Jab1 is a target of EGFR signaling in ERα-negative breast cancer
c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERα-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERα- phenotype.
MDA-MB-231 and MDA-MB-468 ERα-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry.
EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERα- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004).
Jab1 is a target of EGFR signaling in ERα- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERα- breast cancer.
KeywordsBreast Cancer Epidermal Growth Factor Receptor Epidermal Growth Factor Epidermal Growth Factor Receptor Signaling S100A7 Expression
ductal carcinoma in situ
ethylene diamine tetraacetic acid
epidermal growth factor
epidermal growth factor receptor
ethylene glycol tetraacetic acid
extracellular signal-regulated kinase
glyceraldehyde 3-phosphate dehydrogenase
epidermal growth factor receptor 2
c-Jun activation domain-binding protein-1
c-jun N-terminal kinase
nuclear factor-kappa B
phosphate-buffered saline + 0.05% Tween-20
phosphorylated extracellular signal-regulated kinase
short interfering RNA
Recent therapeutic advances have improved survival for many patients with breast cancer. These advances have been most impressive for targeted therapies, such as those targeting the estrogen receptor (ER) and the human epidermal growth factor receptor (EGFR) 2 (Her2). These advances have specifically benefited the subsets of patients with tumors that exhibit ERα+ or Her2+ phenotypes, respectively. Other subsets of tumors such as the so-called 'triple-negative' breast tumors, ERα-/progesterone receptor-negative (PR-)/Her2-, remain difficult to treat. The ERα- phenotype, which includes the triple-negative phenotype, has dominated clinical and biological consideration of breast cancer for many years and has been reproducibly shown in microarray studies to be distinct from ERα+ breast cancer [1, 2]. Identification of key signaling molecules and pathways relevant to ERα- breast cancer is therefore an important step toward the goal of improving breast cancer therapy [3, 4, 5].
We and others have previously identified genes that are highly associated with the ERα- phenotype, including EGFR and S100A7 [6, 7, 8, 9, 10, 11]. Epidermal growth factors (EGFs) are important in the biology of both normal and malignant breast tissue, exerting their effects through their tyrosine kinase growth factor receptors. EGFR expression is strongly associated with the ERα- phenotype [12, 13, 14] such that there is a strong inverse relationship between EGFR and the steroid receptor, ERα [11, 15]. S100A7 (psoriasin) is a small calcium-binding protein belonging to the S100 gene family [16, 17]. It is highly expressed in some ductal carcinoma in situ (DCIS) [18, 19, 20] and invasive breast  carcinomas. Within both of these stages, S100A7 expression is strongly related to the ERα- phenotype [6, 8].
c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein and is a target of S100A7 that can mediate many of its biological effects, including induction of nuclear factor-kappa B (NF-κB) and promotion of cell survival [21, 22]. Additional evidence that Jab1 is a key gene in breast cancer progression comes from the recent finding that it is a downstream target for Her2 . Furthermore, Jab1 has been found to interact with c-myc to act as a master regulator of the 'wound response' gene signature in breast cells [24, 25]. The wound response signature represents a conserved cluster of gene responses to changes in serum, exclusive of known proliferation response genes. It can be generated in epithelial and fibroblast cells and is associated with poor outcome in invasive breast cancer. Jab1 also interacts with many components of known cell signaling pathways in the context of both phosphorylation and proteasomal activities, typically resulting in translocation of Jab1 to the nucleus and modification of activity in downstream pathways. These interactions result in increased activation protein-1 (AP-1)  and NF-κB  activity and degradation of the cell cycle inhibitor p27 (Kip1)  and the transforming growth factor-β signaling component Smad4 .
Taken together, these findings implicate Jab1 as an important factor in several signaling pathways in breast cancer. Since the S100A7 gene is strongly associated with the ERα- phenotype and our studies have implicated Jab1 as a mediator of S100A7 action [7, 22], we set out to examine the possibility that Jab1 may be an important component of the mechanism of action of other key ERα--associated genes, focusing here specifically on EGFR.
