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Cell and Tumor Biology |
1 Medicity Research Laboratory, Departments of 2 Medical Biochemistry and Molecular Biology and 3 Anatomy, and 4 Turku Postgraduate School of Biomedical Sciences, University of Turku; 5 Department of Oncology, Turku University Hospital, Turku, Finland; 6 Department of Oncology, University of Helsinki, Helsinki, Finland; 7 Laboratory of Cancer Biology, Institute of Medical Technology, Tampere University; and 8 Department of Oncology, Tampere University Hospital, Tampere, Finland
Requests for reprints: Klaus Elenius, Medicity Research Laboratory, University of Turku, Tykistokatu 6 A 4th floor FIN-20520 Turku, Finland. Phone: 358-2-3337569; Fax: 358-2-3337229; E-mail: klaus.elenius{at}utu.fi.
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Abstract |
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(ER) expression (P < 0.001) and a high histologic grade of differentiation (P 
0.002) in real-time reverse transcription-PCR analysis of 62 breast cancer samples. Despite high ErbB4 mRNA expression levels in a subset of samples, ErbB4 gene amplification was not observed. High ErbB4 protein expression levels, as assessed by immunohistochemistry, associated with a favorable outcome in ER-positive cases from a series of 458 breast cancer patients (P = 0.01), whereas no association between ErbB4 expression and survival was found among women with ER-negative cancer (P = 0.86). However, nuclear ErbB4 immunoreactivity was associated with poor survival as compared with women whose cancer had membranous ErbB4 staining (P = 0.04). In vitro, overexpression of a cleavable ErbB4 isoform in ER-positive breast cancer cells resulted in translocation of a proteolytically released intracellular ErbB4 receptor fragment into the nucleus, as well as, enhanced proliferation, anchorage-independent growth, and estrogen response element–mediated transcriptional activity. These results suggest that the association of ErbB4 expression with clinical outcome is dependent on the subcellular localization of ErbB4 and that a proteinase-cleavable ErbB4 isoform promotes growth of ER-positive breast cancer and enhances ER-mediated gene transcription.
Key Words: EGFR • HER4 • hormone-dependent cancer • neuregulins • oncogenes
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Introduction |
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Aberrant ErbB signaling is a frequent phenomenon in various human cancers (1). ErbB1 and ErbB2 have well-characterized roles in tumorigenesis, and cancer drugs that specifically inhibit ErbB1 (such as gefitinib, erlotinib, and cetuximab) or ErbB2 (trastuzumab) have been introduced into clinical practice. Less is known about the tumor biology and clinical relevance of the most recently characterized ErbB family member, ErbB4 (2, 3). The presence of ErbB4 in breast cancer cells has been found to be associated with a well-differentiated phenotype (4, 5) and favorable prognosis (6–8), but contradictory results suggesting an oncogenic role for ErbB4 have also been reported both from cell biological (9–11) and clinical (12, 13) studies.
We have previously shown that ErbB4 is expressed as four alternatively spliced isoforms (14–16). Alternative splicing occurs within the intracellular cytoplasmic tail (isoforms CYT-1 and CYT-2) and within the extracellular juxtamembrane region (isoforms JM-a and JM-b), which generates receptors with different signaling capabilities. The CYT-1 isoform includes an additional exon with docking sites for SH2-domain and putatively WW-domain containing signaling molecules that is absent from the CYT-2 isoform (15, 17, 18) . The JM isoforms differ in their sensitivity to proteinase cleavage. Tumor necrosis factor-–converting enzyme (TACE) can cleave the JM-a isoform whereas the JM-b isoform is proteinase resistant (14, 19). This difference in susceptibility to receptor ectodomain shedding is also reflected in intracellular signaling via ErbB4: cleavage of ErbB4 at the JM domain by TACE-like enzymes triggers a secondary cleavage event by 
-secretase activity, which releases a soluble intracellular domain (ICD) fragment that can translocate into the nucleus and regulate gene transcription (18, 20, 21).9
In this study, we investigated the expression of different ErbB4 isoforms in clinical breast cancer samples and determined their associations with breast cancer patient survival, cancer estrogen receptor (ER) expression, and a few other prognostic markers. Our findings suggest that nuclear translocation of the proteolytically released ErbB4 ICD fragments associates with adverse clinical outcome compared with women who have full-length ErbB4 present at the tumor cell surface. Results from in vitro experiments show that expression of the cleavable ErbB4 isoform in breast cancer cells results in nuclear localization of an intracellular receptor fragment, as well as, enhanced tumor cell growth and estrogen response element (ERE)–mediated transcriptional activity. These findings suggest a tumor-promoting role for the ErbB4 isoform capable of nuclear translocation.
