EGFR and EGFRvIII undergo stress- and EGFR kinase inhibitor-induced mitochondrial translocalization: A potential mechanism of EGFR-driven antagonism of apoptosis

The impact of apoptotic stimuli on EGFR mitochondrial translocalization remains uninvestigated. Using human GBM T98G cells that express high endogenous levels of EGFR, we found that full-length EGFR undergoes increased mitochondrial translocalization after treatments with staurosporine (ST) and Iressa (I) for 15 min (Figure 1A; left panel). Cell fractionation was effective as indicated by the lack of the cytoplasmic marker, β-actin, and the nuclear protein, lamin B, in the mitochondrial extracts, as well as, by the absence of the mitochondrial protein, Cox IV, in the non-mitochondrial extracts. As shown by the right panel of Figure 1A, both treatments have led to an increased level of total EGFR, suggesting that they may regulate EGFR expression at the post-translational level. The extent of EGFR mitochondrial translocalization was subsequently computed and expressed as an mtEGFR index. Notably, staurosporine and Iressa increased the mtEGFR index by approximately 2.5- to 4-fold (Figure 1A). This was further confirmed using immunofluorescence staining and confocal microscopy (Figure 1B), in which mitochondrial EGFR was marked by the yellow fluorescence signals (arrows) merged from the green fluorescence (EGFR) and red fluorescence (mitochondria-specific dye, mitotracker). The bottom panels of Figure 1B show high-resolution images in which arrows point to mitochondrial EGFR (yellow signals). The observations made in T98G cells were similarly found in cells of the human breast cancer, MDA-MB-468, that express endogenous EGFR (Figures 1C-1E), in which the two apoptosis-inducers, staurosporine and anisomycin, were used to stress the cells. As shown by the right panel of Figure 1C, staurosporine has modestly increased the level of total EGFR. Using electron microscopy (Figure 1E), we observed that EGFR was readily detected in the mitochondria of EGF-treated tumor cells in which arrows mark gold particles that label EGFR. As shown in Figure 1F, we further found that staurosporine and Iressa induced EGFR mitochondrial transport in a time-dependent fashion peaking at 2 hrs after treatments. Notably, the majority of T98G cells did not survive 24 hr staurosporine treatment as indicated by the lack of β-actin and EGFR expression. Noticeably, both the GBM and breast cancer cell lines we analyzed contained a basal level of mitochondrial EGFR without stimulation. This may be due to an autocrine effect given the ability of EGF to induce EGFR mitochondrial translocalization [26]. Together, these results indicate that apoptotic stimuli enhance EGFR mitochondrial translocalization in human cancer cells.

It remains uninvestigated whether EGFRvIII translocates into the mitochondria. Using cell fractionation and western blotting, we show that EGFRvIII is present in the mitochondria even under unstressed condition but that the level is enhanced by apoptotic stress (Figure 2A). The molecular weight of the mitochondrially sequestrated EGFRvIII is approximately 145 kD, similar to the full-length EGFRvIII. In these studies, we used U87MG-EGFRvIII cells that we previously established to express stably transfected EGFRvIII [12] because endogenous EGFRvIII expression is not maintained in vitro [28]. As indicated by the mtEGFRvIII indices, staurosporine (ST) and anisomycin (AN) increased the levels of mitochondrial EGFRvIII by approximately 3-4 fold, while Iressa resulted in a 7.5-fold increase in EGFRvIII mitochondrial transport (Figure 2B). These observations were confirmed by immunofluorescence staining and confocal microscopy (Figure 2C). In the immunofluorescence staining experiments, we used an EGFR antibody that recognized the N-terminal epitope present in both EGFR and EGFRvIII (Figure 2C) and a Myc-tag antibody that bound to the C-terminal Myc-tag of the EGFR/EGFRvIII fusion proteins (data not shown). The results showed that both antibodies labeled mitochondrial EGFRvIII. This observation combined with that of the western blotting (Figure 2A) strongly indicate that the mitochondrially localized EGFRvIII is likely to be the full-length EGFRvIII.

