The cyclic (c)AMP responsive element binding protein (CREB) plays a key role in many cellular processes, including differentiation, proliferation, and signal transduction. Furthermore, CREB overexpression was found in tumors of distinct origin and evidence suggests an association with tumorigenicity. To establish a mechanistic link between HER-2/neu–mediated transformation and CREB protein expression and function, in vitro models of HER-2/neu–overexpressing and HER-2/neu–negative/silenced counterparts as well as human mammary carcinoma lesions with defined HER-2/neu status were used. HER-2/neu overexpression resulted in the induction and activation of CREB protein in vitro and in vivo, whereas short hairpin RNA (shRNA)–mediated inhibition of HER-2/neu correlated with downregulated CREB activity. CREB activation in HER-2/neu–transformed cells enhanced distinct signal transduction pathways, whereas their inhibition negatively interfered with CREB expression and/or activation. CREB downregulation in HER-2/neu–transformed cells by shRNA and by the inhibitors KG-501 and lapatinib caused morphologic changes, reduced cell proliferation with G0–G1 cell-cycle arrest, which was rescued by CREB expression. This was accompanied by reduced cell migration, wound healing, an increased fibronectin adherence, invasion, and matrix metalloproteinase expression. In vivo shCREB-HER-2/neu+ cells, but not control cells, exerted a significantly decreased tumorgenicity that was associated with decreased proliferative capacity, enhanced apoptosis, and increased frequency of T lymphocytes in peripheral blood mononuclear cells. Thus, CREB plays an important role in the HER-2/neu–mediated transformation by altering in vitro and in vivo growth characteristics.

Implications: These data suggest that CREB affects tumor immunogenicity and is a potential target for cancer therapy. Mol Cancer Res; 11(11); 1462–77. ©2013 AACR.

Cyclic (c)AMP responsive element binding protein (CREB) is a 43-kDa protein that belongs to the leucine-zipper class of transcription factors, and is activated by multiple signal transduction pathways (1). CREB has been demonstrated to be a transcriptional coactivator, thereby acting as a mediator between different signal-transduction cascades and the downstream target-gene transcription. CREB binds to the cAMP response element (CRE), thereby recruiting coactivators of the histone acetylase transferase p300 to the promoter of cAMP-responsive genes, which is accompanied by phosphorylation of CREB at Ser133 (pCREB), which is located in the kinase-inducible domain (KID; refs. 1, 2). CREB can be phosphorylated by many different protein kinases, including protein kinase A (PKA), protein kinase B (PKB)/Akt, mitogen-activated protein kinases (MAPK), and p90 ribosomal S6 kinase (p90RSK; ref. 3). CREB target genes are involved in distinct biologic processes, which include cell metabolism, survival, proliferation, differentiation, cell cycle, DNA repair, immortalization of cells, inflammation, and immune modulation (4). Furthermore, it has been recently demonstrated that CREB has a critical role in the induction and maintenance of malignant transformation and pathogenesis of different cancer types, due to inappropriate activation or inactivation of components of the cAMP signaling pathway (4–6). In this context, CREB is often overexpressed in solid tumors of distinct histology, for example, breast cancer and leukemias, when compared with normal adjacent tissues (4, 7), which might be due to downregulation of CREB by inhibitors, such as the inducible cAMP repressor and the microRNA (miRNA, miR) miR-34b (8, 9). Furthermore, CREB downregulation is directly associated with tumor progression, disease stage, accelerated relapse, and reduced survival of patients (10). Although the exact molecular mechanisms by which CREB contributes to cancer development have not yet been defined, CREB has been shown to regulate a number of genes that are involved in cell proliferation, apoptosis, invasion, and metastasis (4, 11, 12). However, a direct link between oncogenic transformation and CREB has not yet been determined.

HER-2/neu is a member of the EGF receptor (EGFR) family and it has intrinsic tyrosine kinase activity, thereby modulating receptor-mediated signal transduction in the absence of the ligand (13). HER-2/neu can form heterodimers with other HER family members, and depending on the given heterodimer combination, this can activate different intracellular signaling cascades, such as the MAPKs, Akt/phosphoinositide 3-kinase (PI3K), Janus-activated kinase (JAK), STAT3, and protein kinase C (PKC) pathways (14). Thus, similar to CREB, HER-2/neu can affect a large number of cellular processes, including cell differentiation, proliferation, apoptosis, and cell cycle. Under physiologic conditions, HER-2/neu is expressed at low levels in many epithelial cells (15), whereas it is overexpressed and/or amplified in human tumor cells of distinct origin, including those of mammary carcinoma and colorectal cancer (16–18). HER-2/neu–mediated transformation is associated with initiation and maintenance of the transformed phenotype, aggressiveness of tumors, progression of disease, and poor prognosis and outcome for patients (19).

On the basis of the involvement of HER-2/neu in different signal transduction pathways, it might also affect the expression and function of CREB. To gain further insight into the role of CREB in tumorigenesis in combination with the neoplastic properties of HER-2/neu, CREB expression and function were analyzed in vitro using model systems that reflect the HER-2/neu–associated transformation process and in vivo using human mammary carcinoma lesions with defined HER-2/neu status.

This study characterizes for the first time a direct link between CREB and HER-2/neu expression and activity, and it unravels at least some of the molecular mechanisms by which CREB can contribute to HER-2/neu–mediated cancer development, thereby suggesting the targeting of CREB as a novel strategy for cancer therapy.

Cell culture and treatment

The NIH3T3 murine fibroblast cell line was purchased from the American Type Culture Collection (ATCC) and HER-2/neu+–overexpressing NIH3T3 cells have been described in detail previously (20). The human HER-2/neu+ mammary carcinoma cell line MCF-7 was purchased from the ATCC. The cell lines were routinely maintained in Eagle's modified essential medium (EMEM; Lonza) supplemented with 10% (v/v) heat-inactivated FBS, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (all supplements obtained from PAA) at 37°C in 5% (v/v) CO2 humidified air.

Cells were treated for the indicated lengths of time with a range of signal transduction inhibitors, including: H89 (10 μmol/L; PKA inhibitor; Merck); LY294002 (5 μmol/L; PI3K inhibitor; Adipogen); PD98059 (10 μmol/L; MEK1 inhibitor; Axxora); RO31-8220 (5 μmol/L; PKC inhibitor; Cayman), the tyrosine kinase inhibitors (TKI) lapatinib (1.25–10 μmol/L), sunitinib (10 μmol/L), sorafenib (10 μmol/L), pazopanib (10 μmol/L) and axitinib (10 μmol/L; all from LC Laboratories); RAD001 (5 μmol/L; mTOR inhibitor); the chemotherapeutics paclitaxel (10 μmol/L; mitotic inhibitor; Sigma), doxorubicin (10 μmol/L; DNA synthase inhibitor; Sigma), and epoxomicin (10 μmol/L; proteasome inhibitor; Calbiochem); and the KID/KIX inhibitor KG-501 (2.5–10 μmol/L; 2-naphthol-AS-E-phosphate; Sigma), which blocks the CREB–CREB binding protein (CBP) interaction (21).

Generation of shHER-2/neu and shCREB cells, and CREB rescue variants

For the generation of transfectants with short hairpin RNAs (shRNA) directed against CREB (shCREB cells) and HER-2/neu (shHER-2/neu cells), 4 × 105 cells per well were initially seeded into 6-well plates and incubated for 24 to 36 hours to reach approximately 80% confluence. Then, cells were transfected with 1.5 μg/well ScaI-digested murine CREB1-specific (for NIH3T3 and HER-2/neu+ cells) or HER-2/neu–specific (MCF-7 cells) shRNA-encoding plasmid (SABioscience) using PolyFect (Qiagen) according to the manufacturer's instructions. A nonsense (NC) construct served as the control as recently described (22). Twenty-four hours after transfection, puromycin-resistant (purR) colonies were selected in medium supplemented with 3 μg puromycin/mL. The purR bulk cultures and/or individual purR clones were picked, and subsequently expanded for further analysis.