Materials and methods
Cell lines, antibodies, and reagents
Human breast carcinoma cell lines MDA-MB-468 and MDA-MB-231 (both ERα- and EGFR+ and derived from invasive breast carcinomas) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum under standard conditions as previously described . The antibodies used for immunoblotting and immunoprecipitation were Jab1 (1:3,000) (Sigma-Aldrich, Oakville, ON, Canada); p27 (1:200) (BD Biosciences, Mississauga, ON, Canada); Lamin A/C (1:1,000), pEGFR (phosphorylated EGFR) (1:1,000), extracellular signal-regulated kinase (ERK) (1:1,000), phosphorylated ERK (pERK) (1:1,000), AKT (1:2,000), and pAKT (1:1,000) (Cell Signaling Technology, Inc., Danvers, MA, USA); EGFR (1:1,000) (VWR International, Mississauga, ON, Canada); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:4,000; Advanced ImmunoChemical Inc., Long Beach, CA, USA). The antibody to S100A7 was a rabbit polyclonal generated and described previously [6, 21]. Goat anti-mouse and goat anti-rabbit IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All EGF treatments were for 4 hours and, with the exception of the EGF dose experiments, were 50 ng/mL (Millipore Corporation, Billerica, MA, USA). Treatments with ERK inhibitor PD98059 were at 20 μM for 4 hours (Cell Signaling Technology, Inc.).
Immunofluorescence, nuclear extraction, and immunoblotting
Following treatment with selected reagent (EGF or PD98059), cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 0.2% bovine serum albumin. Cells then were stained for Jab1 (1:50) using the primary antibodies described above and Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:100) (Invitrogen Corporation, Carlsbad, CA, USA). For double-immunostaining of Jab1 and pERK or p27, cells first were stained for Jab1 as described above and then were stained for pERK (1:100) or p27 (1:100) using the primary antibodies described above and Alexa Fluor 594-conjugated chicken anti-mouse IgG secondary antibody (1:100) (Invitrogen Corporation). Immunofluorescence images were captured using a Leica DM 6000B immunofluorescence microscope (Leica, Wetzlar, Germany), and image analysis was performed using OpenLab 4.0.4 software (Improvision Ltd., Coventry, UK).
Nuclear extracts were prepared by rinsing culture dishes with phosphate-buffered saline (PBS) and then resuspending cells in a nuclear extracting lysis buffer (10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] [pH 7.9], 10 mM KCl, 0.1 mM EDTA [ethylene diamine tetraacetic acid], and 0.1 mM EGTA [ethylene glycol tetraacetic acid]) and incubating for 20 minutes on ice. Cells were further fractionated by adding 25 μL of Nonidet P-40 (10%), vortexing for 10 seconds, and centrifuging at 15,000 g for 10 minutes at 4°C. The pellet (nuclei) was then resuspended in 50 mM HEPES (pH 7.3), 150 mM NaCl, 2.5 mM EGTA, 10% glycerol, 0.1% Tween-20, 1 mM NaF, 1 mM DTT (dithiothreitol), 0.1 mM Na3VO3, and one tablet of EDTA-free protease inhibitor (Roche Diagnostics, Basel, Switzerland) per 10 mL, incubated 20 minutes on ice, and then boiled prior to loading.
Protein samples were separated by SDS-PAGE (4% to 12% acrylamide) and transferred to 0.2 μm nitrocellulose (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking in 5% skim milk powder in PBST (PBS + 0.05% Tween-20) for 30 minutes, blots were rinsed in PBST and then incubated with the primary antibody overnight in PBST at 4°C. Blots were washed in PBST for 10 minutes, three times, and then were incubated with the appropriate secondary antibody for 1 hour, followed by washing in PBST for 10 minutes, three times. Blots were developed by chemiluminescence (enhanced chemiluminescence; made in house) and were exposed to X-OMAT Kodak film (Eastman Kodak Company, Rochester, NY, USA). For all assays, at least three separate experiments were performed.
