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Clinical Research |
Departments of 1 Biochemistry and 2 Structural and Cellular Biology, Tulane University Health Sciences Center, Tulane Cancer Center; 3 Gene Therapy Program, The Morphology and Imaging Core Laboratory, Louisiana State University Health Sciences Center, New Orleans, Louisiana; 4 Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina; and 5 Endocrine Cancer Group, Section of Surgical and Translational Research, Glasgow University, Glasgow, Scotland, United Kingdom
Requests for reprints: Frank E. Jones, Department of Biochemistry, SL43, Tulane University Health Sciences Center, Tulane Cancer Center, 1430 Tulane Avenue, New Orleans, LA 70112-2699. Phone: 504-988-6585; Fax: 504-584-2739; E-mail: fjones{at}tulane.edu or John M.S. Bartlett, E-mail: j.m.bartlett{at}clinmed.gla.ac.uk.
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Introduction |
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Three members of the epidermal growth factor receptor (EGFR) family, EGFR, ERBB2/HER-2/neu (referred to here as ERBB2), and ERBB3, are associated with mitogenic pathways directly contributing to aggressive tumor phenotypes (2); however, the final member of this family, ERBB4, suppresses tumor cell proliferation (3, 4), possibly through modulation of cell death pathways (4). The influence of the EGFR family on human cancer is most apparent in breast cancer where ERBB2 is a key player during the progression of primary tumors to widespread and often lethal metastatic disease (2). In contrast, the levels of ERBB4 protein expression in breast cancer inversely correlate with tumor grade (5, 6), disease recurrence (7), and overall worsening patient prognosis (8, 9). Paradoxically, ERBB4 expression is essential for normal breast function (10, 11), prompting us to investigate the molecular mechanisms underlying the loss of ERBB4 expression during breast tumor progression. Consistent with clinical observations, initial studies of ligand-activated ERBB4 in breast cancer cell lines identified an antiproliferative function for ERBB4 (3), and our most recent findings suggest that ERBB4 induces apoptosis when reintroduced into transformed cells (4).
In addition to its unique ability to suppress malignant cell growth, ERBB4 is also the only EGFR family member to undergo ligand induced proteolytic processing. Ligand activation of ERBB4 results in tumor necrosis factor-
converting enzyme (TACE) cleavage and shedding of the 120-kDa receptor ectodomain, whereas the 80-kDa intracellular domain remains tethered to the cell membrane (12). Subsequent intramembrane cleavage by presenilin-dependent 
-secretase activity results in release of the ERBB4 intracellular domain (4ICD; refs. 4, 13, 14). Interestingly, 4ICD accumulates within several subcellular compartments, including endosomes (15), the nucleus (16), and mitochondria (4). A physiologic role for 4ICD has been identified in the nucleus where 4ICD directly regulates gene expression (4, 16, 17) by functioning as a nuclear chaperone for the transcription factor signal transducers and activators of transcription 5A (STAT5A) followed by 4ICD binding to DNA at STAT5A target promoters (16). Although both ERBB4 transcriptional coregulation and cell-killing activity require proteolytic processing at the cell surface to release 4ICD (4), the molecular mechanisms underlying ERBB4 apoptotic activity remain to be established.
Here, we show that ligand activation and proteolytic processing of ERBB4 in multiple breast cancer cell lines results in mitochondrial accumulation of 4ICD, cytochrome c release from permeabilized mitochondria, and apoptotic cell death. We further show that 4ICD harbors a BH3 domain, which is essential for 4ICD proapoptotic activity. Thus, 4ICD represents a novel BH3-only protein member of the BCL-2 family, capable of directly coupling a cell surface signal to mitochondrial dysfunction.