For confirmation of shCREB function, a CREB expression plasmid was generated using Turbo Pfu-PCR cloning of full-length CREB (GI:82546873) from the cDNA of HER-2/neu cells using adaptor primers containing the BamHI or NsiI restriction sites (forward 5′-AAAGGATCCAATGACCATGGAATCTGGAGC-3′ and reverse 5′-AAAATGCATATCTGATTTGTGGCAGTAAAGGTC-3′), respectively, in frame into the C-terminal Human Influenza Hemagglutinin tandem tag into a bicistronic pCMV IRES expression plasmid (23). By exchanging nucleotides from the shRNA target sequence of CREB, a rescue version of CREB (ΔshCREB) was obtained with a two-step PCR mutagenesis approach, using the QuickChange PCR mutagenesis protocol (Stratagene) with the following primers: first step forward 5′-GTCCGTCTAATGAAGAATCGAGAGGCAGCAAGAGAATG-3′ and reverse 5′-CATTCTCTTGCTGCCTCTCGATTCTTCATTAGACGGACC-3′, and second step forward 5′-GAATCGAGAGGCCGCCCGAGAATGTCGTAG-3′ and reverse 5′-CTACGACATTCTCGGGCGGCCTCTCGATTC-3′. The integrity of the ΔshRNACREB1 expression plasmid was verified by sequencing (Eurofins). CREB expression was reconstituted by transfection of the ΔshCREB construct into HER-2/neu+ shCREB cells, and the rescue variants were selected in the presence of 1 mg/mL G418 (PAA) and 3 μg/mL puromycin (Sigma), to generate double-resistant cells.

cDNA synthesis, and quantitative and semiquantitative PCR

RNA was isolated using the NucleoSpin II Kits (Macherey-Nagel), according to the manufacturer's instructions, and then it was subjected to cDNA synthesis. Quantitative real-time PCR (qPCR) or reverse transferase PCR (RT-PCR) was performed with target-specific primers (Supplementary Tables S1A and S1B) as recently described (24). For qPCR, the relative mRNA expression levels of the given target genes within the respective samples were normalized to the corresponding expression levels of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For RT-PCR analysis, β-actin served as a house-keeping gene.

Western blotting

For Western blotting, 50 μg protein per lane were separated in 10% SDS-PAGE gels (19), transferred onto nitrocellulose membranes (Schleicher & Schuell), and stained with Ponceau S as recently described (24). Immunodetection was independently performed with the following target-specific primary monoclonal antibodies (mAb): anti-CREB, anti-phospho(Ser133)-CREB, anti-Akt, anti-phospho(Ser473)-Akt, anti-extracellular signal–regulated kinase (anti-ERK), and anti-phospho(Thr202/Tyr204)-ERK (Cell Signaling Technology). Staining with anti-β-actin (Sigma) and anti-GAPDH (Cell Signaling Technology) served as loading controls. The membranes were then stained with suitable horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology), before the signal was visualized with the LumiLight Western Blotting Substrate (Roche Diagnostics), and recorded with a LAS3000 system (Fuji). The immunostaining signals were subsequently analyzed using the ImageJ software (NIH, Bethesda, MD). Relative protein expression levels are provided as arbitrary units by setting the peak values of the corresponding β-actin or GAPDH signals to 1.

Flow cytometry

The mAbs used for flow cytometry were the phycoerythrin (PE)-labeled anti-HER-2/neu mAb (Becton Dickinson) and the respective PE-labeled isotype mouse immunoglobulin (Beckman Coulter). Flow-cytometric analysis was performed as described recently (25). Briefly, 5 × 105 cells were incubated with the appropriate amounts of antibodies at 4°C for 30 minutes, and the stained cells were measured on a FACScan unit (Becton Dickinson), and subsequently analyzed with the CellQuest software (Becton Dickinson).

Foci formation assay

The cells were plated (1 × 106 cells/9-cm dish) in complete medium containing 5% FBS and insulin–transferrin–selenium (Invitrogen) and cultured for 7 days, followed by incubation in medium supplemented with 2% FBS for a further 2 weeks. During the culture period, the medium was changed every 72 hours. The cells were then fixed in methanol and stained using 1% (w/v) methylene blue solution, to evaluate foci formation.

Cell proliferation assays

The proliferation rate was determined using an XTT-based assay system. Briefly, 5 × 103 cells per well were seeded in 96-well plates in complete medium without phenol red, and incubated at 37°C for the times indicated before addition of the XTT reagent (Cell Proliferation Kit II; Roche Diagnostics) for 4 hours, according to the manufacturer's instructions. Cell proliferation was colorimetrically quantified, by determination of the absorbance of the oxidized XTT solution (formazan) at 570 nm, using an ELISA reader system (Dynex). Alternatively, cells were stained with the CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kits (Invitrogen), harvested 24, 48, and 72 hours after seeding, and directly subjected to flow-cytometric analysis (FACSCalibur; Becton Dickinson), according to the manufacturer's instructions. The CSFE profiles were analyzed using the CellQuest software (Becton Dickinson).

Cell-cycle analysis

For synchronization, the cells were first starved in complete EMEM containing 0.5% FBS for 48 hours, before EMEM with 10% FBS was added for the indicated times. Following trypsinization, the cells were washed twice with PBS, fixed overnight in 70% ethanol at 4°C, and washed twice with PBS. The RNA was digested by incubation with RNase A (30 μg/mL) for 30 minutes in the dark at room temperature, followed by the addition of 20 μg/mL 7-aminoactinomycin D (Sigma) for 1 hour in the dark at 4°C. The cell-cycle distribution was analyzed by flow cytometry on a FACSCalibur (Becton Dickinson) using the ModFit software (Verity Software House).

Apoptosis assays

For quantification of the apoptosis rate, annexin V kits (MBL) were used according to the manufacturer's instructions using 1 × 105 cells. In addition, cleaved caspase-3 was determined by flow cytometry, using an antibody that specifically recognizes the active form of caspase-3 (FITC Active Caspase-3 Apoptosis Kits; Becton Dickinson). Cells treated with the caspase-3 inhibitor Z-DEVD-FMK (5 and 10 μmol/L; Becton Dickinson) for 24 hours served as the control.

Soft agar assay

Soft agar assays were carried out in Dulbecco's modified Eagle medium (PAA) containing 20% FBS as previously described (24). For this, 2 × 104 cells/35-mm plate were seeded in 0.3% agar and incubated at 37°C for 21 days. The number of colonies was determined by staining with iodonitrotetrazolium chloride (Sigma).

Wound-healing assay

Confluent cells were incubated for 24 hours in complete medium containing 0.5% FBS, and a wound was generated by scratching the cell monolayer with a 100 μL pipette tip. After washing with PBS, fresh culture medium supplemented with 0.5% FBS was added to the wounded cell layer. Photographs of the initial wounded cell layer site were taken at 0, 24, and 48 hours using a 10× (1.2 NA) objective on a fluorescence microscope (Leica DM IRB) equipped with a digital camera (Diagnostic Instruments). The wound closure was determined at different times using the MetaVue software (Molecular Devices).

Migration and invasion assay

For determination of the cell migration, 5 × 104 cells cultured in medium supplemented with 1% FBS were seeded into the upper well of the Transwell chamber system (Corning), with medium containing 10% FBS added to the lower chamber. After incubation for 18 hours at 37°C, the nonmigrated cells of the top insert were completely removed, and the cells on the bottom insert surface were lysed with CellTiter-Glo (Promega) before the ATP content was measured in a luminometer (Berthold; ref. 26).

For invasion assays, the polycarbonate membranes were coated with Matrigel (30 μg/well; Becton Dickinson) before cell seeding, which was polymerized for 2 hours at 37°C. The influence of inhibitors on the migration and invasion capacity was determined by incubation of cells for 24 hours with 5 μmol/L KG-501 and 5 μmol/L of the matrix metalloproteinase (MMP) inhibitor GM-6001 (Calbiochem).

Cell-adhesion assays

Varying concentrations of fibronectin prepared in PBS with Ca2+ and Mg2+ were used to coat 96-well plates (50 μL/well). After overnight incubation at 4°C, the plates were rinsed four times with 200 μL/well PBS with Ca2+ and Mg2+, and blocked with 200 μL/well 1% bovine serum albumin for 1 hour at room temperature. Then, 5 × 103 cells per well were allowed to adhere to the plate for 1.5 hours at 37°C. The nonadherent cells were removed by gently washing the plate four times with PBS. The number of adherent cells per well was quantified by measuring the cellular ATP content, as described above.

For analysis of cell–cell interactions, the cells were stained with calcein (Molecular Probes) and seeded onto a confluent layer of NIH3T3 cells. These cells were incubated for 1.5 hours and the nonadherent cells were removed. Staining was analyzed using a fluorescence reader (Tecan).

