Oncogene (2015) 34, 1105–1115 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

The E3 ubiquitin ligase NEDD4 negatively regulates HER3/ErbB3 level and signaling Z Huang, B-K Choi, K Mujoo, X Fan, M Fa, S Mukherjee, N Owiti, N Zhang and Z An HER3/ErbB3, a member of the epidermal growth factor receptor (EGFR) family, has a pivotal role in cancer and is emerging as a therapeutic antibody target. In this study, we identified NEDD4 (neural precursor cell expressed, developmentally downregulated 4) as a novel interaction partner and ubiquitin E3 ligase of human HER3. Using molecular and biochemical approaches, we demonstrated that the C-terminal tail of HER3 interacted with the WW domains of NEDD4 and the interaction was independent of neuregulin-1. Short hairpin RNA knockdown of NEDD4 elevated HER3 levels and resulted in increased HER3 signaling and cancer cell proliferation in vitro and in vivo. A similar inverse relationship between HER3 and NEDD4 levels was observed in prostate cancer tumor tissues. More importantly, the upregulated HER3 expression by NEDD4 knockdown sensitized cancer cells for growth inhibition by an anti-HER3 antibody. Taken together, our results suggest that low NEDD4 levels may predict activation of HER3 signaling and efficacies of anti-HER3 antibody therapies. Oncogene (2015) 34, 1105–1115; doi:10.1038/onc.2014.56; published online 24 March 2014

INTRODUCTION HER3 is a member of the human epidermal growth factor receptor (EGFR) family of tyrosine kinases.1,2 Unlike other family members, HER3 possesses no or low intrinsic kinase activity, and activation of HER3 requires heterodimerization with other EGFR family members such as EGFR, HER2 and HER4.3–5 This heterodimerization leads to phosphorylation of HER3 at its C-terminal tail and activation of downstream signaling.6–8 HER3 signaling has been shown to result in increased cell migration and proliferation.6,9 High HER3 expression has been correlated with poor prognosis in various cancers,10–13 and oncogenic HER3 mutations have been reported in human colon and gastric cancers.14 Moreover, elevation of HER3 expression is associated with resistance to therapeutic agents targeting EGFR and HER2.15–20 There are currently intense efforts toward developing anti-HER3 antibody therapeutics for cancer treatment.21–23 However, the mechanisms regulating HER3 levels and the underlying biology of HER3 in the development and progression of cancer are not well understood. Ubiquitination has been shown to regulate the levels of EGFR family receptors by targeting these receptors to proteasomes or lysosomes.24 Three classes of enzymes are involved in ubiquitination: ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) that determines substrate specificity.25 E3 ubiquitin ligases can be further divided into three types based on domain structures, which include N-end rule E3s, E3s containing the HECT (homology to the E6-AP C terminus) domain and E3s with the RING (really interesting new gene) finger.26 E3 ubiquitin ligases have vital roles in regulating the levels of EGFR family receptors.27 For example, RING finger family E3 ligase Cbl associates with EGFR to mediate its lysosomal degradation,28 and CHIP associates with HER2 to promote its ubiquitination and degradation.29

