Purpose:

Trastuzumab-based chemotherapy has shown remarkable clinical benefits for patients with HER2-positive breast cancer. However, treatment regimens involving trastuzumab had little or no effect for a subset of patients. Preliminary studies revealed WW-binding protein 2 (WBP2), an oncogenic transcription coactivator, to be coamplified with HER2 in 36% of HER2-positive breast cancers. We hypothesize that WBP2 regulates and correlates with the response of HER2-positive breast cancer to trastuzumab.

Experimental Design:

The coexpression of WBP2 and HER2 in breast tumors was validated using IHC. The role and mechanism of WBP2 in regulating breast cancer response to trastuzumab was elucidated using in vitro, patient-derived xenograft and murine xenograft models. A multicenter retrospective study involving 143 patients given neoadjuvant trastuzumab-based chemotherapy was conducted to determine whether WBP2 expression correlates with pathologic complete response (pCR).

Results:

Elevated expression of WBP2 significantly enhanced breast cancer's response to trastuzumab by augmenting trastuzumab-induced HER2 downregulation and cell-cycle arrest via inhibition of cyclin D expression. High level of WBP2 correlated with better pCR (67.19%) compared with low WBP2 level (26.58%). The highest response was observed in subgroups of patients with high WBP2–expressing tumors also aged below 50 years (77.78%) or were premenopausal in status (73.33%). Retrospectively, WBP2 demonstrated sensitivity of 80% to 81% and specificity of 76.5% to 80% in discriminating between patients showing pCR and non-pCR.

Conclusions:

WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab-based neoadjuvant chemotherapy.

This article is featured in Highlights of This Issue, p. 2361

Translational Relevance

Not all patients with HER2-positive breast cancer respond well to trastuzumab-based chemotherapy. Preclinical clinical studies involving in vitro, patient-derived xenografts, and animal models revealed that WBP2 augmented the inhibitory effects of trastuzumab in HER2-positive breast cancer cells by enhancing trsatuzumab's effect on cell-cycle arrest. Patients with tumor expressing high WBP2 level had higher pathologic complete response (pCR) of 67.19% compared with those with low WBP2 (26.58%). The pCR was even higher in subgroups of patients whose tumors had high WBP2 and were aged below 50 years (77.78%) or were premenopausal in status (73.33%). Retrospective analysis revealed WBP2 to have sensitivity of 80% to 81% and specificity of 76.5% to 80% in discriminating between patients showing pCR and non-pCR. WBP2 is a potential companion diagnostics for the management of HER2-positve breast cancer with trastuzumab-based therapies.

Breast cancer is the second leading cause of cancer-related deaths in women, especially among women aged 40–59 years (1). Breast cancer can be molecularly subclassified into four groups depending on the estrogen (ER)/progesterone (PR) and human epidermal growth factor receptor 2 (HER2) status: luminal A (ER and/or PR positive with low Ki67), luminal B (ER and/or PR positive and either HER2 positive or HER2 negative with high Ki67), HER2 (HER2 positive), and basal-like (usually but not always ER/PR/HER2 negative/triple negative; ref. 2). HER2 (also known as ErbB2/neu) is amplified and overexpressed in 15%–20% of breast cancer. Patients with HER2-positive tumors have poorer prognosis and significantly lower overall survival compared with patients with HER2-negative tumors (3, 4).

Trastuzumab humanized mAb is the first HER2-targeted therapy that was approved by the United States Food and Drug Administration (US FDA) for metastatic HER2-overexpressing breast cancer as the first-line therapy often in combination with chemotherapy (5–7). HER2 or EGFR2 is one of the 4 members of the EGF receptor (EGFR) family of tyrosine kinases. It is an orphan receptor and can either dimerize with itself (especially when overexpressed) or with other members such as EGFR (HER1) to initiate downstream signaling events. Trastuzumab inhibits HER2-positive breast cancer cells by binding to the extracellular domain of HER2. The exact mechanism is unknown although trastuzumab-induced receptor endocytosis among others has been implicated (8). Historically, when trastuzumab was used alone in nonselected patients with metastatic breast cancer, only about 10%–15% of patients had a partial response (7). The objective response rate improved to 26% when patients with HER2-overexpressing metastatic disease receiving first-line treatment were selected (9). In the current setting, the response rate of patients to neoadjuvant trastuzumab in combination with chemotherapy ranges between 30% and 53% (10, 11). These results reveal an important gap in clinical oncology–HER2 is the only approved biomarker for anti-HER2–based therapy and the administration of anti-HER2 drugs based on HER2 status alone is insufficient in achieving a good response rate. Better and/or complementary predictors are required.

WW-domain binding protein 2 (WBP2) was first identified to be a cognate ligand of the WW domain of Yes kinase-associated protein (YAP; refs. 12, 13). It was subsequently shown to be an adapter protein for Pax8 thyroid-specific transcription factor (14) and a transcription coactivator in ER/PR signaling (15). The function of WBP2 in cancer was not known until its discovery as a novel breast cancer–associated protein that is posttranslationally modified by EGFR through tyrosine phosphorylation (16). Eventually, WBP2 was demonstrated to be an oncogene whose overexpression transformed normal mammary epithelial cells and conferred aggressive traits to cancer cells (17). At the same time, WBP2 was shown to be required for the oncogenic and growth-promoting function of TAZ (18) and YAP (19). Recently, it was found that Wnt signaling promotes breast cancer by blocking ITCH E3 ligase-mediated degradation of WBP2 (20). In the same study, analysis of >400 clinical specimens revealed that WBP2 is overexpressed in breast tumors and high WBP2 expression level correlated with disease aggression and poor patient survival (20). These studies highlighted the important role of WBP2 to breast cancer.

Two observations led to the hypothesis that WBP2 is a molecular determinant/predictor for anti-HER2–based therapies: first, WBP2 is within the EGFR signaling network in which HER2 plays an intimate role; second, WBP2 is a prognostic factor and potent oncogene that regulates cancer cell growth. In this study, we established WBP2 as a downstream substrate of EGFR/HER2 signaling and investigated the role of WBP2 in regulating the response of breast cancer cells to trastuzumab and the mechanism through which this occurs in preclinical studies. Finally, we conducted a retrospective study to determine the correlation of WBP2 levels to response of HER2-positive breast tumors to neoadjuvant trastuzumab-based chemotherapy.

Reagents

In-house WBP2 polyclonal antibody was generated as described previously (17). Monoclonal anti-WBP2 (MABS441-clone 4C8H10) was obtained from EMD Millipore. Anti-phosphotyrosine antibody-HRP (PY20; MA1-12445) and anti-HER2 antibody (554299) were purchased from BD Biosciences. Anti-phospho-HER2 (PA5-17696) and anti-β-tubulin (MA5-16308-1MG) antibodies were purchased from Thermo Scientific. Anti-phospho-EGFR (2231), anti-EGFR (2232), anti-phospho-AKT (9271), and anti-AKT (9272) antibodies were purchased from Cell Signaling Technology. Anti-cyclin D1 antibody (sc-20044) was purchased from Santa Cruz Biotechnology. Trastuzumab (Herceptin) was obtained from Roche. HER2 siRNAs, WBP2 siRNAs, and luciferase siRNA were purchased from Thermo Scientific. WBP2 overexpression plasmid and knock down (shRNA) constructs were described previously (17, 20).