Promoter assay

The murine MMP-2 and -9 promoter regions were amplified from genomic DNA of NIH3T3 cells using promoter-specific primers (Supplementary Table S1C). For MMP-2, the promoter sequence was digested with XhoI/NheI (−1482 bp to +69 bp), XhoI/KpnI (−695 bp to +69 bp), XhoI/HindIII (−644 bp to +69 bp), and XhoI/SacI (−298 bp to +69 bp) to provide distinct promoter fragments, which were then cloned into the pGl3 luciferase (luc) vector (Promega). In addition, site-directed mutations were introduced into the wild-type (wt) MMP-9 promoter (pGl3-MMP-9wt) by deleting one of the three half CRE elements (pGl3-MMP-9 CRE X) using the QuikChange Site-Directed Mutagenesis Kits (Stratagene), according to the manufacturer's instructions.

For transient transfections, 1 × 104 cells per well were incubated overnight in 100 μL EMEM, followed by transfection with 0.3 μg promoter construct and 0.016 μg β-galactosidase (β-gal) SV40 vector (Promega) using Effectene (Qiagen), according to the manufacturer's instructions. Forty-eight hours after transfection, the luc activity was determined with the luc substrate (Promega) using a luminometer and normalized to the transfection efficiency determined by β-gal enzyme activity (25).

Gelatin zymography

The activities of the MMPs were determined in aliquots of cell-conditioned medium using gelatin zymography. Samples were dissolved in nonreducing Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels (27) containing 1 mg/mL gelatin (Merck). After separation, the gels were washed twice with 2.5% Triton X-100 for 30 minutes, to remove the SDS. The gels were then incubated at 37°C for 20 to 22 hours in 50 mmol/L Tris–HCl containing 5 mmol/L CaCl2, 1 μmol/L ZnCl2, 1% Triton X-100, and 0.02% NaN3, pH 7.6. The gels were stained with 1% (w/v) Coomassie Brilliant Blue R250 in 10% acetic acid and 30% methanol, followed by destaining with 10% acetic acid and 30% methanol.

MMP-2 ELISA

The amounts of MMP-2 in the culture supernatant were measured using mouse-specific ELISA kits (Raybiotech), according to the manufacturer's instructions.

In vivo tumorigenicity

All animal experiments described here were approved by the Regional Council of Halle (Germany). The animals were maintained in accordance with the Guides for the Care and Use of Laboratory Animals. Adult (2–3-month old, 20 ± 4 g body weight, male and female), specific pathogen-free (FELASA) in-bred and immunocompetent DBA/10IaHsd mice (Harlan Laboratories) were used for the cell transplantations. These mice were randomly split into three groups, with 10 mice in groups I and II, and 5 mice in group III [group I, HER-2/neu+ cells; group II, shCREB-transfected HER-2/neu+ cells (shCREB clone 1); group III, NC-transfected HER-2/neu+ cells], and 1 × 106 cells in 200 μL PBS/mouse were subcutaneously injected into the left lateral abdominal wall (regio abdominalis lateralis). The right lateral abdominal wall was used for sham injections with PBS. Tumor growth was monitored three times a week by caliper measurements: length (greatest longitudinal diameter) and width (greatest transverse diameter) were measured, and the tumor volume was calculated by the modified ellipsoidal formula: tumor volume = (length × width2) × 0.5.

Patient selection and tissue microarrays

Eligible patients presenting with primary unilateral breast carcinoma were extracted from 200 cases diagnosed between 2009 and 2011 at the Surgical Units of the “Gaetano Bernabeo” Ortona Hospital, Ortona, Italy. From the original series, only N0 patients (n = 128) and among these, only those with T1/T2 tumors (n = 112), were included in the study. Informed consent was given by the patients.

Tissue microarrays (TMA) were constructed by removing 2-mm diameter cores of histologically confirmed invasive breast carcinoma areas from each original paraffin block, and reembedding these cores into gridded paraffin blocks using a precision instrument (manual tissue arrayer; Beecher Instruments). The patient and tumor characteristics are summarized in Supplementary Table S2.

Tissue embedding, histology, and immunhistochemistry

For the paraffin-embedded human tissues samples, antigen retrieval was performed by microwave treatment for 10 minutes at 750 W in 10 mmol/L of sodium citrate buffer, pH 6.0, and then the 5-μm sections were incubated overnight at 4°C with the pCREB-specific rabbit antibody (clone 87G3; Cell Signaling Technology) at a 1:50 dilution. Anti-rabbit EnVision kits (K4003, Dako) were used for signal amplification. In addition, the TMA was stained with mAbs against the estrogen receptor-α (ER-α; MoAb 6F11; Novocastra), progesterone receptor (PR; MoAb 1A6; Menarini), Ki-67 (MIB-1; Dako), and HER-2/neu (Herceptest Dako). In control sections, the specific primary antibody was omitted or replaced with nonimmune serum. The slides were evaluated by two pathologists (M. Iezzi and R. Lattanzio) without the knowledge of the clinicopathologic data.

For analysis of murine samples, the tumor tissues and organs were fixed in 4.5% neutral-buffered formalin and embedded in paraffin. Then 5-μm slices were cut and stained with hematoxylin and eosin following deparaffinization. Different histologic staining was used according to the tissue sources, for example, azan for collagen staining in the lung (28), and PAS for basement membrane staining in the kidney were performed (29). Then the tumor tissues were stained overnight with antibodies directed against Ki-67 (Novus Biologicals) and CREB (Cell Signaling Technology), using suitable secondary antibodies (SignalStain Boost IHC Detection Reagent; Cell Signaling Technology) in combination with diaminobenzidine (Applichem) as a substrate and counterstained with methylene blue. The frequency of apoptotic cells was determined using the ApopTag Plus Peroxidase In Situ Apoptosis Kits (Merck) according the manufacturer's instructions.

Blood preparation and analysis

Between days 36 and 41, the tumor-bearing mice were anesthetized with 2.5% (v/v) isofluran, and blood was collected by cardiac puncture into heparin-containing tubes. Following lysis of erythrocytes in erythrocyte lysis buffer (c-c-pro GmbH), the cells were incubated with rat anti-mouse CD16/32 (Beckman Coulter), to block nonspecific antibody binding. To identify the different immune cell subpopulations, the following antibody combinations were used: anti-CD8α fluorescein isothiocyanate (FITC; Beckman Coulter), PE-labeled anti-CD49b (DX5; BioLegend Inc.), anti-CD3ϵ PeCy5, and anti-CD4 PeCy7 (both from eBioscience/NatuTec GmbH). Before acquisition on a Navios flow cytometer (Beckman Coulter), the cells were stained with propidium iodide, to remove dead cells. Analysis was performed using the Kaluza software package (Beckman Coulter).

Statistical analysis

ANOVA was used for the statistical analysis of data, with P < 0.05 considered as significant. *, P < 0.05; **, P < 0.01.

Tumor size and tumor grade were dichotomized according to the St. Gallen criteria (30) for the definition of risk categories (T ≤ 2 cm vs. T > 2 cm; grade 1 vs. grade 2–3). ERs and PRs were classified as positive if 10% or more of the cells showed positive staining. A specimen was considered to have high levels of Ki-67 expression if 14% or more of the tumor nuclei stained positively (30). HER-2/neu membrane staining was scored according to Herceptest (Dako) and classified as positive if the intensity was scored 3+, with more than 30% of cells showing complete membrane staining (31), or if the intensity was scored 2+ in the presence of an amplification of the HER-2/neu gene as assessed by FISH. A tumor was considered positive for pCREB if 10% or more of the cell nuclei showed positive staining. The relationships between pCREB expression and clinicopathologic parameters were assessed by Pearson χ2. SPSS version 15.0 was used for analysis, and P < 0.05 was considered statistically significant.