HECT family E3 ligases WWP1 (WW domain containing protein 1) and ITCH (itchy E3 ubiquitin protein ligase) ubiquitinate HER4 to mediate both proteasomal and lysosomal degradation.30,31 Previous studies reported that steady levels of HER3 could also be regulated by ubiquitination. RING finger family E3 ligase Nrdp1 was shown to ubiquitinate HER3, which led to proteasomal degradation of HER3 independent of NRG-1 (neuregulin-1) activation.32,33 In this study, we identified the HECT family E3 ligase NEDD4 (neural precursor cell expressed, developmentally downregulated 4) as a novel E3 ligase of HER3. NEDD4 has been reported as an E3 ligase of fibroblast growth factor-1 and epithelial sodium channels, regulating their ubiquitination, degradation, and signaling.31,34–36 Recently, NEDD4 has also been reported as an E3 ligase of EGFR family receptor HER4 in Madin–Darby canine kidney II cells.31 The human NEDD4 contains an N-terminal C2 domain that is involved in protein localization and trafficking,37,38 a C-terminal HECT domain that is involved in ubiquitin transfer25 and four WW domains 37 that are involved in substrate recognition by interacting with the PPXY motifs located on substrates.38 Our results demonstrated that NEDD4 is a novel interaction partner of HER3 and the C-terminal tail of HER3 interacts with the WW domains of NEDD4. NEDD4 overexpression resulted in decreased HER3 levels and increased HER3 ubiquitination. Conversely, short hairpin RNA (shRNA) knockdown of NEDD4 increased HER3 levels and resulted in elevated HER3 signaling. The elevated HER3 signaling caused by NEDD4 shRNA knockdown led to increased cancer cell proliferation in vitro and tumor growth in vivo, and this effect could be antagonized by an anti-HER3 monoclonal antibody (HER3Mab). The inverse correlation between HER3 and NEDD4 levels was also detected in ductal cells of prostate tumor tissues.

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA. Correspondence: Dr Professor N Zhang or Dr Professor Z An, Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA. E-mail: [email protected] or [email protected] Received 15 October 2013; revised 6 January 2014; accepted 12 January 2014; published online 24 March 2014

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RESULTS NEDD4 is a novel interaction partner of HER3 In an effort to identify novel HER3 interaction partners, we conducted HER3 immunoprecipitation (IP) from lysates of Chinese hamster ovary (CHO) cells that stably overexpress human HER3 (CHO-HER3).39 CHO cells with pcDNA control vector (CHO-pcDNA) showed no detectable level of endogenous HER3, whereas CHOHER3 expressed high level of HER3 and possessed robust NRG-1dependent HER3 phosphorylation and downstream AKT1 (protein kinase B) and ERK1/2 (extracellular-regulated kinase-1/2) phosphorylation (Supplementary Figure S1a). Analysis of HER3 IP by mass spectrometry (MS) identified a panel of candidate proteins that associated with HER3 in the Co-immunoprecipitation (co-IP) complex, including two known interaction partners of HER3: HER2 and the p85 subunit of PI3K (Supplementary Figure S1b and Supplementary Table S1). E3 ubiquitin ligase NEDD4 was among the novel HER3 interacting proteins identified by IP-MS. We subsequently confirmed the association of HER3 and NEDD4 using IP-WB (western blotting) detection in two breast cancer cell lines MDAMB-453 and MCF-7. As NRG-1 ligand is known to induce HER3 phosphorylation and the recruitment of binding partners,6–8 the interaction between NEDD4 and HER3 was examined by IP-WB with and without NRG-1 treatment (Figure 1a). Although NRG-1 treatment increased both the HER3 signaling and the association of HER3 with HER2 and p85 (Supplementary Figures S1a and b), HER3-NEDD4 interaction was independent of NRG-1, as indicated in the two-way co-IP and WB detection of NEDD4 and HER3 interaction, respectively, (Figure 1a). Immunofluorescence staining showed co-localization of HER3 and NEDD4 in CHO-HER3 and MCF-7 cells (Supplementary Figure S2). In situ proximity ligation assay (PLA),40,41 a method that measures close association of two proteins, was also used to further confirm the interaction between NEDD4 and HER3. Using HER2 and HER3 dimerization pairs in the presence of NRG-1 as the positive assay control (Figure 1b, left column), we noticed that the NEDD4-HER3 pair produced similar level of fluorescent signals (red dots; Figure 1b, middle column), indicating the abundance of HER3-NEDD4 pairs in proximity. As a negative control, NEDD4 antibody was replaced with an isotype control antibody in the assay and it produced no fluorescence background (Figure 1b, right column). We then sought to determine which regions of HER3 and NEDD4 were required for their interaction. Previous studies have shown that WW domains of NEDD4 family E3 ligases interact with PPXY motifs of their targeted proteins.37,42 NEDD4 has four WW domains and HER3 has one PPXY motif (AA 972-975: PPRY) in its C-terminal tail. To determine whether the WW domains of NEDD4 contribute to its interaction with the C-terminal tail of HER3, we transiently expressed either the full-length NEDD4, the fragment consisting of the WW and HECT domains, or the fragment consisting of the HECT domain alone in HEK293T cells together with the full-length human HER3 (Figure 1c). A haematoxylin (HA) tag was added to the NEDD4 constructs for WB detection. Both the full-length NEDD4 and the WW–HECT fragment were detected in the HER3 co-IP, indicating their association with HER3 (Figure 1d). HECT domain alone was not detected in the HER3 co-IP by WB (Figure 1d), suggesting that the WW domains of NEDD4 are required for interacting with HER3. To determine whether NEDD4 interacts with the kinase domain or the C-terminal tail of HER3, we performed IP using antibodies against the extracellular domains of HER3 (N-HER3) in lysates of CHO cells expressing different chimeras of HER3 and HER2 (Choi et al.39; Figure 1e). Replacing the kinase domain of HER3 with the HER2 kinase domain (HER3-2-3, Figure 1f) did not abolish the NEDD4-HER3 interaction, suggesting that the kinase domain of HER3 was not essential for the interaction. However, replacing the HER3 kinase domain and C-terminal tail with HER2 Oncogene (2015) 1105 – 1115

