Cancer Research Prevention Award Advances in Breast Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J.
Right arrow Articles by Freeman, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J.
Right arrow Articles by Freeman, M. R.
[Cancer Research 65, 8242-8249, September 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Trafficking of Nuclear Heparin-Binding Epidermal Growth Factor–like Growth Factor into an Epidermal Growth Factor Receptor–Dependent Autocrine Loop in Response to Oxidative Stress

Jayoung Kim, Rosalyn M. Adam and Michael R. Freeman

The Urological Diseases Research Center, Childrens Hospital, and Departments of Surgery and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Michael R. Freeman Enders Research Laboratories, Room 1161, 300 Longwood Avenue, Boston, MA 02115. Phone: 617-919-2644; Fax: 617-730-0238; E-mail: michael.freeman{at}childrens.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heparin-binding epidermal growth factor (EGF)–like growth factor (HB-EGF) accumulates in the nucleus in aggressive transitional cell carcinoma (TCC) cells and this histologic feature is a marker of poor prognosis in human bladder cancer tissues. Here we report that HB-EGF can be exported from the nucleus during stimulated processing and secretion of the growth factor. Production of reactive oxygen species (ROS) resulted in mobilization of the HB-EGF precursor, proHB-EGF, from the nucleus of TCCSUP bladder cancer cells to a detergent-resistant membrane compartment, where the growth factor was cleaved by a metalloproteinase-mediated mechanism and shed into the extracellular space. Inhibition of nuclear export suppressed HB-EGF shedding. Production of ROS resulted in EGF receptor (EGFR) and Akt1 phosphorylation in HB-EGF–expressing cells. HB-EGF also stimulated cell proliferation and conferred cytoprotection when cells were challenged with cisplatin. These findings show that the nucleus can serve as an intracellular reservoir for a secreted EGFR ligand and, thus, can contribute to an autocrine loop leading to cell proliferation and protection from apoptotic stimuli.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Overexpression of the epidermal growth factor receptor (EGFR/ErbB1/HER1), a transmembrane protein tyrosine kinase of the ErbB family, has been observed in many epithelial neoplasms, including bladder cancers (1–4). EGFR expression in bladder cancer correlates with histologic grade, tumor stage, and recurrence (5). EGFR signaling activates Ras-dependent [e.g., Raf-mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase-Akt] and Ras-independent (e.g., signal transducers and activators of transcription 3) mechanisms, resulting in pleiotropic effects on bladder cancer cells including induction of proliferation, angiogenesis, motility, invasion, and metastasis (6, 7). Because inhibition of EGFR activity may be a means to improve the prognosis for patients, therapeutic agents that inactivate EGFR are currently being developed (8). These strategies include humanized, function-blocking antibodies [cetuximab (Erbitux; ref. 9), h-R3 (10), and ABX-EGF (11)], small molecule inhibitors directed against the kinase domain (12), and immunotoxin conjugates (13).

Heparin-binding EGF-like growth factor (HB-EGF) is a direct activating ligand for the EGFR and the related tyrosine kinase, ErbB4 (14). HB-EGF is initially expressed as a transmembrane precursor, with the soluble form of the growth factor generated by regulated ectodomain shedding (15, 16). Both the transmembrane and secreted HB-EGF forms have been shown to possess biological activity independently of the other form (17–19). Secreted HB-EGF activates mitogenic and cell survival functions in multiple cell types. The membrane-bound form, proHB-EGF, is the cell surface receptor for diphtheria toxin (DT) and is the primary effector of DT entry into cells (20). ProHB-EGF can also mediate cell survival activity in an EGFR-independent manner by direct interaction with the co-chaperone protein BAG-1 (17) and is capable of mediating cell adhesion and activation of its cognate receptors on adjacent cells by a juxtacrine mechanism (21). ProHB-EGF also serves as a reservoir for rapid mobilization of soluble HB-EGF following a metalloproteinase cleavage step, a process that mediates transactivation of EGFR by G-protein–coupled receptors (6). Finally, the cleaved proHB-EGF cytoplasmic tail, which can transit to the nucleus after shedding of the ectodomain, mediates yet another cell survival function by triggering export from the nucleus of a transcriptional repressor (22).

The observation that HB-EGF resides in cells in multiple forms, and in different subcellular compartments, suggests that the traditional model for processing of EGF-like growth factors, where cytoplasmic synthesis is followed by membrane processing, is not sufficient to fully explain the growth factor's regulatory repertoire.

ProHB-EGF immunohistochemically localized to the nucleus was recently shown to be a prognostic marker for transitional cell carcinoma (TCC; ref. 23). In multivariate analysis, nuclear-localized proHB-EGF of >20% was an independent prognostic indicator of disease-specific mortality. Cox regression analysis indicated that in patients with >20% nuclear localized proHB-EGF, the monthly risk of death from TCC was increased more than 13-fold. In a separate study, soluble HB-EGF was recently shown to mediate tumor growth and angiogenesis in the EJ human bladder cancer model (6). Collectively, these findings suggest that HB-EGF may perform a tumor-promoting role in TCC; however, a mechanistic role specifically for nuclear proHB-EGF has not been described.