Cell lines and culture conditions

The human breast cancer cell lines, BT-474 and SK-BR-3, were purchased from ATCC. ZR-75–30 was a kind gift from Dr. Boon Tin Chua (Institute of Molecular and Cell Biology, Singapore). All cell lines were tested using MycoAlert PLUS Mycoplasma Detection Kit (Lonza) and verified to be free of Mycoplasma and used after 2–5 passages from thawing. BT-474, SK-BR-3, and ZR-75-30 were maintained in RPMI1640 containing 10% FBS (Thermo Fisher Scientific) and 100U penicillin/streptomycin (Invitrogen). For transient expression, cells were transfected with HER2 siRNA or WBP2 siRNA using jetPRIME transfection reagent (Polyplus Transfection), according to the manufacturer's recommendations. For stable expression, cells were transduced with each expression lentivirus, followed by selection with 400μg/mL hygromycin B (Invitrogen) for BT-474 or 0.5 μg/mL puromycin (Invitrogen) for SK-BR-3. After 2–3 weeks, selected colonies were pooled and expanded.

Cell proliferation assay

Cells were plated in 96-well plates for 2D culture or 96-well ultra-low attachment plates for 3D culture (Corning) at 10,000 cells per well. After 3 or 5 days of incubation with trastuzumab, the viability of cells was measured using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega).

Cell-cycle analysis

Cells were collected and fixed in 70% ethanol at 4°C overnight. After washing with PBS, the cells were stained with PI/RNase staining buffer (PBS containing 0.1% triton X-100, 0.2 mg/mL RNase A, 20 μg/mL PI) for 15 minutes at room temperature. The DNA content of cells was measured by flow cytometry (LSR Fortessa Flow Cytometry Analyser, BD Biosciences). Proportions of cells in G1, S, and G2–M phases were analyzed using FACSDiva Software (BD Biosciences).

Subcellular fractionation

Membrane and cytoplasmic extracts were prepared using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific), as per manufacturer's instructions.

Western blot analyses and immunoprecipitation assays

Cell lysis, Western blot analysis, and immunoprecipitation assay were performed as described previously (17, 20). The detail methods can be found in Supplementary Information.

Reverse transcription and real-time PCR

Total RNA was isolated using PureLink RNA Mini Kit (Thermo Fisher Scientific) and reverse transcribed using random hexamer primers and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qPCR reactions were performed using QuantiFast Probe PCR Kit (Qiagen) as per manufacturer's instructions. Predesigned primers/probes were obtained from IDT.

Bioinformatics analysis

Copy number variation (CNV) data and RNA-sequencing (RNA-Seq) data from TCGA-BRCA (21) was downloaded from the GDC Data Portal (22). The data were cleaned to remove samples with incomplete data. The HER2 expression status were assessed from the RNA-Seq data. The differences in CNV profile between primary tumor and solid tissue normal were then computed as described in Supplementary Information. Gene list analysis was performed using PANTHER (23). A custom Python script was developed to query PubMed (Supplementary Methods). The word cloud was then generated using the Text Analytics Toolbox of MATLAB R2018b (MathWorks, 2018).

Xenograft model

All animal experiments were performed in accordance with the institutional guidelines of and were approved by Institutional Animal Care and Use Committee of National University of Singapore (Singapore). For the xenograft model, 5-week-old female athymic nude mice (n = 6-7, In Vivos) were implanted with 0.72-mg 60-day release 17β-estradiol pellets (Innovative Research) and after 2 days, BT-474 control (vector) or WBP2-overexpressing cells (1 × 107 in 200 μL of DPBS and Matrigel 1:1 mixture) were injected subcutaneously into a mouse mammary fat pad. When the tumors reached the size of 150–200 mm3, the mice were divided into groups, keeping average tumor size similar between groups, and treated with trastuzumab (Herceptin; 10 mg/kg, Roche) or PBS (control) by intraperitoneal (IP) route twice weekly for three weeks. The tumor size was measured twice weekly with calipers and tumor volumes were calculated as follow: volume = (width2 × length)/2.

Patient-derived xenografts

Trastuzumab-sensitive breast cancer PDX (PDX118) and trastuzumab-resistant breast cancer PDXs (PDX118TRs) were established by Arribas and colleagues as described previously (24).

Human specimens

The study was conducted in accordance with the Declaration of Helsinki and written informed consent was obtained from all participants. All specimens obtained after protocol approval by Institutional Review Boards.

Retrospective study cohort 1: correlation of WBP2 and HER2 to patients' outcomes.

Breast tumor tissues from patients who received surgery between 1997 and 2007 as well as the clinical/histopathologic information (including HER2 IHC scores) were obtained from National Cancer Centre, Singapore (NCC; n = 52), National University Hospital, Singapore (NUH; n = 205), and Singapore General Hospital (SGH; n = 126). The information for some cases is not available. Therefore, the total number of cases analyzed may not be the same as the original number available. Patients received the treatment and surgery in accordance with each institution's standard treatment, but the treatment information was not available for this cohort. The samples in this cohort were randomized and include HER2-positive and -negative breast cancer cases.

Retrospective study cohort 2: correlation of WBP2 with pCR to trastuzumab-based neoadjuvant therapy.

The cohort here includes patients with stage I–III HER2-positive breast cancer who received neoadjuvant trastuzumab-based chemotherapy between 2005 and 2017 and from whom archival tumor specimens and clinical information were available. Patients received anthracycline or nonanthracycline-containing regimens in combination with trastuzumab as part of each institution's standard treatment. Neoadjuvant trastuzumab with anthracycline-containing chemotherapy regimens included AC-TH regimen (doxorubicin/cyclophosphamide followed by paclitaxel or docetaxel/trastuzumab) and FEC-TH regimen (fluorouracil/epirubicin/cyclophosphamide followed by paclitaxel or docetaxel/trastuzumab). Neoadjuvant trastuzumab with nonanthracycline-containing chemotherapy regimens included TCH regimen (paclitaxel or docetaxel/carboplatin combined with trastuzumab) and TH regimen (paclitaxel or docetaxel combined with trastuzumab). Specimens comprised FFPE biopsies that were collected before neoadjuvant treatment. A total of 143 cases complete with clinical information such as pathologic response information were obtained from NUH, Kandang Kerbau Women's and Children's Hospital (KKH), and Yonsei Cancer Center (YCC).

IHC analysis

The detailed protocol for IHC was previously described (17, 20). IHC of pretreatment biopsy specimens was done in the NUH pathology department core using BenchMark ULTRA IHC Autostainer (Roche Ventana). All IHC slides were scored in a blinded fashion by pathologists. A scale of 0–3 was used for the stain intensity (0, no staining; 1+, mild; 2+, moderate; and 3+, strong staining.

Statistical analysis

All in vitro experiments were repeated at least three times and the results were presented as mean ± SD. The comparisons between each group were determined by Student t test. P values of <0.05 were considered statistically significant and expressed as *, P < 0.05; **, P < 0.01; ***, P < 0.001. For in vivo and retrospective study, the data represent mean ± SEM and the significance of differences or association was evaluated using Mann–Whitney U test. Kaplan–Meier method was used to plot the overall survival (OS) and disease-free survival (DFS) curves. DFS and OS were defined as time from date-of-diagnosis to date-of-first-recurrence and death or last follow-up date, respectively. Survival between groups was compared using log-rank test. The associations between pCR and WBP2 expression or/with clinical characteristics were investigated by contingency tables and analyzed by Fisher exact test. All statistical analyses were performed using SPSS and GraphPad Prism.