counterparts (HER3-2-2) abolished the NEDD4-HER3 interaction (Figure 1f), indicating that the C-terminal tail domain of HER3 was required for NEDD4-HER3 interaction. Further, the effect of NEDD4 expression on HER3 levels was also investigated. In HEK293T cells co-transfected with pcDNA-HER3 and pCEP4-NEDD4 vectors, we noticed a significant decrease in HER3 levels as compared with the cells transfected with pcDNA-HER3 alone (Figure 1g). When the PPRY motif of the transfected HER3 was mutated to AAAA, NEDD4-HER3 interaction was abolished and HER3 levels were no longer reduced by NEDD4, suggesting that the PPRY motif of HER3 is critical for NEDD4’s regulation on HER3 levels (Figure 1g). NEDD4 negatively regulates HER3 receptor level E3 ligases are known to regulate protein levels of EGFR family receptors27,29,30,43–46 that prompted us to investigate if altering NEDD4 levels can affect HER3 receptor levels. Four independent stable NEDD4 shRNA knockdown (shNEDD4) MCF-7 cell lines were generated. shNEDD4 knockdown resulted in ~ 80% reductions in total NEDD4 protein levels. As all four stable NEDD4 shRNA knockdown constructs exhibited similar results, we selected construct number 3 for all downstream experiments. Meanwhile, the amount of total HER3 protein levels were more than doubled as detected by WB (Figure 2a), despite HER3 mRNA levels that remained unchanged (Supplementary Figure S3). Flow cytometry analysis was performed to examine the HER3 levels at the cell surface in the shNEDD4 knockdown MCF-7 cells. This analysis showed higher mean fluorescence intensity in the shNEDD4 knockdown cells than that in the scramble shRNA (shScramble) control cells (29 vs 14), indicating a significant increase of HER3 levels at the cell surface of shNEDD4 knockdown MCF-7 cells (Figure 2b). In MCF-7 cells, shNEDD4 knockdown had no effect on the protein levels of HER2 as compared with the shScramble control (Figure 2c). We also examined whether shNEDD4 knockdown had any off-target effect on known regulators of HER3 such as the E3 ligase Nrdp1, which has been shown to regulate HER3 levels.32,33 Nrdp1 level remained unchanged upon shNEDD4 knockdown, suggesting NEDD4 regulates HER3 levels independent of Nrdp1 (Figure 2c). In addition, shNEDD4 knockdown MCF-7 cells exhibited increased HER3 half-life compared with the shScramble control MCF-7 cells as demonstrated by treating the cells for 0, 3 and 6 h with the protein biosynthesis inhibitor cycloheximide that blocked de novo HER3 protein biosynthesis (Figure 2d). NEDD4 regulates HER3 ubiquitination and degradation through the proteasomal pathway To investigate the proteasomal vs lysosomal degradation of HER3, we performed experiments using the proteasomal inhibitor MG132 and the lysosomal inhibitor chloroquine. Consistent with previous studies,33 MG132 treatments led to the accumulation of HER3 but chloroquine treatments did not (Figure 2e). HER3 accumulation caused by MG132 treatment was significantly less in shNEDD4 knockdown MCF-7 cells compared with the shScramble control (Figure 2e). In contrast, no effect of chloroquine on HER3 protein levels were observed when compared with the controls in both shScramble and shNEDD4 MCF-7 cells (Figure 2e). Taking together, these results suggest that NEDD4 regulate HER3 degradation primarily through the proteasomal pathway instead of the lysosomal pathway. To investigate the role of NEDD4 on HER3 ubiquitination, in vitro ubiquitination assays were carried out. In the presence of E1, E2 (UbcH5b) and ubiquitin, recombinant NEDD4 led to polyubiquitination of HER3 but not the HER3 PPRY-AAAA mutant protein (Supplementary Figure S4). To evaluate endogenous HER3 ubiquitination, cell lysates of shNEDD4 and shScramble control cells were immunoprecipitated by an anti-HER3Mab and HER3 ubiquitination was detected © 2015 Macmillan Publishers Limited

