Elevated WBP2 Expression in HER2-Positive Breast Cancers

Published OnlineFirst December 28, 2018; DOI: 10.1158/1078-0432.CCR-18-3228

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Elevated WBP2 Expression in HER2-positive Breast Cancers Correlates with Sensitivity to Trastuzumab-based Neoadjuvant Therapy: A Retrospective and Multicentric Study Shin-Ae Kang1, Jye Swei Guan1, Hock Jin Tan1, Tinghine Chu1,2, Aye Aye Thike3, Cristina Bernado4, Joaquín Arribas4, Chow Yin Wong5, Puay Hoon Tan3, Mihir Gudi6, Thomas Choudary Putti7, Joohyuk Sohn8, Swee Ho Lim9, Soo Chin Lee10, and Yoon Pin Lim1,2

Abstract

Purpose: Trastuzumab-based chemotherapy has shown whether WBP2 expression correlates with pathologic complete remarkable clinical benefits for patients with HER2-positive response (pCR). breast cancer. However, treatment regimens involving Results: Elevated expression of WBP2 significantly trastuzumab had little or no effect for a subset of patients. enhanced breast cancer's response to trastuzumab by aug- Preliminary studies revealed WW-binding protein 2 menting trastuzumab-induced HER2 downregulation and (WBP2), an oncogenic transcription coactivator, to be cell-cycle arrest via inhibition of cyclin D expression. High coamplified with HER2 in 36% of HER2-positive breast level of WBP2 correlated with better pCR (67.19%) compared cancers. We hypothesize that WBP2 regulates and correlates with low WBP2 level (26.58%). The highest response was with the response of HER2-positive breast cancer to observed in subgroups of patients with high WBP2–expressing trastuzumab. tumors also aged below 50 years (77.78%) or were premen- Experimental Design: The coexpression of WBP2 and opausal in status (73.33%). Retrospectively, WBP2 demon- HER2 in breast tumors was validated using IHC. The role and strated sensitivity of 80% to 81% and specificity of 76.5% to mechanism of WBP2 in regulating breast cancer response to 80% in discriminating between patients showing pCR and trastuzumab was elucidated using in vitro, patient-derived non-pCR. xenograft and murine xenograft models. A multicenter retro- Conclusions: WBP2 expression correlates with the response spective study involving 143 patients given neoadjuvant tras- of HER2-positive breast cancer to trastuzumab-based neoad- tuzumab-based chemotherapy was conducted to determine juvant chemotherapy.

Introduction 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 2NUS Graduate School for Integrative Breast cancer is the second leading cause of cancer-related Sciences and Engineering, National University of Singapore, Singapore. 3Division deaths in women, especially among women aged 40–59 years (1). of Pathology, Singapore General Hospital, Singapore. 4Preclinical Research Breast cancer can be molecularly subclassified into four groups Program, Vall d'Hebron Institute of Oncology (VHIO) and CIBERONC, Barcelona, depending on the estrogen (ER)/progesterone (PR) and human 5 Spain. Department of General Surgery, Singapore General Hospital, Singapore. epidermal growth factor receptor 2 (HER2) status: luminal A (ER 6Department of Pathology and Laboratory Medicine, KK Women's and Chil- and/or PR positive with low Ki67), luminal B (ER and/or PR dren's Hospital, Singapore. 7Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 8Department of Medical positive and either HER2 positive or HER2 negative with high Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Ki67), HER2 (HER2 positive), and basal-like (usually but not Korea. 9KK Breast Department, KK Women's and Children's Hospital, Singapore. always ER/PR/HER2 negative/triple negative; ref. 2). HER2 (also 10Department of Haematology-Oncology, National University Cancer Institute, known as ErbB2/neu) is amplified and overexpressed in 15%– Singapore. 20% of breast cancer. Patients with HER2-positive tumors have Note: Supplementary data for this article are available at Clinical Cancer poorer prognosis and significantly lower overall survival com- Research Online (http://clincancerres.aacrjournals.org/). pared with patients with HER2-negative tumors (3, 4). Corresponding Author: Yoon Pin Lim, Yong Loo Li School of Medicine, National Trastuzumab humanized mAb is the first HER2-targeted ther- University of Singapore, MD4, Level 1, 5 Science Drive 2, Singapore 117545, apy that was approved by the United States Food and Drug Singapore. Phone: 656-601-2040; Fax: 656-779-1453; E-mail: Administration (US FDA) for metastatic HER2-overexpressing [email protected] breast cancer as the first-line therapy often in combination with doi: 10.1158/1078-0432.CCR-18-3228 chemotherapy (5–7). HER2 or EGFR2 is one of the 4 members of 2018 American Association for Cancer Research. the EGF receptor (EGFR) family of tyrosine kinases. It is an orphan

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WBP2 Level Correlates with Trastuzumab Neoadjuvant Therapy