HER2 amplification–associated gene network in breast cancer

The upregulation of HER2 in breast cancer is usually associated with an amplification of the HER2 locus (25). Inadvertently, other genes along chromosome 17q (C17q) arm are coamplified with HER2 (26). It is conceivable that HER2 and its associated genes along C17q not only form a molecular network but also cooperatively and functionally contribute to the phenotype of HER2-positive breast cancer. The HER2-associated genes may also regulate the response of HER2-positive breast cancer to drugs and are therefore potential companion diagnostics for HER2-based therapeutics. Hence, we attempted to map the genes that are coamplified with HER2, performed preclinical tests to investigate their interactions with HER2 in the presence of trastuzumab, and validate whether the expression of these candidate biomarkers correlate with response of breast tumors to trastuzumab-based treatment via a retrospective study.

First, we obtained the copy number variation (CNV) and RNA-Seq data from the TCGA-BRCA project (21), and employed the workflow shown in Fig. 1A to identify regions of chromosome 17 that were amplified in HER2 upregulated breast cancer. RNA-Seq data was used to identify cases with HER2 upregulated, while CNV data were used to identify regions of chromosome 17 that were amplified or deleted. As shown in Fig. 1B, the p-arm of chromosome 17 experienced a significant deletion, while 3 regions along q-arm of chromosome 17 were significantly amplified in HER2-overexpressing breast cancer. A total of 1,145 genes were identified to reside in these 3 significantly amplified regions. Gene list analysis using the PANTHER Classification System (23) was performed to provide a general understanding of the genes coamplified with HER2. Interestingly, cellular processes (GO: 0009987) and metabolic processes (GO: 0008152) accounted for about half of the hits (Fig. 1C). Amplification of genes involved in these critical processes is likely to result in dysregulation and cancer phenotype.

Figure 1.

A–D, Bioinformatic analysis of TCGA-BRCA (Breast Invasive Carcinoma) dataset. A, Flowchart describing the analysis pipeline of copy number variation (CNV) profile of HER2-overexpressing breast cancers. B, Differences in CNV profile between primary tumor and non-cancer solid tissue. Positive values indicate amplification in primary tumor, while negative values indicate deletion in primary tumors. Colormap indicates the statistical significance (P value) of the difference between primary tumor and solid tissue normal. C, Pie chart showing biological processes associated with genes coamplified with HER2. Panther Classification System was used for the analysis. D, Word cloud showing the frequency of association between each coamplified genes with breast cancer, and EGFR or HER2. E–G, IHC and correlation analyses of WBP2 and HER2 expression in human breast tumors to survival outcomes. E, Kaplan–Meier survival analysis of the OS and DFS survival of patients segregated into HER2+ and HER2 groups (n = 221). F, Boxplot of WBP2 expression in HER2+ and HER2 breast tumors (n = 296). G, Kaplan–Meier analysis of OS and DFS of patients segregated based on WBP2 and HER2 expression status (n = 221). High WBP2 expression is defined as nuclear IHC score of more than 1. Statistical significance was determined by Mann–Whitney U test and log-rank test (***, P < 0.001). H and I, Phosphorylation of WBP2 is dependent on HER2. HER2 was knocked down in human breast cancer cells, SK-BR-3 (H) and ZR-751 (I), via HER2-specific siRNAs. Luciferase siRNA was used as negative control. Cells were treated with 50 ng/mL EGF for 10 minutes after 24-hour serum starvation. Cell lysates were immunoprecipitated (IP) with anti-WBP2 antibody and phosphorylation of endogenous WBP2 was analyzed by Western blot analysis (IB) using anti-phosphotyrosine (PY20) and anti-WBP2 antibodies. HER2 was analyzed by Western blot analysis (IB) with indicated antibodies. β-Tubulin was used as a protein-loading control.

Figure 1.

A–D, Bioinformatic analysis of TCGA-BRCA (Breast Invasive Carcinoma) dataset. A, Flowchart describing the analysis pipeline of copy number variation (CNV) profile of HER2-overexpressing breast cancers. B, Differences in CNV profile between primary tumor and non-cancer solid tissue. Positive values indicate amplification in primary tumor, while negative values indicate deletion in primary tumors. Colormap indicates the statistical significance (P value) of the difference between primary tumor and solid tissue normal. C, Pie chart showing biological processes associated with genes coamplified with HER2. Panther Classification System was used for the analysis. D, Word cloud showing the frequency of association between each coamplified genes with breast cancer, and EGFR or HER2. E–G, IHC and correlation analyses of WBP2 and HER2 expression in human breast tumors to survival outcomes. E, Kaplan–Meier survival analysis of the OS and DFS survival of patients segregated into HER2+ and HER2 groups (n = 221). F, Boxplot of WBP2 expression in HER2+ and HER2 breast tumors (n = 296). G, Kaplan–Meier analysis of OS and DFS of patients segregated based on WBP2 and HER2 expression status (n = 221). High WBP2 expression is defined as nuclear IHC score of more than 1. Statistical significance was determined by Mann–Whitney U test and log-rank test (***, P < 0.001). H and I, Phosphorylation of WBP2 is dependent on HER2. HER2 was knocked down in human breast cancer cells, SK-BR-3 (H) and ZR-751 (I), via HER2-specific siRNAs. Luciferase siRNA was used as negative control. Cells were treated with 50 ng/mL EGF for 10 minutes after 24-hour serum starvation. Cell lysates were immunoprecipitated (IP) with anti-WBP2 antibody and phosphorylation of endogenous WBP2 was analyzed by Western blot analysis (IB) using anti-phosphotyrosine (PY20) and anti-WBP2 antibodies. HER2 was analyzed by Western blot analysis (IB) with indicated antibodies. β-Tubulin was used as a protein-loading control.

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To shortlist candidates that play role in regulating HER2 in breast cancer, we queried PubMed and found that 862 of the 1145 amplified genes are not implicated in breast cancer. We further reasoned that candidates that influence the response of HER2-positive breast cancer to anti-HER2 drugs are likely to be proteins that are associated with EGFR and/or HER2 signaling. From the remaining 283 genes, only 101 genes are related to either EGFR signaling or HER2 signaling. Out of the 101 genes associated with EGFR/HER2 and breast cancer, 55 of them have 2 or more publications and are represented in a word cloud. The size of the words reflects the number of publications (in a Log2 scale) that relate the gene to EGFR or HER2 in breast cancer (Fig. 1D). As expected, HER2 (ERBB2) was the top hit, with 9,947 publications (as of September 2018). Found in this HER2 network of genes is WW-domain Binding Protein 2 (WBP2), an emerging oncogene recently implicated in breast cancer development (17, 20) and a phosphotyrosine substrate of EGFR signaling pathway (16). Our data revealed that WBP2 and HER2 are coamplified in 36% of the HER2-positive breast cancers. It is conceivable that tyrosine phosphorylation of WBP2 by EGFR is HER2-dependent and aberrant WBP2 expression may be a novel mechanism that regulates cancer cellular response to anti-HER2 drugs.