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Figure 1. NEDD4 interacts with HER3. (a) Immunoprecipitations (IPs) using control antibody, anti-HER3 antibody, or anti-NEDD4 antibody was performed using whole-cell lysates (WCL) of breast cancer cell lines MCF-7 and MDA-MB-453, which revealed similar HER3 levels but different HER2 levels. The interaction between HER3 and NEDD4 was examined by immunoblotting with antibodies against respective proteins. N-HER3: antibody against N-terminal extracellular domains of HER3; and C-HER3: antibody against C-terminal intracellular HER3. (b) The interaction between NEDD4 and HER3 was probed by proximity ligation assays (PLAs) in MCF-7 and CHO-HER3 cells using anti-NEDD4 and anti-HER3 antibodies. Red fluorescence dots indicated that NEDD4 and HER3 were in proximity. A control antibody was included as a negative control and an anti-HER2 antibody served as a positive control. (c) NEDD4 is a HECT family E3 ubiquitin ligase containing an N-terminal C2 domain that induces membrane localization, four WW domains that binds to specific substrates, and a C-terminal HECT domain that confers ubiquitin transfer. One or more WW domains can bind to peptides containing the PPXY motif, such as the PPRY motif found in HER3 at the C terminus of its kinase domain (PDB ID: 2PEX). A HA tag was added to the C terminus of full-length NEDD4 (C2+WW+HECT) and NEDD4 truncation fragments (WW+HECT and HECT) in the pCEP4 expression vector. (d) HEK293T cells were transfected with vectors encoding the HA-tagged NEDD4, HA-tagged NEDD4 fragments and full-length HER3. Cells were lysed 24 h following transfection, subjected to IP using an anti-HER3 antibody (HER3Mab) and analyzed with anti-HA antibody. (e) HER3 and HER3/HER2 chimeras have different domain compositions. ECD, extracellular domain; KD kinase domain; and Tail: C-terminal tail. The PPRY motif found in HER3 is missing in corresponding region of HER2. (f) CHO cells overexpressing wild type HER3 and engineered HER2/HER3 chimeras were created.39 IP of antibody against the N-terminal portion of HER3 (anti-N-HER3) was performed using lysates from the HER2/HER3 chimeras. The interaction between NEDD4 and HER2/HER3 chimeras was evaluated by immunoblotting using an anti-NEDD4 antibody. (g) HEK293T cells were transfected with pcDNA-HER3 (or HER3 with PPRY-AAAA mutation) and pCEP4-NEDD4. WCL and NEDD4-HER3 interactions were analyzed by WB analysis using corresponding antibodies.