Altered CREB status in HER-2/neu–overexpressing cells arises from activation of different signal transduction pathways

To determine any links between CREB and HER-2/neu, the expression of CREB was analyzed in HER-2/neu NIH3T3 fibroblasts and in the equivalent HER-2/neu+ transfectants. Although CREB mRNA increased in HER-2/neu+ as compared with HER-2/neu fibroblasts, this effect was modest (data not shown). HER-2/neu expression resulted in a 1.7-fold upregulation of total CREB protein and a strong 3.4-fold induction of pCREB levels (Fig. 1A). The correlation between HER-2/neu and CREB expression and activity was confirmed in MCF-7 cells, in which HER-2/neu silencing resulted in an approximately 60% downregulation of pCREB (Supplementary Fig. S1A and S1B). Furthermore, immunohistochemical analysis of mammary carcinoma lesions with known HER-2/neu status using a TMA demonstrated an immunoreactivity for pCREB in the nucleus of tumor cells. As shown in Fig. 1B and Supplementary Fig. S1C, immunohistochemical stainings for pCREB and HER-2/neu in breast tumors showed a positive correlation between pCREB and HER-2/neu expression levels. Statistical analysis using the Pearson χ2 test demonstrated that the relationship between pCREB and HER-2/neu expression was significant (P = 0.037; Fig. 1B; Supplementary Table S3).

Figure 1.

Constitutive and signal transduction inhibitor-regulated CREB expression and activity in HER-2/neu–expressing fibroblasts and mammary carcinoma lesions. A, representative Western blotting for CREB and phospho-CREB. Total protein extracts from HER-2/neu–transformed mouse fibroblasts and control cells were separated on 10% SDS-PAGE gels and blotted onto nitrocellulose membranes before staining with specific antibodies was performed staining with the anti-β-actin mAb served as loading control. Protein expression was quantified using the ImageJ software. The data shown are immunoblots using p-CREB- and CREB-specific mAbs (left), that are quantified and normalized to β-actin (right; n = 3). B, correlation of HER-2/neu and pCREB expression in mammary carcinoma lesions. The data are from a representative immunohistochemical analysis of HER-2/neu and pCREB expression levels (left), that are quantified for 12 mammary carcinoma lesions performed as described in Materials and Methods. C, HER-2/neu+ cells were treated with dimethyl sulfoxide (DMSO) or with the indicated concentrations of lapatinib for 24 hours with the indicated concentrations of lapatinib. After cell harvest, 50 μg protein extract was analyzed on a 10% SDS gel following Western blotting and incubation with the indicated antibodies. The results of staining are presented as immunoblots (left), that are quantified (pCREB/CREB) and normalized to β-actin (right; n = 4). D, HER-2/neu+ cells were left untreated or treated for 24 hours with the four different signal transduction inhibitors as described in Materials and Methods. Western blotting was performed using respective antibodies (left) with quantification of the ratios between the phosphorylated form and total protein expression (right; n = 3). E, HER-2/neu+ cells were left untreated or treated for 24 hours with TKIs or chemotherapeutics, and the phosphorylation and total protein expression of CREB was analyzed by Western blotting as described above. GAPDH served as a loading control. Fifty microgram protein extract from following inhibitor-treated cells were used: lane 1, paclitaxel (10 μmol/L); lane 2, epoxomicin (10 μmol/L); lane 3, doxorubicin hydrochloride (10 μmol/L); lane 4, sunitinib (10 μmol/L); lane 5, sorafenib (10 μmol/L); lane 6, pazopanib (10 μmol/L); lane 7, RAD001 (5 μmol/L); lane 8, axitinib (10 μmol/L); lane 9, dimethyl sulfoxide (DMSO). Left, representative immunostaining; right, quantification of the pCREB/CREB (n = 3). n.s., not significant.

Figure 1.

Constitutive and signal transduction inhibitor-regulated CREB expression and activity in HER-2/neu–expressing fibroblasts and mammary carcinoma lesions. A, representative Western blotting for CREB and phospho-CREB. Total protein extracts from HER-2/neu–transformed mouse fibroblasts and control cells were separated on 10% SDS-PAGE gels and blotted onto nitrocellulose membranes before staining with specific antibodies was performed staining with the anti-β-actin mAb served as loading control. Protein expression was quantified using the ImageJ software. The data shown are immunoblots using p-CREB- and CREB-specific mAbs (left), that are quantified and normalized to β-actin (right; n = 3). B, correlation of HER-2/neu and pCREB expression in mammary carcinoma lesions. The data are from a representative immunohistochemical analysis of HER-2/neu and pCREB expression levels (left), that are quantified for 12 mammary carcinoma lesions performed as described in Materials and Methods. C, HER-2/neu+ cells were treated with dimethyl sulfoxide (DMSO) or with the indicated concentrations of lapatinib for 24 hours with the indicated concentrations of lapatinib. After cell harvest, 50 μg protein extract was analyzed on a 10% SDS gel following Western blotting and incubation with the indicated antibodies. The results of staining are presented as immunoblots (left), that are quantified (pCREB/CREB) and normalized to β-actin (right; n = 4). D, HER-2/neu+ cells were left untreated or treated for 24 hours with the four different signal transduction inhibitors as described in Materials and Methods. Western blotting was performed using respective antibodies (left) with quantification of the ratios between the phosphorylated form and total protein expression (right; n = 3). E, HER-2/neu+ cells were left untreated or treated for 24 hours with TKIs or chemotherapeutics, and the phosphorylation and total protein expression of CREB was analyzed by Western blotting as described above. GAPDH served as a loading control. Fifty microgram protein extract from following inhibitor-treated cells were used: lane 1, paclitaxel (10 μmol/L); lane 2, epoxomicin (10 μmol/L); lane 3, doxorubicin hydrochloride (10 μmol/L); lane 4, sunitinib (10 μmol/L); lane 5, sorafenib (10 μmol/L); lane 6, pazopanib (10 μmol/L); lane 7, RAD001 (5 μmol/L); lane 8, axitinib (10 μmol/L); lane 9, dimethyl sulfoxide (DMSO). Left, representative immunostaining; right, quantification of the pCREB/CREB (n = 3). n.s., not significant.

Close modal

Because CREB expression and activity can be targeted by different signal transduction pathways, the effects of inhibitors of the EGF-R/HER-2/neu (lapatinib), PKA, PKC, PI3K/Akt, and MAP–ERK kinase (MEK)/ERK on CREB expression and phosphorylation was compared in HER-2/neu+ cells demonstrating distinct effects of the different inhibitors (Fig. 1). Although lapatinib decreased the phosphorylation of CREB in a concentration-dependent matter (Fig. 1C), the PKA inhibitor H89 and the MEK1 inhibitor PD98059 had no effects on pCREB (Fig. 1D). The strongest inhibition of CREB activation was seen for the PI3K/Akt inhibitor LY294002, followed by the PKC inhibitor RO31-8220, which suggested that CREB can be activated by HER-2/neu+ overexpression via different signal transduction pathways (Fig. 1D). Interestingly, the TKIs sorafenib and sunitinib strongly blocked pCREB, but not total CREB expression, in HER-2/neu transformants, whereas the other TKIs and chemotherapeutics did not or only marginally (doxorubicin) affect pCREB levels (Fig. 1E).

HER-2/neu–controlled CREB expression is associated with a transformed phenotype and growth suppression

To investigate CREB function, CREB was silenced in murine HER-2/neu+ cells using four distinct CREB-targeting shRNA (shCREB) constructs in parallel, with a scrambled shRNA construct (NC) that served as the control. As shown in Supplementary Fig. S2A, the four shCREB constructs differentially influenced CREB expression, whereas the NC construct had no effect. For further analysis, the shRNA construct with the greatest effects on CREB (plasmid 2) was chosen, and individual shCREB bulk cultures (n = 3) and clones (n = 5) were generated. As shown for shCREB clones 1 and 2, the CREB transcript levels were reduced with an efficacy ranging from 50% to 90%. This effect was even more pronounced at the CREB protein expression and phosphorylation levels (Fig. 2A). The specificity of the construct was determined by analyzing the mRNA expression of CREB-like transcription factors, namely the activating transcription factor 1 (ATF-1) and the CRE modulator (CREM). Neither of these transcription factors showed significant changes in their expression patterns in the shCREB-HER-2/neu+ cells nor in the NC when compared with the HER-2/neu+ cells (Supplementary Fig. S2B). Furthermore, transfection with ΔshCREB rescued CREB expression and activity, as shown for two rescue batch cultures (RB1 and RB2), was even more pronounced in representative clones (RC1 and RC2) of the rescue batch cultures (Fig. 2A).