Two observations led to the hypothesis that WBP2 is a molec- Translational Relevance ular determinant/predictor for anti-HER2–based therapies: first, Not all patients with HER2-positive breast cancer respond WBP2 is within the EGFR signaling network in which HER2 plays well to trastuzumab-based chemotherapy. Preclinical clinical an intimate role; second, WBP2 is a prognostic factor and potent studies involving in vitro, patient-derived xenografts, and ani- oncogene that regulates cancer cell growth. In this study, we mal models revealed that WBP2 augmented the inhibitory established WBP2 as a downstream substrate of EGFR/HER2 effects of trastuzumab in HER2-positive breast cancer cells by signaling and investigated the role of WBP2 in regulating the enhancing trsatuzumab's effect on cell-cycle arrest. Patients response of breast cancer cells to trastuzumab and the mechanism with tumor expressing high WBP2 level had higher pathologic through which this occurs in preclinical studies. Finally, we complete response (pCR) of 67.19% compared with those conducted a retrospective study to determine the correlation of with low WBP2 (26.58%). The pCR was even higher in sub- WBP2 levels to response of HER2-positive breast tumors to groups of patients whose tumors had high WBP2 and were neoadjuvant trastuzumab-based chemotherapy. 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 Materials and Methods discriminating between patients showing pCR and non-pCR. Reagents WBP2 is a potential companion diagnostics for the manage- In-house WBP2 polyclonal antibody was generated as ment of HER2-positve breast cancer with trastuzumab-based described previously (17). Monoclonal anti-WBP2 (MABS441- therapies. 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-b-tubulin (MA5- receptor and can either dimerize with itself (especially when 16308-1MG) antibodies were purchased from Thermo Scientific. overexpressed) or with other members such as EGFR (HER1) to Anti-phospho-EGFR (2231), anti-EGFR (2232), anti-phospho- initiate downstream signaling events. Trastuzumab inhibits AKT (9271), and anti-AKT (9272) antibodies were purchased HER2-positive breast cancer cells by binding to the extracellular from Cell Signaling Technology. Anti-cyclin D1 antibody (sc- domain of HER2. The exact mechanism is unknown although 20044) was purchased from Santa Cruz Biotechnology. Trastu- trastuzumab-induced receptor endocytosis among others has zumab (Herceptin) was obtained from Roche. HER2 siRNAs, been implicated (8). Historically, when trastuzumab was used WBP2 siRNAs, and luciferase siRNA were purchased from Thermo alone in nonselected patients with metastatic breast cancer, only Scientific. WBP2 overexpression plasmid and knock down about 10%–15% of patients had a partial response (7). The (shRNA) constructs were described previously (17, 20). objective response rate improved to 26% when patients with HER2-overexpressing metastatic disease receiving first-line treat- Cell lines and culture conditions ment were selected (9). In the current setting, the response rate of The human breast cancer cell lines, BT-474 and SK-BR-3, were patients to neoadjuvant trastuzumab in combination with che- purchased from ATCC. ZR-75–30 was a kind gift from Dr. Boon motherapy ranges between 30% and 53% (10, 11). These results Tin Chua (Institute of Molecular and Cell Biology, Singapore). All reveal an important gap in clinical oncology–HER2 is the only cell lines were tested using MycoAlert PLUS Mycoplasma Detec- approved biomarker for anti-HER2–based therapy and the tion Kit (Lonza) and verified to be free of Mycoplasma and used administration of anti-HER2 drugs based on HER2 status alone after 2–5 passages from thawing. BT-474, SK-BR-3, and ZR-75-30 is insufficient in achieving a good response rate. Better and/or were maintained in RPMI1640 containing 10% FBS (Thermo complementary predictors are required. Fisher Scientific) and 100U penicillin/streptomycin (Invitrogen). WW-domain binding protein 2 (WBP2) was first identified to For transient expression, cells were transfected with HER2 siRNA be a cognate ligand of the WW domain of Yes kinase-associated or WBP2 siRNA using jetPRIME transfection reagent (Polyplus protein (YAP; refs. 12, 13). It was subsequently shown to be an Transfection), according to the manufacturer's recommendations. adapter protein for Pax8 thyroid-specific transcription factor (14) For stable expression, cells were transduced with each expression and a transcription coactivator in ER/PR signaling (15). The lentivirus, followed by selection with 400mg/mL hygromycin B function of WBP2 in cancer was not known until its discovery (Invitrogen) for BT-474 or 0.5 mg/mL puromycin (Invitrogen) for as a novel breast cancer–associated protein that is posttransla- SK-BR-3. After 2–3 weeks, selected colonies were pooled and tionally modified by EGFR through tyrosine phosphoryla- expanded. tion (16). Eventually, WBP2 was demonstrated to be an oncogene whose overexpression transformed normal mammary epithelial Cell proliferation assay cells and conferred aggressive traits to cancer cells (17). At the Cells were plated in 96-well plates for 2D culture or 96-well same time, WBP2 was shown to be required for the oncogenic and ultra-low attachment plates for 3D culture (Corning) at 10,000 growth-promoting function of TAZ (18) and YAP (19). Recently, cells per well. After 3 or 5 days of incubation with trastuzumab, the it was found that Wnt signaling promotes breast cancer by viability of cells was measured using CellTiter 96 Aqueous Non- blocking ITCH E3 ligase-mediated degradation of WBP2 (20). In Radioactive Cell Proliferation Assay (Promega). the same study, analysis of >400 clinical specimens revealed that WBP2 is overexpressed in breast tumors and high WBP2 expres- Cell-cycle analysis sion level correlated with disease aggression and poor patient Cells were collected and fixed in 70% ethanol at 4 C overnight. survival (20). These studies highlighted the important role of After washing with PBS, the cells were stained with PI/RNase WBP2 to breast cancer. staining buffer (PBS containing 0.1% triton X-100, 0.2 mg/mL

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RNase A, 20 mg/mL PI) for 15 minutes at room temperature. The Human specimens DNA content of cells was measured by ﬂow cytometry (LSR The study was conducted in accordance with the Declaration of Fortessa Flow Cytometry Analyser, BD Biosciences). Proportions Helsinki and written informed consent was obtained from all of cells in G1, S, and G2–M phases were analyzed using FACSDiva participants. All specimens obtained after protocol approval by Software (BD Biosciences). Institutional Review Boards.

Subcellular fractionation Retrospective study cohort 1: correlation of WBP2 and HER2 to Membrane and cytoplasmic extracts were prepared using the patients' outcomes. Breast tumor tissues from patients who Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scien- received surgery between 1997 and 2007 as well as the clinical/ tific), as per manufacturer's instructions. histopathologic information (including HER2 IHC scores) were obtained from National Cancer Centre, Singapore (NCC; n ¼ 52), Western blot analyses and immunoprecipitation assays National University Hospital, Singapore (NUH; n ¼ 205), and Cell lysis, Western blot analysis, and immunoprecipitation Singapore General Hospital (SGH; n ¼ 126). The information for assay were performed as described previously (17, 20). The detail some cases is not available. Therefore, the total number of cases methods can be found in Supplementary Information. analyzed may not be the same as the original number available. Patients received the treatment and surgery in accordance with Reverse transcription and real-time PCR each institution's standard treatment, but the treatment informa- Total RNA was isolated using PureLink RNA Mini Kit tion was not available for this cohort. The samples in this cohort (Thermo Fisher Scientific) and reverse transcribed using ran- were randomized and include HER2-positive and -negative breast dom hexamer primers and RevertAid First Strand cDNA Syn- cancer cases. thesis Kit (Thermo Fisher Scientific). qPCR reactions were performed using QuantiFast Probe PCR Kit (Qiagen) as per Retrospective study cohort 2: correlation of WBP2 with pCR to manufacturer's instructions. Predesigned primers/probes were trastuzumab-based neoadjuvant therapy. The cohort here includes obtained from IDT. patients with stage I–III HER2-positive breast cancer who received neoadjuvant trastuzumab-based chemotherapy between 2005 Bioinformatics analysis and 2017 and from whom archival tumor specimens and clinical Copy number variation (CNV) data and RNA-sequencing information were available. Patients received anthracycline or (RNA-Seq) data from TCGA-BRCA (21) was downloaded from nonanthracycline-containing regimens in combination with tras- the GDC Data Portal (22). The data were cleaned to remove tuzumab as part of each institution's standard treatment. Neoad- samples with incomplete data. The HER2 expression status were juvant trastuzumab with anthracycline-containing chemotherapy assessed from the RNA-Seq data. The differences in CNV profile regimens included AC-TH regimen (doxorubicin/cyclophospha- between primary tumor and solid tissue normal were then com- mide followed by paclitaxel or docetaxel/trastuzumab) and FEC- puted as described in Supplementary Information. Gene list TH regimen (fluorouracil/epirubicin/cyclophosphamide fol- analysis was performed using PANTHER (23). A custom Python lowed by paclitaxel or docetaxel/trastuzumab). Neoadjuvant script was developed to query PubMed (Supplementary Meth- trastuzumab with nonanthracycline-containing chemotherapy ods). The word cloud was then generated using the Text Analytics regimens included TCH regimen (paclitaxel or docetaxel/carbo- Toolbox of MATLAB R2018b (MathWorks, 2018). platin combined with trastuzumab) and TH regimen (paclitaxel or docetaxel combined with trastuzumab). Specimens comprised Xenograft model FFPE biopsies that were collected before neoadjuvant treatment. A All animal experiments were performed in accordance with the total of 143 cases complete with clinical information such as institutional guidelines of and were approved by Institutional pathologic response information were obtained from NUH, Animal Care and Use Committee of National University of Kandang Kerbau Women's and Children's Hospital (KKH), and Singapore (Singapore). For the xenograft model, 5-week-old Yonsei Cancer Center (YCC). female athymic nude mice (n ¼ 6-7, In Vivos) were implanted with 0.72-mg 60-day release 17b-estradiol pellets (Innovative IHC analysis Research) and after 2 days, BT-474 control (vector) or WBP2- The detailed protocol for IHC was previously described (17, overexpressing cells (1 107 in 200 mL of DPBS and Matrigel 1:1 20). IHC of pretreatment biopsy specimens was done in the NUH mixture) were injected subcutaneously into a mouse mammary pathology department core using BenchMark ULTRA IHC Auto- fat pad. When the tumors reached the size of 150–200 mm3, the stainer (Roche Ventana). All IHC slides were scored in a blinded mice were divided into groups, keeping average tumor size similar fashion by pathologists. A scale of 0–3 was used for the stain between groups, and treated with trastuzumab (Herceptin; 10 intensity (0, no staining; 1þ, mild; 2þ, moderate; and 3þ, strong mg/kg, Roche) or PBS (control) by intraperitoneal (IP) route twice staining. weekly for three weeks. The tumor size was measured twice weekly with calipers and tumor volumes were calculated as follow: Statistical analysis volume ¼ (width2 length)/2. All in vitro experiments were repeated at least three times and the results were presented as mean SD. The comparisons Patient-derived xenografts between each group were determined by Student t test. P values Trastuzumab-sensitive breast cancer PDX (PDX118) and of <0.05 were considered statistically significant and expressed trastuzumab-resistant breast cancer PDXs (PDX118TRs) as , P < 0.05; , P < 0.01; , P < 0.001. For in vivo and were established by Arribas and colleagues as described retrospective study, the data represent mean SEM and the previously (24). significance of differences or association was evaluated using