WBP2 and HER2 expression in combination correlate with a worse prognosis in breast cancer patients than either alone

Because WBP2 and HER2 oncogenes are demonstrated to be coamplified in C17q, we hypothesized that WBP2 coexpress with HER2 in clinical breast cancers at the protein level and in combination they give rise to poorer prognosis. To test these hypotheses, a retrospective study was performed. IHC of HER2 and WBP2 was performed on 296 resected breast tumor tissues. Consistent with previous studies, patients with HER2-positive tumors had worse overall survival (P = 0.011) and disease-free survival (P = 0.137) than the HER2-negative group (Fig. 1E; refs. 4, 27). Next, we analyzed the correlation of WBP2 with HER2 status. Both nuclear and cytoplasmic WBP2 expression was significantly higher in HER2-positive breast tumors compared with HER2-negative tumors (Fig. 1F). Because WBP2 expression and HER2 status were positively correlated, we proceeded to examine whether patients' outcome is associated with WBP2 and HER2 in combination. We found that patients whose tumors showed high nuclear WBP2 and HER2-positive expression had the worst overall (HR: 4.49; P < 0.001) and disease-free survival (HR: 2.58, P = 0.004) than other groups (Fig. 1G). WBP2 expression in combination with HER2 appears to be more powerful than either alone for breast cancer prognosis. It suggests that WBP2 can be used to further prognosticate patients with HER2-positive breast cancer.

HER2 is required for EGF-induced WBP2 tyrosine phosphorylation

The association of WBP2 with HER2 in clinical specimens, coupled to the previous finding that WBP2 is a downstream target of EGF signaling, led to the postulation that tyrosine phosphorylation of WBP2 by EGFR requires HER2, because EGFR and HER2 are known to heterodimerize in the presence of EGF (16). To test this hypothesis, HER2 expression was knocked down using two different siRNAs in HER2+/WBP2+ breast cancer cell lines. Knockdown of HER2 abolished EGF-induced tyrosine phosphorylation of WBP2 in SK-BR-3 and ZR-751 breast cancer cells (Fig. 1H and I). This indicates that HER2 is required for tyrosine phosphorylation and activation of WBP2 by EGF.

WBP2 expression enhances trastuzumab response in breast cancer cells

Given that WBP2 is downstream of HER2, aberrant expression of oncogenic WBP2 (20) may influence cancer cellular response to trastuzumab. To investigate the effect of WBP2 expression on the response of HER2-positive breast cancer cells to trastuzumab as a single agent, SK-BR-3, ZR-75-30, and BT-474 breast cancer cell lines were selected because they are HER2-positive and trastuzumab sensitive as shown by our preliminary study and other reports (28). While trastuzumab is rarely used as a single agent in the clinic, a clean/simplistic system involving only trastuzumab was needed to prove a direct link and to study the interactions between WBP2, HER2, and trastuzumab. As would be evident later, this “reductionist” approach in the laboratory would be complemented with an actual and more complex clinical setting in which the correlation of WBP2 expression with breast tumor response to trastuzumab with chemotherapy treatment would be tested.

WBP2 expression was silenced using two different shRNAs in SK-BR-3 and two different siRNAs in ZR-75-30, while the overexpression of WBP2 was achieved using a lentiviral system in BT-474. Each cell line was subsequently treated with increasing doses of trastuzumab (1, 10, 100 μg/mL) and the cell growth analyzed for 3 days (for SK-BR-3) or 5 days (for ZR-75-30 and BT-474) after incubation in 2D or 3D culture conditions. In WBP2-overexpressing BT-474 cells, the response to trastuzumab (100 μg/mL) increased by 2.1-fold for 2D culture (P = 0.001) and 2.2-fold for 3D culture (P = 0.003) compared with vector control (Fig. 2A and B). On the other hand, knockdown of WBP2 significantly reduced the response to trastuzumab in SK-BR-3 (by ∼37 %; Fig. 2C and D) and ZR-75-30 cells (by ∼45%; Fig. 2E and F) compared with the controls. These data provide evidence that WBP2 expression level influences the response of breast cancer cells to trastuzumab.

Figure 2.

A–F, Dose-dependent effect of trastuzumab on the proliferation of HER2-positive breast cancer cells with WBP2 overexpressed or knocked down. WBP2 was overexpressed in BT-474 using lentivirus (A and B) or knocked down using two different WBP2-specific shRNA in SK-BR-3 (C and D) or two different WBP2-specific siRNAs in ZR-75-30 (E and F). Cells were plated in 96-well plates for 2D culture (A, C, E) or 96-well ultra-low attachment plates for 3D culture (B, D, F) at 10,000 cells per well. After 3 days (SK-BR-3) or 5 days (BT-474 and ZR-75-30) of incubation with trastuzumab, cell viability was measured using CellTiter 96 aqueous nonradioactive cell proliferation assay. Cell viability was calculated as fold change compared with trastuzumab-untreated control cells. The data represent mean ± SD (n = 3). Statistical significance was determined by Student t test (* or +P < 0.05; ** or ++P < 0.01; *** or +++P < 0.001 vs. vector or control). G–I, Dose-dependent effect of trastuzumab on HER2 and its downstream signaling pathway. WBP2 was overexpressed in BT-474 using WBP2-expressing lentivirus (G) or knockdown using two different shRNAs targeting WBP2 in SK-BR-3 (H) or two different siRNAs targeting WBP2 in ZR-75-30 (I). Cells were treated with different concentrations of trastuzumab (0, 1, 10, 100 μg/mL) for 3 days (SK-BR-3) or 5 days (BT-474 and ZR-75-30). Expression and phosphorylation of proteins were analyzed by Western blot analysis using the indicated antibodies. J and K, Effect of WBP2 on cell surface HER2 level in the absence or presence of trastuzumab. BT-474 cells expressing WBP2 or vector (J) and SK-BR-3 cells expressing WBP2 shRNA or scramble shRNA (K) were treated with different concentrations of trastuzumab (0, 1, 10, 100 μg/mL). After 3 days (SK-BR-3) or 5 days (BT-474) of treatment, cells were separated into membrane and cytosol fraction using Mem-PERTM Plus Membrane Protein Extraction Kit. HER2 level in the membrane and cytosol fractions were analyzed by Western blot analysis. L–N, Effect of WBP2 on trastuzumab-induced G1 arrest. Two different siRNAs targeting WBP2 were transfected into ZR-75-30 cells that were subsequently treated with 100 μg/mL of trastuzumab for 5 days. L, Cells were stained with propidium iodide (PI) and cell-cycle distribution was analyzed by flow cytometry. Cyclin D1 protein level (M) and mRNA level (N) were analyzed. Protein expression level of cyclin D1 was quantitated and normalized to β-tubulin using ImageJ software and cyclin D1 expression in control (Luc siRNA or Vector) without trastuzumab was defined as 1. WBP2 or cyclin D1 mRNA expression level was analyzed by qRT-PCR and normalized to 18s rRNA. The data represent mean ± SD from three independent experiments (n = 3). Statistical significance was determined by Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control).

Figure 2.