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Materials and Methods |
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Cell Culture. MCF-7 breast cancer cells (24, 25) were maintained in RPMI, supplemented with 10% FCS (Autogen Bioclear UK Ltd., Wiltshire, United Kingdom), 1% L-glutamine-penicillin-streptomycin solution (Sigma, St. Louis, MO), 1 nmol/L 17-ß-estradiol (Sigma), and for the transfected cell lines with 150 mg/mL Hygromycin B (Roche, Indianapolis, IN). For the experiments that addressed the effects of estradiol, the cells were grown for 48 hours in a phenol red- and estradiol-free medium supplemented with dextran-charcoal stripped FCS before treatment.
Quantitative Reverse Transcription-PCR Analysis of ErbB mRNA Expression. Total RNA was extracted from breast carcinoma tissues using a GenElute mammalian total RNA purification kit (Sigma; ref. 22). cDNA synthesis and quantitative real-time reverse transcription-PCR (RT-PCR, Taqman) was done as previously described (16). ß-Actin mRNA expression was used as a reference for ErbB mRNA quantification. Samples were analyzed in triplicate, and in each measurement, the SD of the CT values was <5% of the mean. ErbB mRNA expression was presented as a percentage of the ß-actin mRNA expression measured from the same sample.
ErbB Gene Amplification Analysis. ErbB4 gene amplification was analyzed by PCR of genomic DNA extracted from 33 patients with known ErbB mRNA expression levels using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). PCR primers and probes were designed to measure the gene copy number of ErbB4 and the reference gene transforming growth factor- (TGF-). Relative copy number of the ErbB4 gene was determined as the ratio of PCR signals obtained for ErbB4 and TGF-. TGF- gene is located on the same chromosome as ErbB4 (2q33) but in the short arm (2p12; refs. 26, 27). Comparison of signals from two genes in opposing arms of the same chromosome allowed detection of isolated gene amplification, as opposed to aneuploidy or polyploidy. The oligonucleotide primers and probes used for analysis of ErbB4 were 5'-TCAAGCATTGGATAATCCCGA-3', 5'-AGTGGCTCATTCACATACTCATCCT-3', and 5'-6-FAM-TATCACAATGCATCCAATGGTCCACCC-TAMRA-3'; and for analysis of TGF-, 5'-AGCCAGAAGAAGCAGGCCAT-3', 5'-GATGATAAGGACAGCCAGGGC-3', and 5'-6-FAM-ACCGCCTTGGTGGTGGTCTCCATC-TAMRA-3'. Real-time PCR was done as previously described (16). Samples were analyzed in duplicate, and in each measurement, the range of the CT values was <5% of the mean.
ErbB2 gene amplification was analyzed by fluorescence in situ hybridization as described (28). ErbB2 was considered to be amplified when the copy number was 
6 in a minimum of 50 counted cells.
ErbB4 Immunohistochemistry. Immunostaining for ErbB4 was done using 1:50 dilution of a monoclonal HFR-1 antibody (NeoMarkers, Fremont, CA) that recognizes the intracellular tail of ErbB4, as described elsewhere (16). The tumor tissue array consisted of 0.6-mm-diameter samples from representative tumor regions obtained from 458 breast cancers patients with a median of 10 years of clinical follow-up after the diagnosis of breast cancer. The preparation of the tumor tissue microarrays, histopathology of tumors, and analysis of ER protein expression have been described (23).
