inc.; ref. 17). Immunodetection was done using the Vectastain kit (Vector Laboratories) according to the manufacturer’s directions. Specificity of the anti-ErbB3 antibody was determined using ErbB3-null tissues described herein. The specificity of the ErbB2 and P-Akt antibodies has been described previously (10, 19).

Mice

All mice were generated on, or crossed more than 10 generations into, an FVB inbred background. ErbB3FL/FL, MMTV-Cre, MMTV-rtTA, TetOp-Cre, MMTV-Neu, MMTV-HER2, and MMTV-NIC mice have been previously described (9, 19–22). ErbB3FL/FL X MMTV-Cre is referred to herein as ErbB3MMTV-KO, ErbB3FL/FL X MMTV-rtTA X TetOp-Cre as ErbB3DOX-KO, and ErbB3FL/FL X MMTV-Neu as NiB3. Where indicated, mice were treated with doxycycline (DOX, 2 mg/mL in 4% sucrose in drinking water; Sigma-Aldrich), or lapatinib (100 mg/kg daily for 4 weeks via orogastric gavage, LC Laboratories). Mammary glands were harvested from age-matched virgin siblings. Where indicated, tumor cells (1 × 106) were injected into left and right inguinal fat pads of 6-week-old female nu/nu (Balb/C) mice. Mice were examined twice weekly for palpable tumor formation, and tumors measured twice weekly with calipers. Tumor volume was calculated as (length − width2/2).

Cell culture

Primary tumor cells were harvested from collagenase-digested NiB3 tumors, and cultured as previously described (19). Cells were infected with retroviral particles generated in 293T cells using pMSCV-ErbB3WT, pMSCV-ErbB36F, or empty pMSCV. After viral infection, cells were selected in 1 μg/mL puromycin. Pooled puromycin-resistant clones were used for analysis. For colony forming assays, cells were seeded in 6-well dishes (104 cells per dish), and cultured 10 to 21 days in Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% FBS (Atlanta Biologicals). Cells were crystal violet stained, and imaged on a flatbed scanner. For cell growth assays, cells were cultured in 10% serum ± lapatinib (1 μg/mL). Viable cells were counted using trypan blue exclusion with a TC-10 automated cell counter (Bio-Rad).

Western blotting

Cells and tissues were homogenized as described previously (19). The following primary antibodies were used: ErbB3 (C-17, Santa Cruz Biotechnology; 1:2,000), α-actin (Sigma-Aldrich; 1:5,000), Y1197 P-ErbB3, p85, AKT, and S473 P-Akt (Cell Signaling Technology; 1:1,000. 1:1,000, 1:2,000, and 1:500, respectively).

Quantitative reverse transcriptase

Total RNA (1 μg), harvested from tumors using RNeasy (Qiagen), was used for cDNA synthesis (High Capacity cDNA RT; Applied Biosystems) followed by qPCR (iQ SYBR Green Supermix (Bio-Rad) and Bio-Rad iCycler) using the following primers: ErbB3 (5′-GGGCAACTCTCAGGCAGTATGT-3′ and 5′-CGGATCCATTGCAGGAAGGAAAGA-3′) and Rplp0 (5′-TCCTATAAAAGGCACACGCGGGC-3′ and 5′-AGACGATGTCACTCCAACGAGGACG-3′). Ct values were calculated as (CtErbB3 − CtRplp0) ErbB3FL/FLNIC − (CtErbB3 − CtRplp0)ErbB3 +/+ NIC.

Results

HER2-driven hyperplasia of the mammary epithelium requires ErbB3

The MMTV-HER2 transgenic mouse model expresses HER2, the human homolog of mouse ErbB2, in the mammary epithelium, resulting in mammary adenocarcinoma formation preceded by a prolonged period of diffuse epithelial hyperplasia (21). Little is known about the molecular events promoting ErbB2/HER2-driven mammary hyperplasia, or what causes hyperplastic lesions to convert to invasive malignant cancers. To determine whether ErbB3 affects early events in the continuum of ErbB2-driven tumor formation, we used transgenic mammary specific Cre recombinase (MMTV-Cre; ref. 22) to drive recombination at floxed ERBB3 alleles (ErbB3FL/FL) in genetically engineered mice (17, 21) referred to hereafter as ErbB3MMTV-KO mice. Whole-mount hematoxylin staining of mammary glands harvested from 12-week-old virgin female mice revealed that ErbB3 loss impaired HER2-driven hyperplasia (Fig. 1A). Decreased hyperplasia was not due to changes in human HER2 expression levels (Fig. 1A, bottom panels). Because ErbB2/HER2 potently activates PI3K signaling through ErbB3, we examined Akt, a primary downstream PI3K effector (23). P-Akt was not seen in ErbB3MMTV-KO X MMTV-HER2 samples (Fig. 1B). P-Akt signaling activates antiapoptotic pathways in numerous cancer cell types. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analysis showed increased cell death in ErbB3-deficient samples (Fig. 1B). Similar results were obtained using the MMTV-Neu transgenic model of breast cancer, which expresses Neu, the rat ErbB2/HER2 homolog, in the mammary epithelium (20), in which ErbB3MMTV-KO X MMTV-Neu mammary glands displayed decreased hyperplasia (Supplementary Fig. S1A), cell proliferation, and increased cell death (Supplementary Fig. S1B).

Figure 1.