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WBP2 Level Correlates with Trastuzumab Neoadjuvant Therapy

Mann–Whitney U test. Kaplan–Meier method was used to plot the recently implicated in breast cancer development (17, 20) and a overall survival (OS) and disease-free survival (DFS) curves. DFS phosphotyrosine substrate of EGFR signaling pathway (16). Our and OS were defined as time from date-of-diagnosis to date-of- data revealed that WBP2 and HER2 are coamplified in 36% of the first-recurrence and death or last follow-up date, respectively. HER2-positive breast cancers. It is conceivable that tyrosine phos- Survival between groups was compared using log-rank test. The phorylation of WBP2 by EGFR is HER2-dependent and aberrant associations between pCR and WBP2 expression or/with clinical WBP2 expression may be a novel mechanism that regulates cancer characteristics were investigated by contingency tables and ana- cellular response to anti-HER2 drugs. lyzed by Fisher exact test. All statistical analyses were performed using SPSS and GraphPad Prism. 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 Results coamplified in C17q, we hypothesized that WBP2 coexpress with HER2 amplification–associated gene network in breast cancer HER2 in clinical breast cancers at the protein level and in com- The upregulation of HER2 in breast cancer is usually associated bination they give rise to poorer prognosis. To test these hypoth- with an amplification of the HER2 locus (25). Inadvertently, other eses, a retrospective study was performed. IHC of HER2 and WBP2 genes along chromosome 17q (C17q) arm are coamplified with was performed on 296 resected breast tumor tissues. Consistent HER2 (26). It is conceivable that HER2 and its associated genes with previous studies, patients with HER2-positive tumors had along C17q not only form a molecular network but also coop- worse overall survival (P ¼ 0.011) and disease-free survival (P ¼ eratively and functionally contribute to the phenotype of HER2- 0.137) than the HER2-negative group (Fig. 1E; refs. 4, 27). Next, positive breast cancer. The HER2-associated genes may also reg- we analyzed the correlation of WBP2 with HER2 status. Both ulate the response of HER2-positive breast cancer to drugs and are nuclear and cytoplasmic WBP2 expression was significantly therefore potential companion diagnostics for HER2-based ther- higher in HER2-positive breast tumors compared with HER2- apeutics. Hence, we attempted to map the genes that are coam- negative tumors (Fig. 1F). Because WBP2 expression and HER2 plified with HER2, performed preclinical tests to investigate their status were positively correlated, we proceeded to examine wheth- interactions with HER2 in the presence of trastuzumab, and er patients' outcome is associated with WBP2 and HER2 in validate whether the expression of these candidate biomarkers combination. We found that patients whose tumors showed high correlate with response of breast tumors to trastuzumab-based nuclear WBP2 and HER2-positive expression had the worst over- treatment via a retrospective study. all (HR: 4.49; P < 0.001) and disease-free survival (HR: 2.58, P ¼ First, we obtained the copy number variation (CNV) and RNA- 0.004) than other groups (Fig. 1G). WBP2 expression in combi- Seq data from the TCGA-BRCA project (21), and employed the nation with HER2 appears to be more powerful than either alone workflow shown in Fig. 1A to identify regions of chromosome 17 for breast cancer prognosis. It suggests that WBP2 can be used to that were amplified in HER2 upregulated breast cancer. RNA-Seq further prognosticate patients with HER2-positive breast cancer. data was used to identify cases with HER2 upregulated, while CNV data were used to identify regions of chromosome 17 that were HER2 is required for EGF-induced WBP2 tyrosine amplified or deleted. As shown in Fig. 1B, the p-arm of chromo- phosphorylation some 17 experienced a significant deletion, while 3 regions along The association of WBP2 with HER2 in clinical specimens, q-arm of chromosome 17 were significantly amplified in HER2- coupled to the previous finding that WBP2 is a downstream target overexpressing breast cancer. A total of 1,145 genes were identi- of EGF signaling, led to the postulation that tyrosine phosphor- fied to reside in these 3 significantly amplified regions. Gene list ylation of WBP2 by EGFR requires HER2, because EGFR and HER2 analysis using the PANTHER Classification System (23) was are known to heterodimerize in the presence of EGF (16). To test performed to provide a general understanding of the genes this hypothesis, HER2 expression was knocked down using two þ þ coamplified with HER2. Interestingly, cellular processes (GO: different siRNAs in HER2 /WBP2 breast cancer cell lines. Knock- 0009987) and metabolic processes (GO: 0008152) accounted down of HER2 abolished EGF-induced tyrosine phosphorylation for about half of the hits (Fig. 1C). Amplification of genes of WBP2 in SK-BR-3 and ZR-751 breast cancer cells (Fig. 1H and I). involved in these critical processes is likely to result in dysregula- This indicates that HER2 is required for tyrosine phosphorylation tion and cancer phenotype. and activation of WBP2 by EGF. To shortlist candidates that play role in regulating HER2 in breast cancer, we queried PubMed and found that 862 of the 1145 WBP2 expression enhances trastuzumab response in breast amplified genes are not implicated in breast cancer. We further cancer cells reasoned that candidates that influence the response of HER2- Given that WBP2 is downstream of HER2, aberrant expression positive breast cancer to anti-HER2 drugs are likely to be proteins of oncogenic WBP2 (20) may influence cancer cellular response to that are associated with EGFR and/or HER2 signaling. From the trastuzumab. To investigate the effect of WBP2 expression on the remaining 283 genes, only 101 genes are related to either EGFR response of HER2-positive breast cancer cells to trastuzumab as a signaling or HER2 signaling. Out of the 101 genes associated with single agent, SK-BR-3, ZR-75-30, and BT-474 breast cancer cell EGFR/HER2 and breast cancer, 55 of them have 2 or more lines were selected because they are HER2-positive and trastuzu- publications and are represented in a word cloud. The size of the mab sensitive as shown by our preliminary study and other fl words re ects the number of publications (in a Log2 scale) that reports (28). While trastuzumab is rarely used as a single agent relate the gene to EGFR or HER2 in breast cancer (Fig. 1D). As in the clinic, a clean/simplistic system involving only trastuzumab expected, HER2 (ERBB2) was the top hit, with 9,947 publications was needed to prove a direct link and to study the interactions (as of September 2018). Found in this HER2 network of genes is between WBP2, HER2, and trastuzumab. As would be evident WW-domain Binding Protein 2 (WBP2), an emerging oncogene later, this "reductionist" approach in the laboratory would be

