Published OnlineFirst May 14, 2014; DOI: 10.1158/1541-7786.MCR-13-0637 Molecular Cancer Genomics Research Convergent and Divergent Cellular Responses by ErbB4 Isoforms in Mammary Epithelial Cells Vikram B. Wali, Jonathan W. Haskins, Maureen Gilmore-Hebert, James T. Platt, Zongzhi Liu, and David F. Stern Abstract Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions. Implications: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer. Mol Cancer Res; 12(8); 1140–55. Ó2014 AACR. Introduction Inconsistent associations of ERBB4 with cancer may be The four receptor kinases in the epidermal growth factor explained by the diversity of ERBB4-regulated signaling (EGF) family, EGFR, ERBB2, ERBB3, and ERBB4 regu- processes enabled by mRNA splice variants. JM-a and late developmental processes in the nervous system, cardio- JM-b isoforms differ in the extracellular juxtamembrane vascular system, and in epithelia. EGFR, ERBB2, and domain (8). JM-b isoforms are conventional receptor tyro- ERBB3 are common drivers in human carcinoma and sine kinases (RTK): The ligands, including neuregulin 1 glioblastoma and are targets for cancer therapeutics approved (NRG1), induce receptor phosphorylation and activate by the US FDA. But, ERBB4 has a more ambiguous subsequent signal transduction. In contrast, JM-a isoforms influence on cancer. ERBB4 is overexpressed in medullo- have a metalloproteinase cleavage site that is clipped by blastoma, and candidate ERBB4–activating mutations have tumor necrosis factor-a-converting enzyme (TACE) in been identified in lung cancer, melanoma, and other cancers response to NRG1 binding. This releases the extracellular (1–4). Nonetheless, conflicting reports have been published domain, leaving the membrane-anchored m80 form. on ERBB4 as a prognostic marker, with both positive and ERBB4 m80 can then undergo intramembrane cleavage by negative clinical outcome correlations (5–7). g-secretase to release the soluble s80 form comprising the intracellular domain (ICD). s80 relocalizes to mitochondria and the nucleus (9, 10), in which it binds transcriptional Authors' Affiliation: Department of Pathology, Yale School of Medicine, coregulators and transcription factors. New Haven, Connecticut A second alternatively spliced region in the ICD includes Note: Supplementary data for this article are available at Molecular Cancer (CYT-1) or excludes (CYT-2) an exon that encodes a binding Research Online (http://mcr.aacrjournals.org/). site for the p85 adaptor subunit of phosphatidyl inositol (30) V.B. Wali and J.W. Haskins contributed equally to this work. kinase, and an overlapping WW domain PPXY-binding site. Corresponding Author: Vikram B. Wali, Yale University School of Med- Divergence of signaling processes incited by the four ERBB4 icine, 300 George Street, New Haven, CT 06511. Phone: 203-737-6491; isoforms may explain the discordance in the ERBB4 cancer Fax: 203-785-7232; E-mail: [email protected] literature: Most studies fail to consider these isoforms sepa- doi: 10.1158/1541-7786.MCR-13-0637 rately, and the isoform(s) expressed and subcellular localiza- Ó2014 American Association for Cancer Research. tion of ERBB4 have an impact on prognosis (11, 12). 1140 Mol Cancer Res; 12(8) August 2014 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 14, 2014; DOI: 10.1158/1541-7786.MCR-13-0637 Biologic Effects of ERBB4 Isoforms in Mammary Epithelial Cells We previously identified binding of both ERBB4 ICD aged as lentivirus by cotransfecting 293T cells with pLP/ isoforms (CYT-1 and CYT-2) with the transcriptional VSV-G, pLP1(Gag/Pol), pLP2(rev), and pcTat (tat) using corepressor KAP1, and identified 16 other candidate inter- Lipofectamine 2000 (Invitrogen Corporation). MCF10A actors, including ubiquitin ligases ITCH and WWP2 (13). cells were infected with a multiplicity of infection of approx- The ERBB4 ICD has been reported by others to associate imately 5 in the presence of 8 mg/mL polybrene. Expression with transcription factors ERa and Stat5, with transcrip- of ERBB4 in these MCF10A cells was tested 24 and 72 hours tional coregulators, including YAP, WWOX, ETO2, and a after infection. Polyclonal stable cell lines were selected with TAB2/N-CoR complex, and with ubiquitin ligases Itch and puromycin. The above