Figure 1

Open in a new tab

ErbB2-driven hyperplasia of the mammary epithelium requires ErbB3. A, histologic analysis by whole-mount hematoxylin staining of mammary glands harvested from 12-week-old virgin female mice revealed that diffuse epithelial hyperplasias induced upon overexpression of ErbB2 are decreased upon loss of ErbB3 (as shown by IHC) despite continued expression of ErbB2 (as shown by IHC). B, decreased phosphorylation of Akt (as shown by IHC) and increased cell death (as shown by TUNEL analysis) were evident in ErbB3-deficient mammary epithelia. Arrows indicate TUNEL+ cells. Values represent the average TUNEL+ epithelial cells per total epithelial cells, N = 4 mammary glands per genotype, 5 random fields per sample. P value calculated using Student t test.

ErbB2/HER2-expressing cells escaping Cre-mediated ErbB3 ablation give rise to ErbB3-expressing tumors in ErbB3MMTV-KO mice

Inhibition of ErbB2/HER2-driven hyperplasia was maintained in ErbB3MMTV-KO X MMTV-HER2 mammary glands through 27 weeks of age (Fig. 2A). However, focal neoplasias were present in ErbB3MMTV-KO X MMTV-HER2 mammary glands, even in the context of suppressed hyperplasia. Average tumor latency was statistically similar in ErbB3MMTV-KO X MMTV-HER2 mice and ErbB3FL/+ X MMTV-HER2 mice (P > 0.05, log-rank test; Fig. 2B). Similar findings were seen in ErbB3MMTV-KO X MMTV-Neu mice versus ErbB3FL/+ X MMTV-Neu (P > 0.05, log-rank test; Fig. 2C).

Figure 2.

Figure 2

Open in a new tab

ErbB2-expressing cells escaping Cre-mediated ErbB3 ablation give rise to ErbB3-expressing tumors in ErbB3MMTV-KO mice. A, whole-mount hematoxylin–stained mammary glands from 27-week-old virgin female mice revealed focal neoplasias in hypoplastic ErbB3MMTV-KO mammary glands. H&E-stained tumors revealed similar histologic characteristics of MMTV-HER2 tumors from ErbB3MMTV-KO and ErbB3FL/+ mice. B and C, Kaplan–Meier analysis of MMTV-HER2 (B) and MMTV-Neu (C) tumor latency in ErbB3MMTV-KO and ErbB3FL/+ mice. T50 = average tumor latency. P value calculated using log-rank test. D, IHC analysis of primary tumor sections and lung metastases revealed ErbB3 expression in tumors, but not in normal adjacent mammary epithelium (inset), of ErbB3MMTV-KO mice. E, β-galactosidase activity assessed in ErbB3MMTV-KO X MMTV-HER2 X Rosa26 mice revealed a large number of cells negative for Cre activity. F, tumor distribution across the 10 mammary glands was altered in ErbB3MMTV-KO mice as compared with ErbB3FL/+ mice, suggesting nonoverlapping expression of MMTV-Cre and MMTV-HER2 transgenes, despite the similarities in their promoter sequences.

These results suggested 2 scenarios, one in which ErbB3 is lost but is not required for ErbB2-mediated focal tumor formation, or another in which some cells escape Cre-mediated recombination, leaving ErbB3 intact to support ErbB2-induced tumor formation. Immunohistochemical analysis of tumor sections revealed that ErbB3 expression was maintained in 100% of ErbB3MMTV-KO X MMTV-HER2 tumors (N = 13), suggesting that tumors arise from epithelial cells escaping Cre-mediated genomic recombination (Fig. 2D). ErbB3 was not detected in normal adjacent mammary epithelium of ErbB3MMTV-KO X MMTV-HER2 mice (Fig. 2D, magnified inset). These data suggested that ErbB3 loss continues to suppress ErbB2/HER2-mediated growth of adjacent epithelial structures.

Nonhomogenous expression of Cre recombinase from the MMTV promoter has been previously reported (24–26) and may contribute to ErbB3 retention in this model. We assessed Cre activity using Rosa26 transgenic mice, which irreversibly express β-galactosidase upon Cre-mediated recombination (27). Whole-mount β-galactosidase staining revealed numerous ErbB3MMTV-KO X MMTV-HER2 X Rosa26 MECs lacking β-galactosidase (Fig. 2E). Furthermore, we detected numerous ErbB3MMTV-KO X MMTV-HER2 epithelial structures with only partial ErbB3 ablation (Supplementary Fig. S2), although the average number of MMTV-HER2 tumors per mouse was not different between ErbB3MMTV-KO and ErbB3FL/+mice, the tumor distribution was (Fig. 2F). Ninety percent of all ErbB3FL/+ X MMTV-HER2 mice occurred in thoracic mammary glands (9 of 10 mice), but occurred at a lesser frequency in ErbB3MMTV-KO X MMTV-HER2 (5 of 13), but cervical and inguinal mammary tumors occurred with higher frequency. These data underscored the nonoverlapping expression patterns of MMTV-Cre and MMTV-HER2 transgenes. Because ErbB3 expression is maintained in this model, an alternate model would be required to determine whether ErbB3 is required for malignant transformation of the breast epithelium by ErbB2.