Figure 2.

shRNA-mediated CREB downregulation in HER-2/neu–transformed cells. A, CREB mRNA and protein expression of phospho-CREB and CREB in HER-2/neu+ cells, NC, a shCREB-HER-2/neu+ cell bulk culture (shBatch) and different shCREB-HER-2/neu+ cell clones from another shBatch culture, as well as bulk cultures (RB1 and RB2) and clones (RC1) of the ΔshCREB-HER-2/neu+ cell rescue variant was determined as described in Materials and Methods. Top, quantified mRNA data (n = 3); bottom, representative immunostaining. Black bars represent HER-2/neu and NC cell line; white bars, the shCREB cells; and gray bars, the rescue cell lines. The dotted line represents “1.” B, representative morphology of HER-2/neu+ cells, shCREB-HER-2/neu+ cell, and the ΔshCREB-HER-2/neu+ cell rescue variant as analyzed by phase-contrast microscopy 4 days after cell seeding is shown. Primary magnification: ×100. C, representative focus formation of HER-2/neu+ cells and shCREB-HER-2/neu+ cells was determined after 3 weeks of culture as described in Materials and Methods. The cells were fixed in methanol and stained with methylene blue. D, representative morphology of HER-2/neu+ cells treated with lapatinib and KG-501 for 24 hours, respectively, is shown. E, representative surface expression of HER-2/neu in HER-2/neu+ cells, shCREB-HER-2/neu+ cells, and ΔshCREB-HER-2/neu+ cell rescue variants was determined using an anti-HER-2/neu–specific antibody (shaded areas). The unshaded areas represent the IgG1 control, and the dotted lines indicate the means. F, representative HER-2/neu surface expression as determined upon treatment with KG-501 for 24 hours using flow cytometry, and a HER-2/neu–specific antibody (shaded area). The unshaded area represents the unspecific IgG control. The ratio between the HER-2/neu expression and the IgG control is given. n.s., not significant.

Figure 2.

shRNA-mediated CREB downregulation in HER-2/neu–transformed cells. A, CREB mRNA and protein expression of phospho-CREB and CREB in HER-2/neu+ cells, NC, a shCREB-HER-2/neu+ cell bulk culture (shBatch) and different shCREB-HER-2/neu+ cell clones from another shBatch culture, as well as bulk cultures (RB1 and RB2) and clones (RC1) of the ΔshCREB-HER-2/neu+ cell rescue variant was determined as described in Materials and Methods. Top, quantified mRNA data (n = 3); bottom, representative immunostaining. Black bars represent HER-2/neu and NC cell line; white bars, the shCREB cells; and gray bars, the rescue cell lines. The dotted line represents “1.” B, representative morphology of HER-2/neu+ cells, shCREB-HER-2/neu+ cell, and the ΔshCREB-HER-2/neu+ cell rescue variant as analyzed by phase-contrast microscopy 4 days after cell seeding is shown. Primary magnification: ×100. C, representative focus formation of HER-2/neu+ cells and shCREB-HER-2/neu+ cells was determined after 3 weeks of culture as described in Materials and Methods. The cells were fixed in methanol and stained with methylene blue. D, representative morphology of HER-2/neu+ cells treated with lapatinib and KG-501 for 24 hours, respectively, is shown. E, representative surface expression of HER-2/neu in HER-2/neu+ cells, shCREB-HER-2/neu+ cells, and ΔshCREB-HER-2/neu+ cell rescue variants was determined using an anti-HER-2/neu–specific antibody (shaded areas). The unshaded areas represent the IgG1 control, and the dotted lines indicate the means. F, representative HER-2/neu surface expression as determined upon treatment with KG-501 for 24 hours using flow cytometry, and a HER-2/neu–specific antibody (shaded area). The unshaded area represents the unspecific IgG control. The ratio between the HER-2/neu expression and the IgG control is given. n.s., not significant.

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The CREB status was associated with altered morphology and growth characteristics. Although HER-2/neu+ cells showed a typically transformed morphology with many foci, the shCREB-HER-2/neu+ cells were more fibroblast-like and could only grow in monolayers, which was rescued by CREB reconstitution (Fig. 2B). Furthermore, the shCREB-induced morphologic alterations were in line with their ability to form foci (Fig. 2C). In addition, treatment with lapatinib caused changes in the morphology to an untransformed phenotype, whereas the KID/KIX inhibitor KG-501 had no effect (Fig. 2D). It is noteworthy that KG-501 did not affect CREB transcription nor pCREB levels in the HER-2/neu+ cells, but decreased CBP mRNA expression after 48 hours of treatment (Supplementary Fig. S3A and S3B). Furthermore, CREB silencing by shRNA and its rescue by ΔshCREB (Fig. 2E) and treatment with KG-501 (Fig. 2F) or lapatinib (data not shown) did not influence HER-2/neu surface expression.

As CREB promotes the proliferation of cancer cells, the influence of CREB silencing on the growth properties of the HER-2/neu+ cells was analyzed. The shCREB-HER-2/neu+ cells exerted an increased generation time (Fig. 3A) that was associated with a reduced proliferation rate (data not shown) and a cell-cycle delay (G0–G1 arrest; Fig. 3B) when compared with the HER-2/neu+ cells. Furthermore, there was a dose-dependent inhibition of cell proliferation with lapatinib (Fig. 3C) and KG-501 (Supplementary Fig. S3C) treatments. The G0–G1 cell-cycle arrest correlated with transcriptional downregulation of cyclin B and D (Fig. 3D). Serum starvation reduced the proliferation of both HER-2/neu+ cells and shCREB-HER-2/neu+ cells, although the shCREB derivatives were more sensitive (Fig. 3E).

Figure 3.

Effects of shCREB and lapatinib on the proliferative capacity of HER-2/neu+ cells. A, for the determination of the generation time, 5 × 103 cells per well of HER-2/neu+ cells, NC, two shCREB-HER-2/neu+ cell clones, a shCREB-HER-2/neu+ cell batch culture, and a ΔshCREB-HER-2/neu+ cell rescue culture were seeded into 96-well plates and proliferation was determined 24, 48, 72, and 96 hours after seeding using the XTT assay according to the manufacturer's protocol. Data are mean ± SD (n = 3, each performed in triplicate). B, cell-cycle analysis as assessed with starved cells (HER-2/neu+, NC, shCREB-HER-2/neu+ cl1) cultured in 0.5% FBS followed by treatment with complete medium supplemented with 10% FBS. Data are mean ± SD (n = 3), for the three different cell-cycle phases. C, HER-2/neu cells were incubated with lapatinib and every 24 hours the proliferation was analyzed using the XTT assay. Data are mean ± SD (n = 2, each performed in triplicate). D, mRNA expression of cyclin B1 and D1 was analyzed in HER-2/neu+ cells, NC, shCREB HER-2/neu+ cells and ΔshCREB-HER-2/neu+ cell rescue batch by qPCR using target-specific primers, as described in Materials and Methods. Data are mean ± SD (n = 3). E, proliferation of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells was analyzed under various FBS concentrations, with 5 × 103 cells per well incubated in media containing the indicated FBS concentration for different times, and cell proliferation was determined using the XTT assay. Data are mean ± SD (n = 2, each performed in triplicate). n.s., not significant.

Figure 3.

Effects of shCREB and lapatinib on the proliferative capacity of HER-2/neu+ cells. A, for the determination of the generation time, 5 × 103 cells per well of HER-2/neu+ cells, NC, two shCREB-HER-2/neu+ cell clones, a shCREB-HER-2/neu+ cell batch culture, and a ΔshCREB-HER-2/neu+ cell rescue culture were seeded into 96-well plates and proliferation was determined 24, 48, 72, and 96 hours after seeding using the XTT assay according to the manufacturer's protocol. Data are mean ± SD (n = 3, each performed in triplicate). B, cell-cycle analysis as assessed with starved cells (HER-2/neu+, NC, shCREB-HER-2/neu+ cl1) cultured in 0.5% FBS followed by treatment with complete medium supplemented with 10% FBS. Data are mean ± SD (n = 3), for the three different cell-cycle phases. C, HER-2/neu cells were incubated with lapatinib and every 24 hours the proliferation was analyzed using the XTT assay. Data are mean ± SD (n = 2, each performed in triplicate). D, mRNA expression of cyclin B1 and D1 was analyzed in HER-2/neu+ cells, NC, shCREB HER-2/neu+ cells and ΔshCREB-HER-2/neu+ cell rescue batch by qPCR using target-specific primers, as described in Materials and Methods. Data are mean ± SD (n = 3). E, proliferation of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells was analyzed under various FBS concentrations, with 5 × 103 cells per well incubated in media containing the indicated FBS concentration for different times, and cell proliferation was determined using the XTT assay. Data are mean ± SD (n = 2, each performed in triplicate). n.s., not significant.