In this study, we show that nuclear proHB-EGF can be exported from the nucleus of human bladder cancer cells in response to the intracellular production of reactive oxygen species (ROS). We also show that this stimulus triggers membrane localization of proHB-EGF and regulates shedding, EGFR activation, and resistance to chemotherapeutic challenge. These findings indicate that nuclear proHB-EGF serves as a reservoir for the secreted growth factor, with nuclear HB-EGF capable of participating in an autocrine signaling mechanism that mediates tumor cell proliferation and protection from apoptotic signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Hydrogen peroxide (H2O2), metalloproteinase inhibitor (BB3489), and EGFR inhibitor (AG1478) were purchased from Calbiochem, Inc. (San Diego, CA). 2',7'-Dichlorofluorescein diacetate, N-acetyl-L-cysteine, filipin-III, and propidium iodide were from Sigma Chemical Co. (St. Louis, MO). The following monoclonal antibodies (mAb) and polyclonal antibodies were used: HB-EGF polyclonal antibody (R&D Systems, Inc., Minneapolis, MN); Gi{alpha}-2 polyclonal antibody and ß-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA); ß-actin mAb and alkaline phosphatase mAb (Sigma). All other antibodies including phospho-EGFR polyclonal antibody, phospho-Akt, phospho-p70S6 kinase, phospho-I{kappa}B, phospho-Erk/MAPK, phospho-p38/MAPK, and phospho-c-jun NH2-terminal kinase were from Cell Signaling Technology (Beverly, MA).

Cell culture and transfection. TCCSUP human bladder cancer cells stably expressing the HB-EGF precursor (proHB-EGFWT-myc) or control vector encoding ß-galactosidase (LacZ) were described (23). TCCSUP cells were transiently transfected with the following constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer: proHB-EGFWT-myc (24), a noncleavable form of proHB-EGF (proHB-EGFNC-myc; ref. 25), proHB-EGF-alkaline phosphatase fusion construct (proHB-EGFWT-alkaline phosphatase; ref. 26), and proHB-EGF with the cytoplasmic tail domain deleted (proHB-EGF{Delta}-tail). Transfected cells were cultured in DMEM/10% fetal bovine serum at 37°C/5% CO2, supplemented with 0.6 mg/mL G-418.

Indirect immunofluorescence microscopy. ProHB-EGF–transfected TCCSUP cells seeded at low density in chamber slides were treated with various reagents, fixed with 3% paraformaldehyde, and incubated with anti–HB-EGF or anti–alkaline phosphatase antibodies in 1% bovine serum albumin solution, followed by species-specific secondary antibodies conjugated to FITC or Texas red fluorophores. Slides were mounted in Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA) and analyzed with oil immersion objectives using an LSM 510 META NLO laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY).

Nuclear extracts. Cells were incubated in hypotonic lysis buffer [20 mmol/L HEPES (pH 7.0), 10 mmol/L KCl, 2 mmol/L MgCl2, 0.5% Nonidet P-40, protease inhibitors] and homogenized by 30 strokes in a tightly fitting Dounce homogenizer. The homogenate was centrifuged at 1,500 x g for 5 minutes, and the clear pellet was boiled completely in SDS-containing lysis buffer. The supernatant was assayed for protein concentration determination and equal amounts analyzed by SDS-PAGE.

Membrane staining analysis by fluorescence-activated cell sorting. TCCSUP/proHB-EGFWT-myc transfectants were treated with 0.01% H2O2 and harvested at the indicated times. Cells were stained with anti–HB-EGF antibody and FITC-conjugated secondary antibody, and visualized by flow cytometry.

Detergent-resistant membrane isolation. A modified membrane raft (detergent-resistant membrane) extraction method was done as described (27). Briefly, membranes were fractionated, resuspended in buffer A [25 mmol/L 2-(N-morpholino)-ethanesulfonic acid, 150 mmol/L NaCl (pH 6.5)], and an equal volume of buffer containing 2% Triton X-100 was added. After 30-minute incubation on ice, lysates were centrifuged and supernatants, containing the nonraft membrane fraction, were removed. Triton-insoluble pellets were resuspended in 10 mmol/L Tris-Cl (pH 7.6), 500 mmol/L NaCl, 1% Triton X-100, 60 mmol/L ß-octylglucoside and incubated for 30 minutes on ice. Fractions corresponding to rafts were collected following centrifugation for 20 minutes at 15,000 x g. Detergent-resistant membranes were also isolated by sucrose gradient ultracentrifugation. Briefly, cells were lysed in 25 mmol/L 2-(N-morpholino)-ethanesulfonic acid, 150 mmol/L NaCl (pH 6.5), 1% Triton X-100, followed by mechanical disruption with 8 strokes of a Dounce homogenizer. Lysates were diluted 1:1 with 60% sucrose (final sucrose concentration of 30%) and layered on a 40% sucrose cushion, followed by 25% to 0% sucrose solution. Ultracentrifugation was done at 100,000 x g for 20 hours in a Beckman SW28 rotor. All experimental steps were done on ice or at 4°C. To measure cholesterol levels, lipids were solubilized in chloroform, extracted through H2O, dried, and subjected to cholesterol determination using the Infinity cholesterol assay kit (Sigma Diagnostics).

Measurement of cell survival. TCCSUP/LacZ and TCCSUP/proHB-EGFWT-myc transfectants were incubated in the absence or presence of 0.01% H2O2 for 30 minutes. Cell viability was determined by uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described (23).