FL CYT-1 and CYT-2 ERBB4 Mdm2 (14–20). To better understand the diverse biologic constructs were also packaged into pInducer20 DOX-induc- outcomes associated with activity of the full-length (FL) and ible expression plasmids that were used to infect ERBB4 KD truncated ERBB4 isoforms, we have explored the pheno- T47D stables to reexpress specific JM-a ERBB4 CYT-1 or typic, transcriptional and signaling consequences of intro- CYT-2 isoform. The pInducer20 DOX-inducible expres- duction and activation of ERBB4 isoforms, and identified sion plasmid used for cloning was generously provided by candidate gene target interactions by chromatin immuno- Dr. Stephen Elledge, Department of Genetics, Harvard precipitation-sequencing (ChIP-seq). Medical School (21). ICD expression cDNAs encoding CYT-1 (amino acids 676–1308) and CYT-2 (amino acids 676–1294) isoforms Materials and Methods (GeneCopoeia) were cloned into the lentiviral TA cloning Cell culture vector Lenti6.3-V5 in frame with the 30 V5 epitope tag (Life MCF10A cells were maintained in DMEM/F12 supple- Sciences Technologies). Stable cell lines for the ICDs and the mented with 5% horse serum, 20 ng/mL EGF, 0.5 mg/mL vector control (vector) were selected with blasticidin. hydrocortisone, 100 ng/mL cholera toxin, 10 mg/mL insu- lin, 100 U/mL penicillin, and 100 mg/mL streptomycin. Immunoblotting MCF10A cells stably expressing FL JM-a CYT-1–ERBB4 For NRG1 stimulation, cells were plated at 1 Â 106 cells isoform (CYT-1 MCF10A) or JM-a CYT-2–ERBB4 iso- per 100-mm plate. The following day, cells were incubated form (CYT-2 MCF10A) or vector only (V-MCF10A) were in serum-free OptiMEM medium for 48 hours, followed by generated by lentiviral infection and selection with 10 incubation with 100 ng/mL NRG1. Sample buffer lysates mg/mL puromycin and maintained in 1 mg/mL puromycin. normalized for protein concentration were analyzed by MCF10A cells stably expressing either of the ICD ERBB4 electrophoresis in 4% to 12% NuPAGE SDS–polyacryl- isoforms: CYT-1 or CYT-2 were produced by lentiviral amide midigels (Life Technologies Corporation). For immu- infection, selection in with 10 mg/mL blastocidin and noblotting, polyvinylidene difluoride membranes were maintenance in 7 mg/mL blastocidin. T47D and MDA- blocked with 2% BSA in 10 mmol/L Tris-HCl, 50 MB-231 cells were cultured in RPMI-1640 with glutamate mmol/L NaCl, 0.1% Tween 20, pH 7.4 (TBST), and (Gibco) containing 100 U/mL penicillin, 100 mg/mL strep- incubated with anti–phospho-ERBB4 (Tyr 1056; ref. 22), tomycin, and 10% fetal bovine serum (BioWest). FuGENE phospho-ERBB4 Tyr 1284 (Cell Signaling Technology; 6 (Roche) or Lipofectamine 2000 reagent (Invitrogen Cor- #4757), ERBB4 (sc-283), GAPDH (Santa Cruz Biotech- poration) were used for transfections. T47D cells were nology), phospho-MAPK (Thr202/Tyr204), or phospho- 0 transduced with pLKO ERBB4 3 untranslated region AKT (Ser473;Cell Signaling Technology) diluted 1:5,000 to (UTR)–directed shRNA (Sigma; TRCN0000314628) or 1:20,000 in TBST/2% BSA for 2 hours. Membranes were scrambled control and selected in 1 mg/mL puromycin. washed five times with TBST, incubated with horseradish These ERBB4 knockdown (KD) T47D stable cell lines peroxide–conjugated secondary antibodies in TBST/2% were subsequently infected with pInducer20 ERBB4 BSA for 1 hour, rinsed with TBST, and detected by chemi- JM-a CYT-1, CYT-2, or vector control and selected in luminescence (SuperSignal West Pico Chemiluminescent 400 mg/mL G418. T47D ERBB4 KD, pInducer20 CYT- Substrate; Pierce). 1 or CYT-2 stable cell lines were maintained in 1 mg/mL puromycin, 200 mg/mL G418, and ERBB4 KD and doxy- Cell proliferation assays cycline (DOX)-inducible ERBB4 isoform reexpression was In Fig. 2A–C, cells were plated at 1,000 cells per well in confirmed by Western blot analysis. 96-well plates. The next day, four wells per group were fed either serum-free OptiMEM medium or 5% horse serum Plasmids containing medium, Æ 100 ng/mL NRG1, and incubated Lentiviral expression plasmids for JM-a FL CYT-1 for 5 days with refeeding day 2. Proliferation was assayed ERBB4 (EX-A0212-Lv105), CYT-2 ERBB4 (EX-Z4265- daily using the ATP-based CellTiter-Glo Luminescent Cell Lv105), and negative control vector (EX-EGFP-Lv105), Viability Assay (Promega).