Polycistronic expression of ErbB2 and Cre reveals ErbB3 is required for ErbB2-induced hyperplasia

We employed an alternative transgenic approach, in which a single polycistronic transcript encodes both Neu (the rat ErbB2 homolog) and Cre recombinase, separated by an internal ribosomal entry site (IRES). Mammary specific expression of the MMTV-Neu-IRES-Cre (NIC) transgene results in mammary tumor formation (28, 29). Mammary glands harvested from 12-week-old virgin female NIC mice revealed ErbB2-induced diffuse hyperplasia and focal neoplasia in ErbB3FL/+ X NIC samples, but not ErbB3FL/+ X NIC (Fig. 3A). Cell proliferation in ErbB3FL/FL X NIC mammary glands was decreased as compared with ErbB3FL/+ X NIC, whereas cell death was increased. Real-time reverse transcriptase PCR experiments revealed that ErbB3 mRNA was eliminated in ErbB3FL/FL samples.

Figure 3.

Figure 3

Open in a new tab

Decreased cell growth, cell survival, hyperplasia, and tumor formation in MMTV-NIC mice, expressing ErbB2 and Cre from the same polycistronic transcript. A, histologic analysis of mammary glands harvested from 12-week-old virgin female mice by whole-mount hematoxylin staining, IHC detection of Ki67, and TUNEL analysis revealed that loss of ErbB3 in ErbB3FL/FL X NIC mice decreased diffuse hyperplasia and focal neoplasia, epithelial proliferation, and cell survival. RNA harvested from ErbB3+/+NIC (n = 8), ErbB3+/FLNIC (n = 7), and ErbB3FL/FLNIC (n = 7) was used to assess ErbB3 mRNA expression levels. Values are expressed as mean fold differences (±SE) in comparison with ErbB3+/+NIC, and normalized to the Rplp0 housekeeping gene. B, DCIS too small for detection by manual palpation became evident in 2/15 ErbB3FL/FL X NIC mice by 6 months of age. Analysis of these 2 DCIS revealed decreased cell proliferation (as seen by Ki67 IHC) and increased cell death (as seen by TUNEL analysis). Comparable DCIS lesions were evident in ErbB3FL/+ X NIC mice, as were frank adenocarcinomas, but DCIS lesions found in control samples displayed profoundly elevated cell proliferation and very low levels of cell death. Higher power magnifications of boxed areas are shown at the right. Bottom, Western analysis of whole mammary lysates using the antibodies indicated at the right of each panel. Genotype for ErbB3 is indicated as +/+ (ErbB3+/+ X NIC), FL/+ (ErbB3FL/+ X NIC) and F/FL (ErbB3FL/FL X NIC). C, Kaplan–Meier analysis of tumor latency in MMTV-NIC mice. Average tumor latency could not be determined (n.d.) in ErbB3FL/FL mice, as the majority of these mice did not develop tumors within the course of this study. Loss of a single ErbB3 allele (ErbB3FL/+ X NIC) did not affect tumor latency as compared with ErbB3+/+. D, average tumor burden per mouse, as determined by the average number of tumors per mouse (top) or total tumor volume (bottom) at 180 days of age, was measured. P value calculated using Student t test. E, lung metastases were identified upon histologic examination. Average number of lung metastases per mouse is shown, N = 8 (ErbB3+/+), 7 (ErbB3FL/+), and 15 (ErbB3FL/FL). P value, Student t test. Representative lung sections are shown.

ErbB3 promotes ErbB2-induced DCIS and adenocarcinoma

Female NIC mice were examined by manual palpation twice weekly through 180 days of age for tumor formation. Palpable tumors were not detected in ErbB3FL/FL X NIC mice. However, 2 of 15 ErbB3FL/FL X NIC mice developed DCIS that were too small for detection by manual palpation, but were detected at necropsy at 180 days. Analysis of these 2 DCIS revealed decreased cell proliferation (as seen by Ki67 IHC) and increased cell death (as seen by TUNEL analysis) as compared with similar DCIS lesions found in ErbB3FL/+ X NIC mice (Fig. 3B). Western blot analysis showed loss of ErbB3 expression in ErbB3FL/FL X NIC whole tissue lysates, which did not affect Akt phosphorylation at S473, but drastically reduced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). Therefore, ErbB3 loss profoundly decreased premalignant changes in the ErbB2-overexpressing breast epithelium, including hyperplastic lesions and DCIS. Palpable mammary tumors were evident in 7 of 8 ErbB3+/+ X NIC mice, and in 7 of 8 ErbB3FL/+ X NIC mice, occurring with similar average tumor latencies (Fig. 3C, 165 days vs. 145 days, respectively, P > 0.05, log-rank test). Average total tumor burden per mouse was decreased in ErbB3FL/FL X NIC mice as compared with ErbB3+/+ X NIC and ErbB3FL/+ X NIC (Fig. 3D), as was the number of lung metastases per mouse (Fig. 3E). Therefore, ErbB3 ablation greatly decreases progression of ErbB2+ premalignant lesions to metastatic breast cancers.