A–F, Dose-dependent effect of trastuzumab on the proliferation of HER2-positive breast cancer cells with WBP2 overexpressed or knocked down. WBP2 was overexpressed in BT-474 using lentivirus (A and B) or knocked down using two different WBP2-specific shRNA in SK-BR-3 (C and D) or two different WBP2-specific siRNAs in ZR-75-30 (E and F). Cells were plated in 96-well plates for 2D culture (A, C, E) or 96-well ultra-low attachment plates for 3D culture (B, D, F) at 10,000 cells per well. After 3 days (SK-BR-3) or 5 days (BT-474 and ZR-75-30) of incubation with trastuzumab, cell viability was measured using CellTiter 96 aqueous nonradioactive cell proliferation assay. Cell viability was calculated as fold change compared with trastuzumab-untreated control cells. The data represent mean ± SD (n = 3). Statistical significance was determined by Student t test (* or +P < 0.05; ** or ++P < 0.01; *** or +++P < 0.001 vs. vector or control). G–I, Dose-dependent effect of trastuzumab on HER2 and its downstream signaling pathway. WBP2 was overexpressed in BT-474 using WBP2-expressing lentivirus (G) or knockdown using two different shRNAs targeting WBP2 in SK-BR-3 (H) or two different siRNAs targeting WBP2 in ZR-75-30 (I). Cells were treated with different concentrations of trastuzumab (0, 1, 10, 100 μg/mL) for 3 days (SK-BR-3) or 5 days (BT-474 and ZR-75-30). Expression and phosphorylation of proteins were analyzed by Western blot analysis using the indicated antibodies. J and K, Effect of WBP2 on cell surface HER2 level in the absence or presence of trastuzumab. BT-474 cells expressing WBP2 or vector (J) and SK-BR-3 cells expressing WBP2 shRNA or scramble shRNA (K) were treated with different concentrations of trastuzumab (0, 1, 10, 100 μg/mL). After 3 days (SK-BR-3) or 5 days (BT-474) of treatment, cells were separated into membrane and cytosol fraction using Mem-PERTM Plus Membrane Protein Extraction Kit. HER2 level in the membrane and cytosol fractions were analyzed by Western blot analysis. L–N, Effect of WBP2 on trastuzumab-induced G1 arrest. Two different siRNAs targeting WBP2 were transfected into ZR-75-30 cells that were subsequently treated with 100 μg/mL of trastuzumab for 5 days. L, Cells were stained with propidium iodide (PI) and cell-cycle distribution was analyzed by flow cytometry. Cyclin D1 protein level (M) and mRNA level (N) were analyzed. Protein expression level of cyclin D1 was quantitated and normalized to β-tubulin using ImageJ software and cyclin D1 expression in control (Luc siRNA or Vector) without trastuzumab was defined as 1. WBP2 or cyclin D1 mRNA expression level was analyzed by qRT-PCR and normalized to 18s rRNA. The data represent mean ± SD from three independent experiments (n = 3). Statistical significance was determined by Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control).

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WBP2 enhances the modulation of EGFR/HER2 signaling in the presence of trastuzumab

The mechanism of trastuzumab as an antitumor agent remains an area of active research. Some groups have shown that trastuzumab induced downregulation of HER2 level (29–31). To determine whether WBP2 affects trastuzumab-induced downregulation of HER2 expression, WBP2-overexpressing BT-474 cells and WBP2 knocked down SK-BR-3 or ZR-75-30 cells were treated with increasing doses of trastuzumab (1, 10, 100 μg/mL) and HER2 protein expression analyzed using Western blot analysis. In WBP2-overexpressing BT-474 cells, both HER2 and EGFR levels were substantially decreased when exposed to a low dose of trastuzumab, compared with control cells (Fig. 2G). Consistently, knockdown of WBP2 inhibited trastuzumab-dependent downregulation of HER2 and EGFR levels in SK-BR-3 and ZR-75-30 breast cancer cells. In contrast, HER2 levels were decreased by trastuzumab in a concentration-dependent manner in control cells (scrambled shRNA or Luc siRNA; Fig. 2H and I). Like HER2 and EGFR, inactivation of AKT by trastuzumab, which has been reported by previous studies (32–34), was enhanced by WBP2 overexpression and diminished by WBP2 knockdown. Figure 2J and K demonstrated that it was the cell-surface species of HER2 that was affected by WBP2 in the presence of trastuzumab. Collectively, our data suggest that WBP2 enhances the inhibitory effect of trastuzumab on cancer cell proliferation by augmenting trastuzumab-induced downregulation of HER2/EGFR signaling.

WBP2 promotes trastuzumab-induced G1 arrest by enhancing downregulation of cyclin D1 expression

Previous studies showed that trastuzumab treatment induced G1 cell-cycle arrest, leading to inhibition of cell proliferation (35). To investigate the involvement of WBP2 in trastuzumab-induced G1 arrest, we performed cell-cycle FACS analysis of WBP2 knocked down versus control ZR-75-30 cells. Trastuzumab increased the number of cells at G0–G1 phase by 14.5% compared with control (no trastuzumab treatment; P = 0.0003) while trastuzumab decreased the number of cells at S and G2–M phase by 52.4% and 37.0%, respectively. Knockdown of WBP2 using two different WBP2 siRNAs abolished the effects of trastuzumab on cell cycle (Fig. 2L). We further examined WBP2's involvement in trastuzumab-mediated G1 arrest by analyzing cyclin D expression. Trastuzumab decreased cyclin D protein level by 87.6% (P < 0.001) in ZR-75-30 cells as compared with untreated cells (Luc si). This effect was only 44.6% to 56.2% when WBP2 was silenced with two separate siRNAs (Fig. 2M). Similarly, the decrease in cyclin D mRNA level by trastuzumab was abolished when WBP2 was knocked down (Fig. 2N). Collectively, these results suggest that WBP2 promotes trastuzumab-induced G1 arrest via downregulation of cyclin D.

WBP2 overexpression sensitizes breast tumor to trastuzumab in vivo

Our in vitro observations indicated that cancer cellular response to trastuzumab depended on WBP2 level. To confirm observation, an in vivo tumor xenograft model was used. BT-474 cells stably expressing WBP2 were injected into the mammary fat pad of athymic nude mice. When the size of tumors reached 150–200 mm3, the mice were divided into two groups and treated with trastuzumab (10 mg/kg) or PBS by intraperitoneal injection twice weekly for three weeks. The size of WBP2-overexpressing tumor after trastuzumab treatment was reduced to 5.23% as compared with no treatment (100%), whereas the size of tumor with vector control was reduced only to 38.33% (Fig. 3A and B). As the difference between the two trastuzmab-treated conditions appeared small due to the scale used in the plot shown in Fig. 3A and B, separate plots are provided in Supplementary Fig. S1 to show their differences more clearly. Hence, overexpression of WBP2 increased tumor response to trastuzumab by 7.32 times (P = 0.0047). The results support the notion that high WBP2 level sensitized breast cancer to trastuzumab.

Figure 3.