Statistical analysis. Data were compared using paired Student's t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ProHB-EGF was previously shown to localize to the nucleus as well as the cytoplasm in TCCSUP bladder cancer cells (23). This is shown in Fig. 1A, where a proHB-EGF-alkaline phosphatase fusion protein (proHB-EGFWT-alkaline phosphatase) is shown to be present in the nucleus. Treatment of TCCSUP cells with H2O2, an inducer of HB-EGF shedding (16), increased ROS production (20-fold by 12 minutes, returning to basal levels by 15 minutes; data not shown) and resulted in the clearance of HB-EGF from nuclei, as assessed using proHB-EGFWT-alkaline phosphatase (Fig. 1A) and a noncleavable form of proHB-EGF (proHB-EGFNC-myc; Fig. 1B). Blotting of nuclear fractions verified this result and indicated that the nuclear form of the growth factor retained the ectodomain (Fig. 1C). Retention of the ectodomain with export from the nucleus is also shown in Fig. 1A because the alkaline phosphatase moiety is fused upstream of the EGF-like motif (28). H2O2 treatment reduced the level of proHB-EGF in nuclear fractions within 30 minutes (Fig. 1C), consistent with the results of immunofluorescent cell staining analysis. A similar result was obtained in the presence of the proteasome inhibitor MG132 (Fig. 1C), indicating that proteasome-mediated degradation is an unlikely explanation for the reduction in nuclear HB-EGF levels. Identical results were obtained with lactacystin, another proteasome inhibitor (not shown).



View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Translocation of nuclear HB-EGF. A, TCCSUP bladder cancer cells were transiently transfected with an expression construct encoding proHB-EGFWT-alkaline phosphatase. Expression was confirmed by Western blot using alkaline phosphatase antibody (lane 1, TCCSUP/vector; lane 2, TCCSUP/proHB-EGFWT-alkaline phosphatase). Indirect immunofluorescence staining with anti–alkaline phosphatase antibody was done to assess HB-EGF localization in the absence (control) or presence of treatment with 0.01% H2O2. Nuclei were counterstained with DAPI. B, TCCSUP cells transiently expressing proHB-EGFNC-myc (bottom:1, TCCSUP/vector alone; 2, TCCSUP/proHB-EGFNC-myc) were treated with 0.01% H2O2 or vehicle and HB-EGF expression was determined by immunofluorescence as in A but using an antibody to the HB-EGF ectodomain. Expression of proHB-EGFNC-myc was confirmed by immunoblot (bottom). To quantitate the subcellular distribution of proHB-EGFNC-myc, at least 100 cells per condition were scored from three independent experiments, and the data expressed as percent of total cells counted. C, TCCSUP/proHB-EGFWT-myc transfectants were treated for the indicated times with 0.01% H2O2 and nuclear fractions isolated as described. Nuclear fractions were blotted with antibodies to the HB-EGF ectodomain or nuclear lamin. D, subconfluent TCCSUP/proHB-EGFWT-myc transfectants were treated with H2O2 for the indicated times and levels of membrane-localized HB-EGF determined by flow cytometry using anti–HB-EGF ectodomain antibody. E, conditioned media from H2O2-treated TCCSUP/proHB-EGFWT-myc or TCCSUP/LacZ cells were incubated with heparin-sepharose beads to enrich for heparin-binding proteins. Eluates were subjected to Western blot with an anti–HB-EGF ectodomain antibody. Arrow, soluble HB-EGF (sHB-EGF) secreted from TCCSUP/proHB-EGF-myc cells.

 
Fluorescence-activated cell sorting (FACS) analysis was done to assess changes in plasma membrane HB-EGF levels immediately following H2O2 treatment. HB-EGF on the cell membrane increased ~2-fold 15 minutes following H2O2 treatment, then returned to baseline at 30 minutes, suggesting cleavage of the cell-surface form of the growth factor (Fig. 1D). Consistent with this interpretation, soluble HB-EGF was detected in conditioned medium of proHB-EGF-myc–transfected cells treated with H2O2 but not in conditioned medium of H2O2-treated TCCSUP cells containing only the plasmid vector (Fig. 1E).

The subcellular distribution of proHB-EGF was verified in TCCSUP/proHB-EGFWT-myc cells. Consistent with the expression pattern illustrated in Fig. 1, immunofluorescence staining showed the presence of proHB-EGFWT-myc in multiple locations within cells including the cytoplasm, nucleus, and membrane (Fig. 2A). Some proHB-EGF was found to colocalize with the lipid raft (detergent-resistant membrane) marker, cholera toxin B fragment (29), which recognizes the raft-localized ganglioside GM1 (Fig. 2A). We confirmed these findings using biochemical methods to isolate raft and nonraft membrane fractions (data not shown). ProHB-EGF was found to accumulate in raft membrane fractions within 15 minutes of H2O2 treatment (Fig. 2B). Membrane HB-EGF levels decreased at 30 minutes, consistent with growth factor shedding from the membrane compartment. In contrast, HB-EGF levels increased within 30 minutes following H2O2 treatment of cells expressing the noncleavable proHB-EGFNC-myc construct (Fig. 2C), consistent with the resistance of this form of proHB-EGF to shedding. We observed no detectable change in either HB-EGF mRNA or protein levels after H2O2 treatment (Fig. 2D), indicating that new protein synthesis is an unlikely explanation for the accumulation of HB-EGF in the membrane fraction. Lipid raft disruption using the cholesterol-binding compound filipin suppressed oxidative HB-EGF enrichment in membrane rafts, suggesting that raft integrity is required for proHB-EGF accumulation on the membrane (Fig. 2E).