Inducible Cre-mediated ErbB3 ablation decreases growth of preexisting HER2-overexpressing mammary tumors

To determine the potential use of therapeutic ErbB3 targeting, we developed a transgenic model system in which mammary specific Cre expression could be temporally regulated by doxycycline resulting in doxycycline-inducible ErbB3 ablation (ErbB3DOX-KO) as described previously (19). Treatment of 12-week-old virgin female ErbB3DOX-KO X MMTV-Neu mice with doxycycline for 14 days decreased ErbB3 expression and ErbB2-induced hyperplastic structures (Fig. 4A). Next, we maintained ErbB3DOX-KO X MMTV-Neu naive to doxycycline until their tumors surpassed 200 mm3, at which point mice were treated ±doxycycline for 4 weeks. Doxycycline-treated tumors grew more slowly than untreated tumors (Fig. 4B), and after 4 weeks harbored decreased tumor cellularity (Fig. 4C). IHC confirmed decreased ErbB3, P-Akt, Ki67, and cell survival after 7 days of doxycycline (Fig. 4D and E).

Figure 4.

Figure 4

Open in a new tab

Inducible Cre-mediated ErbB3 ablation decreases growth of ErbB2-overexpressing mammary tumors. A, treatment of 12-week-old virgin female ErbB3DOX-KO X MMTV-Neu mice with doxycycline for 14 days decreased mammary epithelial ErbB3 expression and ErbB2-induced lobuloalveolar density. B, ErbB3DOX-KO X MMTV-Neu female mice remained without doxycycline until tumors surpassed 200 mm3, at which point mice were treated with doxycycline (2 mg/mL in drinking water). Tumor volume was measured twice weekly. Values shown represent the average ± SD. P value calculated using Student t test, N = 8. C, histologic analysis of tumors treated 28 days with doxycycline show decreased tumor cellularity and increased acellular debris. D, ErbB3, P-Akt, Ki67, and cell survival were decreased in doxycycline-treated samples. TUNEL analysis revealed increased cell death in doxycycline-treated tumors. E and F, values shown indicate the average number of Ki67+ cells (E) or TUNEL+ cells (F) per total tumor cells. N = 4 per group, 5 fields per sample. P values calculated using Student t test. DOX, doxycycline.

Inhibition of ErbB3 expression or ErbB3-to-PI3K signaling improves response of ErbB2-overexpressing breast cancers to lapatinib

We treated ErbB3DOX-KO X MMTV-Neu mice bearing tumors more than 200 mm3 for 3 weeks with control vehicles, doxycycline, lapatinib (100 mg/kg once daily), or doxycycline + lapatinib. ErbB3DOX-KO tumors treated with either lapatinib or doxycycline grew more slowly than untreated tumors (Fig. 5A). Tumor growth was reduced to a greater extent when treated with doxycycline and lapatinib together, suggesting that ErbB3 inhibition improves therapeutic response of ErbB2-overexpressing breast cancers to lapatinib.

Figure 5.

Figure 5

Open in a new tab

Inhibition of ErbB3 expression or ErbB3-to-PI3K signaling improves response of ErbB2-overexpressing breast cancers to lapatinib. A, ErbB3DOX-KO mice bearing tumors more than 200 mm3 were randomized into groups treated ±doxycycline (2 mg/mL in drinking water) and ±lapatinib (100 mg/kg once daily). Tumor volume was measured twice weekly. Left, average tumor volume ± SE. Right, fold-increase in tumor volume calculated individually for each tumor, then used to calculate average fold increase in tumor volume for each group. P values, Student t test. N = 8. B, primary NiB3 cells were infected with Ad.Cre or Ad.LacZ. Cells (1 × 104) were cultured in 10% serum ± lapatinib (1 μg/mL) or dimethyl sulfoxide (DMSO) for 96 hours. Viable cells were counted (Bio-Rad TC-10). Values represent average cell number ± SD. *, P < 0.01. C and D, NiB3 cells were infected with pBMN retrovirus expressing the empty vector, ErbB3WT, or ErbB36F. Cells were infected with Ad.Cre or Ad.LacZ. Ten days after infection, cell lysates were used for Western blot analysis (C) or ErbB3 immunoprecipitation followed by Western blot analysis (D). E, 24 hours after infection with Ad.Cre or Ad.LacZ, NiB3 cells (1 × 104) were cultured in 10% serum for 96 hours. Viable cells were counted. Values shown represent the average ± SD, N = 3 per group analyzed in duplicate. *, P < 0.01, Student t test. F, 24 hours after infection with Ad.Cre, NiB3 cells (1 × 104) were cultured with lapatinib (1 μmol/L), BKM120 (2 μmol/L), or DMSO for 21 days, stained with crystal violet and imaged. G, NiB3 tumors cells stably expressing ErbB3WT or ErbB6F were infected with Ad.Cre and cultured for 7 days. Cells (106) were injected into the left (NiB3-ErbB36F) and right (NiB3-ErbB3WT) inguinal mammary fat pads. Tumor bearing mice were treated daily 5 days with lapatinib or BKM120. TUNEL analysis of tumors collected 1 hour after final treatment was quantitated as average number of apoptotic bodies per ×400 field (N = 9–15 per group). P values were calculated using Student t test. H, representative photomicrograph of TUNEL-stained NiB3 tumors. DOX, doxycycline.

ErbB3 is a powerful activator of PI3K/Akt signaling (23). To understand the role of ErbB3 in total PI3K signaling output in ErbB2-overexpressing breast cancer cells, we eliminated endogenous ErbB3 expression in primary NiB3 cells (ErbB3FL/FL X MMTV-Neu) using adenoviral Cre (Ad.Cre), thus generating an ErbB3-deficient background for adding back wild-type (WT) ErbB3 or a mutant ErbB3 isoform unable to engage PI3K. Ad.Cre-infected NiB3 cells grew more slowly than Ad.LacZ-infected cells, and exhibited greater lapatinib-mediated growth inhibition (Fig. 5B), suggesting that these primary tumor cells mirror the phenotype of the tumors from which they were derived.