Effect of WBP2 on tumor response to trastuzumab in vivo. Five-week-old female athymic nude mice (n = 6–7) were implanted with 0.72-mg 60-day release 17β-estradiol pellets. After 2 days, BT-474 control (vector) or WBP2-overexpressing cells (1 × 107 of cells in 200 μL of DPBS and Matrigel 1:1 mixture) were injected subcutaneously into a mouse mammary fat pad. When the tumor size reached 150–200 mm3, the mice were divided into groups, keeping average tumor size similar between groups, and treated with trastuzumab (10 mg/kg, Roche) or PBS (control) intraperitoneally twice weekly for three weeks. A and B, The tumor size was measured twice weekly with calipers and tumor volumes calculated as follow: volume = (width2 × length)/2. Tumor growth was presented in time-course line plot (A) and endpoint dot plot (B). The data represent mean ± SEM. Statistical significance was determined by Mann–Whitney test. C and D, HER2 and WBP2 expression in PDX model of isogenic trastuzumab-sensitive and -resistant breast cancer cells. C, HER2 and WBP2 expression in tumor from trastuzumab-sensitive or resistant PDXs that were established in J. Arribas's research group (24) were analyzed by IHC. Scale bars, 250 μm. D, Statistical correlation studies of the IHC scores for HER2 and WBP2 for each tumor was performed using the Pearson correlation test.

Figure 3.

Effect of WBP2 on tumor response to trastuzumab in vivo. Five-week-old female athymic nude mice (n = 6–7) were implanted with 0.72-mg 60-day release 17β-estradiol pellets. After 2 days, BT-474 control (vector) or WBP2-overexpressing cells (1 × 107 of cells in 200 μL of DPBS and Matrigel 1:1 mixture) were injected subcutaneously into a mouse mammary fat pad. When the tumor size reached 150–200 mm3, the mice were divided into groups, keeping average tumor size similar between groups, and treated with trastuzumab (10 mg/kg, Roche) or PBS (control) intraperitoneally twice weekly for three weeks. A and B, The tumor size was measured twice weekly with calipers and tumor volumes calculated as follow: volume = (width2 × length)/2. Tumor growth was presented in time-course line plot (A) and endpoint dot plot (B). The data represent mean ± SEM. Statistical significance was determined by Mann–Whitney test. C and D, HER2 and WBP2 expression in PDX model of isogenic trastuzumab-sensitive and -resistant breast cancer cells. C, HER2 and WBP2 expression in tumor from trastuzumab-sensitive or resistant PDXs that were established in J. Arribas's research group (24) were analyzed by IHC. Scale bars, 250 μm. D, Statistical correlation studies of the IHC scores for HER2 and WBP2 for each tumor was performed using the Pearson correlation test.

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WBP2 expression correlates with trastuzumab response in PDX model

To obtain more evidence that high WBP2 expression is associated with positive response to trastuzumab treatment, we examined WBP2 and HER2 expression in a PDX model of drug resistance to trastuzumab. Tumor samples from trastuzumab-sensitive breast cancer PDX (PDX118) and trastuzumab-resistant breast cancer PDXs (PDX118TRs) established by Arribas and colleagues were used (24). The IHC staining shows that trastuzumab-sensitive sample (PDX118) was HER2-positive and WBP2-positive. Interestingly, 3 (TR2-4) of 5 tumors that acquired trastuzumab resistance had lower HER2 and WBP2 expression (Fig. 3C). Pearson correlation test revealed that HER2 and WBP2 expression had a correlation coefficient (Pearson r) of 0.8944 (P = 0.0161), indicating strong association between HER2 and WBP2 expression (Fig. 3D). These data suggest that WBP2 in combination with HER2 expression is a determinant of response to trastuzumab.

High WBP2 expression is associated with pCR of HER2-positive breast cancer patients to neoadjuvant trastuzumab-based treatment

To investigate whether WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab in the clinical setting where chemotherapies are used in conjunction, a retrospective study was conducted. A total of 143 cases of pretreatment biopsy specimens from patients with breast cancer who received trastuzumab-based neoadjuvant chemotherapy were collected from three different cohorts; NUH, KKH, and YCC. The characteristics of the patients before treatment are provided in Table 1. Overall, 64 (44.76%) patients achieved pCR after trastuzumab-based neoadjuvant chemotherapy which is similar to previous studies (30%–53%; refs. 10, 11). The ER- or PR-negative group showed higher pCR than the ER- or PR-positive group, which is also consistent with previous reports (10). No statistically significant relationship between pCR and other clinical factors was obtained (Supplementary Table S1).

Table 1.

Patients characteristics

CharacteristicsOverall populationN (%) 143
Pathologic response pCR 64 (44.8) 
Age Median age [range] (years) 53 [25–76] 
 <50 56 (39.2) 
 ≥50 86 (60.1) 
 NA 1 (0.7) 
Menopausal status Premenopause 61 (42.7) 
 Postmenopause 71 (49.7) 
 Perimenopause 10 (7.0) 
 NA 1 (0.7) 
ER status Negative 77 (53.8) 
 Positive 63 (44.1) 
 Null (NA) 3 (2.1) 
PR status Negative 83 (58.0) 
 Positive 57 (39.9) 
 Null (NA) 3 (2.1) 
Tumor grade – 
 49 (34.3) 
 79 (55.2) 
 NA 15 (10.5) 
T stage T1 19 (13.3) 
 T2 80 (55.9) 
 T3 29 (20.3) 
 T4 13 (9.1) 
 NA 2 (1.4) 
N stage N0 40 (28.0) 
 N1 22 (15.4) 
 N2 64 (44.8) 
 N3 13 (9.1) 
 NA (NX) 4 (2.8) 
Overall TNM stage 8 (5.6) 
 II 84 (58.7) 
 III 49 (34.3) 
 NA 2 (1.4) 
CharacteristicsOverall populationN (%) 143
Pathologic response pCR 64 (44.8) 
Age Median age [range] (years) 53 [25–76] 
 <50 56 (39.2) 
 ≥50 86 (60.1) 
 NA 1 (0.7) 
Menopausal status Premenopause 61 (42.7) 
 Postmenopause 71 (49.7) 
 Perimenopause 10 (7.0) 
 NA 1 (0.7) 
ER status Negative 77 (53.8) 
 Positive 63 (44.1) 
 Null (NA) 3 (2.1) 
PR status Negative 83 (58.0) 
 Positive 57 (39.9) 
 Null (NA) 3 (2.1) 
Tumor grade – 
 49 (34.3) 
 79 (55.2) 
 NA 15 (10.5) 
T stage T1 19 (13.3) 
 T2 80 (55.9) 
 T3 29 (20.3) 
 T4 13 (9.1) 
 NA 2 (1.4) 
N stage N0 40 (28.0) 
 N1 22 (15.4) 
 N2 64 (44.8) 
 N3 13 (9.1) 
 NA (NX) 4 (2.8) 
Overall TNM stage 8 (5.6) 
 II 84 (58.7) 
 III 49 (34.3) 
 NA 2 (1.4) 

To analyze WBP2 expression in pretreatment biopsy specimens, IHC of WBP2 was performed using a WBP2 polyclonal antibody (17, 20). The specificity of the WBP2 polyclonal antibody for IHC was validated previously (20). The IHC score for WBP2 was higher in the pCR group than non-pCR group (mean WBP2 IHC score, 2.63 vs. 2.05, P < 0.0001; Fig. 4A). Next, the breast tumors were segregated into high (=3) and low WBP2 IHC score (<3). The high WBP2–expressing group had significantly higher pCR of 2.5 times more than the low WBP2–expressing group (67.19% vs. 26.58%, respectively; Fig. 4B; Table 2). We further examined whether there were clinical factors that might be associated with pCR and WBP2 expression. When the patients were first stratified by age (<50 and ≥50 years old) and menopausal status and the breast tumors further stratified on the basis of WBP2 IHC score (=3 and <3), the pCR of patients with high WBP2 and age below 50 years was 77.78% while patients with high WBP2 and premenopausal was 73.33%, both of which are higher than the pCR of 67.19% for tumors with high WBP2 alone (Fig. 4C and 4D).

