The E3 Ubiquitin Ligase NEDD4 Negatively Regulates HER3/Erbb3 Level and Signaling

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 HECT family E3 ligases WWP1 (WW domain containing HER3 is a member of the human epidermal growth factor receptor protein 1) and ITCH (itchy E3 ubiquitin protein ligase) ubiquitinate (EGFR) family of tyrosine kinases.1,2 Unlike other family members, HER4 to mediate both proteasomal and lysosomal 30,31 HER3 possesses no or low intrinsic kinase activity, and activation degradation. of HER3 requires heterodimerization with other EGFR family Previous studies reported that steady levels of HER3 could also members such as EGFR, HER2 and HER4.3–5 This hetero- be regulated by ubiquitination. RING finger family E3 ligase Nrdp1 dimerization leads to phosphorylation of HER3 at its C-terminal was shown to ubiquitinate HER3, which led to proteasomal tail and activation of downstream signaling.6–8 HER3 signaling has degradation of HER3 independent of NRG-1 (neuregulin-1) 32,33 been shown to result in increased cell migration and activation. In this study, we identified the HECT family E3 proliferation.6,9 High HER3 expression has been correlated with ligase NEDD4 (neural precursor cell expressed, developmentally poor prognosis in various cancers,10–13 and oncogenic HER3 downregulated 4) as a novel E3 ligase of HER3. NEDD4 has been mutations have been reported in human colon and gastric reported as an E3 ligase of fibroblast growth factor-1 and 14 epithelial sodium channels, regulating their ubiquitination, degra- cancers. Moreover, elevation of HER3 expression is associated – with resistance to therapeutic agents targeting EGFR and dation, and signaling.31,34 36 Recently, NEDD4 has also been HER2.15–20 There are currently intense efforts toward developing reported as an E3 ligase of EGFR family receptor HER4 in anti-HER3 antibody therapeutics for cancer treatment.21–23 How- Madin–Darby canine kidney II cells.31 The human NEDD4 contains ever, the mechanisms regulating HER3 levels and the underlying an N-terminal C2 domain that is involved in protein localization biology of HER3 in the development and progression of cancer are and trafficking,37,38 a C-terminal HECT domain that is involved in not well understood. ubiquitin transfer25 and four WW domains 37 that are involved in Ubiquitination has been shown to regulate the levels of substrate recognition by interacting with the PPXY motifs located EGFR family receptors by targeting these receptors to protea- on substrates.38 somes or lysosomes.24 Three classes of enzymes are involved in Our results demonstrated that NEDD4 is a novel interaction ubiquitination: ubiquitin activating enzyme (E1), ubiquiti- partner of HER3 and the C-terminal tail of HER3 interacts with the n-conjugating enzyme (E2) and ubiquitin ligase (E3) that WW domains of NEDD4. NEDD4 overexpression resulted in determines substrate specificity.25 E3 ubiquitin ligases can be decreased HER3 levels and increased HER3 ubiquitination. further divided into three types based on domain structures, Conversely, short hairpin RNA (shRNA) knockdown of NEDD4 which include N-end rule E3s, E3s containing the HECT (homology increased HER3 levels and resulted in elevated HER3 signaling. The to the E6-AP C terminus) domain and E3s with the RING elevated HER3 signaling caused by NEDD4 shRNA knockdown led (really interesting new gene) finger.26 E3 ubiquitin ligases have to increased cancer cell proliferation in vitro and tumor growth vital roles in regulating the levels of EGFR family receptors.27 in vivo, and this effect could be antagonized by an anti-HER3 For example, RING finger family E3 ligase Cbl associates with monoclonal antibody (HER3Mab). The inverse correlation between EGFR to mediate its lysosomal degradation,28 and CHIP associates HER3 and NEDD4 levels was also detected in ductal cells of with HER2 to promote its ubiquitination and degradation.29 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 NEDD4 regulates HER3 level and signaling Z Huang et al 1106 RESULTS counterparts (HER3-2-2) abolished the NEDD4-HER3 interaction NEDD4 is a novel interaction partner of HER3 (Figure 1f), indicating that the C-terminal tail domain of HER3 was In an effort to identify novel HER3 interaction partners, we required for NEDD4-HER3 interaction. Further, the effect of NEDD4 conducted HER3 immunoprecipitation (IP) from lysates of Chinese expression on HER3 levels was also investigated. In HEK293T cells co-transfected with pcDNA-HER3 and pCEP4-NEDD4 vectors, we hamster ovary (CHO) cells that stably overexpress human HER3 fi (CHO-HER3).39 CHO cells with pcDNA control vector (CHO-pcDNA) noticed a signi cant decrease in HER3 levels as compared with showed no detectable level of endogenous HER3, whereas CHO- the cells transfected with pcDNA-HER3 alone (Figure 1g). HER3 expressed high level of HER3 and possessed robust NRG-1- When the PPRY motif of the transfected HER3 was mutated to dependent HER3 phosphorylation and downstream AKT1 (protein AAAA, NEDD4-HER3 interaction was abolished and HER3 levels were no longer reduced by NEDD4, suggesting that the kinase B) and ERK1/2 (extracellular-regulated kinase-1/2) phos- ’ phorylation (Supplementary Figure S1a). Analysis of HER3 IP by PPRY motif of HER3 is critical for NEDD4 s regulation on HER3 mass spectrometry (MS) identified a panel of candidate proteins levels (Figure 1g). that associated with HER3 in the Co-immunoprecipitation (co-IP) complex, including two known interaction partners of HER3: HER2 NEDD4 negatively regulates HER3 receptor level and the p85 subunit of PI3K (Supplementary Figure S1b and E3 ligases are known to regulate protein levels of EGFR family Supplementary Table S1). receptors27,29,30,43–46 that prompted us to investigate if altering E3 ubiquitin ligase NEDD4 was among the novel HER3 NEDD4 levels can affect HER3 receptor