Next, NiB3 cells (with endogenous ErbB3 still intact) were infected with pMSCV-based retroviral particles encoding WT rat ErbB3 (ErbB3WT), or mutant rat ErbB3 in which tyrosine-to-phenylalanine mutations were introduced into the 6 PI3K interaction motifs of ErbB3 (ErbB36F; ref. 4). Pools of puromycin-resistant cells were infected with Ad.Cre to remove endogenous ErbB3, leaving expression of ErbB3WT or ErbB36F (Fig. 5C). P-ErbB3 and total ErbB3 expression were rescued in Ad.Cre-infected NiB3-ErbB3WT cells as compared with vector controls. ErbB3 expression, but not Tyr1197 phosphorylation (a PI3K-interaction site), was rescued in Ad.Cre-infected NiB3-ErbB36F cells. Akt phosphorylation was also substantially reduced but not eliminated in NiB3 vector (i.e., ErbB3 deficient) and NiB3-ErbB36Fcells, consistent with the idea that ErbB3 facilitates PI3K activation in ErbB2-overexpressing cells, but that ErbB2 retains alternative mechanisms to activate the PI3K/Akt signaling pathway outside of ErbB3, as previously reported (3). Interestingly, we found that Erk1/2 MAPK phosphorylation was lost in cells lacking ErbB3, but was restored in cells expressing either ErbB3WT or ErbB36F, suggesting that ErbB3 is required for ErbB2-induced MAPK activation through mechanisms that do not involve tyrosine phosphorylation at these 6 residues.

Immunoprecipitation of ErbB3 from whole-cell lysates confirmed that Ad.Cre infection eliminated endogenous ErbB3 (Fig. 5D). Total tyrosine phosphorylation of ErbB36F was detected, consistent with the presence of remaining phosphorylated tyrosine residues in the ErbB3 C-terminal tail that are not involved in p85 interaction. Importantly, coprecipitation of p85 with ErbB3 was eliminated in ErbB36F-expressing cells, despite the presence of tyrosine phosphorylated ErbB3, confirming that ErbB3-PI3K interactions were disrupted.

In cell growth assays, acute loss of ErbB3 expression by Ad.Cre infection of NiB3-vector cells decreased cell number nearly 10-fold, which was rescued by exogenous ErbB3WT expression (Fig. 5E). NiB3-ErbB36F cells grew more slowly than NiB3-ErbB3WT, but more rapidly than NiB3-vector cells, suggesting ErbB36F maintains some mitogenic properties, despite loss of ErbB3-PI3K interactions. In long-term colony assays, Ad.Cre-infected NiB3 cells expressing ErbB36F were more sensitive to lapatinib-induced growth inhibition than ErbB3WT-expressing cells (Fig. 5F), suggesting that ErbB3-induced PI3K activity is a critical barrier to achieving full response to lapatinib. However, complete loss of ErbB3 improved response to lapatinib over ErbB36F-expressing cells. In addition, treatment of cells with the pan-PI3K inhibitor BKM120 decreased colony formation in lapatinib-treated NiB3-ErbB3WT cells and in NiB3-ErbB36F cells.

We carried out orthotopic transplant of NiB3-ErbB3WT and NiB3-ErbB36F into contralateral mammary fat pads of mice, using a protocol in which Ad.Cre eliminated endogenous ErbB3 ex vivo, 7 days before tumor inoculation. Tumor latency was similar for each group, occurring within 10 days after tumor cell injection. Tumor-bearing mice were treated daily for 5 days with lapatinib or BKM120 (20 mg/kg). Relative levels of cell proliferation (as measured by mitotic index) in tumors harvested 1 hour after the final treatment were not statistically different in NiB3-ErbB36F and NiB3-ErbB3WT tumors (Supplementary Fig. S3A). However, basal and BKM120-induced rates of cell death were upregulated in NiB3-ErbB36F tumors (Fig. 5G and H). More cell death was also observed in lapatinib-treated NiB3-ErbB36F tumors as compared with lapatinib-treated NiB3-ErbBWT tumors (Supplementary Fig. S3B), but was not greater than basal cell death levels in NiB3-ErbB6F tumors (Supplementary Fig. S3C). Therefore, ErbB2-driven tumors can activate PI3K signaling pathways in the absence of canonical ErbB3-p85 interactions, although loss of this interaction leaves cells more susceptible to PI3K inhibition. Importantly, these studies highlighted the importance of ErbB3-induced signaling beyond PI3K.

Discussion

Nearly 25% of all breast cancers overexpress HER2/ErbB2. Therapeutic strategies targeting ErbB2 have improved breast cancer mortality for patients with ERBB2/HER2-amplified breast cancers. However, clinical success of ErbB2 inhibitors is limited by tumor resistance to these inhibitors (11). It is important to understand the signaling mechanisms driving anti-ErbB2 resistance, which is often associated with recovery of PI3K signaling (12, 19, 30, 31). ErbB2 inhibitors initially decrease P-ErbB3/PI3K signaling (10, 30, 31), but resurgent ErbB3 signaling after sustained lapatinib exposure increases PI3K signaling, and is the rationale for clinical trials of pertuzumab–trastuzumab combinations in advanced ErbB2+ breast cancers, which have seen clinical success (32). We report herein that ErbB3, a heterodimeric partner of ErbB2, drives the response of ErbB2-overexpressing breast cancers to ErbB2 inhibitors. Our results suggest that complete ablation of ErbB3 decreases growth and survival of ErbB2-expressing mammary tumors in mice. Importantly, our results expand the role of ErbB3 in ErbB2-expressing breast cancers beyond PI3K activation, and highlight the critical importance PI3K-indendent pathways in driving tumor cell growth and survival.