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Materials and Methods |
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Cell lines. The cell line hTERT-HME was purchased from Clontech (Palo Alto, CA), and the human breast cancer cell lines MDA-MB-231, MDA-MB-361, T47D, MCF-7, and SKBr3 were purchased from the American Type Cell Culture (Manassas, VA). All cell lines were maintained according to the manufacturer's recommendations. The MCF-7/BCL-2 cell line stably overexpressing human BCL-2 has been described elsewhere (18). Generation and growth conditions of the mouse embryonic fibroblast (MEF) cell lines with deletions of BAX (BAX–/–), BAK (BAK–/–), or both (BAX/BAK–/–) have been described elsewhere (19).
Apoptosis assays. Apoptosis was determined visually by examining cells at 40 hours after transfection using an inverted Leica DMIRB fluorescent microscope and calculating the percentage of EGFP-positive cells displaying morphologic signs of apoptosis following 4',6-diamidino-2-phenylindole staining. All samples were prepared in duplicate, and each experiment was repeated at least thrice. Significant differences between data sets was determined using the paired Student's t test.
Apoptosis was also determined by Annexin V-APC/7-AAD (BD PharMingen, San Diego, CA) staining of cells cotransfected with pEGFPN3 and pLXSN, pLXSN-ERBB4, or pLXSN-ERBB4KD exactly as described by the manufacturer. The level of apoptosis was determined by flow cytometry analysis of EGFP-positive cells using a Becton Dickinson FACSCalibur. Data was analyzed using CellQuest (Becton Dickinson, Mountain View, CA) software according to the manufacturer's instructions. All samples were prepared in duplicate and each experiment was repeated at least thrice. Significant differences between data sets was determined using the paired Student's t test.
Apoptosis induced DNA fragmentation of heregulin ß1 (R&D Systems, Minneapolis, MN)–stimulated T47D cells was determined by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assay. In these experiments, T47D cells were stimulated with 50 ng/mL of heregulin ß1 for 24 or 48 hours, and DNA fragmentation was detected using ApopTag Fluorescein In situ Apoptosis Detection kit (Chemicon) exactly as described by the manufacturer. Where indicated, T47D cells were preincubated for 1 hour before heregulin ß1 stimulation with 20 µmol/L of the broad spectrum caspase inhibitor Z-VAD-FMK (zVAD; BD PharMingen).
Suppression of ERBB4 expression. To suppress expression of endogenous ERBB4, T47D cells were transfected with erbB-4/HER-4 siRNA SMARTpool or Nonspecific siRNA Negative Control Pool using siIMPORTER transfection reagent (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions. Suppression of ERBB4 was confirmed by Western blot analysis of total cell lysates as described previously (20) using antibodies directed against ERBB4 (Santa Cruz Biotechnology, Santa Cruz, CA) and -tubulin (Upstate Biotechnology) as a loading control.
In vitro transcription/translation and pull-down assay. In vitro transcription/translation was done with linearized pBluescript II SK, pBl4ICD-Flag, pBl4ICDdelBH3-Flag, or pcDNA-BCL-2 (generously supplied by Stanley Korsmeyer, Harvard Medical School, Boston, MA) using the TnT Quick Coupled Transcription/Translation System (Promega, Madison, WI) supplemented with 20 µCi of Redivue L-[35S]methionine (Amersham, Arlington Heights, IL) exactly as described by the manufacturer. The pull-down assay was done using standard procedures with ANTI-FLAG M2-Agarose (Sigma, St. Louis, MO) as the affinity reagent.
Mitochondrial staining in situ. Mitochondrial localization of ERBB4-EGFP in transfected SKBr3 cells was visualized at 24 hours after transfection by incubating transfected cells in growth media containing 250 nmol/L Mito Tracker Red (Molecular Probes, Eugene, OR) for 30 minutes at 37°C. Cells were fixed in 4% paraformaldehyde, counterstained with Hoechst, and coverslipped with Prolong Antifade Media (Molecular Probes). The slides were analyzed by deconvolution microscopy on a Leica DMRXA automated upright epifluorescent microscope (Leica Microsystems, Bannockburn, IL).