levels. Four independent interacting proteins identified by IP-MS. We subsequently stable NEDD4 shRNA knockdown (shNEDD4) MCF-7 cell lines were confirmed the association of HER3 and NEDD4 using IP-WB generated. shNEDD4 knockdown resulted in ~ 80% reductions in (western blotting) detection in two breast cancer cell lines MDA- total NEDD4 protein levels. As all four stable NEDD4 shRNA MB-453 and MCF-7. As NRG-1 ligand is known to induce HER3 knockdown constructs exhibited similar results, we selected phosphorylation and the recruitment of binding partners,6–8 the construct number 3 for all downstream experiments. Meanwhile, interaction between NEDD4 and HER3 was examined by IP-WB the amount of total HER3 protein levels were more than doubled with and without NRG-1 treatment (Figure 1a). Although NRG-1 as detected by WB (Figure 2a), despite HER3 mRNA levels that treatment increased both the HER3 signaling and the association remained unchanged (Supplementary Figure S3). Flow cytometry of HER3 with HER2 and p85 (Supplementary Figures S1a and b), analysis was performed to examine the HER3 levels at the cell HER3-NEDD4 interaction was independent of NRG-1, as indicated surface in the shNEDD4 knockdown MCF-7 cells. This analysis in the two-way co-IP and WB detection of NEDD4 and HER3 showed higher mean fluorescence intensity in the shNEDD4 interaction, respectively, (Figure 1a). Immunofluorescence staining knockdown cells than that in the scramble shRNA (shScramble) showed co-localization of HER3 and NEDD4 in CHO-HER3 and control cells (29 vs 14), indicating a significant increase of HER3 MCF-7 cells (Supplementary Figure S2). In situ proximity ligation levels at the cell surface of shNEDD4 knockdown MCF-7 cells assay (PLA),40,41 a method that measures close association of two (Figure 2b). In MCF-7 cells, shNEDD4 knockdown had no effect on proteins, was also used to further confirm the interaction between the protein levels of HER2 as compared with the shScramble NEDD4 and HER3. Using HER2 and HER3 dimerization pairs in the control (Figure 2c). We also examined whether shNEDD4 knock- presence of NRG-1 as the positive assay control (Figure 1b, left down had any off-target effect on known regulators of HER3 such as the E3 ligase Nrdp1, which has been shown to regulate HER3 column), we noticed that the NEDD4-HER3 pair produced similar 32,33 level of fluorescent signals (red dots; Figure 1b, middle column), levels. Nrdp1 level remained unchanged upon shNEDD4 indicating the abundance of HER3-NEDD4 pairs in proximity. As a knockdown, suggesting NEDD4 regulates HER3 levels indepen- negative control, NEDD4 antibody was replaced with an isotype dent of Nrdp1 (Figure 2c). In addition, shNEDD4 knockdown control antibody in the assay and it produced no fluorescence MCF-7 cells exhibited increased HER3 half-life compared with the background (Figure 1b, right column). shScramble control MCF-7 cells as demonstrated by treating the We then sought to determine which regions of HER3 and cells for 0, 3 and 6 h with the protein biosynthesis inhibitor NEDD4 were required for their interaction. Previous studies cycloheximide that blocked de novo HER3 protein biosynthesis have shown that WW domains of NEDD4 family E3 ligases interact (Figure 2d). 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 NEDD4 regulates HER3 ubiquitination and degradation through its C-terminal tail. To determine whether the WW domains of the proteasomal pathway NEDD4 contribute to its interaction with the C-terminal tail of To investigate the proteasomal vs lysosomal degradation of HER3, HER3, we transiently expressed either the full-length NEDD4, the we performed experiments using the proteasomal inhibitor fragment consisting of the WW and HECT domains, or the MG132 and the lysosomal inhibitor chloroquine. Consistent with fragment consisting of the HECT domain alone in HEK293T cells previous studies,33 MG132 treatments led to the accumulation of together with the full-length human HER3 (Figure 1c). A HER3 but chloroquine treatments did not (Figure 2e). HER3 haematoxylin (HA) tag was added to the NEDD4 constructs for accumulation caused by MG132 treatment was significantly less in WB detection. Both the full-length NEDD4 and the WW–HECT shNEDD4 knockdown MCF-7 cells compared with the shScramble fragment were detected in the HER3 co-IP, indicating their control (Figure 2e). In contrast, no effect of chloroquine on HER3 association with HER3 (Figure 1d). HECT domain alone was not protein levels were observed when compared with the controls in detected in the HER3 co-IP by WB (Figure 1d), suggesting that the both shScramble and shNEDD4 MCF-7 cells (Figure 2e). Taking WW domains of NEDD4 are required for interacting with HER3. together, these results suggest that NEDD4 regulate HER3 To determine whether NEDD4 interacts with the kinase domain or degradation primarily through the proteasomal pathway instead the C-terminal tail of HER3, we performed IP using antibodies of the lysosomal pathway. To investigate the role of NEDD4 against the extracellular domains of HER3 (N-HER3) in lysates of on HER3 ubiquitination, in vitro ubiquitination assays were CHO cells expressing different chimeras of HER3 and HER2 carried out. In the presence of E1, E2 (UbcH5b) and ubiquitin, (Choi et al.39; Figure 1e). Replacing the kinase domain of HER3 recombinant NEDD4 led to polyubiquitination of HER3 but not with the HER2 kinase domain (HER3-2-3, Figure 1f) did not abolish the HER3 PPRY-AAAA mutant protein (Supplementary Figure S4). the NEDD4-HER3 interaction, suggesting that the kinase domain To evaluate endogenous HER3 ubiquitination, cell lysates of of HER3 was not essential for the interaction. However, replacing shNEDD4 and shScramble control cells were immunoprecipitated the HER3 kinase domain and C-terminal tail with HER2 by an anti-HER3Mab and HER3 ubiquitination was detected