Although genetic ErbB3 ablation decreased tumor growth as a single therapeutic strategy (Fig. 4), treatment of BT474 xenografts with U3-1287 did provide any benefit as a single-agent therapy (10, 12). The reasons for this apparent discrepancy remain unclear, but may include model differences (xenografts vs. endogenous tumors, human cells vs. mouse cells). Also, exposure of tumor cells to the targeting agent may have been a contributing factor, as ErbB3 loss in the genetic model was tumor cell-intrinsic upon continuous exposure to doxycycline, as opposed to pulsatile treatment with exogenous antibodies twice weekly as previously reported. Finally, it is possible that U3-1287 may leave ErbB3 intact at the cell surface or in recycling endosomes, allowing low levels of ErbB3 signaling. In contrast, genetic ablation of ErbB3 removes all ErbB3. The observation that genetic ErbB3 ablation effectively limits tumor cell growth as a single-agent strategy is consistent with previous reports that siRNA sequences targeting ErbB3 decreased growth of ERBB2/HER2-amplified breast cell lines (8) and the hormone-independent growth of MCF-7 cells (33). Furthermore, genetic ablation of ErbB3, or treatment of mice with EZN-3920, an antisense oligonucleotide that was used to knock down ErbB3 in vivo, decreased PI3K signaling and tumor growth in the MMTV-PyVmT model of breast cancer (19).

It is also important to understand the underlying signaling pathways contributing to the earliest events in ERBB2/HER2-amplified breast cancer formation, to eventually develop strategies to prevent ErbB2-overexpressing breast cancers. We report herein that ErbB3 drives ErbB2-mediated preneoplastic hyperplasia, the transition of DCIS to malignant adenocarcinoma, and the response of ErbB2-overexpressing breast cancers to ErbB2 inhibitors. Large phase III clinical trials have identified selective estrogen receptor (ER) modulators (tamoxifen, raloxifene, and lasofoxifene) and aromatase inhibitors (exemestane) that reduce the risk of breast cancer in moderate-to-high risk women without prior breast cancer (34, 35). Although these drugs only prevent ER-positive tumors, their success is precedence for prevention of other types of breast cancers. Given that ErbB2 is overexpressed in up to 45% of premalignant DCIS (36, 37), lapatinib or other ErbB family inhibitors may have potential for breast cancer prevention. Lapatinib suppressed ER-negative mammary tumor development in MMTV-Neu mice through decreased epithelial cell proliferation without affecting cell death (38). We show herein that ErbB3 loss prevented formation of ErbB2-overexpressing breast cancers in mice, even at earliest stages of ErbB2-induced epithelial hyperplasia. ErbB3 loss decreased cell proliferation, but unlike lapatinib, also increased cell death, suggesting that drugs targeting ErbB3, including monoclonal antibodies U3-1287 and MM-121 (targeting ErbB3 extracellular domain), or pertuzumab (preventing ErbB2-ErbB3 heterodimerization), may be effective pharmacologic strategies to prevent malignant conversion of ErbB2+ DCIS.

In summary, ErbB3 is expressed in preneoplastic ErbB2-overexpressing breast epithelium and is required for cell growth and malignant progression of ErbB2-driven hyperplasias. Furthermore, targeted inhibition of ErbB3 decreased growth of preexisting ErbB2-driven tumors and improved response to the ErbB2 inhibitor lapatinib. These data support future studies examining novel ErbB3-targeting agents in prevention and treatment of ErbB2+ breast cancers.

Supplementary Material

Supp Fig 1
Supp Fig 2
Supp Fig. 3
Suuplemental Figure Legends

Acknowledgments

Grant Support

This work was supported by NIH R01s CA143126 (R.S. Cook), CA80195 (C.L. Arteaga), Breast Cancer SPORE P50 CA98131 (VICC), Susan G. Komen for the Cure KG100677 (R.S. Cook), ACS Clinical Research Professorship grant CRP-07-234 (C.L. Arteaga), Lee Jeans Translational Breast Cancer Research Program (C.L. Arteaga), and C.L Arteaga is supported by a Stand Up To Cancer Innovative Research grant, a Program of the Entertainment Industry Foundation (SU2C-AACR-DT0209).