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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 HER2þ and HER2 groups (n ¼ 221). F, Boxplot of WBP2 expression in HER2þ and HER2 breast tumors (n ¼ 296). G, Kaplan–Meier analysis of OS and DFS of patients segregated based on WBP2 and HER2 expression status (n ¼ 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. b-Tubulin was used as a protein-loading control.

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complemented with an actual and more complex clinical setting mab-mediated G1 arrest by analyzing cyclin D expression. Tras- in which the correlation of WBP2 expression with breast tumor tuzumab decreased cyclin D protein level by 87.6% (P < 0.001) in response to trastuzumab with chemotherapy treatment would be ZR-75-30 cells as compared with untreated cells (Luc si). This tested. effect was only 44.6% to 56.2% when WBP2 was silenced with two WBP2 expression was silenced using two different shRNAs in separate siRNAs (Fig. 2M). Similarly, the decrease in cyclin D SK-BR-3 and two different siRNAs in ZR-75-30, while the over- mRNA level by trastuzumab was abolished when WBP2 was expression of WBP2 was achieved using a lentiviral system in knocked down (Fig. 2N). Collectively, these results suggest that BT-474. Each cell line was subsequently treated with increasing WBP2 promotes trastuzumab-induced G1 arrest via downregula- doses of trastuzumab (1, 10, 100 mg/mL) and the cell growth tion of cyclin D. 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- WBP2 overexpression sensitizes breast tumor to trastuzumab overexpressing BT-474 cells, the response to trastuzumab (100 mg/ in vivo mL) increased by 2.1-fold for 2D culture (P ¼ 0.001) and 2.2-fold Our in vitro observations indicated that cancer cellular for 3D culture (P ¼ 0.003) compared with vector control response to trastuzumab depended on WBP2 level. To confirm (Fig. 2A and B). On the other hand, knockdown of WBP2 observation, an in vivo tumor xenograft model was used. BT-474 significantly reduced the response to trastuzumab in SK-BR-3 (by cells stably expressing WBP2 were injected into the mammary fat 37 %; Fig. 2C and D) and ZR-75-30 cells (by 45%; Fig. 2E pad of athymic nude mice. When the size of tumors reached 150– and F) compared with the controls. These data provide evidence 200 mm3, the mice were divided into two groups and treated with that WBP2 expression level influences the response of breast trastuzumab (10 mg/kg) or PBS by intraperitoneal injection twice cancer cells to trastuzumab. weekly for three weeks. The size of WBP2-overexpressing tumor after trastuzumab treatment was reduced to 5.23% as compared WBP2 enhances the modulation of EGFR/HER2 signaling in the with no treatment (100%), whereas the size of tumor with vector presence of trastuzumab control was reduced only to 38.33% (Fig. 3A and B). As the The mechanism of trastuzumab as an antitumor agent remains difference between the two trastuzmab-treated conditions an area of active research. Some groups have shown that trastu- appeared small due to the scale used in the plot shown zumab induced downregulation of HER2 level (29–31). To in Fig. 3A and B, separate plots are provided in Supplementary determine whether WBP2 affects trastuzumab-induced downre- Fig. S1 to show their differences more clearly. Hence, overexpres- gulation of HER2 expression, WBP2-overexpressing BT-474 cells sion of WBP2 increased tumor response to trastuzumab by 7.32 and WBP2 knocked down SK-BR-3 or ZR-75-30 cells were treated times (P ¼ 0.0047). The results support the notion that high with increasing doses of trastuzumab (1, 10, 100 mg/mL) and WBP2 level sensitized breast cancer to trastuzumab. HER2 protein expression analyzed using Western blot analysis. In WBP2-overexpressing BT-474 cells, both HER2 and EGFR levels WBP2 expression correlates with trastuzumab response in PDX were substantially decreased when exposed to a low dose of model trastuzumab, compared with control cells (Fig. 2G). Consistently, To obtain more evidence that high WBP2 expression is asso- knockdown of WBP2 inhibited trastuzumab-dependent down- ciated with positive response to trastuzumab treatment, we exam- regulation of HER2 and EGFR levels in SK-BR-3 and ZR-75-30 ined WBP2 and HER2 expression in a PDX model of drug breast cancer cells. In contrast, HER2 levels were decreased by resistance to trastuzumab. Tumor samples from trastuzumab- trastuzumab in a concentration-dependent manner in control sensitive breast cancer PDX (PDX118) and trastuzumab-resistant cells (scrambled shRNA or Luc siRNA; Fig. 2H and I). Like HER2 breast cancer PDXs (PDX118TRs) established by Arribas and and EGFR, inactivation of AKT by trastuzumab, which has been colleagues were used (24). The IHC staining shows that trastu- reported by previous studies (32–34), was enhanced by WBP2 zumab-sensitive sample (PDX118) was HER2-positive and overexpression and diminished by WBP2 knockdown. Figure 2J WBP2-positive. Interestingly, 3 (TR2-4) of 5 tumors that acquired and K demonstrated that it was the cell-surface species of HER2 trastuzumab resistance had lower HER2 and WBP2 expression that was affected by WBP2 in the presence of trastuzumab. (Fig. 3C). Pearson correlation test revealed that HER2 and WBP2 Collectively, our data suggest that WBP2 enhances the inhibitory expression had a correlation coefficient (Pearson r) of 0.8944 (P ¼ effect of trastuzumab on cancer cell proliferation by augmenting 0.0161), indicating strong association between HER2 and WBP2 trastuzumab-induced downregulation of HER2/EGFR signaling. expression (Fig. 3D). These data suggest that WBP2 in combination with HER2 expression is a determinant of response to WBP2 promotes trastuzumab-induced G1 arrest by enhancing trastuzumab. downregulation of cyclin D1 expression Previous studies showed that trastuzumab treatment induced High WBP2 expression is associated with pCR of HER2-positive G1 cell-cycle arrest, leading to inhibition of cell proliferation (35). breast cancer patients to neoadjuvant trastuzumab-based To investigate the involvement of WBP2 in trastuzumab-induced treatment G1 arrest, we performed cell-cycle FACS analysis of WBP2 knocked To investigate whether WBP2 expression correlates with the down versus control ZR-75-30 cells. Trastuzumab increased the response of HER2-positive breast cancer to trastuzumab in the number of cells at G0–G1 phase by 14.5% compared with control clinical setting where chemotherapies are used in conjunction, a (no trastuzumab treatment; P ¼ 0.0003) while trastuzumab retrospective study was conducted. A total of 143 cases of pre- decreased the number of cells at S and G2–M phase by 52.4% treatment biopsy specimens from patients with breast cancer who and 37.0%, respectively. Knockdown of WBP2 using two different received trastuzumab-based neoadjuvant chemotherapy were WBP2 siRNAs abolished the effects of trastuzumab on cell cycle collected from three different cohorts; NUH, KKH, and YCC. The (Fig. 2L). We further examined WBP2's involvement in trastuzu- characteristics of the patients before treatment are provided