IP PLA assay MCF-7 MDA-MB-453 anti-HER2 anti-NEDD4 anti-HER3 NRG-1: --+--+ + anti-HER3 + anti-HER3 + control Ab Control Ab: +-- +-- IP Ab: -++ -++

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CHO HER3 HER3-2-2 HER3-2-3 HEK293T HEK293T NRG-1: --+--+--+ Control Ab: +--+--+-- HER3 DNA: HER3 HER3 (PPRY-AAAA) 130 N-HER3 IP Ab: -++-++-++ HER3 IP: HER3 93 NEDD4 DNA: -++ N-HER3 IB: HA 70 WB: 53 C-HER3 41 HER3

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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 overexpression showed reduced HER3 levels and increased HER3 ubiquitination was observed in the shNEDD4 knockdown HER3 ubiquitination (Figure 2g). Taken together, these data cells when compared with shScramble control cells (Figure 2f), suggest that NEDD4 contributes to HER3 ubiquitination and suggesting that NEDD4 functions as an E3 ligase of HER3. In modulates HER3 protein levels through proteasomal degradation another experiment, HEK293T cells with recombinant NEDD4 pathway.

MCF-7 MCF-7 shNEDD4 MCF-7 1234 100 WB:

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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 anti- ubiquitin 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 phosphorylation each reached a plateau in shScramble MCF-7 It is well documented that HER3 phosphorylation activates control cells (at 25 ng/ml NRG-1 treatment), but they did not reach downstream PI3K/AKT and ERK signaling pathways.7,8 To deter- a plateau in shNEDD4 knockdown MCF-7 cells (Figures 3d–f). mine whether increased HER3 levels in shNEDD4 knockdown Together, these data suggested that the elevated HER3 upon cancer cells would trigger an increase in HER3-mediated down- shNEDD4 knockdown rendered cells more responsive to NRG-1 stream signaling, we measured phosphorylation of HER3, AKT1 activation, which mediated increased HER3, AKT1 and ERK1/2 and ERK1/2 by WB. In both breast cancer MCF-7 and prostate phosphorylation. 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, NEDD4 knockdown increases HER3-mediated cell migration and both AKT1 and ERK1/2 phosphorylation also increased (Figure 3a). proliferation in vitro and tumor growth in vivo In shNEDD4 knockdown DU145 cells, an increase in NRG-1- The elevated HER3 levels and signaling we observed upon induced AKT1 phosphorylation was observed, but ERK1/2 shNEDD4 knockdown prompted us to investigate whether phosphorylation was not affected (Figure 3b). Unlike HER3 levels, shNEDD4 knockdown would also impact downstream events total protein levels of AKT1 and ERK1/2 were not affected by such as cell migration, proliferation and tumor growth in vivo. shNEDD4 knockdown in both MCF-7 and DU145 cells (Figures 3a While investigating cell migration, we noticed that HER3 shRNA and b). In shNEDD4 knockdown MCF-7 cells, the HER3, AKT1 and knockdown (shHER3) in DU145 cells led to reduced cell migration ERK1/2 phosphorylation were elevated at various NRG-1 concen- and the loss of response to NRG-1 stimulation (Figure 4a). trations but the increase was more pronounced at higher NRG-1 Compared with the shScramble DU145 cells, shNEDD4 knockdown concentrations (Figure 3c). Moreover, HER3, AKT1 and ERK1/2 DU145 cells not only displayed increased migration, but these