View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Localization of HB-EGF to raft microdomains. A, TCCSUP/proHB-EGFWT-myc transfectants were stained with FITC-conjugated cholera toxin B subunit (green) and anti–HB-EGF ectodomain antibody (red). Nuclei were counterstained with DAPI (blue). B, detergent-resistant membrane (raft) fractions from TCCSUP/proHB-EGFWT-myc cells treated with 0.01% H2O2 for the indicated times were blotted for HB-EGF and Gi{alpha}-2, a raft marker. C, TS and raft fractions from H2O2-treated TCCSUP/proHB-EGFNC-myc cells were blotted for HB-EGF and Gi{alpha}-2. D, total protein and RNA were prepared from H2O2-treated TCCSUP/proHB-EGFWT-myc cells and expressions of HB-EGF protein and mRNA determined by Western blot and reverse transcription-PCR, respectively. E, cells were treated with 2 µg/mL filipin before stimulation with 0.01% H2O2 for 15 minutes, and raft fractions were blotted for HB-EGF.

 
H2O2 treatment of TCCSUP/proHB-EGFWT-myc cells led to accumulation of proHB-EGF in rafts at 15 minutes after initiation of treatment, and to secretion of HB-EGF by 30 minutes (Fig. 2B). H2O2-induced shedding of proHB-EGF from rafts was reversed by matrix metalloproteinase (MMP) inhibition (Fig. 3A), consistent with the known dependence of HB-EGF secretion on MMP activity (25). Accumulation of proHB-EGF in the raft fraction following H2O2 treatment was suppressed by the general trafficking inhibitor brefeldin A (Fig. 3B, top, note 15 minute time point), although nuclear export of HB-EGF was not altered by this drug (Fig. 3B, bottom). ProHB-EGF accumulation in the raft membrane was not blocked by the actin polymerization inhibitor latrunculin A (not shown). Collectively, the above results indicate that H2O2 stimulates cytoplasmic trafficking of proHB-EGF and accumulation of the growth factor precursor in a detergent-resistant membrane compartment, and culminates in proteolytic processing of the membrane-anchored protein to the soluble form.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Accumulation of HB-EGF in membranes is dependent on ROS production, MMP activity, and brefeldin-sensitive trafficking. A, TCCSUP/proHB-EGFWT-myc cells were pretreated with 10 µmol/L BB3489 (MMP inhibitor) and exposed to 0.01% H2O2 for 30 minutes. HB-EGF levels in rafts were determined by Western blot. B, TCCSUP/proHB-EGFWT-myc cells were pretreated with 1 µg/mL brefeldin A and exposed to H2O2 for the indicated times. The level of HB-EGF in nuclear (bottom panel) and raft (top panel) fractions was determined by Western blot.

 
The time-dependent correlation between trafficking/shedding and nuclear export of HB-EGF suggests that the nuclear-to-cytoplasmic shuttling process supplies growth factor to the cytoplasmic pool that is then mobilized for display on the membrane before proteolytic processing. To test this, we used a nuclear export blocker, leptomycin B, which specifically inhibits nuclear export of proteins (30), to see if HB-EGF nuclear export could be inhibited with H2O2 treatment. Western blot analysis showed that when leptomycin B was present, HB-EGF was largely retained in the nucleus in the oxidative condition (Fig. 4A). Notably, a substantial decrease in raft-localized HB-EGF was also evident at 15 minutes in the presence of leptomycin B, suggesting that export from the nucleus is a requirement for accumulation of proHB-EGF in membrane rafts. Indirect fluorescence staining of TCCSUP cells expressing proHB-EGFWT-myc showed a substantial reduction in nuclear and cytosolic HB-EGF following H2O2 treatment; however, this was not evident in the presence of leptomycin B (Fig. 4B). These results were repeated using a cytoplasmic tail–deleted proHB-EGF construct, indicating that the cytoplasmic domain is not required for nuclear export (Fig. 4C).



View larger version (42K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Inhibition of nuclear export blocks HB-EGF secretion. A, TCCSUP/proHB-EGFWT-myc cells were treated with 10 µmol/L leptomycin B, followed by 0.01% H2O2 for the indicated times. HB-EGF distribution in nuclear and raft fractions was determined by Western blot. B, TCCSUP/proHB-EGFWT-myc cells were treated with H2O2 for 30 minutes in the absence or presence of 10 µmol/L leptomycin B. HB-EGF was visualized by indirect immunofluorescence microscopy with anti–HB-EGF antibody. C, TCCSUP transiently expressing proHB-EGF{Delta}-tail were treated as in B; inset, expression of proHB-EGF{Delta}-tail (1, vector alone; 2, proHB-EGF{Delta}-tail). D, conditioned media (CM) from TCCSUP/proHB-EGFWT-myc cells treated with 0.01% H2O2 for 30 minutes in the absence or presence of 10 µmol/L leptomycin B were incubated with heparin-sepharose beads and the eluates blotted for HB-EGF; protein loading was confirmed by Coomassie blue staining of the gel.

 
To test directly whether nuclear HB-EGF can contribute to the growth factor pool destined for shedding, we examined whether leptomycin B affected HB-EGF secretion induced by H2O2. HB-EGF levels were assessed in conditioned medium collected after H2O2 stimulation in the absence or presence of leptomycin B. As shown in Fig. 4D, leptomycin B significantly inhibited HB-EGF secretion into the medium, indicating that nuclear HB-EGF is a component of the intracellular trafficking mechanism leading to shedding.