Figure 4.

Correlation between WBP2 expression and pCR. A–D, Analyses of WBP2 expression (based on IHC) in breast tumors of neoadjuvant trastuzumab + chemotherapy-treated patients segregated into those that showed pCR and partial/non-pCR (non-pCR; n = 143). A, WBP2 IHC scores in pCR or non-pCR groups. The data represent mean ± SEM. Statistical significance was determined by Mann–Whitney test. B–D, WBP2 expression was segregated into IHC score of 3 or <3 and pCR or non-pCR in the overall group (B), in the age <50 years group (C), and premenopausal group (D). Statistical significance was determined by Fisher exact test. E, ROC analysis was conducted for overall group, premenopausal group, and aged below 50 years group. Area under the curve (AUC) of overall, premenopausal group, and aged below 50 years group was calculated (***, P < 0.0001).

Figure 4.

Correlation between WBP2 expression and pCR. A–D, Analyses of WBP2 expression (based on IHC) in breast tumors of neoadjuvant trastuzumab + chemotherapy-treated patients segregated into those that showed pCR and partial/non-pCR (non-pCR; n = 143). A, WBP2 IHC scores in pCR or non-pCR groups. The data represent mean ± SEM. Statistical significance was determined by Mann–Whitney test. B–D, WBP2 expression was segregated into IHC score of 3 or <3 and pCR or non-pCR in the overall group (B), in the age <50 years group (C), and premenopausal group (D). Statistical significance was determined by Fisher exact test. E, ROC analysis was conducted for overall group, premenopausal group, and aged below 50 years group. Area under the curve (AUC) of overall, premenopausal group, and aged below 50 years group was calculated (***, P < 0.0001).

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Table 2.

Association between WBP2 IHC score and pCR

pCRNon-pCR
CharacteristicsWBP2 IHC scoreN%N%PaSensitivity (%)95% CISpecificity (%)95% CI
Overall population  64 44.76 79 55.24      
 =3 43 67.19 21 32.81 ***, <0.0001 67.19 54.31%–78.41% 73.42 62.28%–82.73% 
 <3 21 26.58 58 73.42      
Age           
 <50 =3 21 77.78 22.22 ***, <0.0001 80.77 60.65%–93.45% 80.00 61.43%–92.29% 
 <3 17.24 24 82.76      
 ≥50 =3 22 59.46 15 40.54 *, 0.017 57.89 40.82%–73.69% 68.75 53.75%–81.34% 
 <3 16 32.65 33 67.35      
Menopausal status           
 Premenopause =3 22 73.33 26.67 ***, <0.0001 81.48 61.92%–93.70% 76.47 58.83%–89.25% 
 <3 16.13 26 83.87      
 Postmenopause =3 16 57.14 12 42.86 n.s., 0.088 51.61 33.06%–69.85% 70.00 53.47%–83.44% 
 <3 15 34.88 28 65.12      
pCRNon-pCR
CharacteristicsWBP2 IHC scoreN%N%PaSensitivity (%)95% CISpecificity (%)95% CI
Overall population  64 44.76 79 55.24      
 =3 43 67.19 21 32.81 ***, <0.0001 67.19 54.31%–78.41% 73.42 62.28%–82.73% 
 <3 21 26.58 58 73.42      
Age           
 <50 =3 21 77.78 22.22 ***, <0.0001 80.77 60.65%–93.45% 80.00 61.43%–92.29% 
 <3 17.24 24 82.76      
 ≥50 =3 22 59.46 15 40.54 *, 0.017 57.89 40.82%–73.69% 68.75 53.75%–81.34% 
 <3 16 32.65 33 67.35      
Menopausal status           
 Premenopause =3 22 73.33 26.67 ***, <0.0001 81.48 61.92%–93.70% 76.47 58.83%–89.25% 
 <3 16.13 26 83.87      
 Postmenopause =3 16 57.14 12 42.86 n.s., 0.088 51.61 33.06%–69.85% 70.00 53.47%–83.44% 
 <3 15 34.88 28 65.12      

aFisher exact test.

Retrospective ROC curve analysis was performed to determine the optimal sensitivity and specificity of WBP2 expression in discriminating pCR from non-pCR cases. The areas under the curve (AUC) was 0.72 with the sensitivity and specificity at 67.19% and 73.42%, respectively, when a WBP2 IHC cut-off score of 2.5 was used (Fig. 4E, black line; Table 2). Both the sensitivity and specificity were higher in patients aged below 50 years (80.77% and 80.00%) and those in the premenopausal group (81.48% and 76.47%; Table 2). The AUC for the premenopausal patients (0.80) and patients aged below 50 years (0.81) were higher compared with overall group (0.72; Fig. 4E, red line and blue line, respectively). No significant correlation was observed for other clinical factors including ER/PR status, tumor grade, TNM stage, chemotherapy regimen (data not shown). Collectively, the retrospective study indicates that high WBP2 expression in pretreatment biopsy is positively correlated with pCR, especially for those aged below 50 years group and in the premenopausal group.

Chromosome 17 aneusomy often occurs in breast cancer and is associated with poor prognosis in invasive breast carcinoma (36). Clinical trials showed that polysomy of chromosome 17 might be associated with trastuzumab response in HER2 FISH-negative but HER2 IHC-positive breast cancer (37, 38). These studies indicate that other genes in chromosome 17 might be involved in trastuzumab response in HER2-positive breast cancer. Through bioinformatic analysis of chromosome 17q, WBP2 was found to be coamplified with HER2 in human breast tumors. We previously reported that WBP2 is an oncogene that promotes cell growth, proliferation, and invasion via EGFR/Wnt pathway crosstalk in breast cancer (17, 20). WBP2 is phosphorylated by c-Src and c-Yes through EGF/EGFR signaling and phosphorylated/activated WBP2 translocates to the nucleus, acting as a transcriptional coactivator to promote growth of breast cancer cells. WBP2 is overexpressed in breast cancer and its expression is correlated with poor OS/DFS. In this study, we further discovered that patients with tumors that coexpress WBP2 and HER2 have the worst prognosis compared with either alone. EGF-induced tyrosine phosphorylation of WBP2 was also found to be dependent on HER2 in this study. Taking these observations into account, we tested the hypothesis that WBP2 expression regulates the response of breast cancer to trastuzumab-based treatment.

