Reagents and chemicals

ImClone Systems, Inc. (New York, NY) generously provided cetuximab (C225), a selective antihuman EGFR antagonist monoclonal antibody in a stock concentration of 2 mg/mL. Iressa (Astra-Zeneca) and Erlotinib/Tarceva (Genentech), which are specific EGFR kinase inhibitors have been generously provided by the manufacturer or purchased from the UT MD Anderson Pharmacy. The following antibodies were purchased from the manufacturer's list and were used for western blot analysis: EGFR (Lab Vision Corp., Fremont, CA); E-cadherin and P-cadherin (BD Transduction Laboratories, Lexington, KY); actin, and vinculin (Sigma-Aldrich Corporation, St. Louis, MO); ErbB receptor inhibitor-1 (ERRFI-1) and pEGFR (Tyr¹⁰⁶⁸; Cell Signaling Technology, Danvers, MA). Pre-miRNAs and antagomirs were purchased from Ambion, Inc. (Austin, TX). The TaqMan gene expression assays, miRNA expression assays, and ERRFI-1. The short hairpin RNA (shRNA) against ERRFI-1 and non-targeting controls, as well as miR-200b RFP lentiviral system were all obtained from Open Biosystems-Thermo Scientific.

Cell proliferation assay

Cells (5 × 10³) were plated in 96-well plates for 24 hours, serum-starved for 24 hours in 2% fetal calf serum supplemented with MEM, and then treated with or without EGFR inhibitors at increasing concentrations in an EGF-nonstimulated environment for 30 hours. We measured the effect of cetuximab (C225), Erlotinib (Tarceva) and Iressa on DNA synthesis by pulse-labeling cells with ^[3H]thymidine (MP Biomedicals, Santa Ana, CA) for 2 hours followed by lysis in 100 μL of 0.1 mol/L KOH. Cells were harvested onto fiberglass filters and incorporated tritium was quantified by β-counter.

Wound-healing assay

UMUC3 cells (4 × 10⁶) were plated in 6-well plates for 24 to 48 hours (until they reached confluence). A diametric scratch was performed using a pipette tip followed by 2 culture media changes. Cells were photographed in several pre-marked spots as time zero. Multiple photographs were then taken at 24 and 48 hours in the same spots for comparison.

Plasmid construction and luciferase reporter assays

3′UTR reporter plasmids for miR-200c were constructed via insertion of miR-200c seed sequence into the XbaI restriction site 3′ to luciferase gene in the pGL3-control plasmid (Promega Corp., Madison, WI). An ERRFI-1 3′ UTR segment of 648bp was amplified by PCR from human cDNA and inserted into the pMIR-REPOR (Ambion) with MluI and SpeI sites. For point mutations we used the QuikChange® II Site-Directed Mutagenesis Kit (Qiagen) following manufactory's instruction. The primers for point mutations (U:C) are Mutant 1-F: 5′-ccttgtgttgctggttcctattcagtacctcctggggattgttt-3′; Mutant 1-R: 5′-aaacaatccccaggaggtactgaataggaaccagcaacacaagg-3′; Mutant 2-F: 5′-cactgatttctgcattatgtgtacagtaccggacaaaggattttattcattttgtt-3′; Mutant 2-R: 5′-aacaaaatgaataaaatcctttgtccggtactgtacacataatgcagaaatcagtg-3′. The sequences of the recombinant plasmids were confirmed by DNA sequencing.

Reporter vector transfection was performed using Lipofectamine-2000 reagent (Invitrogen) as described previously (25). miRNA transfections were performed using 20 nmol/L Lipofectamine-2000. Plasmid transfections were carried out similarly but with 50nM/L of reporter plasmid in 24-well plates plus 0.02 μg cytomegalovirus-renilla.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) to quantify mature, miRNAs

Total RNA was extracted using a Mirvana extraction reverse transcription kit (Applied Biosystems) and 10 ng total RNA along with miR-specific primer miRNA were used for expression analysis. cDNA was synthesized using Taqman miRNA specific kit (Ambion-Applied Biosystems).