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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-specificshRNAinSK-BR-3(C and D) or two different WBP2-specificsiRNAsinZR-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 ¼ 3). Statistical significance was determined by Student t test ( or þP < 0.05; or þþP < 0.01; or þþþP < 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 mg/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)weretreatedwith different concentrations of trastuzumab (0, 1, 10, 100 mg/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 mg/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)andmRNAlevel(N) were analyzed. Protein expression level of cyclin D1 was quantitated and normalized to b-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 ¼ 3). Statistical significance was determined by Student t test (, P < 0.05; , P < 0.01; , P < 0.001 vs. control).

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Figure 3. Effect of WBP2 on tumor response to trastuzumab in vivo. Five-week-old female athymic nude mice (n ¼ 6–7) were implanted with 0.72-mg 60-day release 17b-estradiol pellets. After 2 days, BT-474 control (vector) or WBP2-overexpressing cells (1 107 of cells in 200 mL 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 ¼ (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 mm. 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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Table 1. Patients characteristics curve (AUC) was 0.72 with the sensitivity and specificity at Characteristics Overall population N (%) 143 67.19% and 73.42%, respectively, when a WBP2 IHC cut-off Pathologic response pCR 64 (44.8) score of 2.5 was used (Fig. 4E, black line; Table 2). Both the – Age Median age [range] (years) 53 [25 76] sensitivity and specificity were higher in patients aged below 50 <50 56 (39.2) 50 86 (60.1) years (80.77% and 80.00%) and those in the premenopausal NA 1 (0.7) group (81.48% and 76.47%; Table 2). The AUC for the premen- Menopausal status Premenopause 61 (42.7) opausal patients (0.80) and patients aged below 50 years (0.81) Postmenopause 71 (49.7) were higher compared with overall group (0.72; Fig. 4E, red line Perimenopause 10 (7.0) and blue line, respectively). No significant correlation was NA 1 (0.7) observed for other clinical factors including ER/PR status, tumor ER status Negative 77 (53.8) Positive 63 (44.1) grade, TNM stage, chemotherapy regimen (data not shown). Null (NA) 3 (2.1) Collectively, the retrospective study indicates that high WBP2 PR status Negative 83 (58.0) expression in pretreatment biopsy is positively correlated with Positive 57 (39.9) pCR, especially for those aged below 50 years group and in the Null (NA) 3 (2.1) premenopausal group. Tumor grade 1 – 2 49 (34.3) 3 79 (55.2) Discussion NA 15 (10.5) T stage T1 19 (13.3) Chromosome 17 aneusomy often occurs in breast cancer and is T2 80 (55.9) associated with poor prognosis in invasive breast carcinoma (36). T3 29 (20.3) Clinical trials showed that polysomy of chromosome 17 might be T4 13 (9.1) associated with trastuzumab response in HER2 FISH-negative but NA 2 (1.4) N stage N0 40 (28.0) HER2 IHC-positive breast cancer (37, 38). These studies indicate N1 22 (15.4) that other genes in chromosome 17 might be involved in trastu- N2 64 (44.8) zumab response in HER2-positive breast cancer. Through bioin- N3 13 (9.1) formatic analysis of chromosome 17q, WBP2 was found to be NA (NX) 4 (2.8) coamplified with HER2 in human breast tumors. We previously Overall TNM stage I 8 (5.6) reported that WBP2 is an oncogene that promotes cell growth, II 84 (58.7) III 49 (34.3) proliferation, and invasion via EGFR/Wnt pathway crosstalk in NA 2 (1.4) 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 in Table 1. Overall, 64 (44.76%) patients achieved pCR after coactivator to promote growth of breast cancer cells. WBP2 is trastuzumab-based neoadjuvant chemotherapy which is similar overexpressed in breast cancer and its expression is correlated with to previous studies (30%–53%; refs. 10, 11). The ER- or PR- poor OS/DFS. In this study, we further discovered that patients negative group showed higher pCR than the ER- or PR-positive with tumors that coexpress WBP2 and HER2 have the worst group, which is also consistent with previous reports (10). No prognosis compared with either alone. EGF-induced tyrosine statistically significant relationship between pCR and other clin- phosphorylation of WBP2 was also found to be dependent on ical factors was obtained (Supplementary Table S1). HER2 in this study. Taking these observations into account, we To analyze WBP2 expression in pretreatment biopsy speci- tested the hypothesis that WBP2 expression regulates the response mens, IHC of WBP2 was performed using a WBP2 polyclonal of breast cancer to trastuzumab-based treatment. antibody (17, 20). The specificity of the WBP2 polyclonal anti- Trastuzumab is the first-line therapy for patients with HER2- body for IHC was validated previously (20). The IHC score for positive breast cancer. However, not all of these patients respond WBP2 was higher in the pCR group than non-pCR group (mean to treatments that involved trastuzumab (9). It is conceivable that WBP2 IHC score, 2.63 vs. 2.05, P < 0.0001; Fig. 4A). Next, the breast cancer's response to trastuzumab does not solely depend breast tumors were segregated into high (¼3) and low WBP2 IHC on HER2 expression, but also on other biomarkers that are score (<3). The high WBP2–expressing group had significantly molecularly networked with HER2. Several studies have reported higher pCR of 2.5 times more than the low WBP2–expressing potential biomarkers that are associated with positive or negative group (67.19% vs. 26.58%, respectively; Fig. 4B; Table 2). We response to trastuzumab (39, 40). For example, loss of PTEN and further examined whether there were clinical factors that might be PIK3CA mutations has been observed in trastuzumab-resistant associated with pCR and WBP2 expression. When the patients breast cancers and both are linked to poor prognosis and lower were first stratified by age (<50 and 50 years old) and meno- overall survival (39). Patients with HER2-positive breast cancer pausal status and the breast tumors further stratified on the basis with PIK3CA mutation showed lower pCR (28.6%) to HER2- of WBP2 IHC score (¼3 and <3), the pCR of patients with high targeting neoadjuvant therapies, as compared with wild-type WBP2 and age below 50 years was 77.78% while patients with PIK3CA (53.1%; ref. 40). In this study, we provide evidence that high WBP2 and premenopausal was 73.33%, both of which are WBP2 is a potential predictor of trastuzumab response in HER2- higher than the pCR of 67.19% for tumors with high WBP2 alone positive breast cancer. (Fig. 4C and 4D). Trastuzumab plus chemotherapy in the neoadjuvant setting is Retrospective ROC curve analysis was performed to determine given to HER2-positive breast cancer with locally advanced or the optimal sensitivity and specificity of WBP2 expression in inflammatory breast cancer. This treatment strategy has shown discriminating pCR from non-pCR cases. The areas under the significantly improved pathologic response compared with