The intensity of ErbB4 staining was scored from all 458 samples as negative, weak, or strong without knowledge of the clinicopathologic data. The cancer cell staining pattern was scored from 410 samples and classified either as membranous or nuclear. When 5% to 100% of the cancer cell membranes stained for ErbB4 (weak or strong intensity), the staining pattern was considered to be membranous. When 5% to 100% of the nuclei stained for ErbB4 (weak or strong intensity), the staining pattern was classified as nuclear. Cytosolic immunoreactivity was observed in conjunction with both the membranous and the nuclear staining, and was not considered a distinct defined staining pattern.
ErbB4 Expression Vectors. The expression vectors encoding the full-length ErbB4 isoform JM-a CYT-2 and JM-b CYT-2 (pCEP4ErbB4JM-aCYT-2 and pCEP4ErbB4JM-bCYT-2) were generated by cloning 4,345- and 4,315-bp XbaI-PmeI fragments from pcDNA3.1ErbB4JM-aCYT-2 and pcDNA3.1ErbB4JM-bCYT-2 plasmids,10 respectively, into NheI and XhoI sites in the pCEP4 vector (Invitrogen, Carlsbad, CA). The XhoI site was filled to blunt-end with Klenow before ligation. Construction of the vector encoding the ICD of ErbB4 of the CYT-2 type (pcDNA3.1ErbB4ICDCYT-2) will be described elsewhere.11
Western Blot Analysis of ErbB4 Cleavage. MCF-7 cells were transfected with pCEP4ErbB4JM-aCYT-2 as previously described (25). Cells were starved overnight in the absence of FCS and stimulated for indicated periods of time with neuregulin-1 (NRG-1; 20 ng/mL; R&D systems, Minneapolis, MN) or phorbol 13-myristate 12-acetate (100 ng/mL; Sigma). Full-length ErbB4 and its cleaved intracellular domain were visualized by Western blotting using an antibody that recognizes the intracellular tail of ErbB4 (Santa Cruz Biotechnology, Santa Cruz, CA), as described (17).
Nuclear Translocation of ErbB4. MCF-7 breast cancer cells (24) were grown on coverslips in the presence of 1 nmol/L estradiol and transiently transfected with 0.5 µg of ErbB4 expression plasmids using FuGENE 6 Transfection Reagent (Roche). Twenty-four hours after transfection, the cells were treated for 1 hour at 37°C with or without leptomycin B (100 ng/mL; Sigma) and phorbol 12-myristate 13-acetate (100 ng/mL). The cells were fixed with methanol, stained with a mouse monoclonal anti-ErbB4 antibody (HFR-1; NeoMarkers; 1:50 dilution) followed by Alexa Fluor goat anti-mouse IgG (Molecular Probes, Eugene, OR). TO-PRO-3 iodide (Molecular Probes) was used to visualize the nuclei. Coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA). Images were obtained by LSM 510 Meta confocal microscope (Carl Zeiss, Inc., Thornwood, NY).
Cell Proliferation Assay. MCF-7 breast cancer cell lines transfected with a vector encoding ErbB4 (pEBS7ErbB4JM-aCYT-2) or an empty vector (pEBS7; ref. 25) were plated at a density of 5 x 104 cells per well on 24-well plates and cultured in the presence or absence of 10 nmol/L estradiol as described above. The cell numbers were counted with hemocytometer.
Anchorage-Independent Growth Assay. Before analysis of growth in soft agar, MCF-7 breast cancer cell lines transfected with a vector encoding ErbB4 (pEBS7ErbB4JM-aCYT-2) or an empty vector (pEBS7; ref. 25) were cultured for 2 days in phenol red–free RPMI supplemented with 10% dextran-charcoal stripped FCS and 1% L-glutamine-penicillin-streptomycin solution. Bottom layers (2 mL) composed of phenol red–free RPMI containing 0.5% agar (Bacto-agar; Difco, Detroit, MI), 10% dextran-charcoal stripped FCS, and 1% L-glutamine-penicillin-streptomycin solution were prepared into 6-well plate wells. Top layers (1.2 mL) consisting of 30,000 MCF-7 cells and phenol red–free RPMI containing 0.33% agar were added onto the solidified bottom layers. The effect of estradiol was tested by including 1 nmol/L estradiol to bottom and top layers in half of the wells. After 3 days of incubation, 200 µL per well of fresh phenol red–free RPMI supplemented with 5% dextran-charcoal stripped FCS and 1% L-glutamine-penicillin-streptomycin solution were added on wells to maintain humidity. Cells were incubated at 37°C for 16 days. Colonies consisting of eight or more cells were counted under a microscope from five arbitrary fields from each of three parallel wells.