Subcellular fractionation. Subcellular fractions were prepared from ca. 3 x 107 cells using modifications of procedures described elsewhere (21, 22). Cells were harvested by scraping and resuspended into 600 µL of hypotonic RSB buffer [10 mmol/L Tris-HCl (pH 7.5), 10 mmol/L NaCl, 1.5 mmol/L MgCl2, with Complete Protease Inhibitors (Roche Diagnostics, Indianapolis, IN)] and incubated on ice for 20 minutes. Cells were disrupted with 25 strokes of a Dounce homogenizer fitted with a B pestle followed by 20 passages through a 27-gauge needle. A 2.5x MS buffer [12.5 mmol/L Tris-HCl (pH 7.5), 525 mmol/L mannitol, 175 mmol/L sucrose, 2.5 mmol/L EDTA (pH 7.5)] was added to the iso-osmotic concentration of 1x MS (400 µL). Cell lysates were centrifuged at 1,300 x g for 6 minutes at 4°C. In some experiments, this cleared lysate was processed, as described below, to assay for BAK oligomerization. A crude mitochondria pellet was obtained by centrifugation of the cleared lysate at 16,100 x g for 20 minutes at 4°C. The supernatant was centrifuged at 100,000 x g for 1 hour at 4°C. The supernatant from this spin was saved as the cytosolic fraction (Cyto), and the pellet was dissolved in 200 µL of radioimmunoprecipitation assay buffer [10 mmol/L Tris (pH 8), 140 mmol/L NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% NP40] and saved as the endoplasmic reticulum/microsomal fraction. The crude mitochondria pellet from above was resuspended in 1 mL of ice-cold 1x MS buffer, and mitochondria were purified at the interface of a 1.0:1.5 mol/L sucrose step gradient by centrifugation in a swinging bucket rotor at 60,000 x g for 1 hour at 4°C. The isolated mitochondria were diluted to 0.25 mol/L sucrose by adding 4 volumes of dilution buffer [5 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA (pH 7.5)] and then pelleted by centrifugation at 16,100 x g for 20 minutes at 4°C. The mitochondria pellet (Mito) was dissolved in 200 µL of RIPA buffer. Fifty micrograms of total protein were separated by PAGE and analyzed by Western blot using antibodies directed against ERBB4 (Santa Cruz Biotechnology), BAX (Upstate Biotechnology), BAK (Upstate Biotechnology), calnexin (Stressgen Bioreagents), cytochrome c (BD Biosciences, San Jose, CA), and TOM40 (Santa Cruz Biotechnology).
Assay for BAK oligomerization. The cleared cell lysate from above was cross-linked with 1 mmol/L bismaleimidohexane (Pierce Chemical Co., Rockford, IL) for 30 minutes. The reaction was quenched by incubating with 1 mmol/L ß-mercaptoethanol for 15 minutes, and cross-linked mitochondria were pelleted by centrifugation at 16,100 x g for 20 minutes. BAK oligomerization was analyzed by Western blot.
Western blot analysis of poly(ADP-ribose) polymerase cleavage. Cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP) was examined in heregulin ß1–stimulated T47D cells by Western blot analysis. Briefly, T47D cells were mock stimulated or stimulated with 50 ng/mL heregulin ß1 for 12, 24, or 48 hours, and cell lysates were prepared in a high salt extraction buffer [20 mmol/L HEPES (pH 7.9), 350 mmol/L NaCl, 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.5 mmol/L DTT, 1% NP40, 20% glycerol, and supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, and Complete Protease Inhibitor (Roche Diagnostics)]. Fifty micrograms of each sample were separated by PAGE and analyzed by Western blot as described elsewhere (20) using rabbit anti-PARP at 1:1,000 (Roche Diagnostics).