Immunoblots

Cells were treated with cetuximab (C225) for 3 hours, harvested at approximately 75% to 80%, and lysed. Protein concentration was assayed using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). To prepare cell extracts, cells were washed 3 times with phosphate-buffered saline, and then lysed with RIPA buffer [50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 0.5% NP-40; 0.1% sodium dodecyl sulfate; 0.1% sodium deoxycholate; protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN); and 1 mM sodium ortho-vanadate] for 20 minutes on ice. To measure the activity (phosphorylation) of EGFR, we used an anti-Tyr-1068--specific antibody (Cell Signaling Technololgy) raised against the kinase, which measures the level of autophosphorylated EGFR. Western blot analyses were performed as previously described (25). Antibodies anti-phosphorylated p42/44 MAPKinase and pp42/44 were purchased from Cell Signaling Technology. All chemicals were purchased from Sigma Immunochemicals.

Indirect immunofluorescence staining

Cellular localization of EGFR and ERRFI1 was determined using indirect immunofluorescence as previously described (24). Briefly, cells grown on glass coverslips were fixed (without permeabilization) in 3.7% paraformaldehyde at room temperature for 10 minutes and then extracted with ice-cold acetone. Cells were treated with or without anti-EGFR (LabVision, Inc.) and ERRFI-1 antibodies (Cell Signaling Technology) and then treated with Alexa-488--labeled goat anti-rabbit and Alexa-546--labeled goat anti-mouse secondary antibodies (Molecular Probes, Inc., Eugene, OR). Confocal analysis was carried out using a Zeiss laser-scanning confocal microscope and established methods involving processing of the same section for each detector (2 excitations corresponding to 546 and 488). Co-localization of the 2 proteins (EGFR and ERRFI-1) was indicated by the presence of yellow color as a result of overlapping red and green pixels.

RNA isolation, microarray platform, and statistics

All transcriptome data were generated from duplicates of the cell lines. Cells were plated and total RNA was isolated independently using Trizol reagent (Roche), followed by clean-up with RNeasy® Mini kit (Qiagen, Valencia, CA). RNA was used for the synthesis of biotin-labeled cRNA, which was prepared using the Illumina RNA amplification kit (Ambion, Inc.). Briefly, 500 ng of total RNA from each specimen was converted to cDNA then to cRNA by in vitro transcription. After purification, 1.5 μg of cRNA was fragmented and hybridized to Illumina human-6 v 2 (Illumina, Inc., San Diego, CA) chip. After being washed, the slides were scanned with Bead Station 500X (Illumina, Inc.), and the signal intensities were quantified with Bead Studio (Illumina, Inc.). Quantile normalization was used to normalize the data.

ZEB1, ZEB2, and ERRFI-1 are inversely associated with miR-200c expression

We inquired the TargetScan and PictarVert databases for potential direct targets of miR-200b/c/429 that would associate to an EMT phenotype and targets involved in the EGFR pathway. Several targets were identified according to their ranking in terms of content of conserved miR-200 sites and their expression pattern in our panel of bladder cancer cell lines as a result of gene expression profiling of the 9 cell lines. This list included ZEB1, ZEB2, ERRFI-1, MSN (a modulator of the cytoskeleton), and an unknown Zn finger transcription factor (ZNF532). A heat map showing the relative differences between the cell lines with respect to these potential miR-200 direct targets is shown in Fig. 2F. Further scrutiny for known direct modulators of EGFR-related signaling eliminated all of the targets except ERRFI-1. Real-time RT-PCR analysis revealed that ERRFI-1 mRNA levels were very low in 3 of 5 epithelial cell lines and high in all 3 resistant cell lines (Fig. 3A), suggesting that it is a good candidate target of miR-200 and that its higher baseline expression may be a component of EGFR-resistance.

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Figure 3

Relation between the differentially expressed ERRFI-1 and TGF-α and cell sensitivity to EGFR-targeted therapy

A) Measurement by real-time westernblot of ERRFI-1 and E-cadherin. B) Measurement by real-time RT-PCR of mRNA relative levels of TGF-α. The data are means of measurements from triplicate experiments. C) Cell proliferation measurement (DNA index) of 9 bladder cancer cell lines in the absence or presence of different cetuximab (C225) concentrations. Data are means of at least 3 triplicate experiments.