Analysis of ERE-Mediated Transcription. MCF-7 cell lines (24) with and without ErbB4 overexpression were generated using an ErbB4 expression vector (pCEP4ErbB4JM-aCYT-2) and the empty vector (pCEP4), as previously described (25). Cells were cultured in 24-well plates and transiently transfected with 0.5 µg of a firefly luciferase reporter construct including two ERE and an SV40 promoter (pGL2-2xERE). A herpes simplex virus-TK promoter driven Renilla luciferase reporter plasmid (pRL-TK; Promega, Madison WI) was cotransfected for normalization of the ERE-regulated luciferase signal. Luminescence from firefly and Renilla luciferases was detected 24 hours after transient transfection and treatment with or without 10 nmol/L estradiol using a Dual-Luciferase Reporter Assay System kit (Promega).
Statistical Analysis. Associations of ErbB mRNA expression levels with other variables were analyzed using nonparametric, tie-corrected, Spearman's rank test (SPSS for Windows; SPSS, Inc., Chicago, IL). Association of ErbB4 protein expression with survival was analyzed with Kaplan-Meier statistics, and survival between groups was compared using the log-rank test. Distant disease-free survival was used for statistical analyses and was defined as the time computed from the date of the diagnosis of breast cancer to the first detection of distant recurrence or to death from breast cancer. All Ps are two tailed.
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Results |
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Expression of mRNAs Encoding Cleavable ErbB4 Isoforms in Breast Cancer Is Associated with Estrogen Receptor Positivity and High Histologic Grade of Differentiation. ErbB4 overexpression has been associated with prognostic variables, such as the tumor ER content in breast cancer (5, 6). In this series, the mean ErbB4 JM-a mRNA expression was 5.7-fold greater in ER-positive than in ER-negative carcinoma patients (4% versus 0.7% of ß-actin mRNA, respectively; Fig. 1B). ErbB4 JM-a mRNA expression associated with expression of the ER and progesterone receptor (P < 0.001 and P = 0.003, respectively; Table 1). Similarly, expression of both cytoplasmic isoforms CYT-1 and CYT-2 associated with ER and progesterone receptor expression (P 0.001 for all comparisons). Expression of JM-a, CYT-1, and CYT-2 isoforms also associated with a high histologic grade of differentiation (P 0.002 for all comparisons). These results indicate that expression of the two proteinase-sensitive ErbB4 isoforms, JM-a CYT-1 and JM-a CYT-2, in breast cancer, associates with prognostic factors that suggest a favorable outcome: the presence of hormone receptors and high histologic grade of differentiation.
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0.008 for all three ErbB4 transcripts) but not with expression of ErbB3. No significant associations were observed between ErbB2 mRNA expression and expression of other ErbB mRNAs or ER (Fig. 2; Table 1; data not shown).
ErbB4 Isoform mRNAs Are Expressed in Breast Cancer in Similar Maximal Relative Quantities as ErbB2 mRNAs, but only the ErbB2 Gene Is Amplified. ErbB2 gene amplification is predictive for cancer responsiveness to trastuzumab (30). In this series, the highest copy number of ErbB2 was 20 to 40 copies per cell (31) and the presence of 6 copies per cell was used as the cutoff number to define ErbB2 amplification. The greatest ErbB2 mRNA expression levels in patients with gene amplification were up to 220-fold higher than the mean mRNA expression level measured in the normal mammary glands (688% versus 3.2% of ß-actin mRNA, respectively; Fig. 2). Interestingly, the same range was observed for the maximum relative ErbB4 mRNA expression. The highest ErbB4 mRNA levels in cancer samples reached 170-fold above the values measured from the histologically normal mammary glands (16.7% versus 0.1% of ß-actin mRNA, respectively; Fig. 2).