In subsequent experiments, we assessed whether secreted HB-EGF in this context is associated with one or more biological functions. Consistent with this possibility, TCCSUP cells expressing proHB-EGFWT-myc exhibited a distinct signal transduction pattern in comparison with cells transfected with the empty vector (Fig. 5A). EGFR and Akt phosphorylation states were higher in proHB-EGF–transfected cells, whereas Erk- and p38-MAPK signaling was unchanged or possibly reduced. To confirm that this was the result of the presence of HB-EGF in the medium, we did additional studies with pharmacologic MMP and EGFR inhibitors (BB3489 and AG1478, respectively). Both agents blocked the effect on Akt phosphorylation (Fig. 5B). Furthermore, FACS analysis showed that HB-EGF–stimulated cell cycle transit could be inhibited by AG1478-mediated blockade of the EGFR (Fig. 5C).



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Secreted HB-EGF activates EGFR->Akt signaling. A, total cell lysates were prepared from TCCSUP/LacZ or TCCSUP/proHB-EGFWT-myc cells at the indicated times after H2O2 treatment. Lysates were blotted with antibodies to the phosphorylated forms of the indicated proteins. B, TCCSUP/LacZ or TCCSUP/proHB-EGFWT-myc cells were preincubated with 10 µmol/L BB3489 or 10 µmol/L AG1478 before H2O2 treatment for 15 minutes. Activation of signaling molecules was determined as in A. C, following serum starvation for 24 hours, TCCSUP parent cells were treated without or with 10 µmol/L AG1478 for 1 hour, followed by 100 ng/mL HB-EGF and 10 µmol/L nocodazole. Twenty-four hours later, cells were fixed and incubated with propidium iodide and RNase A for FACS analysis.

 
Proliferation rate was substantially increased in TCCSUP cells stably engineered to express only the soluble form of HB-EGF, but this effect was not seen with cells expressing either proHB-EGFWT-myc or proHB-EGFNC-myc (Fig. 6A). ProHB-EGFWT-myc cells, however, did exhibit enhanced survival under oxidative stress conditions (Fig. 6B). Consistent with this result, TCCSUP cells expressing proHB-EGFWT-myc showed enhanced resistance to cisplatin-induced apoptosis, shown by FACS analysis and Western blot using the apoptosis markers p21Waf1/Cip1, cleaved caspase-3, and poly(ADP-ribose) polymerase (Fig. 6C).



View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Secretion of HB-EGF stimulates cell proliferation and survival. A, proliferation rates of TCCSUP/vector only, proHB-EGFWT-myc, proHB-EGFNC-myc, or a constitutively secreted variant of HB-EGF (HB-EGFCS-myc) were determined by uptake of MTT. B, TCCSUP/LacZ and TCCSUP/proHB-EGFWT-myc cells were stimulated with H2O2 or vehicle in serum-free medium for 30 minutes. Cells were switched to fresh medium for a further 48 hours, at which time viability was measured by uptake of MTT. C, TCCSUP/LacZ and TCCSUP/proHB-EGFWT-myc cells were incubated in the absence or presence of 10 µmol/L cisplatin for 24 hours. The percentage of apoptotic cells was determined by flow cytometry (left). Cells exposed to the same treatments for 48 hours were harvested for Western blot analysis using antibodies to the apoptosis-related markers p21Waf1 and cleaved caspase-3 (right). To determine the effect of antioxidant on cisplatin-induced apoptosis, cells were coincubated with cisplatin and 1 mmol/L N-acetylcysteine (NAC). Apoptosis was assessed by Western blot for cleaved poly(ADP-ribose) polymerase (c-PARP; bottom).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we identified nuclear-localized HB-EGF as a component of an EGFR-dependent autocrine loop in bladder cancer cells. This conclusion is based on the following lines of evidence: (a) proHB-EGF expressed in TCCSUP cells was rapidly mobilized from the nucleus in response to oxidative treatment; (b) H2O2 treatment increased both membrane accumulation of HB-EGF and subsequent release into the medium; (c) blockade of nuclear export suppressed HB-EGF translocation to the membrane and growth factor shedding; (d) soluble HB-EGF stimulated EGFR, Akt, and p70S6 kinase phosphorylation as well as enhanced cell cycle transit and survival; (e) interference with the HB-EGF->EGFR autocrine loop by inhibition of either HB-EGF shedding or EGFR activation attenuated H2O2-induced signaling; and (f) proHB-EGF partially protected cells from apoptosis induced by oxidative stress or exposure to cisplatin. Collectively, these findings indicate that the nucleus of bladder cancer cells can serve as a reservoir for an EGFR ligand and that nuclear HB-EGF can rapidly transit to the cell surface for secretion and autocrine signaling. Our findings are consistent with previous reports linking nuclear localization of proHB-EGF (23) and increased activation of the EGFR (4) to clinically aggressive bladder cancer.

The demonstration in the present study that full-length HB-EGF resides in the nucleus contrasts somewhat with a recent report that a cleaved, COOH-terminal fragment of HB-EGF (C-HB-EGF) can translocate to the nucleus (31). Nuclear localization of C-HB-EGF was linked to alterations in cell cycle transit that arise from HB-EGF–mediated nuclear export and inhibition of the promyelocytic leukemia zinc finger transcription factor (32). Significantly, C-HB-EGF exerts its effects on promyelocytic leukemia zinc finger in an EGFR-independent manner. Previously, our group showed that proHB-EGF binds via its cytoplasmic tail domain (corresponding to C-HB-EGF) to the co-chaperone protein BAG-1 to promote cell survival, also independently of the EGFR (17). Interestingly, in the present study, we found that trafficking of proHB-EGF to the membrane and regulated shedding were found not to be dependent on the cytoplasmic tail. Taken together, these findings suggest that HB-EGF can promote cell cycle traverse and cell survival by multiple mechanisms, including EGFR-dependent/-independent and COOH-terminal–dependent/–independent pathways.