Knockdown of Jab1
Jab1 expression was inhibited by transfecting cells with a pool of four different Jab1-specific short interfering RNA (siRNA) duplexes (Dharmacon, Inc., Chicago, IL, USA). Scrambled siRNA was used as a non-targeting control (Dharmacon, Inc.). siRNA transfection was carried out using DharmaFECT 1 transfection reagent/vehicle (Dharmacon, Inc.) according to manufacturer recommendations. siRNA was transfected at a concentration of 100 nM, after which cells were cultured for 48 hours prior to lysis and protein harvest. Densitometry of Western blots was conducted using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA). Densitometry results for p27 were normalized to GAPDH within each treatment. Statistical analysis of p27 densitometry was performed with JMP software (version 7.0) (SAS Institute Inc., Cary, NC, USA) using t tests.
Tissue microarray breast cancer cohort
After the institutional research ethics board gave ethical approval, a tissue microarray (TMA) was obtained from the Manitoba Breast Tumor Bank (Winnipeg, MB, Canada) to investigate the relationship between Jab1 and EGFR and S100A7 in breast tumors in vivo. The TMA was constructed from duplicate 0.6-mm tissue cores that were removed from the central portion of a representative paraffin block from each tumor and arrayed within one of seven paraffin blocks, using a tissue arrayer (Beecher Instruments, Inc., Sun Prairie, WI, USA). The TMA included interpretable cores from 424 invasive breast carcinomas. Case selection was designed to mirror the distribution of major prognostic clinical-pathological features (size, grade, and lymph node and ER status) that the entire tumor bank collection accrued over the period 1992 to 2002 and was also based on the following criteria: (a) a minimum patient follow-up of 60 months and tumors that had (b) an invasive component of greater than 20% of the tissue section and (c) less than or equal to 10% of the normal epithelial content. ER- status was defined by ligand-binding assay (LBA) criteria of less than 3 fmol/mg protein. The criteria for interpretation of the variables were as follows: (a) PR+ status was defined as greater than or equal to 15 fmol/mg protein by LBA, (b) tumor grading was according to the Nottingham system, and (c) tumor size was classified as either small (≤ 20 mm) or large (>20 mm). Patients received a range of treatments, including local radiotherapy (n = 163) and systemic hormonal and/or chemotherapy (n = 375). Patient outcome was defined as the time from initial surgery to the date of death attributable to breast cancer only.
Immunohistochemistry and statistical analysis
Immunohistochemistry (IHC) staining for Jab1, EGFR, and S100A7 was performed using an automated tissue immunostainer (Ventana Medical Systems, Inc., Tucson, AZ, USA) and using bulk reagents supplied by the manufacturer (IHC protocol previously described [6, 21]). Primary antibody incubation for Jab1 and S100A7 was 32 minutes. Tumor cell staining was scored for each protein in semi-serial sections by a single observer (PHW) but in independent sessions for each protein to ensure blinded independent scoring. For Jab1 and S100A7, only nuclear expression was scored as cytoplasmic signals were generally weak and difficult to quantify. IHC staining was scored using a semi-quantitative IHC score (IHC score = [percentage of positive neoplastic epithelial cells] × [staining intensity ranked from 0 to 3]) that ranged from 0 to 300. In univariate analysis, cut-points for Jab1 and S100A7 were those used in previous studies [6, 29] to distinguish low from high expression (Jab1 IHC scores of greater than 50, corresponding to nuclear expression in greater than 50% of tumor cells, and S100A7 IHC scores of greater than 0) or EGFR IHC scores of greater than 100, corresponding to 2+ or 3+ intensity as used for the clinical assessment of Her2 . Statistical analysis was performed with JMP software (version 7.0) (SAS Institute Inc.) and GraphPad Prism (version 4.2) (GraphPad Software, Inc., San Diego, CA, USA) using Spearman correlation, chi-square, Mann-Whitney t test, or log-rank test as appropriate.