To determine whether ErbB4 gene was amplified in breast cancer, a real-time PCR analysis was done with primers and probes specific for the ErbB4 gene and the reference gene TGF-. The amplification analysis was done for 33 of the patients from whom ErbB4 mRNA expression data was also available (Fig. 3). The mean ErbB4/TGF- signal ratio, used as an indicator of relative ErbB4 gene copy number, was 1.2 (SD = 0.4), and the maximal ratio was 2.0. These data suggest that ErbB4 gene amplification did not contribute to ErbB4 mRNA overexpresion in the series.
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In univariate survival analysis, breast cancer patients with strong ErbB4 expression tended to have better survival than patients with weak or no ErbB4 staining (Fig. 4A, left; P = 0.09). Because ErbB4 mRNA expression clearly correlated with the presence of ERs (Fig. 1B), the analysis was repeated as stratified by the ER status. In women with ER-positive cancer, strong ErbB4 expression was associated with significantly longer survival than weak expression or no ErbB4 immunoreactivity (P = 0.01). In turn, no significant association was present between ErbB4 protein expression and survival among patients with ER-negative cancer (P = 0.86). These results suggest that ErbB4 protein expression associates with a favorable prognosis among patients with ER-positive breast cancer.
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An Intracellular Fragment of a Cleavable ErbB4 Isoform Translocates into the Nucleus of Breast Cancer Cells. To directly assess whether a proteinase cleavable ErbB4 isoform overexpressed in breast cancer can be translocated to the nucleus, ER-positive MCF-7 breast cancer cells were transiently transfected with cDNAs encoding the proteinase cleavable JM-a CYT-2 isoform, or the noncleavable JM-b CYT-2 isoform. HFR-1 antibody that recognizes the intracellular tail of ErbB4 was used to visualize the ErbB4 ICD by confocal microscopy. Nuclear staining was observed in cells overexpressing JM-a CYT-2, but not in cells expressing JM-b CYT-2, when nuclear export of proteins was blocked by treatment with leptomycin and ErbB4 shedding was stimulated with phorbol 12-myristate 13-acetate (Fig. 5A). To eliminate the need for cellular proteinase activity for analyzing subsequent targeting of the proteolytically released ICD, the MCF-7 cells were transiently transfected with a plasmid encoding only the CYT-2 ICD. This CYT-2 fragment readily translocated and accumulated in the nuclei also in the absence of leptomycin (Fig. 5A). Basal, as well as NRG-1- and phorbol 12-myristate 13-acetate–stimulated cleavage of ErbB4 JM-a CYT-2 in MCF-7 cells was also shown by visualizing the cleavage product, 80-kDa ICD, by Western blotting (Fig. 5B). Taken together, these results indicate that the intracellular domain of ErbB4 can translocate into the nucleus of breast cancer cells.
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Expression of ErbB4 JM-a CYT-2 might promote ER-independent growth by interacting with the ER signaling pathway that regulates transcription of estrogen-responsive genes. The effect of ErbB4 JM-a CYT-2 overexpression on ER-mediated transcription was analyzed by transiently expressing an ERE-driven luciferase reporter gene in MCF-7 cells. Renilla luciferase reporter plasmid was cotransfected to control the transfection efficiency. In the absence of estradiol, overexpression of ErbB4 JM-a CYT-2 increased the ERE-mediated transcriptional activity by 3.7-fold (Fig. 5E). ErbB4 overexpression in the presence of estradiol resulted in a 1.6-fold enhancement as compared with the vector transfected cells.