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Altered apoptosis sensitivity of CREB-deficient cells

To test whether CREB silencing caused increased apoptosis rates, the vitality of HER-2/neu+ cells, their shCREB derivatives (shCREB-HER-2/neu+ cells), and KG501- and lapatinib-treated cells were compared under standard culture conditions. CREB silencing resulted in statistically significant increased apoptosis when compared with controls and the rescue variants (ΔshCREB-HER-2/neu+ cells). In addition, treatment with low concentrations of KG-501 (2.5 μmol/L) and with 25 μmol/L lapatinib enhanced apoptosis sensitivity (Fig. 4A). CREB knockdown was accompanied by reduced transcription of the antiapoptotic markers bcl-2 and bcl-xL and an enhanced expression of the proapoptotic bax gene in the shCREB-HER-2/neu+ cells, when compared with controls (Fig. 4B). Furthermore, the effector caspase-3 was activated to a greater level in the shCREB-HER-2/neu+ cells when compared with control cells (Fig. 4C). Treatment with the caspase-3 inhibitor Z-DEVD-FMK only partially reduced the apoptotic rate of shCREB-HER-2/neu+ cells, suggesting that shCREB-mediated apoptosis is not solely caused by caspase-3 activation (Fig. 4D).

Figure 4.

Enhanced apoptosis of HER-2/neu+ cells after CREB silencing. A, flow cytometry analysis of annexin V and propidium iodide staining was performed in HER-2/neu+ cells, NC, shCREB-HER-2/neu+ cell clones, along with different rescue variants, as described in Materials and Methods. HER-2/neu+ cells were also treated with DMSO, lapatinib or KG-501 for 24 hours. Data are mean ± SD (n = 3) for the frequency of vital cells. B, representative RT-PCR of NC and two different shCREB-HER-2/neu+ cell clones was performed as described in Materials and Methods using bcl-xL-, bcl-2- and bax-specific primers. Amplification with β-actin–specific primers served as control. One representative gel out of three is shown. C, activation of caspase-3 by cleavage was monitored in HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells by representative flow cytometry using a specific antibody against caspase-3 with 5 × 103 cells gated and analyzed using the CellQuest Pro software. The numbers on the histograms represent the amounts of cells with cleaved caspase-3. Representative histograms from one out of three experiments are shown. D, apoptotic shCREB-silenced HER-2/neu+ cells were incubated with the caspase-3 inhibitor Z-DEVD-FMK for 24 hours. Representative flow-cytometric analysis of apoptotic cells determined by annexin V propidium iodide staining is shown. Representative dot blots from one out of two experiments are shown. n.s., not significant; DMSO, dimethyl sulfoxide.

Figure 4.

Enhanced apoptosis of HER-2/neu+ cells after CREB silencing. A, flow cytometry analysis of annexin V and propidium iodide staining was performed in HER-2/neu+ cells, NC, shCREB-HER-2/neu+ cell clones, along with different rescue variants, as described in Materials and Methods. HER-2/neu+ cells were also treated with DMSO, lapatinib or KG-501 for 24 hours. Data are mean ± SD (n = 3) for the frequency of vital cells. B, representative RT-PCR of NC and two different shCREB-HER-2/neu+ cell clones was performed as described in Materials and Methods using bcl-xL-, bcl-2- and bax-specific primers. Amplification with β-actin–specific primers served as control. One representative gel out of three is shown. C, activation of caspase-3 by cleavage was monitored in HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells by representative flow cytometry using a specific antibody against caspase-3 with 5 × 103 cells gated and analyzed using the CellQuest Pro software. The numbers on the histograms represent the amounts of cells with cleaved caspase-3. Representative histograms from one out of three experiments are shown. D, apoptotic shCREB-silenced HER-2/neu+ cells were incubated with the caspase-3 inhibitor Z-DEVD-FMK for 24 hours. Representative flow-cytometric analysis of apoptotic cells determined by annexin V propidium iodide staining is shown. Representative dot blots from one out of two experiments are shown. n.s., not significant; DMSO, dimethyl sulfoxide.

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Reduced migration, invasion potential, and loss of adhesion capacity after CREB silencing

The important characteristics of cancer cells include enhanced migration and invasion abilities, which can be monitored using wound-healing and transwell assays. The shCREB-HER-2/neu+ cells showed strongly reduced wound-healing (Fig. 5A) and migration potential when compared with controls, which could be restored by CREB rescue (Fig. 5B). A reduced migration rate was also obtained by treatment with KG-501 and lapatinib (Fig. 5B). Analogous to shCREB-mediated silencing, KG-501 and lapatinib treatments downregulated the anchorage-independent growth of HER-2/neu+ cells in soft agar (Fig. 5C), whereas the CREB rescue variants demonstrated increased numbers of soft-agar colonies. This was accompanied by a reduced capacity of CREB-deficient cells to adhere to fibronectin-coated plates (Fig. 5D) and to a NIH3T3 cell monolayer (Fig. 5E).

Figure 5.

Reduced migration and cell motility of HER-2/neu transformants upon CREB silencing. A, representative scratch assays of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells were performed to show the wound distance with time. The numbers indicate the scratch area relative to 0 hours as 100% (left), with quantification shown on the right (n = 3). Scale bar, 80 μm. B, quantification of cell motility of untreated, lapatinib- (1.25 μmol/L) and KG-501 (5 μmol/L)–treated HER-2/neu+ cells, NC, shCREB-HER-2/neu+ cells and the ΔshCREB-HER-2/neu+ cell rescue variants were determined by Transwell assays as described in Materials and Methods. The data are represented in a histogram as percentage of migrated cells and are expressed as percentages of migrated cells (n = 3; each performed in triplicate). C, representative images of determination of anchorage-independent growth by soft agar colony formation for 2 × 104 cells per plate of untreated and lapatinib- (1.25 μmol/L) and KG-501 (5 μmol/L)–treated HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells, seeded in 0.3% agar noble and incubated for 21 days at 37°C are shown. The cells were stained with 0.5% iodnitrotetrazolium chloride and stained soft agar colonies were counted (n = 3; each performed in duplicate). D, quantification of adhesion of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells to fibronectin, determined as described in Materials and Methods. Data are mean ± SD (n = 3) and demonstrate that CREB-deficient cells are less adherent to fibronectin-coated wells than the parental or NC cells. E, quantification of adhesion of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells to a confluent cell layer of NIH3T3 cells. Briefly, 5 × 105 cells per well (24-well plates) were stained with calcein and seeded onto the confluent NIH3T3 cell layer. After 1.5 hours of incubation at 37°C, nonadherent cells were removed by washing with PBS, and the bound cells were directly analyzed in an ELISA reader. Data are mean ± SD (n = 3). n.s., not significant.

Figure 5.

Reduced migration and cell motility of HER-2/neu transformants upon CREB silencing. A, representative scratch assays of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells were performed to show the wound distance with time. The numbers indicate the scratch area relative to 0 hours as 100% (left), with quantification shown on the right (n = 3). Scale bar, 80 μm. B, quantification of cell motility of untreated, lapatinib- (1.25 μmol/L) and KG-501 (5 μmol/L)–treated HER-2/neu+ cells, NC, shCREB-HER-2/neu+ cells and the ΔshCREB-HER-2/neu+ cell rescue variants were determined by Transwell assays as described in Materials and Methods. The data are represented in a histogram as percentage of migrated cells and are expressed as percentages of migrated cells (n = 3; each performed in triplicate). C, representative images of determination of anchorage-independent growth by soft agar colony formation for 2 × 104 cells per plate of untreated and lapatinib- (1.25 μmol/L) and KG-501 (5 μmol/L)–treated HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells, seeded in 0.3% agar noble and incubated for 21 days at 37°C are shown. The cells were stained with 0.5% iodnitrotetrazolium chloride and stained soft agar colonies were counted (n = 3; each performed in duplicate). D, quantification of adhesion of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells to fibronectin, determined as described in Materials and Methods. Data are mean ± SD (n = 3) and demonstrate that CREB-deficient cells are less adherent to fibronectin-coated wells than the parental or NC cells. E, quantification of adhesion of HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells to a confluent cell layer of NIH3T3 cells. Briefly, 5 × 105 cells per well (24-well plates) were stained with calcein and seeded onto the confluent NIH3T3 cell layer. After 1.5 hours of incubation at 37°C, nonadherent cells were removed by washing with PBS, and the bound cells were directly analyzed in an ELISA reader. Data are mean ± SD (n = 3). n.s., not significant.