Trastuzumab is the first-line therapy for patients with HER2-positive breast cancer. However, not all of these patients respond to treatments that involved trastuzumab (9). It is conceivable that breast cancer's response to trastuzumab does not solely depend on HER2 expression, but also on other biomarkers that are molecularly networked with HER2. Several studies have reported potential biomarkers that are associated with positive or negative response to trastuzumab (39, 40). For example, loss of PTEN and PIK3CA mutations has been observed in trastuzumab-resistant breast cancers and both are linked to poor prognosis and lower overall survival (39). Patients with HER2-positive breast cancer with PIK3CA mutation showed lower pCR (28.6%) to HER2-targeting neoadjuvant therapies, as compared with wild-type PIK3CA (53.1%; ref. 40). In this study, we provide evidence that WBP2 is a potential predictor of trastuzumab response in HER2-positive breast cancer.

Trastuzumab plus chemotherapy in the neoadjuvant setting is given to HER2-positive breast cancer with locally advanced or inflammatory breast cancer. This treatment strategy has shown significantly improved pathologic response compared with chemotherapy alone (41, 42). Pathologic complete response (pCR) is correlated with better prognosis and shows clinical responsiveness to systemic therapy. The neoadjuvant treatment approach is becoming diverse and the choice of treatment is best personalized, based on the individual's molecular portrait. Identifying novel biomarkers beyond HER2 status that could improve pCR would be helpful for patients and clinicians in choosing a treatment strategy with better clinical outcome. In this study, we observed that patients with HER2-positive breast cancer and high WBP2 expression showed higher pCR (67.19%) to trastuzumab-based neoadjuvant chemotherapy, as compared with the overall HER2-positive breast cancer cases (44.76%). Subgroup analysis showed that pCR was higher in the group of patients aged below 50 years and whose tumors possessed high WBP2 level (77.78%) compared with those above 50 years old. Similarly premenopausal patients with tumors that expressed elevated WBP2 level showed higher pCR (73.33%) than the nonmenopausal group. The sensitivity and specificity of WBP2 in discriminating between pCR and non-pCR were also better in patient groups with age below 50 years (80.77% and 80.00%) and of premenopausal status (81.48% and 76.47%) compared with the overall group (67.19% and 73.42%). Because the average age of menopause is 49–52 years old (43), there would be significant overlap of patients between the two groups. Given that menopausal status is sometimes not well documented in the medical records while age is a more objective parameter, we recommend that age is a more useful clinical factor in conjunction with the molecular determinants WBP2 and HER2 for stratification of patients for precision medicine using trastuzumab. The ability to identify patients who are likely to respond well to neoadjuvant trastuzumab allows clinicians to better plan therapeutic interventions for patients. Patients can also be better counselled regarding what to expect from the neoadjuvant therapy. It would also be possible to predict which patients would attain successful downstaging of their tumors from neoadjuvant therapy, thus allowing for breast conserving surgery, instead of mastectomy in some instances. This will have significant impact on the patients' cosmetic and psychologic outcome.

We showed that WBP2 expression correlated with response to trastuzumab-based neoadjuvant therapy. However, it should be noted that the retrospective study performed in this study involved patients who had received neoadjuvant therapy comprising trastuzumab in combination with chemotherapy regimens that varied according to the physician in attendance and hospital practices. This, therefore, confounds the data generated and possibly the conclusions drawn. Recognizing this weakness, we performed an analysis to investigate whether WBP2 levels correlates with response to chemotherapy regimens. The chemotherapy regimens given can be classified as (i) anthracycline-based regimens, such as adriamycin (doxorubicin) and (ii) nonanthracycline based. As shown in Supplementary Table S1, there was no significant difference in the pCR rate between these groups of patients treated with anthracycline and nonanthracycline regimens (45.36% and 44.44%, respectively) compared with the overall rate of 44.76% in the nonstratified population. Furthermore, we stratified tumors with high WBP2 level into those treated with anthracycline- and nonanthracycline-based regimens. The pCR of patients with high WBP2–expressing tumors treated with anthracycline-based and nonanthracycline-based regimens was 69.23% and 64.00%, respectively (Supplementary Fig. S2A), both of which were similar to the pCR of the group of samples stratified based on high WBP2 alone (67.19%; Supplementary Fig. S2A; Supplementary Table S2).

These results above indicate that the chemotherapy regimens do not influence pCR of patients to trastuzumab-based neoadjuvant therapy. It also supports the notion that WBP2 does not correlate with response to chemotherapy and strengthens the link between WBP2 and trastuzumab-targeted therapy. This is further corroborated by preliminary in vitro data, which argues that WBP2 does not regulate breast cancer response to chemotherapy because overexpression or knockdown of WBP2 did not affect cellular viability upon doxorubicin treatment (Supplementary Fig. S2B–S2D). Nevertheless, it would be necessary to confirm and establish the value of WBP2 in predicting response to trastuzumab-based chemotherapy via a more tightly controlled prospective study. Taken together, the molecular basis for the role of WBP2 as a companion diagnostics for trastuzumab precision medicine is putatively through its interaction with HER2 in breast cancer cells.

Neoadjuvant therapy for HER2-positive breast cancer has improved significantly over time with clinical trials supporting the combinatorial use of different anti-HER2 inhibitors, including lapatinib (tykerb) and pertuzumab (44, 45). Because our data show that WBP2 regulates HER2 and its downstream signaling, it is conceivable that WBP2 also enhances response of HER-positive breast cancer to other HER2-targeting agents such as pertuzumab and lapatinib. Further studies will be needed to test these hypotheses.

In conclusion, there is a clinical need to improve the response rate of patients with HER2-positive breast cancer to HER2-targeted therapeutics especially in the neoadjuvant setting. Our study posits that trastuzumab is more effective in HER2-positive breast cancers that express high levels of WBP2. WBP2 is a potential companion diagnostic for further stratification of patients with HER2-positive breast cancer for more effective anti-HER2 therapies.

J. Arribas is a consultant/advisory board member for Menarini. S. Lee reports receiving speakers bureau honoraria from and is a consultant/advisory board member for Roche. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S.-A. Kang, Y.P. Lim

Development of methodology: S.-A. Kang, J.S. Guan, H.J. Tan, T. Chu, J. Arribas, T.C. Putti, Y.P. Lim

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.-A. Kang, J.S. Guan, H.J. Tan, T. Chu, C. Bernadó, J. Arribas, C.Y. Wong, P.H. Tan, M. Gudi, T.C. Putti, J. Sohn, S.H. Lim, S.-C. Lee

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.-A. Kang, J.S. Guan, H.J. Tan, T. Chu, T.C. Putti, J. Sohn, S.-C. Lee, Y.P. Lim

Writing, review, and/or revision of the manuscript: S.-A. Kang, H.J. Tan, T. Chu, J. Arribas, M. Gudi, T.C. Putti, J. Sohn, S.H. Lim, S.-C. Lee, Y.P. Lim

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.-A. Kang, J.S. Guan, T. Chu, A.A. Thike, C. Bernadó, Y.P. Lim

Study supervision: Y.P. Lim

The study is funded by Exploit Technologies, Agency for Science, Technology and Research (F09/X/037_c) and the National Medical Research Council, Ministry of Health (NMRC/OFIRG/0034/2017), Singapore. J Arribas is supported by grants of the Breast Cancer Research Foundation (BCRF-17-008) and Instituto de Salud Carlos III (PI16/00253) for establishing and maintaining the resistant PDXs.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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