These results show that overexpression of a cleavable ErbB4 isoform can promote proliferation and colony formation of hormone-dependent breast cancer cells both in the presence and absence of estrogen. The findings further suggest that the mechanism of growth promotion by ErbB4 may include enhancement of the transcriptional regulation of ER-responsive genes. Taken together with data from the clinical series, our findings suggest that cleavable ErbB4 isoforms are present in ER-positive breast cancer, and that they are capable of stimulating cellular functions associated with malignant cell behavior.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of transcripts encoding ErbB receptors in human tissue samples was specifically quantitated by real-time RT-PCR, which we have previously shown to be accurate in measuring ErbB mRNA expression, and to reflect ErbB expression in tumor cells at protein level (16). Our results show that only cleavable isoforms of ErbB4 are present in breast cancer. The expression of these isoforms also significantly associated with both the presence of ER and a high histologic grade of differentiation. In particular, the association of ErbB4 expression with ER expression is consistent with previous studies, none of which address the role of specific ErbB4 isoforms (4–6, 8).
Although no isoform-specific antibodies are available, subcellular localization of the ErbB4 ICD can provide information about the presence of the cleavable or noncleavable ErbB4 isoforms. Our immunocytochemistry data showed nuclear ErbB4 staining when a cleavable ErbB4 isoform or its sole ICD was overexpressed in MCF-7 breast cancer cells, but not in cells overexpressing a noncleavable isoform. Consistent with the expression of ErbB4 transcripts encoding cleavable ErbB4 isoforms, 21% of breast cancer tissue samples analyzed showed nuclear ErbB4 immmunoreactivity, suggesting that the cleavable ErbB4 isoforms are indeed proteolytically cleaved in vivo. In accordance with our findings, nuclear ErbB4 staining in breast cancer cells has previously been reported from studies using antibodies that recognize the ICD of ErbB4 (13, 35, 36).
In survival analyses, overall ErbB4 protein expression intensity was associated with a high survival rate. Intriguingly, the association with survival was dependent on the subcellular localization of the receptor. Nuclear ErbB4 staining associated with inferior survival as compared with membranous staining. Hypothetically, this might be due to the tumor-promoting properties of the nuclear ErbB4 fragments, or from the loss of tumor-suppressing activity of the full-length ErbB4 at the cell membrane. Our in vitro studies showing increased cancer cell proliferation rate, growth in soft agar, and reduced estrogen dependency, suggest a growth-promoting role for the cleavable ErbB4 JM-a CYT-2 isoform. These data also imply that the presence and activity of the relevant ErbB4-cleaving proteinase, such as TACE (19), in the tumor tissue may be more biologically relevant than the actual ErbB4 isoform, as cleavable and noncleavable ErbB4 isoforms may function similarly in the absence of proteinases (14). The significance of proteinase-cleavage in the process of translocation of receptor fragments was supported by our in vitro assays, in which the ErbB4 ICD was readily seen in the nucleus. When transfecting the full-length receptor, ErbB4 cleavage and detection of ErbB4 in the nucleus was clearly enhanced by activation of proteinases by phorbol 12-myristate 13-acetate. Furthermore, reports have suggested that TACE expression is up-regulated both at the mRNA and protein levels in breast cancer (37).13
ErbB4 cleavage in breast cancer cells could be stimulated by ligands, such as NRG-1 (38). Enhanced cleavage in response to NRG-1 supplied into culture medium was also shown in our experiments with MCF-7 transfectants. However, the MCF-7 cells did not express detectable levels of NRG-1 mRNA when assessed by real-time RT-PCR (data not shown), ruling out a NRG-1-mediated autocrine loop regulating ErbB4 cleavage in this cellular context. Our experiments with in vivo tissue samples have also indicated that nuclear ErbB4 ICD localization is frequently observed in breast cancer sections with relatively low levels of NRG-1 immunoreactivity,14 suggesting that NRG-1 may not function as a key regulator of ErbB4 processing in vivo. Analysis of NRG-1 mRNA expression levels in the series of 62 breast cancer RNA samples also failed to show significant NRG-1 overexpression when compared with the 20 normal samples, or association of NRG-1 expression levels with ErbB4 expression, hormone receptor status, or histologic grade of differentiation (data not shown).