Tumor microarray construction. The tissue microarrays were generated from retrospectively identified, archival formalin-fixed, paraffin-embedded primary breast cancer cases diagnosed at the Glasgow Royal Infirmary between 1984 and 1993. None of the patients in this sample set received neoadjuvant therapy. Three 0.6-mm2 cores of primary human breast cancer tissue were removed from representative tumor areas on each paraffin block identified by a pathologist. These cores were used to construct tissue microarray blocks in triplicate (80-120 cores per block). Cores of normal skin, smooth muscle, testes, lymph node, placenta, and tonsil were also included in the tissue microarray as controls.
Immunohistologic and statistical analysis of primary human breast tumors. Immunohistochemical staining for ERBB4/HER4 was done on tissue microarray samples using an antibody directed against the ERBB4 COOH terminus (Neomarkers, Fremont, CA) exactly as described elsewhere (9). The in situ TUNEL assay was done using the ApopTag Plus Peroxidase In situ Apoptosis Detection kit (Chemicon International, Temecula, CA) exactly as described by the manufacturer. Membrane or cytosolic ERBB4 staining and TUNEL staining was scored by first determining the intensity of tumor cell staining on a scale of 1 to 4, with 4 being the most intense staining. This number was multiplied by the percentage of tumor cells in each tissue microarray exhibiting positive staining and then divided by 100. Using this criteria, staining was recorded on a scale of 1 to 4 with 4 being the most intense widespread staining (examples of TUNEL staining are presented in Supplementary Fig. S1). Samples with staining of 3 to 4 were considered to be unequivocal positives and used for statistical analysis. In addition, all samples were examined by a total of four observers with a consensus obtained on all staining of 3 to 4. Examples of apoptosis staining from 1 to 4 are included as Supplementary Data.
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Results |
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40% and 80% of transfected SKBr3 and MCF-7 cells, respectively (Fig. 1B).
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-helix, which threads into the hydrophobic binding pocket of BCL-XL (Fig. 3A
). This interaction between BH3-only proteins and antiapoptotic BCL-2 family members suppresses BH3-only protein cell-killing activity (1). We, therefore, determined if the 4ICD BH3 domain mediated a functional interaction with BCL-2. Consistent with the idea that the 4ICD BH3 domain is a protein interaction motif, we coimmunoprecipitated in vitro translated BCL-2 with 4ICD but not with a 4ICD deletion mutant lacking the BH3 domain (4ICDdelBH3; Fig. 3B). Overexpression of the antiapoptotic BCL-2 oncogene disrupts apoptosis, in part, by binding to and sequestering BH3-only proteins (26). Likewise, heregulin ß1–induced apoptosis was suppressed by stable overexpression of BCL-2 in the MCF-7 cell line (Fig. 3C). Furthermore, BCL-2 overexpression also repressed apoptosis induced by ectopic expression of ERBB4 (Fig. 3D). ERBB4-induced cell killing was, however, restored in the presence of BCL-2 by coexpression with BAD (Fig. 3D). BAD also harbors a BH3 domain and regulates apoptosis by binding to BCL-2, thereby releasing BH3-only proteins with intrinsic cell-killing activity (27). Taken together, these results strongly implicate 4ICD as a proapoptotic BH3-only protein and show that 4ICD cell-killing activity can be regulated by members of the BCL-2 family.