Treatment with EGF influences localization of Jab1
Effect of EGF on Jab1 translocation is mediated through the ERK pathway
EGFR signaling regulates genes downstream of Jab1
Jab1 expression correlates with EGFR in breast tumors
Clinicopathological features of the tumor cohort
Correlation between Jab1 expression and clinicopathological features of the estrogen receptor-alpha-negativea subset
Outcome analysis of the ERα- subgroup showed no significant association between survival and Jab1+, EGFR+, or S100A7+ status when each marker was analyzed independently. Comparison of the subset of ERα- tumors that were positive for all three markers, EGFR/S100A7/Jab1, showed that this phenotype was associated with worse outcome compared with EGFR/S100A7/Jab1- tumors (n = 10 versus 13; P = 0.07).
ERα-, and in particular the 'triple-negative' subset of breast cancer lacking detectable ERα, PR, and Her2, has emerged as a challenge for systemic therapy now that successful targeted therapies have become available for the treatment of other phenotypic subgroups. Nevertheless, one prominent feature of the ERα- subgroup is expression of the EGFR [34, 35], raising the possibility that this receptor may offer a target for treatment of this subgroup. However, anti-EGFR therapies, alone or in combination with chemotherapy, have benefited only a small cohort of patients in the face of both de novo and acquired resistance to these therapies . To circumvent this resistance, it will be important to understand more of the signaling pathways downstream of EGFR in ERα- tumors . Recent findings suggest that the Jab1 protein may be the central mediator in several of the biological circuits that promote tumor progression in breast cancer cells . We have therefore set out to explore whether Jab1 is also involved in EGFR signaling. We have shown that EGFR activation in ERα- breast cell lines is associated with Jab1 nuclear localization and that these changes relate to activation of both AKT and ERK pathways and modulation of Jab1 downstream genes. Furthermore, higher Jab1 nuclear expression was associated with EGFR+ status in a cohort of ERα- breast tumors, and this relationship was most significant in tumors that expressed both EGFR and S100A7 markers.
Jab1 is a multifunctional protein that has been shown to interact with several components of cell signaling pathways within in vitro yeast systems and human cell lines. These interactions usually are associated with translocation of Jab1 from the cytoplasm to the nucleus and result in either enhanced activity of transcription factors, including c-Jun [26, 38], AP-1 [26, 38], HIF-1α , steroid receptors, and cofactors , or the promotion of degradation of interacting proteins, including p27 [27, 32], Smad4 , MIF1 , and p53 . Although the physiological relevance of some of these interactions is mostly unknown (specifically, within the context of breast epithelial cells ), they are evidently complex. For example, in documenting that EGF can affect Jab1 localization in breast cells, we have confirmed previous findings that EGF affects a representative Jab1 downstream gene, p27, and that these effects correlate with alterations of PI3K (phosphatidylinositol-3-kinase)/AKT [44, 45]. However, we also show here that changes in the ERK pathway may contribute to the effects of Jab1 in some breast cell lines. Interestingly, others recently have shown that Her2 (EGFR2) signaling can regulate Jab1 through the AKT/β-catenin pathway  and, in a subsequent study, that Her2 modulates p27 through Jab1 . In contrast to our data and other interaction effects, these studies concluded that Her2-mediated Jab1 regulation occurs at the transcriptional level. Others have shown Her2 activation to be associated with relocalization to the cytoplasm rather than nuclear accumulation of Jab1  and that activation of the Her2-ras-MAP kinase pathway can alter Jab1 and stimulate downregulation of p27 . One potential explanation for these apparent incongruities relates to the different cell lines used in these studies .