In line with the association of ErbB4 mRNA expression and ER expression, ErbB4 protein expression associated with survival in breast cancer patients who were diagnosed with ER-positive cancer. The connection between ErbB4 signaling and ER signaling is also supported by our in vitro experiments with the estrogen-dependent breast cancer cells, in which ErbB4 overexpression promoted cellular responses also in the absence of exogenous estrogens. Furthermore, the growth of another ER-positive breast cancer cell line T-47D was suppressed by down-regulating endogenous overexpression of cleavable ErbB4 with ErbB4-specific small interfering RNA (data not shown) or ribozymes (10). Interestingly, ribozyme-mediated ErbB4 down-regulation did not reduce the growth of an ER-negative MDA-MB-453 breast cancer cell line (10), in accordance with our unpublished findings of a lack of effect of ErbB4 JM-a CYT-2 overexpression on basal growth of some ER-negative nonneoplastic cell lines.
ErbB4 overexpression also enhanced ERE-mediated transcription, indicating a functional connection between ErbB4 and ER signaling pathways. Several previous studies have addressed the interaction between the ErbB and ER pathways. For example, activation by NRG or epidermal growth factor, or overexpression of ErbB2 have been shown to lead to ER phosphorylation, modulation of ER-responsive transcription, or hormone-independent growth in ER-positive breast cancer cells (39–42). Although providing support for the general concept for functional ErbB-ER interaction, these previous studies have not specifically addressed whether ErbB4 is involved in the crosstalk or suggested a molecular mechanism by which ErbB4 promotes ER-dependent cellular responses.
Taken together, our findings suggest that targeting ErbB4 in breast cancer could be favorable in cases where cleavable ErbB4 isoforms are overexpressed and proteolytically released ICD fragments have translocated to the nuclei, but not when ErbB4 is located at the cell surface. One approach for specific ErbB4 targeting could be compounds that block the activity of the known ErbB4 processing enzymes (i.e., TACE or -secretase). These results further suggest that ErbB4 inhibitors might enhance the efficacy of ER inhibitors and inactivators, such as tamoxifen and fulvestrant, but these hypotheses require confirmation in clinical trials.
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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 Maria Tuominen for excellent technical assistance and Dr. Mika Savisalo for help in construction of expression plasmids.
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Footnotes |
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10 Määttä et al., submitted for publication.
11 Peri et al., in preparation.
12 Määttä et al., submitted for publication.
13 Määttä et al., submitted for publication.
Received 8/31/04. Revised 11/22/04. Accepted 11/30/04.
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References |
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-converting enzyme is required for cleavage of erbB4/HER4. J Biol Chem 2000;275:10379–87.-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. gene on human chromosome 2 close to the breakpoint of the Burkitt's lymphoma t(2;8) variant translocation. Cancer Res 1985;45:5593–7. in tumors. EMBO J 2003;22:1114–24.[CrossRef][Medline]
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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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 R. S. Muraoka-Cook, M. Sandahl, C. Husted, D. Hunter, L. Miraglia, S.-m. Feng, K. Elenius, and H. S. Earp III The Intracellular Domain of ErbB4 Induces Differentiation of Mammary Epithelial Cells Mol. Biol. Cell, September 1, 2006; 17(9): 4118 - 4129. [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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J. A. Maatta, M. Sundvall, T. T. Junttila, L. Peri, V. J. O. Laine, J. Isola, M. Egeblad, and K. Elenius Proteolytic Cleavage and Phosphorylation of a Tumor-associated ErbB4 Isoform Promote Ligand-independent Survival and Cancer Cell Growth Mol. Biol. Cell, January 1, 2006; 17(1): 67 - 79. [Abstract] [Full Text] [PDF]
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R. R. Arasada and G. Carpenter Secretase-dependent Tyrosine Phosphorylation of Mdm2 by the ErbB-4 Intracellular Domain Fragment J. Biol. Chem., September 2, 2005; 280(35): 30783 - 30787. [Abstract] [Full Text] [PDF]
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). ß-Actin mRNA expression was used to normalize ErbB expression. SDs of three parallel analyses were <5% of the means.