Heregulin ß1–induced mitochondrial accumulation of 4ICD results in mitochondrial permeabilization. We next determined the molecular mechanism(s) underlying ERBB4 cell-killing activity. An important functional characteristic of BH3-only proteins is their ability to regulate apoptosis by localizing to and integrating signals at the mitochondria (1). We, therefore, determined if ectopically expressed ERBB4 localized to the mitochondria of transfected SKBr3 cells. Mitochondria were stained with MitoTracker Red (CMXRos), and CMXRos colocalization with ectopic ERBB4-EGFP was determined by deconvolution microscopy. The results indicate that a substantial perinuclear population of ERBB4 localizes to the mitochondria of SKBr3 cells undergoing apoptosis (Fig. 4A
). We have previously shown that a point mutation introduced into the juxtamembrane region of ERBB4 (V673I) abolished -secretase processing of ERBB4 and subsequent membrane release of 4ICD. Significantly, and in contrast to wild-type ERBB4, this ERBB4 processing mutant (referred to as ERBB4V673I) was not detected in isolated mitochondria and lacks cell-killing activity (4). ERBB4V673I was, therefore, used as a negative control in our mitochondrial localization experiments. Similar to ERBB4, ERBB4V673I also accumulates in the perinuclear region of transfected SKBr3 cells, but ERBB4V673I fails to colocalize with CMXRos (Fig. 4B), providing additional evidence that ERBB4V673I is excluded from mitochondria. We confirmed these observations by Western blot analysis of mitochondria isolated from SKBr3 cells ectopically expressing ERBB4 or ERBB4V673I each lacking an EGFP fusion. Consistent with our previous results (4), 4ICD was the predominant form of ERBB4 localizing to mitochondria of ERBB4-transfected cells, whereas both the ERBB4 holoreceptor and 4ICD were excluded from the mitochondrial fraction of ERBB4V673I-transfected cells (Fig. 4C). We next determined if heregulin ß1 stimulation of endogenous ERBB4 promoted mitochondrial accumulation of 4ICD. Mitochondria were isolated from T47D cells following a 1-hour heregulin ß1 stimulation, and ERBB4 was detected by Western blot analysis. Consistent with ectopic expression of ERBB4 in SKBr3 cells, heregulin ß1 stimulation of T47D breast cancer cells resulted in ERBB4 proteolytic processing, 4ICD membrane release, and mitochondrial accumulation of endogenous 4ICD (Fig. 4D). Low but detectable levels of mitochondrial ERBB4 holoreceptor was also observed (Fig. 4D). These results show that the 4ICD BH3-only protein is the predominant form of ERBB4 localizing to mitochondria in response to heregulin ß1.
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BAK is the essential mediator of 4ICD-induced apoptosis. Cell death signals that activate the intrinsic apoptotic pathway must converge at the mitochondria through the multiple BH domain proteins BAX and BAK (28). Activation and subsequent oligomerization of mitochondrial BAX or BAK results in mitochondrial permeabilization and is the essential and committed step of the intrinsic apoptotic pathway. We, therefore, determined the effect of heregulin ß1 stimulation of ERBB4 in T47D cells on endogenous BAX and BAK activation. When activated, BAX monomers translocate from the cytosol and oligomerize within the endoplasmic reticulum (22) and mitochondrial membranes (29) to initiate mitochondrial permeabilization, whereas activated BAK monomers residing within the mitochondrial membrane oligomerize to induce mitochondrial dysfunction (30). Heregulin ß1 stimulation of T47D cells failed to stimulate endoplasmic reticulum or mitochondrial accumulation of BAX (Fig. 5A ), suggesting that BAX is not involved in the heregulin ß1/4ICD apoptotic pathway. Calnexin and TOM40 were included as controls for endoplasmic reticulum and mitochondrial fractions, respectively. In contrast, heregulin ß1 stimulation of T47D cells promoted a depletion of BAK monomers with a corresponding increase in BAK dimer formation (Fig. 5B, compare lanes 2 and 4). ERBB4-specific RNAi, but not the nonspecific control RNAi pool, abolished heregulin ß1–induced loss of BAK monomers and accumulation of BAK dimers (Fig. 5B, compare lanes 6 and 8). Taken together, these results strongly suggest that heregulin ß1 activation of ERBB4 results in activation of the BAK pore-inducing complex, leading to mitochondrial permeabilization and cytochrome c release. To further substantiate a role for BAK as the exclusive mediator of ERBB4 cell killing, we transfected MEF cells or MEF cells lacking BAX, or BAK, or both with ERBB4-EGFP and determined the levels of apoptosis in each cell line. In direct concordance with our observations of heregulin ß1–stimulated T47D cells, ERBB4 induced apoptosis in >90% of transfected MEF and BAX-null MEF cells but only 10% of transfected BAK-null or BAX/BAK-null MEF cells (Fig. 5C). Collectively, our results indicate that heregulin ß1 activation of ERBB4 and subsequent mitochondrial accumulation of 4ICD promote mitochondrial dysfunction and cell killing through activation of the apoptosis "gateway" protein BAK.