Footnotes

Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors’ Contributions

Conception and design: D.B. Vaught, C.L. Arteaga, R.S. Cook

Development of methodology: D.B. Vaught, J.C. Stanford, V. Sanchez, J. Koland

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.B. Vaught, J.C. Stanford, F. Wheeler, R.S. Cook

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.C. Stanford, D. Hicks, F. Wheeler, C. Rinehart, W.J. Muller, C.L. Arteaga

Writing, review, and/or revision of the manuscript: D.B. Vaught, C.L. Arteaga, R.S. Cook

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Young, F. Wheeler

Study supervision: R.S. Cook

References

  • 1.Hynes NE, Stern DF. The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta. 1994;1198:165–84. doi: 10.1016/0304-419x(94)90012-4. [DOI] [PubMed] [Google Scholar]
  • 2.Holbro T, Civenni G, Hynes NE. The ErbB receptors and their role in cancer progression. Exp Cell Res. 2003;284:99–110. doi: 10.1016/s0014-4827(02)00099-x. [DOI] [PubMed] [Google Scholar]
  • 3.Stern DF. ERBB3/HER3 and ERBB2/HER2 duet in mammary development and breast cancer. J Mammary Gland Biol Neoplasia. 2008;13:215–23. doi: 10.1007/s10911-008-9083-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hellyer NJ, Cheng K, Koland JG. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase. Biochem J. 1998;333:757–63. doi: 10.1042/bj3330757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF, 3rd, Hynes NE. The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A. 2003;100:8933–8. doi: 10.1073/pnas.1537685100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Alimandi M, Romano A, Curia MC, Muraro R, Fedi P, Aaronson SA, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene. 1995;10:1813–21. [PubMed] [Google Scholar]
  • 7.Chakrabarty A, Rexer BN, Wang SE, Cook RS, Engelman JA, Arteaga CL. H1047R phosphatidylinositol 3-kinase mutant enhances HER2-mediated transformation by heregulin production and activation of HER3. Oncogene. 2010;29:5193–203. doi: 10.1038/onc.2010.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lee-Hoeflich ST, Crocker L, Yao E, Pham T, Munroe X, Hoeflich KP, et al. A central role for HER3 in HER2-amplified breast cancer: implications for targeted therapy. Cancer Res. 2008;68:5878–87. doi: 10.1158/0008-5472.CAN-08-0380. [DOI] [PubMed] [Google Scholar]
  • 9.Abramson V, Arteaga CL. New strategies in HER2-overexpressing breast cancer: many combinations of targeted drugs available. Clin Cancer Res. 2011;17:952–8. doi: 10.1158/1078-0432.CCR-09-1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Garrett JT, Olivares MG, Rinehart C, Granja-Ingram ND, Sanchez V, Chakrabarty A, et al. Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase. Proc Natl Acad Sci U S A. 2011;108:5021–6. doi: 10.1073/pnas.1016140108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Rexer BN, Engelman JA, Arteaga CL. Overcoming resistance to tyrosine kinase inhibitors: lessons learned from cancer cells treated with EGFR antagonists. Cell Cycle. 2009;8:18–22. doi: 10.4161/cc.8.1.7324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Garrett JT, Arteaga CL. Resistance to HER2-directed antibodies and tyrosine kinase inhibitors: Mechanisms and clinical implications. Cancer Biol Ther. 2011;11:793–800. doi: 10.4161/cbt.11.9.15045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Siegel PM, Ryan ED, Cardiff RD, Muller WJ. Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer. EMBO J. 1999;18:2149–64. doi: 10.1093/emboj/18.8.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Andrechek ER, Laing MA, Girgis-Gabardo AA, Siegel PM, Cardiff RD, Muller WJ. Gene expression profiling of neu-induced mammary tumors from transgenic mice reveals genetic and morphological similarities to ErbB2-expressing human breast cancers. Cancer Res. 2003;63:4920–6. [PubMed] [Google Scholar]
  • 15.Bartkova J, Barnes DM, Millis RR, Gullick WJ. Immunohistochemical demonstration of c-erbB-2 protein in mammary ductal carcinoma in situ. Hum Pathol. 1990;21:1164–7. doi: 10.1016/0046-8177(90)90154-w. [DOI] [PubMed] [Google Scholar]
  • 16.Howe LR, Brown PH. Targeting the HER/EGFR/ErbB family to prevent breast cancer. Cancer Prev Res (Phila) 2011;4:1149–57. doi: 10.1158/1940-6207.CAPR-11-0334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Qu S, Rinehart C, Wu HH, Wang SE, Carter B, Xin H, et al. Gene targeting of ErbB3 using a Cre-mediated unidirectional DNA inversion strategy. Genesis. 2006;44:477–86. doi: 10.1002/dvg.20243. [DOI] [PubMed] [Google Scholar]
  • 18.Vaught D, Chen J, Brantley-Sieders DM. Regulation of mammary gland branching morphogenesis by EphA2 receptor tyrosine kinase. Mol Biol Cell. 2009;20:2572–81. doi: 10.1091/mbc.E08-04-0378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Cook RS, Garrett J, Sanchez V, Stanford JC, Young C, Chakrabarty A, et al. ErbB3 ablation impairs phosphatidylinositol 3-kinase (PI3K)/AKT-dependent mammary tumorigenesis. Cancer Res. 2011;71:3941–51. doi: 10.1158/0008-5472.CAN-10-3775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Siegel PM, Dankort DL, Hardy WR, Muller WJ. Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors. Mol Cell Biol. 1994;14:7068–77. doi: 10.1128/mcb.14.11.7068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Finkle D, Quan ZR, Asghari V, Kloss J, Ghaboosi N, Mai E, et al. HER2-targeted therapy reduces incidence and progression of midlife mammary tumors in female murine mammary tumor virus huHER2-transgenic mice. Clin Cancer Res. 2004;10:2499–511. doi: 10.1158/1078-0432.ccr-03-0448. [DOI] [PubMed] [Google Scholar]