Another conclusion of our study is that oxidative stress regulates subcellular distribution of proHB-EGF in a manner that promotes cell survival and growth. ROS are constantly generated in cells as a metabolic by-product and can stimulate proliferation or apoptosis in a dose- and cell type–dependent manner (reviewed in ref. 33). Tumor cells exhibit increased intrinsic ROS as a result of oncogenic transformation, elevated metabolic activity, and altered mitochondrial function. Consequently, ROS may contribute to the malignant phenotype exhibited by cancer cells. Exposure of TCCSUP cells to exogenous H2O2 elicited a rapid burst of ROS production that preceded nuclear export of proHB-EGF and accumulation of the growth factor on the plasma membrane. H2O2-induced HB-EGF shedding stimulated EGFR phosphorylation and activation of Akt signaling, but did not appreciably up-regulate MAPK signaling. Ligand-mediated activation of the EGFR itself has previously been shown to increase ROS levels in cells (34). Together with our data, this suggests the existence of a positive feedback loop in tumor cells, in which ROS generation promotes nuclear export and membrane localization of proHB-EGF, growth factor shedding, and autocrine EGFR activation leading to continued ROS production. Moreover, because EGF-like growth factor expression is subject to autoinduction (35), HB-EGF–stimulated EGFR activation may lead to enhanced expression of other EGFR activating ligands, resulting in further augmentation of survival and proliferation signaling pathways.

The association between nuclear HB-EGF and aggressive disease in bladder cancer suggests the possibility that the nuclear-localized growth factor might perform additional functions separate from the autocrine activity we describe in this report. The potential for pleiotropic activity of growth factor isoforms that localize to discrete subcellular compartments has been shown for certain members of the fibroblast growth factor (FGF) family (36, 37). Distinct 18 kDa and 24 kDa FGF-2 isoforms, synthesized from a single mRNA through the use of alternative initiation codons, mediate autocrine/paracrine and intracrine signaling, respectively (36). Nuclear FGF-2 has been associated with increased tumor cell survival and enhanced metastatic capability in vivo (38), direct regulation of transcription (39) and mRNA splicing (40), and activation of Erk-MAPK signaling, leading to expression of the cell cycle regulator c-Jun (41). The EGF-like growth factors TGF-{alpha} and amphiregulin have been reported to reside in nuclei in several cell types, but a specialized role for these nuclear isoforms is still poorly understood. Notably, the ErbB receptors EGFR and ErbB2/HER2 have also recently been shown to localize to nuclei (42–44), where they seem to exert a novel role as direct regulators of gene expression by acting as components of transcription complexes (43, 44). An interesting possibility for further investigation is whether EGF-like growth factors and cognate receptors interact in the nucleus to elicit transcriptional or other nucleus-specific functions.

Our findings also suggest the possibility that nuclear export inhibitors might be useful therapeutically as inhibitors of EGFR activation in vivo. Leptomycin B, the related antifungal antibiotic kazusamycin, and myxobacterial products termed ratjadones inhibit nuclear translocation in tumor cells in vitro (45–47). These agents act by binding to and inhibiting the function of exportin-1/CRM1, a transporter of proteins that contain a leucine-rich nuclear export sequence (48). All three agents display significant antiproliferative activity against a range of tumor cell lines, in addition to inhibiting nuclear export (46, 47). Moreover, leptomycin B and kazusamycin exhibited antitumor activity in experimental tumor models (45, 46). Leptomycin B (also known as CI-940 or elactocin) was tested in phase I clinical trials against a range of tumor types (49) but, due to dose-limiting toxicity, significant side effects, and the lack of any measurable therapeutic effect, no further trials were done.

Despite concerns about toxicity, targeting of nuclear trafficking mechanisms as a strategy to inhibit tumor cell growth still remains attractive (48). Kau and Silver (50) recently described a chemical genetic screen to identify compounds that modulate nuclear localization of the Forkhead family transcription factor FOXO1 or the RevGFP fusion protein. Of >18,000 small molecule compounds tested, several discrete classes of inhibitors were identified, including agents that were general inhibitors of nuclear export as well as those specific for FOXO1 translocation.

In summary, we have identified an EGFR-dependent autocrine signaling loop in bladder cancer cells in which nuclear export of mitogenically active HB-EGF is a key component. The bladder is a "privileged" site for instillation of cancer-targeting drugs because agents that would be highly toxic if administered systemically (e.g., Bacillus Calmette-Guerin and Adriamycin) are tolerated in patients with bladder cancer when given intravesically. This suggests that despite the toxicity observed when nuclear export inhibitors are given systemically (49), intravesical targeting of CRM1 or other components of the nuclear transport apparatus (50) may have therapeutic benefit.


   Acknowledgments
 
Grant support: NIH grants R37 DK47556, R01 DK 57691, and P50 DK65298 (M.R. Freeman). J. Kim and R.M. Adam are American Foundation for Urologic Disease Research scholars.

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.