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2 analysis, P < 0.0001). Cytosolic 4ICD staining was most prominent in perinuclear regions (Fig. 6C, arrowheads), where the majority of mitochondria reside. An additional tumor sample positive for cytosolic 4ICD and TUNEL is illustrated in Supplementary Fig. S1. Interestingly, nearly all tumors with ERBB4 localized to cell membranes were also TUNEL negative (Fig. 6A and B). Indeed, only 1 of the 17 tumors with membrane ERBB4 was TUNEL positive (2 analysis, P > 0.6).
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Discussion |
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Our findings provide evidence that ERBB4 suppresses breast cancer cell growth through activation of the intrinsic apoptotic pathway, by functioning as a proapoptotic BH3-only protein. Although members of the BH3-only protein class of the BCL-2 family are functionally diverse, these proteins share several critical mechanistic properties. For example, an intact BH3 domain is required to initiate apoptotic signals at the mitochondrial membrane through activation of BAX or BAK. The BH3-only protein apoptotic signal may however be disengaged at the mitochondria through interaction with antiapoptotic proteins including BCL-2, BCL-XL, or MCL-1. Likewise, ERBB4 cell-killing activity requires an intact BH3 domain and is inhibited by an interaction with BCL-2. Furthermore, ligand stimulation of endogenous ERBB4 results in mitochondrial accumulation of the BH3 domain–bearing 4ICD, oligomerization of BAK, and subsequent cytochrome c efflux from permeabilized mitochondria (Fig. 7 ).
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Critical for the appropriate function of BH3-only proteins is a mechanism to regulate apoptotic activity, thereby preventing deleterious apoptosis. The activities of several BH3-only proteins, including BIM, HRK, BBC3, NOXA, and PUMA, are transcriptionally regulated, whereas the apoptotic activities of BID, BAD, and BIK are regulated by different posttranslational mechanisms (36, 37). Posttranslational regulation of ERBB4 apoptotic activity seems to involve both activation of its intrinsic tyrosine kinase and subsequent proteolytic processing at the cell surface to release 4ICD. Interestingly, an intact kinase domain, essential for apoptosis induced by the ERBB4 holoreceptor, was dispensable for 4ICD-mediated cell killing, suggesting that signaling pathways coupled to the ERBB4 holoreceptor are not required for cell killing. In support of this contention, we have previously shown that an ERBB4 mutant that lacks -secretase processing, therefore failing to release 4ICD while retaining canonical signal transduction pathways, lacked cell-killing activity (4). Furthermore, here, we show that cytosolic 4ICD but not membrane-tethered ERBB4 was associated with tumor apoptosis, providing additional evidence that ERBB4 signaling from the cell surface has little, if any, effect on tumor apoptosis. Thus, we propose that the ERBB4 tyrosine kinase provides an essential contribution to apoptosis by supplying a mechanism for ERBB4 proteolytic processing, thereby releasing the apoptotic activity of 4ICD.
Although we show a significant association between cytosolic 4ICD and tumor apoptosis, the lack of apoptosis in some primary breast tumors expressing cytosolic 4ICD indicates that cytosolic accumulation of 4ICD is insufficient for tumor cell apoptosis. There exist several possible explanations for the lack of apoptosis in tumors with cytosolic 4ICD. Cytosolic staining of 4ICD indicates successful cleavage of ERBB4 by TACE; however, -secretase-mediated cleavage is required to release 4ICD from cellular membranes. Thus, cytosolic staining of 4ICD may represent endosome-tethered 4ICD in tumors lacking -secretase activity. Alternatively, these tumors may have altered expression of regulatory BCL-2 family members. For example, our results predict that tumor overexpression of BCL-2 or the absence of BAK would disengage the 4ICD apoptotic signal. Nevertheless, our results suggest that proteolytic processing of membrane-bound ERBB4 and subsequent mitochondrial accumulation of the 4ICD BH3-only protein represents a clinically important event contributing to tumor cell death.