  • 22.Wagner KU, Wall RJ, St-Onge L, Gruss P, Wynshaw-Boris A, Garrett L, et al. Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res. 1997;25:4323–30. doi: 10.1093/nar/25.21.4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wong KK, Engelman JA, Cantley LC. Targeting the PI3K signaling pathway in cancer. Curr Opin Genet Dev. 2010;20:87–90. doi: 10.1016/j.gde.2009.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Andrechek ER, White D, Muller WJ. Targeted disruption of ErbB2/Neu in the mammary epithelium results in impaired ductal outgrowth. Oncogene. 2005;24:932–7. doi: 10.1038/sj.onc.1208230. [DOI] [PubMed] [Google Scholar]
  • 25.Lahlou H, Sanguin-Gendreau V, Zuo D, Cardiff RD, McLean GW, Frame MC, et al. Mammary epithelial-specific disruption of the focal adhesion kinase blocks mammary tumor progression. Proc Natl Acad Sci U S A. 2007;104:20302–7. doi: 10.1073/pnas.0710091104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.White DE, Kurpios NA, Zuo D, Hassell JA, Blaess S, Mueller U, et al. Targeted disruption of beta1-integrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell. 2004;6:159–70. doi: 10.1016/j.ccr.2004.06.025. [DOI] [PubMed] [Google Scholar]
  • 27.Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–1. doi: 10.1038/5007. [DOI] [PubMed] [Google Scholar]
  • 28.Northey JJ, Chmielecki J, Ngan E, Russo C, Annis MG, Muller WJ, et al. Signaling through ShcA is required for transforming growth factor beta- and Neu/ErbB-2-induced breast cancer cell motility and invasion. Mol Cell Biol. 2008;28:3162–76. doi: 10.1128/MCB.01734-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Schade B, Rao T, Dourdin N, Lesurf R, Hallett M, Cardiff RD, et al. PTEN deficiency in a luminal ErbB-2 mouse model results in dramatic acceleration of mammary tumorigenesis and metastasis. J Biol Chem. 2009;284:19018–26. doi: 10.1074/jbc.M109.018937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Chakrabarty A, Sanchez V, Kuba MG, Rinehart C, Arteaga CL. Breast cancer special feature: feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors. Proc Natl Acad Sci U S A. 2012;109:2718–23. doi: 10.1073/pnas.1018001108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Rexer BN, Ham AJ, Rinehart C, Hill S, de Matos Granja-Ingram N, Gonzalez-Angulo AM, et al. Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition. Oncogene. 2011;30:4163–74. doi: 10.1038/onc.2011.130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Baselga J, Cortes J, Kim SB, Im SA, Hegg R, Im YH, et al. Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N Engl J Med. 2012;366:109–19. doi: 10.1056/NEJMoa1113216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Miller TW, Perez-Torres M, Narasanna A, Guix M, Stal O, Perez-Tenorio G, et al. Loss of phosphatase and tensin homologue deleted on chromosome 10 engages ErbB3 and insulin-like growth factor-I receptor signaling to promote antiestrogen resistance in breast cancer. Cancer Res. 2009;69:4192–201. doi: 10.1158/0008-5472.CAN-09-0042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Vogel VG, Costantino JP, Wickerham DL, Cronin WM, Cecchini RS, Atkins JN, et al. Update of the national surgical adjuvant breast and bowel project study of tamoxifen and raloxifene (STAR) P-2 trial: preventing breast cancer. Cancer Prev Res (Phila) 2010;3:696–706. doi: 10.1158/1940-6207.CAPR-10-0076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Goss PE, Ingle JN, Ales-Martinez JE, Cheung AM, Chlebowski RT, Wactawski-Wende J, et al. Exemestane for breast-cancer prevention in postmenopausal women. N Engl J Med. 2011;364:2381–91. doi: 10.1056/NEJMoa1103507. [DOI] [PubMed] [Google Scholar]
  • 36.Park K, Han S, Kim HJ, Kim J, Shin E. HER2 status in pure ductal carcinoma in situ and in the intraductal and invasive components of invasive ductal carcinoma determined by fluorescence in situ hybridization and immunohistochemistry. Histopathology. 2006;48:702–7. doi: 10.1111/j.1365-2559.2006.02403.x. [DOI] [PubMed] [Google Scholar]
  • 37.Ramachandra S, Machin L, Ashley S, Monaghan P, Gusterson BA. Immunohistochemical distribution of c-erbB-2 in in situ breast carcinoma–a detailed morphological analysis. J Pathol. 1990;161:7–14. doi: 10.1002/path.1711610104. [DOI] [PubMed] [Google Scholar]
  • 38.Strecker TE, Shen Q, Zhang Y, Hill JL, Li Y, Wang C, et al. Effect of lapatinib on the development of estrogen receptor-negative mammary tumors in mice. J Natl Cancer Inst. 2009;101:107–13. doi: 10.1093/jnci/djn436. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig 1
NIHMS407226-supplement-Supp_Fig_1.pdf (4.8MB, pdf)
Supp Fig 2
NIHMS407226-supplement-Supp_Fig_2.pdf (3.7MB, pdf)
Supp Fig. 3
NIHMS407226-supplement-Supp_Fig__3.pdf (122.8KB, pdf)
Suuplemental Figure Legends
NIHMS407226-supplement-Suuplemental_Figure_Legends.pdf (58.8KB, pdf)

RESOURCES