In this study, we targeted two N-terminal regions of EGFR and EGFRvIII that are homologous to L/I/V/F/M-rich sequences that have been shown to be involved in protein intracellular trafficking [29‐31]. As shown in Figures 3A and 3B, the two N-terminal motifs are present in the extracellular region of EGFR, EGFRvIII and HER2 while the second motif is found in all members of the EGFR family of receptors. Both motifs are conserved among EGFR proteins in different mammalian species (Figure 3C). To determine the role of these motifs in modulating EGFR/EGFRvIII intracellular trafficking, we performed site-directed mutagenesis to substitute leucine/isoleucine residues with alanines and thereby, altered the L/I-rich property of the motifs. Consequently, we generated two EGFR mutants, EGFR-MTS1 (L399A/I402A) and EGFR-MTS2 (L429A/I432A) and two EGFRvIII mutants (Figure 3D), EGFRvIII-MTS1 (L132A/I35A) and EGFRvIII-MTS2 (L162A/I165A). The subcellular location of these EGFR/EGFRvIII mutants was then examined via immunofluorescence staining/confocal microscopy following transfecting the mutant constructs into EGFR/EGFRvIII-null Chinese hamster ovary (CHO) cells. The results (Figure 3E) showed that all four mutant receptors were significantly localized in the mitochondria, as indicated by the yellow signals after merging the green fluorescence (EGFR/EGFRvIII and their mutants) with the red fluorescence (mitochondrion). EGFR and EGFRvIII MTS mutant proteins were also present in the cytoplasm. Interestingly, the MTS mutants were rarely found on the plasma membranes. Given the similarity of the two N-terminal motifs to the reported nuclear-export signals [30], we determined whether the mutations within the motifs block EGFR nuclear export and consequently, facilitate EGFR nuclear accumulation. The results of immunofluorescence staining and confocal microscopy (Figure 3F) showed no increased accumulation of EGFR-MTS1 or EGFR-MTS2 (not shown) in the nucleus, despite their ability to undergo significant mitochondrial import. This was confirmed using nuclear fractionation followed by western blotting (Figure 3G), in which the effectiveness of nuclear fractionation was indicated by the absence of the cytoplasmic protein β-actin in the nucleus and the lack of the nuclear protein histone H3 in the non-nuclear extracts. Collectively, these results indicate that the EGFR/EGFRvIII MTS mutants undergo enhanced mitochondrial translocalization. These data also suggest that these mutants can be used as valuable tools for studies aiming to elucidate the actions of mitochondrial EGFR/EGFRvIII that are largely unknown at the present time.

In light of our observations that apoptotic stimuli induced EGFR/EGFRvIII translocation into the mitochondria, a focal point of intrinsic apoptosis initiation, we speculated that mitochondrial EGFR/EGFRvIII may be involved in mediating mitochondria-mediated apoptosis. Consequently, we created two U87MG stable transfectant cell lines, U87MG-EGFRvIII-MTS1 and U87MG-EGFRvIII-MTS2, to respectively express EGFRvIII-MTS1 and EGFRvIII-MTS2 mutants. As shown in Figure 4A, the expression levels of the transgenes in U87MG-EGFRvIII-MTS1 and U87MG-EGFRvIII-MTS2 stable transfectant cells were similar to that of U87MG-EGFRvIII cells. The two MTS mutants are expressed at high levels in the mitochondria as shown by the results of immunofluorescence staining/confocal microscopy (Figure 4B) and cell fractionation/western blotting (Figure 4C; only EGFR-MTS1 data are shown; ME, mitochondrial extracts; NME, non-mitochondrial extracts). Stability of EGFRvIII, EGFRvIII-MTS1 and EGFRvIII-MTS2 proteins was similar, as indicated by the protein degradation study using the transcription inhibitor, cycloheximide (Figure 4D; data for EGFRvIII-MTS2 are not shown). Given the observation that EGFRvIII-MTS1 and EGFRvIII-MTS2 behave similarly, we focused on EGFRvIII-MTS1 for determining the effects of EGFRvIII mitochondrial accumulation on the response of cancer cells to apoptosis-inducing agents. We observed that U87MG-EGFRvIII-MTS1 cells were more resistant to 48 hr treatments of staurosporine (Figure 5A) and anisomycin (Figure 5B) than U87MG-EGFRvIII cells. Consistent with these observations, anisomycin induced a significantly lower level of apoptosis in U87MG-EGFRvIII-MTS1 cells than in U87MG-EGFRvIII cells (Figure 5C). In the TUNEL assay, the extent of apoptosis was indicated by the yellow fluorescence signals, merged products of the red fluorescence (nuclei) and the green (fluorescence fragmented DNA). Together, these results suggest that EGFRvIII mitochondrial accumulation protects cancer cells from undergoing mitochondria-mediated apoptotic death.