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By comparing the invasion potential of untreated HER-2/neu+ cells, shCREB-HER-2/neu+ cells, and the NC, as well as of inhibitor-treated HER-2/neu+ cells, an approximately 50% to 60% reduced invasion rate was found for the shCREB-HER-2/neu+ cells in the presence of the MMP inhibitor GM-6001, lapatinib, and KG-501, which suggests that EGF-R/HER-2/neu signaling, CREB activity, and MMPs are involved in tumor invasion (Fig. 6A; ref. 32). Furthermore, the expression profiles of MMP-2 and -9 in HER-2/neu+ cells and shCREB-HER-2/neu+ cells were compared. A strong downregulation of MMP-2 and -9 mRNA was detected upon CREB silencing (Fig. 6B), which was associated with reduced MMP expression levels (Fig. 6C) and activity (Fig. 6D) in the culture supernatant.

Figure 6.

Association of transcriptional suppression of MMPs with delayed invasiveness of HER-2/neu+ cells upon CREB silencing. A, invasiveness of untreated, GM-6001- (5 μM), lapatinib- (1.25 μM), and KG-501 (5 μM)–treated HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells and ΔshCREB-HER-2/neu+ cell rescue controls determined using Matrigel invasion assays is shown. Data are mean ± SD (n = 3) given as relative invasiveness. B, expression of different MMPs analyzed in HER-2/neu+ cells and shCREB-HER-2/neu+ cl1 cells was determined by qPCR. Data are mean ± SD (n = 3). C, MMP-2 protein levels in culture supernatants of HER-2/neu+ cells and shCREB-HER-2/neu+ cells were determined using a MMP-2–specific ELISA, where 5 × 104 cells were seeded into 24-well plates with 300 μl serum-free EMEM and incubated for 24 hours at 37°C. Data are mean ± SD (n = 4, each performed in duplicate). D, representative activity of MMPs in culture supernatants of HER-2/neu+ cells and shCREB-HER-2/neu+ cells was determined by gelatin zymography as described in Materials and Methods. After electrophoresis, gels were stained in 1% Coomassie blue. The different gelatin proteolytic MMPs are as indicated. E, wt and mutated MMP-2 and -9 promoter constructs transiently transfected into HER-2/neu+ cells and shCREB-HER-2/neu+ cells (left). The luciferase activity was measured on a luminometer after 48 hours, with the activity normalized to β-gal activity (right). Data are mean ± SD (n = 3).

Figure 6.

Association of transcriptional suppression of MMPs with delayed invasiveness of HER-2/neu+ cells upon CREB silencing. A, invasiveness of untreated, GM-6001- (5 μM), lapatinib- (1.25 μM), and KG-501 (5 μM)–treated HER-2/neu+ cells, NC, and shCREB-HER-2/neu+ cells and ΔshCREB-HER-2/neu+ cell rescue controls determined using Matrigel invasion assays is shown. Data are mean ± SD (n = 3) given as relative invasiveness. B, expression of different MMPs analyzed in HER-2/neu+ cells and shCREB-HER-2/neu+ cl1 cells was determined by qPCR. Data are mean ± SD (n = 3). C, MMP-2 protein levels in culture supernatants of HER-2/neu+ cells and shCREB-HER-2/neu+ cells were determined using a MMP-2–specific ELISA, where 5 × 104 cells were seeded into 24-well plates with 300 μl serum-free EMEM and incubated for 24 hours at 37°C. Data are mean ± SD (n = 4, each performed in duplicate). D, representative activity of MMPs in culture supernatants of HER-2/neu+ cells and shCREB-HER-2/neu+ cells was determined by gelatin zymography as described in Materials and Methods. After electrophoresis, gels were stained in 1% Coomassie blue. The different gelatin proteolytic MMPs are as indicated. E, wt and mutated MMP-2 and -9 promoter constructs transiently transfected into HER-2/neu+ cells and shCREB-HER-2/neu+ cells (left). The luciferase activity was measured on a luminometer after 48 hours, with the activity normalized to β-gal activity (right). Data are mean ± SD (n = 3).

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The underlying molecular mechanisms of decreased MMP expression upon CREB silencing was further characterized by determination of the transcriptional activity of the wtMMP-2 and wtMMP-9 promoters and/or the role of the CRE, half CRE, and CREB binding sites within these promoters. Constitutive wtMMP-2 and wtMMP-9 promoter activity was reduced in shCREB-HER-2/neu+ cells when compared with HER-2/neu+ cells (Fig. 6E). Deletion of the CREB-binding site of the MMP-2 promoter resulted in a 40% reduction of MMP-2 promoter activity when compared with the activity of the −695 bp promoter. This correlated to an 18% reduced wtMMP-2 promoter activity, whereas mutations within the half CRE elements 2 and 3, but not within the half CRE site 1, resulted in a 30% to 50% downregulation of MMP-9 promoter activity. However, the activities of the mutated MMP promoters were comparable in the HER-2/neu+ cells and shCREB-HER-2/neu+ cells, suggesting that MMP transcription is dependent on the CREB-binding sites and half CRE elements (Fig. 6E).

Correlation of HER-2/neu–controlled CREB expression and function with the in vivo tumorigenicity

Because the loss of anchorage-independent growth upon CREB silencing might also have an impact on the in vivo tumorigenicity, HER-2/neu+ cells, shCREB-HER-2/neu+ cells, and the NC were injected into DBA-1 mice, and the frequency of tumor formation and tumor size was monitored over time. Although all the mice injected with the HER-2/neu+ cells developed tumors within 28 days, 60% of the NC cells and 40% of the shCREB-HER-2/neu+ cells formed tumors at this time (Fig. 7A). Furthermore, tumor growth (Fig. 7B) and tumor end volume (Fig. 7C) were strongly delayed in the shCREB-HER-2/neu+ cells, but not in the parental HER-2/neu+ cells and the NC. However, when compared with the control group, the organs of the tumor-bearing mice showed no pathologic changes (Supplementary Fig. S4A). The altered in vivo growth properties were further supported by reduced proliferation and increased apoptosis rates of shCREB-induced tumors, as determined by immunohistochemical staining using the Ki-67 antibody (Fig. 7D and Supplementary Fig. S4B) and by using an in situ apoptosis detection kit (Fig. 7E) despite HER-2/neu expression in these tumors (Supplementary Fig. S4C). Furthermore, reduced frequencies of both CD4+ and CD8+ T cells were found in the mice injected with the HER-2/neu+ cells, which were increased in mice injected with the shCREB-HER-2/neu+ cells (Fig. 7E). This was further supported being the fact that the mice bearing tumors induced by the shCREB-HER-2/neu+ cells showed low frequencies of both CD4+ and CD8+ T cells in peripheral blood.

Figure 7.

Effects of CREB silencing on HER-2/neu–mediated tumor formation in vivo. A, percentage of tumor-free mice after subcutaneous injection of HER-2/neu+ cells (n = 10), NC (n = 5), and shCREB-HER-2/neu+ cl1 cells (n = 10). Mice were routinely monitored at least three times a week. B, development of tumor volumes of the respective cell lines was measured three times per week with a caliper. C, tumor end volumes of injected cells were determined after sacrifice of the mice their removal. D, immunohistochemical staining of paraffin-embedded tumor sections was performed as described in Materials and Methods using the Ki-67–specific antibody, with apoptotic cells measured using the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay. E, peripheral blood isolated from PBS-treated mice and from mice injected with HER-2/neu+ cells and shCREB-HER-2/neu+ cells, was subjected to flow cytometry as described in Materials and Methods. Data are expressed as frequencies of CD4+ and CD8+ T cells. F, schematic diagram of the mode of CREB function in HER-2/neu+ cells. Dotted and smooth lines indicate minor signal-transduction pathways targeting CREB phosphorylation in HER-2/neu+ cells, whereas bold pathways were highly effected by target-specific inhibitors, with strong downregulation of pCREB. CREB driven, important physiologic features such as proliferation are given as an example.