In addition to 4ICD mitochondrial localization and apoptotic function, we have recently shown that 4ICD regulates gene expression as a nuclear protein (4, 16, 17). These divergent 4ICD functional activities can be reconciled in part because in the normal breast 4ICD seems to be retained in the nucleus (10) through interaction with the STAT5A transcription factor at target promoters (16). We propose that cellular transformation disrupts nuclear retention of 4ICD, leading to 4ICD mitochondrial accumulation and eventually apoptosis. This hypothesis is supported by recent experiments showing enhanced cell-killing activity of ERBB4 containing a mutated nuclear localization signal (4). In addition, disruption of 4ICD nuclear retention results in a 4ICD mechanistic switch from a transcriptional coregulator to a mitochondrial proapoptotic protein.6 We are currently investigating the exact molecular mechanisms regulating 4ICD subcellular localization and functional activities influencing normal breast function and transformation.
In conclusion, we have shown that ligand stimulation and subsequent proteolytic processing of ERBB4 results in membrane release and mitochondrial accumulation of 4ICD, a proapoptotic BH3-only protein. Furthermore, 4ICD directly initiates mitochondrial permeabilization through activation of the apoptotic "gateway" protein BAK, distinguishing ERBB4 from transmembrane "death receptors" that must recruit a BH3-only protein to initiate mitochondrial dysfunction (1). This unique apoptotic function for a cell surface receptor provides the first mechanistic description of a cell death pathway directly integrating an activated transmembrane receptor with the tumor cell mitochondrial apoptotic machinery. Moreover, the potent cell-killing activity of 4ICD provides a mechanistic explanation for the selective loss of ERBB4 expression during the metastatic progression of breast cancer and supports a novel tumor suppressor function for ERBB4.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Diane Clark, Laura McDonald, and Lacey Sullivan for excellent laboratory management; other members of the Jones lab for providing intellectual and technical support; Nicolette Solano for outstanding administrative assistance; the late Stanley Korsmeyer and Anthony Letai for helpful and stimulating discussions during the evolution of this project; Stanley Korsmeyer for providing the MEF cell lines and BCL-2 cDNA; Charles Hemenway for helpful suggestions; and Amy Johnson for editing this article.
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Footnotes |
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A. Naresh, W. Long, and G.A. Vidal contributed equally to this work.
This work is dedicated to Dr. Roy S. Weiner for his stalwart commitment to cancer patients and to the physicians and researchers of the Tulane Cancer Center.
6 A. Naresh and F.E. Jones, unpublished observations. 
Received 7/ 6/05. Revised 3/24/06. Accepted 3/30/06.