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WBP2 Level Correlates with Trastuzumab Neoadjuvant Therapy

Figure 4. Correlation between WBP2 expression and pCR. A–D, Analyses of WBP2 expression (based on IHC) in breast tumors of neoadjuvant trastuzumab þ chemotherapy-treated patients segregated into those that showed pCR and partial/non-pCR (non-pCR; n ¼ 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 3or<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).

chemotherapy alone (41, 42). Pathologic complete response observed that patients with HER2-positive breast cancer and high (pCR) is correlated with better prognosis and shows clinical WBP2 expression showed higher pCR (67.19%) to trastuzumab- responsiveness to systemic therapy. The neoadjuvant treatment based neoadjuvant chemotherapy, as compared with the overall approach is becoming diverse and the choice of treatment is best HER2-positive breast cancer cases (44.76%). Subgroup analysis personalized, based on the individual's molecular portrait. Iden- showed that pCR was higher in the group of patients aged below tifying novel biomarkers beyond HER2 status that could improve 50 years and whose tumors possessed high WBP2 level (77.78%) pCR would be helpful for patients and clinicians in choosing a compared with those above 50 years old. Similarly premeno- treatment strategy with better clinical outcome. In this study, we pausal patients with tumors that expressed elevated WBP2 level

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Table 2. Association between WBP2 IHC score and pCR pCR Non-pCR Characteristics WBP2 IHC score N % N % Pa Sensitivity (%) 95% CI Speciﬁcity (%) 95% CI Overall population 64 44.76 79 55.24 ¼3 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 ¼3 21 77.78 6 22.22 , <0.0001 80.77 60.65%–93.45% 80.00 61.43%–92.29% <3 5 17.24 24 82.76 50 ¼3 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 ¼3 22 73.33 8 26.67 , <0.0001 81.48 61.92%–93.70% 76.47 58.83%–89.25% <3 5 16.13 26 83.87 Postmenopause ¼3 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.

showed higher pCR (73.33%) than the nonmenopausal group. of which were similar to the pCR of the group of samples stratified The sensitivity and specificity of WBP2 in discriminating between based on high WBP2 alone (67.19%; Supplementary Fig. S2A; pCR and non-pCR were also better in patient groups with age Supplementary Table S2). below 50 years (80.77% and 80.00%) and of premenopausal These results above indicate that the chemotherapy regimens status (81.48% and 76.47%) compared with the overall group do not influence pCR of patients to trastuzumab-based neoadju- (67.19% and 73.42%). Because the average age of menopause is vant therapy. It also supports the notion that WBP2 does not 49–52 years old (43), there would be significant overlap of correlate with response to chemotherapy and strengthens the link patients between the two groups. Given that menopausal status between WBP2 and trastuzumab-targeted therapy. This is further is sometimes not well documented in the medical records while corroborated by preliminary in vitro data, which argues that WBP2 age is a more objective parameter, we recommend that age is a does not regulate breast cancer response to chemotherapy because more useful clinical factor in conjunction with the molecular overexpression or knockdown of WBP2 did not affect cellular determinants WBP2 and HER2 for stratification of patients for viability upon doxorubicin treatment (Supplementary Fig. S2B– precision medicine using trastuzumab. The ability to identify S2D). Nevertheless, it would be necessary to confirm and establish patients who are likely to respond well to neoadjuvant trastuzu- the value of WBP2 in predicting response to trastuzumab-based mab allows clinicians to better plan therapeutic interventions for chemotherapy via a more tightly controlled prospective study. patients. Patients can also be better counselled regarding what to Taken together, the molecular basis for the role of WBP2 as a expect from the neoadjuvant therapy. It would also be possible to companion diagnostics for trastuzumab precision medicine is predict which patients would attain successful downstaging of putatively through its interaction with HER2 in breast cancer cells. their tumors from neoadjuvant therapy, thus allowing for breast Neoadjuvant therapy for HER2-positive breast cancer has conserving surgery, instead of mastectomy in some instances. This improved significantly over time with clinical trials supporting will have significant impact on the patients' cosmetic and psy- the combinatorial use of different anti-HER2 inhibitors, including chologic outcome. lapatinib (tykerb) and pertuzumab (44, 45). Because our data We showed that WBP2 expression correlated with response to show that WBP2 regulates HER2 and its downstream signaling, it trastuzumab-based neoadjuvant therapy. However, it should be is conceivable that WBP2 also enhances response of HER-positive noted that the retrospective study performed in this study breast cancer to other HER2-targeting agents such as pertuzumab involved patients who had received neoadjuvant therapy com- and lapatinib. Further studies will be needed to test these prising trastuzumab in combination with chemotherapy regi- hypotheses. mens that varied according to the physician in attendance and In conclusion, there is a clinical need to improve the response hospital practices. This, therefore, confounds the data generated rate of patients with HER2-positive breast cancer to HER2-tar- and possibly the conclusions drawn. Recognizing this weakness, geted therapeutics especially in the neoadjuvant setting. Our study we performed an analysis to investigate whether WBP2 levels posits that trastuzumab is more effective in HER2-positive breast correlates with response to chemotherapy regimens. The chemo- cancers that express high levels of WBP2. WBP2 is a potential therapy regimens given can be classified as (i) anthracycline-based companion diagnostic for further stratification of patients with regimens, such as adriamycin (doxorubicin) and (ii) nonanthra- HER2-positive breast cancer for more effective anti-HER2 cycline based. As shown in Supplementary Table S1, there was no therapies. significant difference in the pCR rate between these groups of patients treated with anthracycline and nonanthracycline regi- Disclosure of Potential Conflicts of Interest mens (45.36% and 44.44%, respectively) compared with the J. Arribas is a consultant/advisory board member for Menarini. S. Lee reports overall rate of 44.76% in the nonstratified population. Further- receiving speakers bureau honoraria from and is a consultant/advisory board more, we stratified tumors with high WBP2 level into those treated member for Roche. No potential conflicts of interest were disclosed by the other with anthracycline- and nonanthracycline-based regimens. The authors. pCR of patients with high WBP2–expressing tumors treated with anthracycline-based and nonanthracycline-based regimens was Authors' Contributions 69.23% and 64.00%, respectively (Supplementary Fig. S2A), both Conception and design: S.-A. Kang, Y.P. Lim