Analysis of the 3′UTR region of ERRFI-1 and ZEB1/ZEB2 revealed that although ZEB1 and ZEB2 displayed at least 5 potential binding sites for the miR-200 family members (miR200a/b/c/429/141 and 205), ERRFI-1 displayed 2 such conserved sites (http:TargetScan.org), with 1 additional miRNA conserved site for miR-148/152, which was not found in either ZEB1 or ZEB2 3′UTRs. We analyzed the expression pattern of the miR-148 in all 9 cell lines and found no correlation with an epithelial or a mesenchymal phenotype, which underscored its role as a primary ERRFI-1 regulator (data not shown).

Because an intriguing property of ERRFI-1 is its ability to bind more tightly to activated EGFR than to the unligated receptor, we next determined which EGFR ligands are expressed by our cell lines. Gene expression array analysis revealed that three of the E-cadherin-positive cell lines, 253JBV, UMUC16, and UMUC9 also expressed TGF-α; whereas none of the mesenchymal cell lines expressed this gene; (Fig. 3B). In addition, these “autocrine-positive” and E-cadherin positive cells displayed slightly higher levels of the ERRFI-1 protein (Fig. 3A), suggesting that the “feed-back” mechanism of ligand-induced ERRFI-1 up-regulation is present in these cells, as previously described (27). Importantly, UMUC5, UMUC5 which are E-cadherin positives but do not express the TGF-α have almost undetectable levels of ERRFI-1 protein (Fig. 3A and B). An evaluation of cell growth inhibition by the cetuximab (C225) treatment and expression of ERRFI-1 and TGF-α revealed that KU7, UMUC2, UMUC13, and UMUC3 (high expressers of ERRFI-1) and E-cadherin-negative, were resistant for up to 1 μM cetuximab (C225) concentrations; whereas UMUC9, 253J BV, and UMUC16 (moderate ERRFI-1 expressers and high TGF-α expressers) or UMUC5 and UMUC6 (no detectable expression of ERRFI-1 protein or TFG-α mRNA) cell proliferation was inhibited up to 40% by 1 μM cetuximab (C225) (Fig. 3C). Importantly, these assays were done in EGF-unstimulated or “baseline” growth conditions, in the presence of Dulbecco's MEM supplemented with 2% fetal calf serum and reflect the “lower inhibitory efficiencies” for the ligand antagonist cetuximab (C225).

miR-200c expression induced a mesenchymal to epithelial transition (MET) phenotype

To more directly define the role of miR-200c in maintaining the epithelial phenotype and EGFR dependency in bladder cancer cells, we stably transduced the mesenchymal UMUC3 cells with a retroviral vector containing the miR-200c precursor as shown previously (16). Stable expression of miR-200c in fibroblast-like mesenchymal UMUC3 cells resulted in morphological changes suggestive of epithelial differentiation in that the cells grew in a more compact fashion and displayed cobblestone-like cell morphologies (Fig. 4A). These changes were associated with a reduced invasive phenotype as measured by in vitro wound-healing assays, where the miR-200c--expressing clones showed a significantly reduced wound-closing efficiency at 48 hours (Fig. 4, B). The high miR-200c levels expressed by the UMUC3 clones (Fig. 4C) were associated with the appearance of E-cadherin mRNA transcripts and reduced mRNA transcripts for ZEB1 and ZEB2 (Fig. 4D). These differences were also true at the protein levels, as shown for ZEB1 and ZEB2 using immunofluorescence microscopy and confocal analysis (Fig. 4E) and for E-cadherin by western blot analysis (Fig. 4F). These changes were also associated with a reduced ERRFI-1 protein expression (Fig. 4F). Based on morphological criteria as well as biochemical and biological behavior, we concluded that the UMUC3/200c clones demonstrated a MET phenotype and reduced ERRFI-1 expression.