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Quantified AKT 5000 Quantified ERK Quantified HER3 phosphorylation signal phosphorylation signal phosphorylation signal 0 0 0 0 1020304050 0 1020304050 0 1020304050 NRG-1 concentration NRG-1 concentration NRG-1 concentration (ng/ml) (ng/ml) (ng/ml) Figure 3. NEDD4 knockdown increases ligand-dependent HER3 phosphorylation and its downstream signaling. Cells were seeded at 50% confluency and starved in serum-free media overnight. (a, b) MCF-7 and DU145 shScramble control and shNEDD4 knockdown cells were treated with NRG-1 (50 ng/ml) for 20 min. Whole cell lysates (WCL) were subjected to immunoblotting using phospho-HER3, phospho-AKT, phospho-ERK, HER3, AKT1 and ERK1/2 antibodies. (c) shScramble control and shNEDD4 knockdown MCF-7 cells were treated with different concentrations of NRG-1 (0, 3, 6, 12, 25 and 50 ng/ml) for 20 min. Total protein and phosphorylation levels of HER3, AKT1 and ERK were evaluated by immunoblotting using respective antibodies. (d–f) HER3, AKT1 and ERK1/2 phosphorylation signals from WBs upon NRG-1 activation were quantified by ImageJ. cells also became more responsive to NRG-1 stimulation NEDD4 knockdown cancer cells are more sensitive toward anti- (Figure 4a), which was consistent with the increased HER3 levels HER3 antibody treatments in vitro and in vivo and NRG-1-dependent HER3 signaling upon NEDD4 knockdown. In three-dimensional (3D) cultures of MCF-7 cells, shNEDD4 The effect of shNEDD4 knockdown on NRG-1-dependent cell knockdown cells formed larger colony spheres than the shScram- proliferation was also examined. In MCF-7 and DU145 shScramble ble cells in the presence of NRG-1 (Po0.01; Figures 6a and b). To control cells, NRG-1 treatment increased cell proliferation by 15 investigate whether such increase is a result of elevated HER3 and 16%, respectively (Figures 4b and c). In contrast, in MCF-7 and signaling, HER3Mab treatments (10 μg/ml) were carried out, which DU145 shNEDD4 knockdown cells, NRG-1 increased cell prolifera- significantly reduced the sphere sizes in both shNEDD4 knock- tion by 30 and 28%, respectively, that was significantly higher than o o down and the shScramble control MCF-7 cancer cells (P 0.05), scramble control cells (P 0.01) (Figures 4b and c). but the effect was more prominent in shNEDD4 knockdown In colony formation assays, shNEDD4 knockdown MCF-7 cells MCF-7 cells than shScramble controls (51% vs 27% reduction in formed more colonies than shScramble control cells, whereas sphere diameters; Figures 6a and b). shHER3 knockdown MCF-7 cells formed the lowest number of In comparison, the spheres formed by shNEDD4 knockdown colonies (Figure 4d). We also noticed that the shNEDD4 knock- DU145 cells are of similar diameters to those formed by down and shScramble cells formed protrusions, but shHER3 knockdown cells did not (Figure 4e). Although MCF-7 is shScramble cells in the absence of NRG-1 (P>0.05); however, considered to display low cell migration, these different cell shNEDD4 knockdown DU145 cells displayed higher NRG-1 morphologies caused by shRNA knockdown of NEDD4 prompted dependent growth compared with shScramble controls (100% vs us to investigate whether these changes in morphologies 70% increase in average sphere diameter upon NRG-1 treatment; fi reflected the alternations in migration potential. Indeed, cell Figures 6c and d). HER3Mab treatment signi cantly reduced the migration increased in shNEDD4 knockdown MCF-7 cells average sphere diameters for both shNEDD4 knockdown and (Po0.01) and decreased in shHER3 knockdown MCF-7 cells shScramble control DU145 cells (Po0.05). However, NRG-1- (Po0.01; Supplementary Figure S5). activated shNEDD4 knockdown DU145 cells were more responsive In xenografts in vivo, shNEDD4 knockdown MCF-7 cells formed to the HER3Mab treatment than shScramble control cells (42% vs significantly larger tumors than shScramble MCF-7 cells (Po0.01; 32% reduction in sphere diameters; Figures 6c and d). Figure 5f). WB analysis also revealed elevated HER3 protein levels We investigated whether elevated HER3-mediated signaling in in shNEDD4 knockdown xenografts (Supplementary Figure S6). NEDD4 knockdown cells can be reduced by HER3Mab treatments. Together, our in vivo data was consistent with the in vitro results, In shNEDD4 knockdown MCF-7 cells, HER3Mab significantly showing that shNEDD4 knockdown in MCF-7 cells led to increased reduced the elevated NRG-1-mediated HER3 and AKT1 phospho- cell growth and tumor formation. rylation (Figure 5a). However, the residual HER3 and AKT1