by an anti-ubiquitin antibody in WB. In MCF-7 cells, a reduction in HER3 ubiquitination was observed in the shNEDD4 knockdown cells when compared with shScramble control cells (Figure 2f), suggesting that NEDD4 functions as an E3 ligase of HER3. In another experiment, HEK293T cells with recombinant NEDD4 © 2015 Macmillan Publishers Limited

overexpression showed reduced HER3 levels and increased HER3 ubiquitination (Figure 2g). Taken together, these data suggest that NEDD4 contributes to HER3 ubiquitination and modulates HER3 protein levels through proteasomal degradation pathway. Oncogene (2015) 1105 – 1115

NEDD4 regulates HER3 level and signaling Z Huang et al

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Figure 2. NEDD4 regulates HER3 levels and HER3 ubiquitination. (a) Four independent shNEDD4 knockdown in MCF-7 cell lines resulted in significant increase of HER3 receptor levels as determined by immunoblots. Quantitation of the immunoblot signals via ImageJ showed an average of 80% decrease in NEDD4 levels and >200% increase of total HER3 levels upon shNEDD4 knockdown. shNEDD4#3 was then used in following experiments. (b) Flow cytometry using either control antibody or anti-HER3 monoclonal antibody (HER3Mab)39 followed by fluorescent secondary antibody was performed on shScramble and shNEDD4 knockdown MCF-7 cells. The mean fluorescence intensity for background, shScramble and shNEDD4 were 5, 14 and 29, respectively. (c) The protein levels of NEDD4, HER3, HER2 and Nrdp1 in shScramble and shNEDD4 MCF-7 cells were monitored by immunoblotting using respective antibodies. shNEDD4 knockdown led to a reduction in NEDD4 and an increase in HER3, but did not affect the level of HER2 or Nrdp1. (d) HER3 protein half-life as impacted by NEDD4 shRNA knockdown. MCF-7 cells were treated with cyclohexamide (CHX) at 10 μg/ml and cell lysates were collected at indicated times. HER3 and actin protein levels were assessed by immunoblotting. (e) shScramble and shNEDD4 expressing MCF-7 cells were treated with MG132 (10 μm) and chroloquine (10 μm) for 8 h and the protein levels of HER3 were evaluated by immunoblotting. (f) MCF-7 whole-cell lysates (WCL) were collected following 8 h MG132 (10 μm) treatment, and the protein levels of HER3, actin and NEDD4 were evaluated by immunoblotting using respective antibodies. Cell lysates were subjected to IP using anti-HER3 antibody, and ubiquitinated HER3 were detected with an antiubiquitin antibody. (g) HEK293T cells were transfected with full-length human HER3 and NEDD4, and cell lysates were collected 48 h following transfection. Protein levels of HER3, actin and NEDD4 were evaluated by immunoblotting using respective antibodies. IPs were performed using an anti-HER3 antibody, and ubiquitinated HER3 were detected with an anti-ubiquitin antibody (P4D1).