Received 3/21/05. Revised 6/27/05. Accepted 7/ 6/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Smith K, Fennelly JA, Neal DE, Hall RR, Harris AL. Characterization and quantitation of the epidermal growth factor receptor in invasive and superficial bladder tumors. Cancer Res 1989;49:5810–5.[Abstract/Free Full Text]
  2. Neal DE, Marsh C, Bennett MK, et al. Epidermal-growth-factor receptors in human bladder cancer: comparison of invasive and superficial tumours. Lancet 1985;1:366–8.[CrossRef][Medline]
  3. Messing EM. Clinical implications of the expression of epidermal growth factor receptors in human transitional cell carcinoma. Cancer Res 1990;50:2530–7.[Abstract/Free Full Text]
  4. Highshaw RA, McConkey DJ, Dinney CP. Integrating basic science and clinical research in bladder cancer: update from the first bladder Specialized Program of Research Excellence (SPORE). Curr Opin Urol 2004;14:295–300.[CrossRef][Medline]
  5. Bellmunt J, Hussain M, Dinney CP. Novel approaches with targeted therapies in bladder cancer. Therapy of bladder cancer by blockade of the epidermal growth factor receptor family. Crit Rev Oncol Hematol 2003;46 Suppl:S85–104.
  6. Ongusaha PP, Kwak JC, Zwible AJ, et al. HB-EGF is a potent inducer of tumor growth and angiogenesis. Cancer Res 2004;64:5283–90.[Abstract/Free Full Text]
  7. Schafer B, Gschwind A, Ullrich A. Multiple G-protein-coupled receptor signals converge on the epidermal growth factor receptor to promote migration and invasion. Oncogene 2004;23:991–9.[CrossRef][Medline]
  8. Grunwald V, Hidalgo M. Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst 2003;95:851–67.[Abstract/Free Full Text]
  9. Kies MS, Harari PM. Cetuximab (Imclone/Merck/Bristol-Myers Squibb). Curr Opin Investig Drugs 2002;3:1092–100.[Medline]
  10. Crombet T, Osorio M, Cruz T, et al. Use of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy in the treatment of locally advanced head and neck cancer patients. J Clin Oncol 2004;22:1646–54.[Abstract/Free Full Text]
  11. Foon KA, Yang XD, Weiner LM, et al. Preclinical and clinical evaluations of ABX-EGF, a fully human anti-epidermal growth factor receptor antibody. Int J Radiat Oncol Biol Phys 2004;58:984–90.[CrossRef][Medline]
  12. Janmaat ML, Giaccone G. Small-molecule epidermal growth factor receptor tyrosine kinase inhibitors. Oncologist 2003;8:576–86.[Abstract/Free Full Text]
  13. Schmidt M, Maurer-Gebhard M, Groner B, Kohler G, Brochmann-Santos G, Wels W. Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors. Oncogene 1999;18:1711–21.[CrossRef][Medline]
  14. Nishi E, Klagsbrun M. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a mediator of multiple physiological and pathological pathways. Growth Factors 2004;22:253–60.[CrossRef][Medline]
  15. Higashiyama S. Metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor and its pathophysiological roles. Protein Pept Lett 2004;11:443–50.[CrossRef][Medline]
  16. Kim J, Lin J, Adam RM, Lamb C, Shively SB, Freeman MR. An oxidative stress mechanism mediates chelerythrine-induced heparin-binding EGF-like growth factor ectodomain shedding. J Cell Biochem 2005;94:39–49.[CrossRef][Medline]
  17. Lin J, Hutchinson L, Gaston SM, Raab G, Freeman MR. BAG-1 is a novel cytoplasmic binding partner of the membrane form of heparin-binding EGF-like growth factor: a unique role for proHB-EGF in cell survival regulation. J Biol Chem 2001;276:30127–32.[Abstract/Free Full Text]
  18. Singh AB, Tsukada T, Zent R, Harris RC. Membrane-associated HB-EGF modulates HGF-induced cellular responses in MDCK cells. J Cell Sci 2004;117:1365–79.[Abstract/Free Full Text]
  19. Yamazaki S, Iwamoto R, Saeki K, et al. Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities. J Cell Biol 2003;163:469–75.[Abstract/Free Full Text]
  20. Mitamura T, Higashiyama S, Taniguchi N, Klagsbrun M, Mekada E. Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity. J Biol Chem 1995;270:1015–9.[Abstract/Free Full Text]
  21. Iwamoto R, Mekada E. Heparin-binding EGF-like growth factor: a juxtacrine growth factor. Cytokine Growth Factor Rev 2000;11:335–44.[CrossRef][Medline]
  22. Toki F, Nanba D, Matsuura N, Higashiyama S. Ectodomain shedding of membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the C-terminal fragment in the cell cycle. J Cell Physiol 2005;202:839–48.[CrossRef][Medline]
  23. Adam RM, Danciu T, McLellan DL, et al. A nuclear form of the heparin-binding epidermal growth factor-like growth factor precursor is a feature of aggressive transitional cell carcinoma. Cancer Res 2003;63:484–90.[Abstract/Free Full Text]
  24. Nguyen HT, Bride SH, Badawy AB, et al. Heparin-binding EGF-like growth factor is up-regulated in the obstructed kidney in a cell- and region-specific manner and acts to inhibit apoptosis. Am J Pathol 2000;156:889–98.[Abstract/Free Full Text]
  25. Suzuki M, Raab G, Moses MA, Fernandez CA, Klagsbrun M. Matrix metalloproteinase-3 releases active heparin-binding EGF-like growth factor by cleavage at a specific juxtamembrane site. J Biol Chem 1997;272:31730–7.[Abstract/Free Full Text]