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References |
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-secretase processing regulates multiple ERBB4/HER4 activities. J Biol Chem 2005;280:19777–83.-secretase-like intramembrane cleavage of ErbB4. J Biol Chem 2002;277:6318–23.-secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science 2001;294:2179–81.This article has been cited by other articles:
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  A. D. Thor, S. M. Edgerton, and F. E. Jones Subcellular Localization of the HER4 Intracellular Domain, 4ICD, Identifies Distinct Prognostic Outcomes for Breast Cancer Patients Am. J. Pathol., November 1, 2009; 175(5): 1802 - 1809. [Abstract] [Full Text] [PDF]
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 R. S. Muraoka-Cook, M. A. Sandahl, K. E. Strunk, L. C. Miraglia, C. Husted, D. M. Hunter, K. Elenius, L. A. Chodosh, and H. S. Earp III ErbB4 Splice Variants Cyt1 and Cyt2 Differ by 16 Amino Acids and Exert Opposing Effects on the Mammary Epithelium In Vivo Mol. Cell. Biol., September 15, 2009; 29(18): 4935 - 4948. [Abstract] [Full Text] [PDF]
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 D. Mitra, M. J. Brumlik, S. U. Okamgba, Y. Zhu, T. T. Duplessis, J. G. Parvani, S. M. Lesko, E. Brogi, and F. E. Jones An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance Mol. Cancer Ther., August 1, 2009; 8(8): 2152 - 2162. [Abstract] [Full Text] [PDF]
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 J. J.-C. Sheu, C.-H. Hua, L. Wan, Y.-J. Lin, M.-T. Lai, H.-C. Tseng, N. Jinawath, M.-H. Tsai, N.-W. Chang, C.-F. Lin, et al. Functional Genomic Analysis Identified Epidermal Growth Factor Receptor Activation as the Most Common Genetic Event in Oral Squamous Cell Carcinoma Cancer Res., March 15, 2009; 69(6): 2568 - 2576. [Abstract] [Full Text] [PDF]
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 J. M.S. Bartlett, A. Munro, D. A. Cameron, J. Thomas, R. Prescott, and C. J. Twelves Type 1 Receptor Tyrosine Kinase Profiles Identify Patients With Enhanced Benefit From Anthracyclines in the BR9601 Adjuvant Breast Cancer Chemotherapy Trial J. Clin. Oncol., November 1, 2008; 26(31): 5027 - 5035. [Abstract] [Full Text] [PDF]
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A. Naresh, A. D. Thor, S. M. Edgerton, K. C. Torkko, R. Kumar, and F. E. Jones The HER4/4ICD Estrogen Receptor Coactivator and BH3-Only Protein Is an Effector of Tamoxifen-Induced Apoptosis Cancer Res., August 1, 2008; 68(15): 6387 - 6395. [Abstract] [Full Text] [PDF]
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 A. M. Strohecker, F. Yehiely, F. Chen, and V. L. Cryns Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector J. Biol. Chem., June 27, 2008; 283(26): 18269 - 18282. [Abstract] [Full Text] [PDF]
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 M. Rokavec, C. Justenhoven, W. Schroth, M. A. Istrate, S. Haas, H.-P. Fischer, C. Vollmert, T. Illig, U. Hamann, Y.-D. Ko, et al. A Novel Polymorphism in the Promoter Region of ERBB4 Is Associated with Breast and Colorectal Cancer Risk Clin. Cancer Res., December 15, 2007; 13(24): 7506 - 7514. [Abstract] [Full Text] [PDF]
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H.-G. Kang, J. M. Jenabi, J. Zhang, N. Keshelava, H. Shimada, W. A. May, T. Ng, C. P. Reynolds, T. J. Triche, and P. H.B. Sorensen E-Cadherin Cell-Cell Adhesion in Ewing Tumor Cells Mediates Suppression of Anoikis through Activation of the ErbB4 Tyrosine Kinase Cancer Res., April 1, 2007; 67(7): 3094 - 3105. [Abstract] [Full Text] [PDF]
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 A. J. Law, J. E. Kleinman, D. R. Weinberger, and C. S. Weickert Disease-associated intronic variants in the ErbB4 gene are related to altered ErbB4 splice-variant expression in the brain in schizophrenia Hum. Mol. Genet., January 15, 2007; 16(2): 129 - 141. [Abstract] [Full Text] [PDF]
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Y. Zhu, L. L. Sullivan, S. S. Nair, C. C. Williams, A. K. Pandey, L. Marrero, R. K. Vadlamudi, and F. E. Jones Coregulation of Estrogen Receptor by ERBB4/HER4 Establishes a Growth-Promoting Autocrine Signal in Breast Tumor Cells Cancer Res., August 15, 2006; 66(16): 7991 - 7998. [Abstract] [Full Text] [PDF]
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