NEDD4 knockdown leads to increased HER3 signaling It is well documented that HER3 phosphorylation activates downstream PI3K/AKT and ERK signaling pathways.7,8 To determine whether increased HER3 levels in shNEDD4 knockdown cancer cells would trigger an increase in HER3-mediated downstream signaling, we measured phosphorylation of HER3, AKT1 and ERK1/2 by WB. In both breast cancer MCF-7 and prostate cancer DU145 cells, NEDD4 knockdown resulted in increased levels of HER3 phosphorylation upon stimulation with NRG-1 (50 ng/ml; Figures 3a and b). In shNEDD4 knockdown MCF-7 cells, both AKT1 and ERK1/2 phosphorylation also increased (Figure 3a). In shNEDD4 knockdown DU145 cells, an increase in NRG-1induced AKT1 phosphorylation was observed, but ERK1/2 phosphorylation was not affected (Figure 3b). Unlike HER3 levels, total protein levels of AKT1 and ERK1/2 were not affected by shNEDD4 knockdown in both MCF-7 and DU145 cells (Figures 3a and b). In shNEDD4 knockdown MCF-7 cells, the HER3, AKT1 and ERK1/2 phosphorylation were elevated at various NRG-1 concentrations but the increase was more pronounced at higher NRG-1 concentrations (Figure 3c). Moreover, HER3, AKT1 and ERK1/2 Oncogene (2015) 1105 – 1115

phosphorylation each reached a plateau in shScramble MCF-7 control cells (at 25 ng/ml NRG-1 treatment), but they did not reach a plateau in shNEDD4 knockdown MCF-7 cells (Figures 3d–f). Together, these data suggested that the elevated HER3 upon shNEDD4 knockdown rendered cells more responsive to NRG-1 activation, which mediated increased HER3, AKT1 and ERK1/2 phosphorylation. NEDD4 knockdown increases HER3-mediated cell migration and proliferation in vitro and tumor growth in vivo The elevated HER3 levels and signaling we observed upon shNEDD4 knockdown prompted us to investigate whether shNEDD4 knockdown would also impact downstream events such as cell migration, proliferation and tumor growth in vivo. While investigating cell migration, we noticed that HER3 shRNA knockdown (shHER3) in DU145 cells led to reduced cell migration and the loss of response to NRG-1 stimulation (Figure 4a). Compared with the shScramble DU145 cells, shNEDD4 knockdown DU145 cells not only displayed increased migration, but these © 2015 Macmillan Publishers Limited

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cells also became more responsive to NRG-1 stimulation (Figure 4a), which was consistent with the increased HER3 levels and NRG-1-dependent HER3 signaling upon NEDD4 knockdown. The effect of shNEDD4 knockdown on NRG-1-dependent cell proliferation was also examined. In MCF-7 and DU145 shScramble control cells, NRG-1 treatment increased cell proliferation by 15 and 16%, respectively (Figures 4b and c). In contrast, in MCF-7 and DU145 shNEDD4 knockdown cells, NRG-1 increased cell proliferation by 30 and 28%, respectively, that was significantly higher than scramble control cells (P o 0.01) (Figures 4b and c). In colony formation assays, shNEDD4 knockdown MCF-7 cells formed more colonies than shScramble control cells, whereas shHER3 knockdown MCF-7 cells formed the lowest number of colonies (Figure 4d). We also noticed that the shNEDD4 knockdown and shScramble cells formed protrusions, but shHER3 knockdown cells did not (Figure 4e). Although MCF-7 is considered to display low cell migration, these different cell morphologies caused by shRNA knockdown of NEDD4 prompted us to investigate whether these changes in morphologies reflected the alternations in migration potential. Indeed, cell migration increased in shNEDD4 knockdown MCF-7 cells (P o 0.01) and decreased in shHER3 knockdown MCF-7 cells (P o 0.01; Supplementary Figure S5). In xenografts in vivo, shNEDD4 knockdown MCF-7 cells formed significantly larger tumors than shScramble MCF-7 cells (P o 0.01; Figure 5f). WB analysis also revealed elevated HER3 protein levels in shNEDD4 knockdown xenografts (Supplementary Figure S6). Together, our in vivo data was consistent with the in vitro results, showing that shNEDD4 knockdown in MCF-7 cells led to increased cell growth and tumor formation. © 2015 Macmillan Publishers Limited