The results summarized in Figure 2 indicate that the EGFR inhibitor, Iressa, enhances EGFRvIII mitochondrial translocalization in tumor cells. We, therefore, examined whether the mitochondrial accumulation of EGFRvIII impacts cancer cell sensitivity to Iressa. The results of these studies, summarized in Figure 6A, show that U87MG-EGFRvIII-MTS1 cells with increased mitochondrial EGFRvIII are significantly more resistant to a 48-hr Iressa treatment than control U87MG-EGFRvIII cells. This is further confirmed by the results of the clonogenic cell assay (Figures. 6B and 6C), showing that control U87MG-EGFRvIII-MTS1 cells had a reduced ability to form colonies compared to untreated U87MG-EGFRvIII cells. This interesting observation may be due to the likelihood that EGFRvIII-MTS1 is mitochondrially enriched for the purpose of enhancing survival and that it is rarely localized on the cell-surface where the mitogenic proliferative signal is transmitted. These results combined with those in Figure 2 indicate that Iressa induces EGFRvIII mitochondrial translocalization and that the mitochondrial accumulation of EGFRvIII contributes to the resistance of GBM cells to EGFR inhibition.

Human GBM T98G and U87MG cells, human breast carcinoma MDA-MB-468 cells and Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection (Manassas, VA). U87MG-EGFRvIII stable transfectant cells were established from the parental U87MG cells that expressed a very low level of wild-type EGFR as previously described [19]. All cell lines were cultured in DMEM with 10% fetal calf serum while the U87MG stable transfectant cell lines were additionally supplemented with 0.7 mg/ml G418.

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. Rabbit polyclonal antibody against EGFR (sc-05) used in western blotting was purchased from Santa Cruz Biotech. (Santa Cruz, CA). Monoclonal mouse EGFR antibody used in immunofluorescence staining was obtained from Zymed. Mouse monoclonal Myc-tag antibody was from Roche (Indianapolis, IN). Lamin B mouse monoclonal antibody was from Calbiochem (San Diego, CA). Mouse monoclonal β-actin and α-tubulin antibodies were obtained from Sigma. Rabbit polyclonal Cox IV antibody was from Abcam (Cambridge, MA). Mouse monoclonal anti-EGFR antibody used in immunofluorescence staining was from Zymed/Invitrogen (Carlsbad, CA). Mitotracker was purchased from Invitrogen/Molecular Probes. All transfections were performed with cells in exponential growth using lipofectamine 2000 (Invitrogen) and FuGENE HD (Roche). Iressa was purchased from LC Laboratories (Woburn, MA). CellTiter Blue Cell Viability Assay was obtained from Promega (Madison, WI). Mitochondrial isolation kit was from Pierce (Rockford, IL). QuikChange Mutagenesis Kit was from Stratagene (Madison, WI).

For this, mitochondria were isolated from tumor cells using a commercial kit (Pierce), according to the manufacturer's instructions. Both mitochondrial and non-mitochondrial fractions were subjected to protein extraction using 1% SDS and 0.1% NP-40 and sonication followed by centrifugation at 15,000 × g and 4°C for 20 min. In these studies, we subjected 25% of the mitochondrial proteins and 2.5% of the non-mitochondrial proteins to western blotting. Band signals from mitochondrial EGFR/EGFRvIII and non-mitochondrial EGFR/EGFRvIII were determined densitometrically using the NIH ImageJ software, as we previously described [49], and normalized against loading controls, Cox IV and β-actin/α-tubulin. The extent of EGFR/EGFRvIII mitochondrial translocalization, designated as mtEGFR/EGFRvIII Index, was computed using the equation: (mitochondrial EGFR/EGFRvIII ÷ 25%)/(mitochondrial EGFR/EGFRvIII ÷ 25%) + (non-mitochondrial EGFR/EGFRvIII ÷ 2.5%).

Tumor cells seeded in 8-well Lab-Tek chamber slides (Nunc Inc., Rochester, NY) for 24 hrs were washed with ice-cold PBS, fixed in 4% paraformaldehyde for 15 min and permeablized with 0.2% Triton-X100 for 5 min. Following treatment with 10% normal goat serum/1% BSA for 60 min, the cells were incubated with polyclonal rabbit Myc or monoclonal mouse EGFR antibody overnight at 4°C. After three washes with PBS, the cells were incubated with goat anti-rabbit secondary or donkey anti-mouse antibodies (1:200, Vector Lab) tagged with Texas Red or fluorescein, respectively. To label the mitochondria, the cells were additionally exposed to DMEM containing 200 nM mitotracker for 30 min prior to fixation in 4% paraformaldehyde. They were then mounted with VECTASHIELD Mounting Medium with and without propidium iodide (for nuclei detection) and examined under a Zeiss LSM 510 confocal microscope.