© 2015 Macmillan Publishers Limited Oncogene (2015) 1105 – 1115 NEDD4 regulates HER3 level and signaling Z Huang et al 1110 DU145 migration MCF-7 proliferation DU145 proliferation ** 300% 150% ** ** ** ** ** 150% ** 250% ** ** 200% ** 100% 100% 150%

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Number of colonies 200 DU145 100 m 0 shScramble shNEDD4 shHER3 shHER3

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 phosphate-buffered saline (PBS) and tumor growth was mon- also inhibited the elevated ERK1/2 phosphorylation in shNEDD4 itored in all groups. shNEDD4 knockdown in MCF-7 cells knockdown MCF-7 cells but the inhibition was less effective significantly increased tumor growth in vivo compared with the (Figure 5a). In shNEDD4 knockdown DU145 cells, HER3Mab shScramble control (Po0.01; Figure 5e). HER3Mab significantly completely neutralized the elevated NRG-1-mediated HER3 and reduced tumor growth in both shNEDD4 knockdown (Po0.01) AKT1 phosphorylation to the basal level (Figure 5b). Unlike HER3 and shScramble control MCF-7 cells (Po0.01) but exerted larger and AKT1 phosphorylation that are NRG-1 dependent, ERK1/2 effect in shNEDD4 knockdown MCF-7 cells (59% vs 36% decrease phosphorylation in DU145 cells was not affected by NRG-1 at day 30; Po 0.01) (Figure 5f). These results suggested that treatment or shNEDD4 knockdown, and HER3Mab treatment only shNEDD4 knockdown MCF-7 cells were more responsive to slightly reduced the ERK1/2 phosphorylation (Figure 5b). HER3Mab treatment in vivo. 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 Inverse relationship between NEDD4 and HER3 expression in shNEDD4 knockdown cells compared with shScramble control ductal cells of prostate cancer tumors cells (Po0.01; IC50 of 0.22 vs 0.44 μg/ml; Figure 5c). However, the To investigate whether the inverse correlation between NEDD4 maximum inhibition rate was not significantly different (P = 0.17, and HER3 levels observed in the shNEDD4 knockdown cell lines 43% vs 36%, Figure 5c). In DU145 cells, the maximum inhibition also existed in clinical samples, immunohistochemistry were rate of HER3Mab in shNEDD4 knockdown cells was significantly performed on tissue micro arrays of prostate cancer tumor higher than that in shScramble control cells (Po0.01; 63% vs sections from 60 patients. It was noticed that staining of NEDD4 39%), and the IC50 of HER3Mab was significantly lower in shNEDD4 and HER3 showed a reverse pattern. NEDD4 mostly expressed in knockdown cells (Po0.05; IC50 of 0.20 vs 0.32 μg/ml; Figure 5d). the ductal epithelial cells where HER3 showed higher expression Together, the increased maximum inhibition rate and lower IC50 in regions surrounding the ducts (Figure 7a). Spectral unmixing demonstrated that HER3 antibody treatments are more effective was performed to separate the HA and diaminobenzidine in shNEDD4 knockdown MCF-7 and DU145 cells. channels, which enabled visualization and quantification of the The in vivo efficacy of HER3 antibody was investigated in MCF-7 NEDD4 and HER3 (Supplementary Figure S7) in 60 randomly xenografts. shNEDD4 knockdown and shScramble control MCF-7 selected prostate ducts and surrounding regions (Supplementary cells were injected into two groups of nude mice and both groups Figure S8). To enable comparison, the average NEDD4 and HER3 were randomly divided in half at day 9 before treatments. The staining in the ductal regions were designated values of 1.0 animals received intraperitoneal injections of HER3Mab, or (Figure 7b). In the ductal regions, NEDD4 levels were significantly

MCF-7 DU145 MCF-7 xenografts shRNA: Scr NEDD4 Scr NEDD4 shRNA: Scr NEDD4 Scr NEDD4 HER3Mab: ----++++ HER3Mab: ----++++ 700 NRG-1: -+-+-+-+ NRG-1: -+-+-+-+ shNEDD4 600 NEDD4 NEDD4 + PBS

pHER3 pHER3 ± SEM) 500

(Y1289) (Y1289) 3 ** shScramble ** + PBS HER3 HER3 400

pAKT1 pAKT1 300 ** shNEDD4 (S473) (S473) + HER3Mab 200 AKT1 AKT1

Tumor volume (mm 100 shScramble pERK1/2 pERK1/2 Treatment HER3Mab + HER3Mab 0 duration: ERK1/2 ERK1/2 0 5 10 15 20 25 30 35 Time (Days) Actin Actin