NEDD4 knockdown cancer cells are more sensitive toward antiHER3 antibody treatments in vitro and in vivo In three-dimensional (3D) cultures of MCF-7 cells, shNEDD4 knockdown cells formed larger colony spheres than the shScramble cells in the presence of NRG-1 (P o 0.01; Figures 6a and b). To investigate whether such increase is a result of elevated HER3 signaling, HER3Mab treatments (10 μg/ml) were carried out, which significantly reduced the sphere sizes in both shNEDD4 knockdown and the shScramble control MCF-7 cancer cells (Po 0.05), but the effect was more prominent in shNEDD4 knockdown MCF-7 cells than shScramble controls (51% vs 27% reduction in sphere diameters; Figures 6a and b). In comparison, the spheres formed by shNEDD4 knockdown DU145 cells are of similar diameters to those formed by shScramble cells in the absence of NRG-1 (P>0.05); however, shNEDD4 knockdown DU145 cells displayed higher NRG-1 dependent growth compared with shScramble controls (100% vs 70% increase in average sphere diameter upon NRG-1 treatment; Figures 6c and d). HER3Mab treatment significantly reduced the average sphere diameters for both shNEDD4 knockdown and shScramble control DU145 cells (Po0.05). However, NRG-1activated shNEDD4 knockdown DU145 cells were more responsive to the HER3Mab treatment than shScramble control cells (42% vs 32% reduction in sphere diameters; Figures 6c and d). We investigated whether elevated HER3-mediated signaling in NEDD4 knockdown cells can be reduced by HER3Mab treatments. In shNEDD4 knockdown MCF-7 cells, HER3Mab significantly reduced the elevated NRG-1-mediated HER3 and AKT1 phosphorylation (Figure 5a). However, the residual HER3 and AKT1 Oncogene (2015) 1105 – 1115

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Figure 4. NEDD4 knockdown increased NRG-1-dependent cell migration and proliferation in vitro and tumor growth in vivo. (a) 5 × 105 of shScramble control, shNEDD4 and shHER3 knockdown DU145 cells were seeded into six-well transwell plates with and without NRG-1 (50 ng/ml). After 24 h incubation at 37 °C, migrated cells were stained with 0.5% crystal violet for 5 min, and the migrated cells from five random fields were counted; **Po0.01. (b, c) MCF-7 cells (3000) and DU145 cells were seeded in 96-well plates and cell proliferation were measured using Alamar Blue assay 72 h following NRG-1 treatment (50 ng/ml); **Po0.01. (d) Colony formation assays were performed using shScramble control, shNEDD4 knockdown and shHER3 knockdown MCF-7 cells. Cells (100 or 1000) were seeded in six-well plates. Colonies were allowed to form for 2 weeks and stained using 0.5% crystal violet. The number of colonies formed when 1000 cells were seeded was quantified using ImageJ. (e) Phase contrast images of shScramble control, shNEDD4 and shHER3 knockdown MCF-7 and DU145 cells. Scale bars, 100 μm.