Figure 7.

Effects of CREB silencing on HER-2/neu–mediated tumor formation in vivo. A, percentage of tumor-free mice after subcutaneous injection of HER-2/neu+ cells (n = 10), NC (n = 5), and shCREB-HER-2/neu+ cl1 cells (n = 10). Mice were routinely monitored at least three times a week. B, development of tumor volumes of the respective cell lines was measured three times per week with a caliper. C, tumor end volumes of injected cells were determined after sacrifice of the mice their removal. D, immunohistochemical staining of paraffin-embedded tumor sections was performed as described in Materials and Methods using the Ki-67–specific antibody, with apoptotic cells measured using the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay. E, peripheral blood isolated from PBS-treated mice and from mice injected with HER-2/neu+ cells and shCREB-HER-2/neu+ cells, was subjected to flow cytometry as described in Materials and Methods. Data are expressed as frequencies of CD4+ and CD8+ T cells. F, schematic diagram of the mode of CREB function in HER-2/neu+ cells. Dotted and smooth lines indicate minor signal-transduction pathways targeting CREB phosphorylation in HER-2/neu+ cells, whereas bold pathways were highly effected by target-specific inhibitors, with strong downregulation of pCREB. CREB driven, important physiologic features such as proliferation are given as an example.

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Many human and murine tumors of distinct histology and cells of the tumor microenvironment show increased CREB expression and activity when compared with adjacent nonmalignant tissue (4), which is even more enhanced in metastasis suggesting a role for CREB in the initiation, maintenance, and progression of tumors (33, 34). This is often accompanied by poor prognosis and decreased survival rates of patients with tumor (35). Although CREB dysregulation is a critical factor in cancer development and progression, the underlying molecular mechanisms that lead to its overexpression have not yet been determined in detail. One approach to unravel this process is to determine the link(s) between oncogene expression and CREB using an in vitro model of oncogene-mediated transformation, as well as human tumor lesions with known HER-2/neu status. As shown in Fig. 7F, an important role for HER-2/neu was suggested for CREB expression and activity both in vitro and in vivo. This is based on the data demonstrating that: (i) HER-2/neu overexpression in murine NIH3T3 cells causes upregulation of CREB expression and activity, which is directly associated with transformed morphology, uncontrolled proliferation, altered cell-cycle progression, increased cell invasion and migration, and altered signal transduction; (ii) inhibition of CREB and/or HER-2/neu reverts the transformed phenotype, which can be rescued by CREB overexpression; and (iii) treatments with selected TKIs including lapatinib, sunitinib, and sorafenib known to block components of the HER-2/neu signal pathways resulted in inhibition of pCREB expression. These data support the hypothesis that CREB is an important transcription factor in maintaining the cellular features of HER-2/neu–transformed cells. This is further confirmed by immunohistochemistry staining of mammary carcinoma lesions with known HER-2/neu status, which demonstrates a positive correlation of HER-2/neu and pCREB in breast cancer lesions. This is in line with an upregulation of CREB and pCREB in stage IV HER-2/neu–overexpressing mammary carcinoma lesions (36).

Although inhibition of CREB by shRNA, lapatinib, or KG-501 not only reversed the HER-2/neu–transformed phenotype, the in vitro and/or in vivo growth properties and apoptosis sensitivity of HER-2/neu+ cells was altered. These data are in line with recent studies demonstrating that CREB knockdown reduces cell survival and proliferation, and cell-cycle progression of different tumor cells (37–39), and they were further strengthened by the rescue of CREB expression in the shCREB-HER-2/neu+ cells, which reversed the untransformed morphology and growth characteristics (Fig. 2). Although targeting specific cancer-promoting factors in terms of particular kinases, growth factors and growth factor receptors are currently successfully implemented for the treatment of various cancers (40). The inhibition of the expression of transcription factors, such as CREB, might represent a novel and attractive therapeutic approach for the treament of CREB-overexpressing tumors (41). However, the influence of lapatinib on pCREB expression and function suggests that the tyrosine kinase activity of HER-2/neu is promoting its effects on CREB, as it binds to the intracellular kinase domain (42). So far, initial studies testing the potential to target CREB as a strategy for cancer therapy have focused on approaches that inhibit CREB function by shRNAs or by using dominant-negative CREB mutants, which target the CREB–CBP interaction, thereby preceding the CREB-dependent transcriptional activation of gene expression. Indeed, inhibition of CREB activity by dominant-negative mutants or by shRNAs was associated with a reduced in vitro and in vivo growth potential and increased radiosensitivity (39, 43, 44). In the HER-2/neu model used, shCREB-mediated inhibition also resulted in decreased proliferation of HER-2/neu+ cells, accompanied by cell-cycle arrest, increased apoptosis, and reduced cell migration and invasion, which was directly associated with an altered expression of proapoptotic and antiapoptotic genes as well as a transcriptional downregulation of MMP-2 and -9 expression.

Recently, anticancer drugs targeting CREB have been developed, including the KID/KIX inhibitor KG-501, which blocks the KID–KIX interaction and has a similar activity when compared with the shCREB-silenced or lapatinib-treated cells (21, 41). This is in line with data obtained in the present study demonstrating that CREB-specific shRNA, lapatinib, and KG501 treatment caused reversal of the transformed phenotype and growth characteristics of HER-2/neu+ cells. These data provide further support for the rational to target CREB, or related components directly associated with the CREB pathway, for the treatment of patients with tumor. However, a detailed analysis of the protein/DNA and protein/protein interactions of CREB is required, which might lead to the further identification of novel targets and to the development of small molecules that can interfere with these interactions. In this context, it is of note that some inhibitors, such as lapatinib, LY294002, and RO31-8220, as well as the TKIs sorafenib and sunitinib, can strongly inhibit CREB expression, which might be associated with their antiproliferative and proapoptotic activities, thereby explaining some of the molecular activities of these drugs.

CREB directly regulates a number of critical genes that are involved in cell proliferation, apoptosis, and metastasis formation, and the cell cycle (45–48). This might be associated with an altered expression of CREB target genes, including MMPs (49). Indeed, CREB silencing in HER-2/neu+ cells altered their migration and invasion capacity. This was associated with a reduced activity of the MMP promoters, which was dependent on putative CREB- or CRE-binding sites. Modulation of MMP-2 and -9 promoter activity by CREB has also been shown in different cell systems (49–51). Furthermore, the transcriptional upregulation of MMP-2 and -9 by CREB not only increased their invasion potential in vitro and in vivo, but also enhanced angiogenesis in vivo (52), which was not addressed in the present study. The reduced invasion potential and enhanced apoptotic activity in vitro was confirmed by the significant decrease and delayed tumor growth of the shCREB-HER-2/neu+ cells in vivo. These data are in line with results from a nude-mouse model in which the mice were injected with melanoma cells expressing dominant-negative CREB (12), in glioma cells transfected with shCREB (39), and in an artificial CREB model in which CREB Ser133 is mutated (53). Furthermore, the first own results analyzing the frequency of immune cells upon in vivo application of HER-2/neu+ cells and their counterpart shCREB-HER-2/neu+ cells suggest that CREB silencing also affects the immunogenicity of tumors and the antitumor immune response, which is currently being investigated in detail.

No potential conflicts of interest were disclosed.

Conception and design: A. Steven, J. Bukur, B. Hiebl, B. Seliger

Development of methodology: A. Steven, S. Leisz, J. Bukur, B. Hiebl

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Steven, C. Massa, M. Iezzi, A. Lamolinara, J. Bukur, A. Müller, B. Hiebl, H.-J. Holzhausen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Steven, C. Massa, M. Iezzi, R. Lattanzio, A. Lamolinara, B. Seliger

Writing, review, and/or revision of the manuscript: A. Steven, B. Seliger

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Bukur, B. Hiebl

Study supervision: B. Seliger

The authors thank Dr. Frank Bartel, Mrs. Beer, and Mrs. Ernst for help with the histologic staining, immunohistochemical analysis, and picture acquisition, Sylvi Magdeburg for excellent secreterial help, and Christopher Berris for correcting the language.

This work was supported by the Wilhelm Roux Programme of the Medical Faculty of the Martin-Luther-University Halle-Wittenberg, by the Mildred Scheel Foundation for Cancer Research, and by the Italian Ministry for Universities and Research (PRIN 2009P5JPT4_004).

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.

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Supplementary data