Jab1 recently has been identified as a master regulator of a spectrum of genes (the 'wound response signature') that may promote tumor progression in breast cancer [24, 25]. Jab1 also acts as an essential modulator of c-myc transcriptional activity, regulating c-myc protein ubiquitination and stability. Thus, Jab1 and c-myc together influence the expression of a subset of c-myc-regulated genes that comprise the 'wound response'. Jab1 and c-myc expression and upregulation of the wound response signature do not appear to be limited to specific phenotypic subgroups of breast tumors . However, deregulation of c-myc is known to occur in ERα- breast cell lines and to be associated with PR- breast cancer and resistance to endocrine therapy [49, 50]. We have previously identified Jab1 as a mediator of several intracellular and biological effects of S100A7, which itself may promote breast tumor progression [21, 22]. Pathways downstream of S100A7 are also of interest because of this gene's strong association with the ERα- phenotype in DCIS and, when expression persists, in invasive breast cancer [7, 8, 18, 19].
These observations raise the question of whether Jab1 is a common factor in mediating cell signaling pathways that are important in ERα- breast cancer. Our data presented here suggest that Jab1 may be regulated by the EGFR and S100A7 pathways in ERα- breast cells. Notably, we  and (very recently) others  have shown that there may be crosstalk between S100A7 and EGFR and that S100A7 can regulate EGFR signaling. Jab1 expression in breast cancer has been explored previously by us  and others [23, 29, 53]. High nuclear Jab1 was associated with reduced p27 expression in all of these studies, in both DCIS  and invasive disease [29, 53]. But no consistent association with any prognostic features, including ERα status, has emerged. However, there is some indication that increased Jab1 might be related to poor outcome . Nevertheless, these studies were based on small  and/or highly selected case series (in terms of stage  and nodal status [29, 53]). The present study has now extended these findings by assessing nuclear Jab1 expression in relation to prognostic features and markers in a large cohort of invasive breast tumors representative of the case distribution in a large tumor bank. We have confirmed that Jab1 is not strongly correlated with any prognostic features examined, except in subset analysis in which there was a positive association with nodal metastasis in the ERα- subset. Despite the observation of a possible association between Jab1 and worse outcome in the ERα- subset, this was not statistically significant, and the same was true for EGFR and S100A7. This difference from previous findings [6, 29, 54] may relate to the use of a TMA for the present study. While this format is optimal for examining coexpression of biomarkers within small defined tumor regions, it may not be optimal for outcome analyses of genes that are heterogeneously expressed within tumors. However, the aggregate results from this and other studies support the conclusion that nuclear Jab1 is only weakly related, if at all, to standard prognostic features and outcome as an independent factor.
This lack of clear association with complex phenotypic traits represented by prognostic factors such as tumor grade or with patient outcome is intriguing given the range of potentially important signaling pathways and proteins that Jab1 influences. On the other hand, it is perhaps not surprising given that these multiple factors may influence the equilibrium between nuclear and cytoplasmic Jab1 and its activity. It has also been shown that p53 and c-Jun can compete for Jab1 . These and other interacting proteins might influence its collaborative role with c-myc as a regulator of the wound response. Jab1 can also exist in several different protein complexes within both cellular compartments in breast cells, further complicating analysis and deductions based only on protein localization . It will be interesting to examine Jab1 in relation to c-myc and Jab1 protein complex status in future outcome analyses.
Jab1 lies at the intersection of several signaling pathways that are believed to be important in breast cancer cells and may be a decisive influence on the outcome of specific pathway alterations and their cumulative effects on progression. Our results implicating Jab1 in the EGFR pathway, in addition to its role in the S100A7 pathway, suggest that Jab1 may be particularly important in the ERα- breast cancer cell and provide insight into the application of new therapeutic strategies (for example, proteasome inhibitors) directed to this important and difficult-to-treat subset of breast cancer.
PHW is supported by an operating grant from the Canadian Institutes of Health Research (CIHR) (grant number MOP-64349). JW is supported by a Michael Smith Foundation for Health Research Trainee Fellowship and by a Susan Komen for the Cure Postdoctoral Fellowship grant. This study was also supported by the BC Cancer Agency's Tumor Tissue Repository and the Manitoba Breast Tumor Bank, funded by a grant from the CIHR (grant number PRG80155), and supported by CancerCare Manitoba.
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