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Figure 4

The miR-200c expression induces an EMT phenotype in UMUC3 bladder cancer cell line

A) Phase contrast microscopy of UMUC3 cells transduced with a miR-200c containing retrovirus (UMUC3/200c) or with an empty, control, retroviral construct (UMUC3-E). Scale bars represent 100 μm. B) Measurement of in vitro cell migration by “wound healing” assay. Representative pictures for same single spot are shown. The experiment was performed twice in triplicate experiments. Scale bars represent 400 μm. C) Quantification of miR-200c in empty virus transduced (UMUC3-E) and miR-200c-transduced (UMUC3/200c) cells. The levels of miR-200c in UMUC5 cells are plotted for comparison. D) Measurement by real-time RT-PCR of the epithelial and mesenchymal markers E-cadherin, ZEB1 and ZEB2 in empty virus-transduced cells and miR-200c-transduced UMUC3 cells. E) Confocal microscopy analysis of the UMUC3 series co-stained for ZEB1 or ZEB2 (red pixels) and nuclear DNA (green pixels). Note downregulation of nuclear ZEB1 and ZEB2 in UMUC3 clones expressing the miR-200c. F) Measurement of ERRFI-1 and E-cadherin protein levels by western blot. Actin reprobing served as internal (loading) control. Lower panel, OD relative values expressed as ratios between actin (internal control) and ERRFI-1. Note up-regulation of E-cadherin and down-regulation of ERRFI-1 protein levels upon miR-200c expression.

miR-200c expression reversed the EGFR resistant phenotype

Expression of miR-200c in UMUC3 cells constantly but modestly reduced the mRNA transcripts of ERRFI-1 (data not shown), whereas the ERRFI-1 protein levels were significantly reduced, suggesting an additional post-translational mechanism involved in ERRFI-1 downregulation in MET clones (Fig. 4F). It is well documented that the EGFR is subject to ligand-induced downregulation mediated by receptor internalization and subsequent degradation by lysosome and the ubiquitin/proteasome system (27-29). Thus, it has recently been suggested that ERRFI-1 is able to bind EGFR molecules, including the trafficking EGFR molecules and that ERRFI-1 becomes expressed at a time in which cells contain a sizable population of activated EGFRs that are being routed through the endosomal compartment. The blockade of kinase activity imposed by ERRFI-1 to trafficking EGFR molecules is expected to shift the kinase/phosphatase equilibrium in favor of phosphatases, therefore leading to a decline of phosphorylated EGFR levels and concomitant loss of signaling competence (28).

To interrogate the location of these EGFR/ERRFI-1 complexes we performed immunofluorescence staining and confocal microscopy studies of UMUC3 cells co-stained with antibodies specific for EGFR (Fig. 5A, green pixels) and ERRFI-1 (Fig. 5A, red pixels). The results revealed significant co-localized signals (yellow pixels) inside the cytoplasm, corresponding to high levels of EGFR trafficking, as well as on the cell membrane edges, such as ruffles. To our surprise, the distribution of EGFR molecules in the UMUC3/200c clones was very different, with most of the EGFR localized on the cell membrane, suggesting a higher rate of the surface-recycled EGFR. ERRFI-1 expression (red pixels) was also lower in UMUC3/200c cells compared to UMUC3 controls (Fig. 5 A, middle and right panels, compared to left panel) confirming our immunoblot analysis.

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Figure 5

The miR-200c expression reverses EGFR resistance in UMUC3 cells

A) Confocal microscopy analysis of the UMUC3 series costained for ERRFI-1 (red pixels) and EGFR (green pixels) and a DNA dye, showing the nucleus (blue pixels). Note the yellow pixels (left panel) as a result of red and green pixels co-localization. B) Immunoblot of autophosphorylated EGFR, total EGFR, and total ERRFI-1 in cells transduced with miR-200c from the experiment above. In this experiment, actin served as the internal control. C) Upper panels, Immunoblot of phosphorylated MAPKinase, total MAPKinase and total EGFR of the UMUC3 series. Cells grown in 2% serum-supplemented MEM were left untreated or were treated with increased concentrations of cetuximab (C225) for 3 hours. Lower panel, OD relative values expressed as ratios between EGFR (internal control) and pMAPKinase. D) Cell proliferation measurement of the UMUC3 series using radioactive thymidine incorporation. Each experiment was done in at least two different triplicates.

We thank GJ Brock for his generosity in providing the retrovirus constructs for miR-200c and J. Kuriyan for providing the full-length myc-tagged ERRFI-1 construct. This work was supported by the NCI-Bladder SPORE and the MD Anderson CORE grant to C.D and by the Alliance for Nanohealth seed grants to L.A. Dr. Calin is supported in part, as a Fellow of The University of Texas M. D. Anderson Research Trust, and University of Texas System Regents Research Scholar and as a Ladjevardian Regents Research Scholar Fund.