MCF-7 DU145 MCF-7 xenografts shScrambleIC50: 0.43 g/ml shScramble IC50: 0.32 g/ml shScramble shNEDD4 50% shNEDD4 80% IC50: 0.22 g/ml shNEDD4 IC50: 0.20 g/ml HER3Mab: -+-+ 40% IC50: 60% IC50: ** P<0.01 * P<0.01 30% InhibitionMax: 40% InhibitionMax: 20% P=0.17 ** P=0.17 20% 10% % Proliferation inhibition 0% % Proliferation inhibition 0% 0.01 0.1 1 10 0.01 0.1 110 10 mm HER3Mab (g/ml) HER3Mab (g/ml) Figure 5. Anti-HER3 antibody displayed higher efficacy in inhibiting the signaling and growth of shNEDD4 knockdown cancer cells in vitro and in vivo.(a, b) shScramble control and shNEDD4 knockdown MCF-7 and DU145 cells were treated with anti-HER3 antibody HER3Mab for 2 h, followed by 20 min NRG-1 (50 ng/ml) activation. Cell lysates were collected and the total levels of HER3, AKT1 and ERK1/2 and their phosphorylation levels were examined by WB using respective antibodies. (c, d) shScramble control and shNEDD4 knockdown MCF-7 and DU145 cells were treated with NRG-1 (50 ng/ml) together with different concentrations of HER3Mab in cell culture media containing 0.5% FBS over 72 h. Cell proliferations were measured by the Alamar Blue assay. HER3Mab exhibited bigger inhibitory effects in the proliferation of shNEDD4 knockdown MCF-7 and DU145 cells than the shScramble controls. (e)5×106 shScramble control or shNEDD4 knockdown MCF-7 cells were subcutaneously injected into nude mice (n = 8) and the sizes of the tumor were measured twice every week. At day 9, shNEDD4 and shScramble groups were randomly divided in half (n = 4). Between days 9 and 30, xenografts were treated with PBS control or HER3Mab (5 mg/kg) twice a week. The results shown were mean tumor volume ± s.e.m. Student’s t-test was applied for pairwise statistical comparisons, **Po0.01. (f) Xenografts were excised and imaged at day 30. higher than HER3 (Po0.01; Figure 7c). In contrast, NEDD4 It is common for multiple E3 ligases to regulate a single expression levels in the stromal regions were significantly lower substrate. For example, the proteasome degradation and than that of HER3 levels (Figure 7d). In addition, there was an subcellular localization of p53 have been shown to be regulated inverse correlation (n = 60, Po0.01, R = − 0.59) between NEDD4 by E3 ligases Mdm2, Pirh2 and COP1.49–51 In the EGFR family, and HER3 levels in the ductal regions (Figure 7e). multiple E3 ligases (WWP1, ITCH and NEDD4) are involved in ubiquitination of HER4.31,46 Previously, RING finger E3 ligase Nrdp1 was shown to ubiquitinate HER3 and regulate steady-state DISCUSSION HER3 levels.32,33 Our data indicate that a HECT family E3 ligase This study reports, for the first time, that NEDD4 functions as an E3 NEDD4 is a also an E3 ligase of HER3. Nrdp1 and NEDD4 ligase of HER3 that regulates steady-state HER3 levels. shNEDD4 have different protein structures and interact with HER3 on knockdown in MCF-7 and DU145 cells resulted in elevated HER3 different sites. Although our results showed that NEDD4 interacted levels and enhanced NRG-1-dependent HER3 downstream AKT with the C-terminal tail of HER3, previous study showed that and ERK signaling, and cancer cell proliferation, and migration Nrdp1 did not associate with the C-terminal tail of HER3 but in vitro and in vivo. More importantly, we demonstrated that instead associated with either the juxtamembrane domain or the NEDD4 knockdown resulted in elevated HER3 signaling and kinase domain of HER3.52 Taken together, this study suggests that sensitized the cancer cells to anti-HER3Mab treatment in vitro and Nrdp1 and NEDD4 could offer different mechanism of action in in vivo. Our working model for the HER3/NEDD4 interaction and its the regulation of HER3 levels. Further studies are needed to underlining biology is shown in Figure 7f. elucidate how cells differentially regulate HER3 ubiquitination by NEDD4 has been shown to be an E3 ligase for multiple proteins, the two seemingly independent E3 ligases under different cellular including cell surface receptors IGF1R, FGF1R and HER4 (refs. contexts. 31,36,47,48), and the WW domains of NEDD4 has been shown to There is currently an intense effort toward developing mono- interact with the PPXY motifs of fibroblast growth factor-1.36 A clonal antibodies targeting HER3 for the treatment of various PPXY motif is present in the C-terminal tail of HER3 but not in types of cancer.20,53,54 The regulation of HER3 by NEDD4 E3 ligase HER2. Transient expression of NEDD4 in HEK293 cells markedly has significant implications in our understanding of HER3 in reduced HER3 levels but not the levels of HER2 or the mutated cancer biology. Increase of HER3 expression in prostate cancer was HER3 (PPXY-AAAA), suggesting that the PPXY motif is critical for found in microarray55 and immunohistochemistry studies.56–58 the regulation of NEDD4 on HER3 levels. Our finding of a negative correlation between NEDD4 and HER3