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WBP2 Level Correlates with Trastuzumab Neoadjuvant Therapy

Development of methodology: S.-A. Kang, J.S. Guan, H.J. Tan, T. Chu, J. Arribas, Acknowledgments T.C. Putti, Y.P. Lim The study is funded by Exploit Technologies, Agency for Science, Technology Acquisition of data (provided animals, acquired and managed patients, and Research (F09/X/037_c) and the National Medical Research Council, provided facilities, etc.): S.-A.Kang,J.S.Guan,H.J.Tan,T.Chu,C.Bernado, Ministry of Health (NMRC/OFIRG/0034/2017), Singapore. J Arribas is sup- J. Arribas, C.Y. Wong, P.H. Tan, M. Gudi, T.C. Putti, J. Sohn, S.H. Lim, ported by grants of the Breast Cancer Research Foundation (BCRF-17-008) and S.-C. Lee Instituto de Salud Carlos III (PI16/00253) for establishing and maintaining the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, resistant PDXs. computational analysis): S.-A. Kang, J.S. Guan, H.J. Tan, T. Chu, T.C. Putti, J. Sohn, S.-C. Lee, Y.P. Lim The costs of publication of this article were defrayed in part by the payment of Writing, review, and/or revision of the manuscript: S.-A. Kang, H.J. Tan, page charges. This article must therefore be hereby marked advertisement in T. Chu, J. Arribas, M. Gudi, T.C. Putti, J. Sohn, S.H. Lim, S.-C. Lee, Y.P. Lim accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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. Bernado, Y.P. Lim Received October 3, 2018; revised November 16, 2018; accepted December ﬁ Study supervision: Y.P. Lim 21, 2018; published rst December 28, 2018.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin ulates estrogen receptor a function in breast cancer via the Wnt pathway. 2016;66:7–30. The FASEB J 2011;25:3004–18. 2. Sims AH, Howell A, Howell SJ, Clarke RB. Origins of breast cancer subtypes 18. Chan SW, Lim CJ, Huang C, Chong YF, Gunaratne HJ, Hogue KA, et al. WW and therapeutic implications. Nat Clin Pract Oncol 2007;4:516–25. domain-mediated interaction with Wbp2 is important for the oncogenic 3. Yu D, Hung MC. Overexpression of ErbB2 in cancer and ErbB2-targeting property of TAZ. Oncogene 2011;30:600–10. strategies. Oncogene 2000;19:6115–21. 19. Zhang X, Milton CC, Poon CL, Hong W, Harvey KF. Wbp2 cooperates with 4. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo breast cancer: correlation of relapse and survival with amplification of the pathway. Cell Death Differ 2011;18:1346–55. HER-2/neu oncogene. Science 1987;235:177–82. 20. Lim SK, Lu SY, Kang SA, Tan HJ, Li Z, Adrian Wee ZN, et al. Wnt signaling 5. Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, et al. promotes breast cancer by blocking ITCH-mediated degradation of YAP/ Phase II study of weekly intravenous recombinant humanized anti- TAZ transcriptional coactivator WBP2. Cancer Res 2016;76:6278–89. p185HER2 monoclonal antibody in patients with HER2/neu-overexpres- 21. The Cancer Genome Atlas Network. Comprehensive molecular portraits of sing metastatic breast cancer. J Clin Oncol 1996;14:737–44. human breast tumours. Nature 2012;490:61–70. 6. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. 22. Grossman RL, Heath AP, Ferretti V, Varmus HE, Lowy DR, Kibbe WA, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for Toward a shared vision for cancer genomic data. N Engl J Med 2016;375: metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 1109–12. 344:783–92. 23. Mi H, Dong Q, Muruganujan A, Gaudet P, Lewis S, Thomas PD. 7. Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, PANTHER version 7: improved phylogenetic trees, orthologs and col- et al. First-line Herceptin monotherapy in metastatic breast cancer. laboration with the Gene Ontology Consortium. Nucleic Acids Res. Oncology 2001;61:37–42. 2010;38:D204–10. 8. Barok M, Tanner M, Koninki K, Isola J. Trastuzumab-DM1 causes tumour 24. Vicario R, Peg V, Morancho B, Zacarias-Fluck M, Zhang J, Martinez- growth inhibition by mitotic catastrophe in trastuzumab-resistant breast Barriocanal A, et al. Patterns of HER2 gene amplification and response to cancer cells in vivo. Breast Cancer Res 2011;13:R46. anti-HER2 therapies. PLoS One 2015;10:e0129876. 9. Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, 25. Sircoulomb F, Bekhouche I, Finetti P, Adelaide J, Ben Hamida A, Bonansea et al. Efficacy and safety of trastuzumab as a single agent in first-line J, et al. Genome profiling of ERBB2-amplified breast cancers. BMC Cancer treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 2010;10:539. 2002;20:719–26. 26. Vanden Bempt I, Drijkoningen M, De Wolf-Peeters C. The complexity of 10. Li XB, Krishnamurti U, Bhattarai S, Klimov S, Reid MD, O'Regan R, et al. genotypic alterations underlying HER2-positive breast cancer: an explana- Biomarkers predicting pathologic complete response to neoadjuvant che- tion for its clinical heterogeneity. Curr Opin Oncol 2007;19:552–7. motherapy in breast cancer. Am J Clin Pathol 2016;145:871–8. 27. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. 11. Rimawi MF, Schiff R, Osborne CK. Targeting HER2 for the treatment of Studies of the HER-2/neu proto-oncogene in human breast and ovarian breast cancer. Ann Rev Med 2015;66:111–28. cancer. Science 1989;244:707–12. 12. Chen HI, Sudol M. The WW domain of Yes-associated protein binds a 28. Ginestier C, Adelaide J, Goncalves A, Repellini L, Sircoulomb F, Letessier A, proline-rich ligand that differs from the consensus established for Src et al. ERBB2 phosphorylation and trastuzumab sensitivity of breast cancer homology 3-binding modules. Proc Natl Acad Sci U S A 1995;92: cell lines. Oncogene 2007;26:7163–9. 7819–23. 29. Cuello M, Ettenberg SA, Clark AS, Keane MM, Posner RH, Nau MM, et al. 13. McDonald CB, McIntosh SK, Mikles DC, Bhat V, Deegan BJ, Seldeen KL, Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) et al. Biophysical analysis of binding of WW domains of the YAP2 enhances tumor necrosis factor-related apoptosis-inducing ligand-medi- transcriptional regulator to PPXY motifs within WBP1 and WBP2 adaptors. ated apoptosis in breast and ovarian cancer cell lines that overexpress erbB- Biochemistry 2011;50:9616–27. 2. Cancer Res 2001;61:4892–900. 14. Nitsch R, Di Palma T, Mascia A, Zannini M. WBP-2, a WW domain binding 30. Gajria D, Chandarlapaty S. HER2-amplified breast cancer: mechanisms of protein, interacts with the thyroid-specific transcription factor Pax8. trastuzumab resistance and novel targeted therapies. Expert Rev Biochem J 2004;377:553–60. Anticancer Ther 2011;11:263–75. 15. Dhananjayan SC, Ramamoorthy S, Khan OY, Ismail A, Sun J, Slingerland J, 31. Spankuch B, Kurunci-Csacsko E, Kaufmann M, Strebhardt K. Rational et al. WW domain binding protein-2, an E6-associated protein interacting combinations of siRNAs targeting Plk1 with breast cancer drugs. protein, acts as a coactivator of estrogen and progesterone receptors. Oncogene 2007;26:5793–807. Mol Endocrinol 2006;20:2343–54. 32. Timms JF, White SL, O'Hare MJ, Waterfield MD. Effects of ErbB-2 over- 16. Chen Y, Choong L-Y, Lin Q, Philp R, Wong C-H, Ang B-K, et al. Differential expression on mitogenic signalling and cell cycle progression in human expression of novel tyrosine kinase substrates during breast cancer devel- breast luminal epithelial cells. Oncogene 2002;21:6573–86. opment. Mol Cell Proteomics 2007;6:2072–87. 33. Dubska L, Ande?ra L, Sheard MA. HER2 signaling downregulation by 17. Lim SK, Orhant-Prioux M, Toy W, Tan KY, Lim YP. Tyrosine phosphory- trastuzumab and suppression of the PI3K/Akt pathway: an unexpected lation of transcriptional coactivator WW-domain binding protein 2 reg- effect on TRAIL-induced apoptosis. FEBS Lett 2005;579:4149–58.