  26. Raab G, Kover K, Paria BC, Dey SK, Ezzell RM, Klagsbrun M. Mouse preimplantation blastocysts adhere to cells expressing the transmembrane form of heparin-binding EGF-like growth factor. Development 1996;122:637–45.[Abstract]
  27. Kim J, Adam RM, Solomon KR, Freeman MR. Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology 2004;145:613–9.[Abstract/Free Full Text]
  28. Dethlefsen SM, Raab G, Moses MA, Adam RM, Klagsbrun M, Freeman MR. Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. J Cell Biochem 1998;69:143–53.[CrossRef][Medline]
  29. Harder T, Scheiffele P, Verkade P, Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 1998;141:929–42.[Abstract/Free Full Text]
  30. Fukuda M, Asano S, Nakamura T, et al. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 1997;390:308–11.[CrossRef][Medline]
  31. Nanba D, Mammoto A, Hashimoto K, Higashiyama S. Proteolytic release of the carboxy-terminal fragment of proHB-EGF causes nuclear export of PLZF. J Cell Biol 2003;163:489–502.[Abstract/Free Full Text]
  32. Toki F, Nanba D, Matsuura N, Higashiyama S. Ectodomain shedding of membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the C-terminal fragment in the cell cycle. J Cell Physiol 2005;202:839–48.
  33. Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004;7:97–110.[CrossRef][Medline]
  34. Bae YS, Kang SW, Seo MS, et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J Biol Chem 1997;272:217–21.[Abstract/Free Full Text]
  35. Barnard JA, Graves-Deal R, Pittelkow MR, et al. Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family. J Biol Chem 1994;269:22817–22.[Abstract/Free Full Text]
  36. Chen CH, Poucher SM, Lu J, Henry PD. Fibroblast growth factor 2: from laboratory evidence to clinical application. Curr Vasc Pharmacol 2004;2:33–43.[CrossRef][Medline]
  37. Kiefer P, Acland P, Pappin D, Peters G, Dickson C. Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3. EMBO J 1994;13:4126–36.[Medline]
  38. Thomas-Mudge RJ, Okada-Ban M, Vandenbroucke F, et al. Nuclear FGF-2 facilitates cell survival in vitro and during establishment of metastases. Oncogene 2004;23:4771–9.[CrossRef][Medline]
  39. Shimizu-Sasaki E, Yamazaki M, Furuyama S, Sugiya H, Sodek J, Ogata Y. Identification of FGF2-response element in the rat bone sialoprotein gene promoter. Connect Tissue Res 2003;44 Suppl 1:103–8.
  40. Gringel S, van Bergeijk J, Haastert K, Grothe C, Claus P. Nuclear fibroblast growth factor-2 interacts specifically with splicing factor SF3a66. Biol Chem 2004;385:1203–8.[CrossRef][Medline]
  41. Hortala M, Estival A, Pradayrol L, Susini C, Clemente F. Identification of c-Jun as a critical mediator for the intracrine 24 kDa FGF-2 isoform-induced cell proliferation. Int J Cancer 2005;114:863–9.[CrossRef][Medline]
  42. Lo HW, Xia W, Wei Y, Ali-Seyed M, Huang SF, Hung MC. Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer. Cancer Res 2005;65:338–48.[Abstract/Free Full Text]
  43. Lin SY, Makino K, Xia W, et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 2001;3:802–8.[CrossRef][Medline]
  44. Wang SC, Lien HC, Xia W, et al. Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer Cell 2004;6:251–61.[CrossRef][Medline]
  45. Roberts BJ, Hamelehle KL, Sebolt JS, Leopold WR. In vivo and in vitro anticancer activity of the structurally novel and highly potent antibiotic CI-940 and its hydroxy analog (PD 114,721). Cancer Chemother Pharmacol 1986;16:95–101.[CrossRef][Medline]
  46. Yoshida E, Komiyama K, Naito K, et al. Antitumor effect of kazusamycin B on experimental tumors. J Antibiot (Tokyo) 1987;40:1596–604.[Medline]
  47. Koster M, Lykke-Andersen S, Elnakady YA, et al. Ratjadones inhibit nuclear export by blocking CRM1/exportin 1. Exp Cell Res 2003;286:321–31.[CrossRef][Medline]
  48. Kau TR, Way JC, Silver PA. Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer 2004;4:106–17.[Medline]
  49. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. Br J Cancer 1996;74:648–9.[Medline]
  50. Kau TR, Silver PA. Nuclear transport as a target for cell growth. Drug Discov Today 2003;8:78–85.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
J. Kim, W. J. Jahng, D. Di Vizio, J. S. Lee, R. Jhaveri, M. A. Rubin, A. Shisheva, and M. R. Freeman
The Phosphoinositide Kinase PIKfyve Mediates Epidermal Growth Factor Receptor Trafficking to the Nucleus
Cancer Res., October 1, 2007; 67(19): 9229 - 9237.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
F. M. Kouri and O. Eickelberg
Transforming Growth Factor-{alpha}, a Novel Mediator of Strain-Induced Vascular Remodeling
Circ. Res., August 18, 2006; 99(4): 348 - 350.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J.
Right arrow Articles by Freeman, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J.
Right arrow Articles by Freeman, M. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online