MCF-7 3D cultures DU145 3D cultures shScramble shNEDD4 shScramble shNEDD4

Phalloidin DAPI Control

Control 100 μm μ 100 μm 100 μm 100 μm 100 μm 100 μm 100 μm 100 m NRG-1 NRG-1 μ μ μ 100 μm 100 μm 100 μm 100 m 100 μm 100 μm 100 m 100 m + NRG-1 + NRG-1 HER3Mab μ μ μ 100 μm 100 μm 100 μm 100 μm HER3Mab 100 m 100 m 100 μm 100 m

MCF-7 spheroids diameters DU145 spheroids diameters

200 No treatment 200 No treatment m) NRG-1 m) NRG-1 175 ** 175 * NRG-1 + Ab * NRG-1 + Ab 150 * 150 ** ** * 125 ** * * 125 100 100 ** * 75 75 50 50 25 25 Diameter of 3D spheres ( 0 Diameter of 3D spheres ( 0 shScramble shNEDD4 shHER3 shScramble shNEDD4 shHER3 Figure 6. The increased NRG-1-dependent 3D spheroid growth in NEDD4 knockdown cancer cells was antagonized by an anti-HER3 antibody. (a, c) shScramble control and shNEDD4 knockdown MCF-7 and DU145 cells were seeded on top of Matrigel and was incubated in media only, media with NRG-1 (50 ng/ml), or a combination of NRG-1 and HER3Mab (10 μg/ml) for 10 days. The 3D spheres were visualized by phase contrast microscopy and confocal microscopy. Cells were stained with rhodamine-labeled phalloidin (red) against F-actin and DRAQ5 (blue) against nuclei. Scale bars, 100 μm. (b, d) The sizes of MCF-7 and DU145 3D spheres were quantiﬁed by measuring the diameter of 100 spheres from 10 random ﬁelds. *Po0.05, **Po0.01.

levels in prostate cancer cells and tissues suggests the importance HEK293 freestyle cells (Life Technologies, Carlsbad, CA, USA) and purified of NEDD4 in HER3-driven cancers. Further investigation of the role to above 95% purity using protein A/G affinity chromatography. Antibody of NEDD4 in HER3 regulation and signaling should help determine purity was verified by protein gel electrophoresis and antibody binding whether NEDD4 levels can serve as a responding marker for the was confirmed by ELISA binding assays, flow cytometry analysis. The ability fi development of HER3 targeting cancer antibody therapies. to inhibit HER3 phosphorylation upon NRG-1 activation were veri ed by ELISA and WBs as described by our group previously.39

MATERIALS AND METHODS Transfection, overexpression and stable shRNA knockdown Cells, antibodies and other reagents For stable expression, pcDNA5/FRT vectors containing the human HER3 HEK293T, CHO, MDA-MB-453, MCF-7, DU145, LNCaP, CWR 22RV1 and PC3 and HER3/2 chimera constructs were transfected into CHO cells and cells were obtained from the American Type Culture Collection (Manassas, incorporated into the chromosomes via the ﬂip-in system (Life Technol- VA, USA) and maintained in supplier speciﬁed media. Monoclonal ogies) under hygromycin selection (400 μg/ml). For transient expression, antibodies against phospho-HER3 (Y1289), C-terminal HER3, AKT1, the pCEP4 vector containing human full-length NEDD4 and fragments, as phospho-AKT1, ERK1 and phospho-ERK (202/204) were obtained from well as the pcDNA5/FRT vector containing human HER3, were transfected Epitomics, Burlingame, CA, USA. Monoclonal antibodies against NEDD4 into HEK293T cells using Lipofectamine 2000 (Life Technologies). For stable (C5F5) and ubiquitin (P4D1) were obtained from Cell Signaling Technology, shRNA knockdowns, lentiviral particles encoding shRNA targeting HER3, Danvers, MA, USA. Antibodies against N-terminal HER3 and Nrdp1 were NEDD4 and scramble control (pLKO.1 shRNA system, Sigma-Aldrich) were obtained from Bethyl Labs, Montgomery, TX, USA. Beta-actin antibody was incubated with target cell lines for 24 h and cells were selected in RMPI obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. NRG-1 (R&D media containing puromycin (2 μg/ml) for 3 weeks. Systems, Minneapolis, MN, USA), cycloheximide (Sigma-Aldrich, St Louis, MO, USA) and MG132 (EMD Millipore, Billerica, MA, USA) were also used in our experiments. The HER3 neutralizing antibody HER3Mab is a mono- Cell lysis and IP clonal antibody that has the variable sequences of the U1-59 (US patent Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (Boston 7,705,130 B2) and the backbone of IgG1. HER3Mab was expressed in BioProducts, Ashland, MA, USA) containing protease inhibitor cocktail V

Prostate cancer TMA Stromal Prostate cancer TMA ductal staining Normal pHER3 pAKT pERK Proliferation NEDD4 NEDD4

Ductal High (2.0) Medium (1.0) Low (0.5)