phosphorylation was above the basal level. Similarly, HER3Mab also inhibited the elevated ERK1/2 phosphorylation in shNEDD4 knockdown MCF-7 cells but the inhibition was less effective (Figure 5a). In shNEDD4 knockdown DU145 cells, HER3Mab completely neutralized the elevated NRG-1-mediated HER3 and AKT1 phosphorylation to the basal level (Figure 5b). Unlike HER3 and AKT1 phosphorylation that are NRG-1 dependent, ERK1/2 phosphorylation in DU145 cells was not affected by NRG-1 treatment or shNEDD4 knockdown, and HER3Mab treatment only slightly reduced the ERK1/2 phosphorylation (Figure 5b). We examined the effect of HER3Mab on the cell proliferation in shNEDD4 knockdown cells. In MCF-7 cells, HER3Mab showed increased sensitivity in the inhibition of cell proliferation in shNEDD4 knockdown cells compared with shScramble control cells (P o 0.01; IC50 of 0.22 vs 0.44 μg/ml; Figure 5c). However, the maximum inhibition rate was not significantly different (P = 0.17, 43% vs 36%, Figure 5c). In DU145 cells, the maximum inhibition rate of HER3Mab in shNEDD4 knockdown cells was significantly higher than that in shScramble control cells (Po 0.01; 63% vs 39%), and the IC50 of HER3Mab was significantly lower in shNEDD4 knockdown cells (P o 0.05; IC50 of 0.20 vs 0.32 μg/ml; Figure 5d). Together, the increased maximum inhibition rate and lower IC50 demonstrated that HER3 antibody treatments are more effective in shNEDD4 knockdown MCF-7 and DU145 cells. The in vivo efficacy of HER3 antibody was investigated in MCF-7 xenografts. shNEDD4 knockdown and shScramble control MCF-7 cells were injected into two groups of nude mice and both groups were randomly divided in half at day 9 before treatments. The animals received intraperitoneal injections of HER3Mab, or Oncogene (2015) 1105 – 1115

phosphate-buffered saline (PBS) and tumor growth was monitored in all groups. shNEDD4 knockdown in MCF-7 cells significantly increased tumor growth in vivo compared with the shScramble control (Po 0.01; Figure 5e). HER3Mab significantly reduced tumor growth in both shNEDD4 knockdown (P o0.01) and shScramble control MCF-7 cells (P o0.01) but exerted larger effect in shNEDD4 knockdown MCF-7 cells (59% vs 36% decrease at day 30; P o 0.01) (Figure 5f). These results suggested that shNEDD4 knockdown MCF-7 cells were more responsive to HER3Mab treatment in vivo. Inverse relationship between NEDD4 and HER3 expression in ductal cells of prostate cancer tumors To investigate whether the inverse correlation between NEDD4 and HER3 levels observed in the shNEDD4 knockdown cell lines also existed in clinical samples, immunohistochemistry were performed on tissue micro arrays of prostate cancer tumor sections from 60 patients. It was noticed that staining of NEDD4 and HER3 showed a reverse pattern. NEDD4 mostly expressed in the ductal epithelial cells where HER3 showed higher expression in regions surrounding the ducts (Figure 7a). Spectral unmixing was performed to separate the HA and diaminobenzidine channels, which enabled visualization and quantification of the NEDD4 and HER3 (Supplementary Figure S7) in 60 randomly selected prostate ducts and surrounding regions (Supplementary Figure S8). To enable comparison, the average NEDD4 and HER3 staining in the ductal regions were designated values of 1.0 (Figure 7b). In the ductal regions, NEDD4 levels were significantly © 2015 Macmillan Publishers Limited

NEDD4 regulates HER3 level and signaling Z Huang et al

1111 MCF-7

DU145 shRNA: Scr NEDD4 Scr NEDD4

shRNA: Scr NEDD4 Scr NEDD4 +

-

+

+ -

+ +

+ -

HER3Mab: NRG-1: -

+ +

NEDD4

NEDD4

pHER3 (Y1289)

pHER3 (Y1289)

HER3

HER3

pAKT1 (S473)

pAKT1 (S473)

AKT1

AKT1

pERK1/2

pERK1/2

+

-

+

+ -

+ + + -

700

+ + Tumor volume (mm3 ± SEM)

HER3Mab: NRG-1: -

MCF-7 xenografts

shNEDD4 + PBS

600 500

**

300

**

Actin

Actin

50% 40% 30% 20%

MCF-7 shScramble IC50: 0.43 g/ml shNEDD4 IC50: 0.22 g/ml

Treatment duration:

IC50: ** P

ErbB3 level and signaling.

HER3/ErbB3, a member of the epidermal growth factor receptor (EGFR) family, has a pivotal role in cancer and is emerging as a therapeutic antibody tar...
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