www.aacrjournals.org Clin Cancer Res; 25(8) April 15, 2019 2599

Kang et al.

34. Zhou X, Tan M, Hawthorne VS, Klos KS, Lan K-H, Yang Y, et al. Activation of neoadjuvant human epidermal growth factor receptor 2–targeted therapies the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 over- in breast cancer. J Clin Oncol 2015;33:1334–9. expression predicts tumor progression in breast cancers. Clin Cancer Res 41. Gianni L, Eiermann W, Semiglazov V, Manikhas A, Lluch A, Tjulandin S, 2004;10:6779–88. et al. Neoadjuvant chemotherapy with trastuzumab followed by adjuvant 35. Nahta R. Molecular mechanisms of trastuzumab-based treatment in HER2- trastuzumab versus neoadjuvant chemotherapy alone, in patients with overexpressing breast cancer. ISRN Oncology 2012;2012:428062. HER2-positive locally advanced breast cancer (the NOAH trial): a rando- 36. Watters AD, Going JJ, Cooke TG, Bartlett JM. Chromosome 17 aneusomy is mised controlled superiority trial with a parallel HER2-negative cohort. associated with poor prognostic factors in invasive breast carcinoma. Lancet 2010;375:377–84. Breast Cancer Res Treat 2003;77:109–14. 42. Buzdar AU, Ibrahim NK, Francis D, Booser DJ, Thomas ES, Theriault RL, 37. Hofmann M, Stoss O, Gaiser T, Kneitz H, Heinmoller P, Gutjahr T, et al. et al. Significantly higher pathologic complete remission rate after neoad- Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin) phase II juvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: monotherapy study: assessment of test sensitivity and impact of chromo- results of a randomized trial in human epidermal growth factor receptor some 17 polysomy. J Clin Pathol 2008;61:89–94. 2-positive operable breast cancer. J Clin Oncol 2005;23:3676–85. 38. Kaufman PA, Broadwater G, Lezon-Geyda K, Dressler LG, Berry D, Fried- 43. Takahashi TA, Johnson KM. Menopause. Med Clin North Am 2015;99: man P, et al. CALGB 150002: Correlation of HER2 and chromosome 17 521–34. (ch17) copy number with trastuzumab (T) efficacy in CALGB 9840, 44. Baselga J, Bradbury I, Eidtmann H, Di Cosimo S, de Azambuja E, Aura C, paclitaxel (P) with or without T in HER2þ and HER2- metastatic breast et al. Lapatinib with trastuzumab for HER2-positive early breast cancer cancer (MBC). J Clin Oncol 2007;25:1009. (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial. Lancet 39. Razis E, Bobos M, Kotoula V, Eleftheraki A, Kalofonos H, Pavlakis K, et al. 2012;379:633–40. Evaluation of the association of PIK3CA mutations and PTEN loss with 45. Gianni L, Pienkowski T, Im Y-H, Roman L, Tseng L-M, Liu M-C, et al. efficacy of trastuzumab therapy in metastatic breast cancer. Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women Breast Cancer Res Treat 2011;128:447–56. with locally advanced, inflammatory, or early HER2-positive breast cancer 40. Majewski IJ, Nuciforo P, Mittempergher L, Bosma AJ, Eidtmann H, Holmes (NeoSphere): a randomised multicentre, open-label, phase 2 trial. E, et al. PIK3CA mutations are associated with decreased benefitto Lancet Oncol 2012;13:25–32.

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Elevated WBP2 Expression in HER2-positive Breast Cancers Correlates with Sensitivity to Trastuzumab-based Neoadjuvant Therapy: A Retrospective and Multicentric Study

Shin-Ae Kang, Jye Swei Guan, Hock Jin Tan, et al.

Clin Cancer Res 2019;25:2588-2600. Published OnlineFirst December 28, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3228

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