Stromal

shNEDD4 ↑ pHER3 HER3

HER3 ↑ pAKT pERK ↑ Proliferation Ductal Low (0.5) Very Low (0.2)

Ductal Stromal Ductal: NEDD4 vs HER3 n=60 **: P<0.01 n=60 **: P<0.01 2.5 n=60 ** P<0.01 2 R=-0.59 1.5 NEDD4+HER3Mab 1 ↓ pHER3 0.5 ↓ pAKT pERK ↓ Proliferation 0 NEDD4 relative intensity 0 0.1 0.2 0.3 0.4 0.5 HER3 relative intensity

Figure 7. Differential expression of NEDD4 and HER3 in tissue array slides. (a) Parafﬁn-embedded prostate cancer tissue arrays were stained using NEDD4 and HER3 antibody and counterstained with hematoxylin. In prostate cancer tissue sections, negative correlation between NEDD4 and HER3 expression was observed. NEDD4 expression was found in ductal epithelial cells near the ducts where HER3 expression was found in stromal regions. (b) The intensities of the average ductal NEDD4 and HER3 staining were each given a relative intensity of 1.0. Representative high (2.0) and low (0.5) level staining of NEDD4 and HER3 were shown. (c) Ductal regions exhibited high NEDD4 levels and low HER3 levels (**Po0.01). (d) Stromal regions exhibit high HER3 levels and low NEDD4 levels (**Po0.01). (e) NEDD4 and HER3 levels in the ductal regions show a negative correlation with R value of À0.59. (f) Working model for the HER3/NEDD4 interaction: (top) normal levels of NEDD4 negatively regulate steady-state HER3 levels and maintain normal HER3, AKT1 and ERK1/2 phosphorylation upon NRG-1 activation. (Middle) shNEDD4 knockdown compromises the negative regulation of HER3 levels, therefore leading to increased HER3 levels and enhanced NRG-1 dependent HER3, AKT1 and ERK1/2 phosphorylation. (Bottom) The increased HER3, AKT1 and ERK1/2 phosphorylation caused by shNEDD4 knockdown can be neutralized by an anti-HER3Mab.

(Calbiochem, Billerica, MA, USA) and phosphatase inhibitor cocktail II Ubiquitination assays (Sigma-Aldrich). NRG-1 treatments were for 20 min at 50 ng/ml. For co-IP, To detect HER3 ubiquitination, cells were lysed in RIPA buffer with an 750 μg of cell lysates were incubated with 1 μg of anti-HER3 (HER3Mab) additional 1% SDS and heated at 95 °C for 5 min to dissociate protein and anti-NEDD4 (C5F5, Cell Signaling Technology) antibodies at 4 °C complexes. The heated lysates were diluted in 10 × volume of RIPA buffer. followed by 2 h incubation with protein G Dynabeads (Life Technologies) HER3 were immunoprecipitated from 500 μg of total cell lysates using anti- at 4 °C. Samples were washed three times with 500 μl RIPA buffer, HER3 antibody (Epitomics) and blotted with anti-ubiquitin antibodies re-suspended in 30 μl of 2 × SDS sample buffer and heated at 90 °C for (P4D1, Cell Signaling Technology). 10 min. For in vitro ubiquitination assays, each 30 μl reaction contained 300 ng of NEDD4 protein (Sigma-Aldrich) and immunoprecipitated HER3 from HEK293T cells that transiently expresses HER3 and HER3 PPRY-AAAA Mass spectrometry mutants. Reactions were performed following the manufacturer’s IP cell extracts were separated on 4–15% SDS–PAGE under reducing instructions (Boston Biochem, Cambridge, MA, USA), which included 50 condition. Gel lanes between 20–150 kDa were divided into 10 consecutive ng of E1, 200 ng of E2 (UbcH5b), 2 μg ubiquitin and ubiquitin reaction μ sections and subjected to in-gel digestions by trypsin (0.12 ng/ l) at 37 °C buffer (40 mM Tris, 5 mM MgCl2,2mM ATP and 2 mM DTT; pH 7.4). Reactions overnight. Tryptic digests were analyzed as described in the Supplemental were incubated at 30 °C for 2 h, stopped by 2 × SDS loading buffer, heated Information. at 95 °C for 10 min, separated on a 10% SDS–PAGE gel, and visualized by immunoblotting using an anti-ubiquitin antibody (P4D1, Santa Cruz Biotechnology). In situ PLAs For PLA, cells incubated with anti-NEDD4 (Cell Signaling Technology) and anti-HER3 (Epitomics) antibodies were incubated with PLA secondary Cell proliferation, colony formation, cell migration assays and 3D antibodies and substrates (Olink Biosciences, Uppsala, Sweden) as cell cultures described.40 Images were captured by a Carl Zeiss ﬂuorescence For cell proliferation assay, 1000 cells were seeded into 96-well clear microscope, Thornwood, NY, USA. bottom black plates (Fisher Scientiﬁc, Pittsburgh, PA, USA). Seventy-two

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