MDA-MB-468 breast cancer cells were subjected to EM analysis. Briefly, following incubation with mouse IgG, cell sections were treated with mouse monoclonal EGFR antibody. The cells were then incubated with gold particle (15 nm) labeled goat anti-mouse secondary antibody (Amersham Biosciences, USA) for 45 min. Sections were washed, stained with uranyl acetate for 2 min and Reynolds's lead citrate for 1 min and examined in a Jeol 1200EX microscope.

This was performed using QuikChange Mutagenesis Kit (Stratagene) and PCR, according to the manufacturer's instructions [24]. The plasmids carrying EGFR and EGFRvIII cDNAs, namely, pCMV-Tag5A-EGFR and pCMV-Tag5A-EGFRvIII [12], were used as templates in the PCR-mutagenesis reactions. Primers used to generate EGFR-MTS1 and EGFRvIII-MTS1 mutants are 5'-ATGCCTTTGAGAACGCAGAAATCGCACGCGGCAGGA-3' (forward) and 5'-TCCTGCCGCGTGCG ATTTCTGCGTTCTCAAAGGCAT-3' (reverse). To produce EGFR-MTS2 and EGFRvIII-MTS2, primers with the sequences of 5'-GATTACGCTCCGCAAGGAGGCAAGTGATGGAGATG-3' (forward) and 5'-CATCTCCATCACTTGCCTCCTTGGCGGAGCGTAATC-3' (reverse) were used. Subsequently, the EGFRvIII-MTS1 and EGFRvIII-MTS2 constructs were transfected into U87MG GBM cells to establish U87MG-EGFRvIII-MTS1 and U87MG-EGFRvIII-MTS2 stable transfectant cell lines.

This was performed as previously described [24, 50]. Briefly, serum-starved cells treated with EGF (100 ng/ml) for 0 and 10 mins were collected, washed with PBS, and swelled in hypotonic buffer (25 mM Tris-HCl, pH 7.5, 5 mM KCl, 0.5 mM dithiothreitol, 1 mM PMSF and 0.15 m/ml aprotinin) for 20 min on ice. Following homogenization using a Dounce homogenizer, nuclei were pelleted and washed. Ultrasonic disruption was used to extract nuclear proteins from the isolated nuclei. To isolate non-nuclear extracts, the supernatant was exposed to 1% SDS and 0.1% NP-40, centrifuged at 15,000 g to remove cell debris and the resulting supernatant collected. Western blotting was conducted. Histone H3 serves a nuclear marker.

Tumor cells exposed to 10 ug/ml cycloheximide for 0, 30, 60, 120 and 180 min were harvested, total proteins extracted and subjected to western blotting. A Myc-tagged mouse monoclonal antibody (Roche) was used to detect Myc-tagged EGFRvIII and EGFRvIII-MTS fusion proteins. Membranes were also blotted with an anti-α-tubulin antibody (Sigma).

This was performed using a TUNEL assay kit (Invitrogen), according to the manufacturer's instructions [20]. Briefly, the cells were fixed in 70% ethanol, washed and incubated with the labeling solution containing 5-bromodeoxyuridine 5'-triphosphate and deoxynucleotidyl terminal transferase for 1 hr at 37°C. The DNA incorporated bromodeoxyuridine was detected by Alexa Fluor^® 488 dye-labeled anti-BrdU antibody at room temperature for 30 min. The cells were then counterstained with propidium iodide and observed under a Zeiss LSM 510 confocal microscope. Yellow signals indicate nuclear fragmented DNA, the merged products of fragmented DNA (green fluorescence) and nuclei (red fluorescence). A total of 250-300 cells were examined in each experiment and three independent experiments were conducted to derive means and standard deviations. Extent of apoptosis was subsequently computed using the equation, (# of nuclei with fragmented DNA)/(# of total nuclei).

Tumor cells in exponential growth were seeded in 96-well culture plates and treated with vehicle control (1% DMSO) and various agents. After 48 hrs, the cells were subjected to cell survival analyses using the CellTiter Blue Cell Viability Assay (Promega). Briefly, 25 μl of the CellTiter Blue reagent was added to each well containing 100 ul media, incubated for 4 hrs at 37°C, and then the absorbance measured at 560 nm/590 nm using a plate reader (Synergy-HT, BIO-TEK, Winooski, VT). Clonogenic growth assay was performed in 6-well cell culture plates with 1,000 cells seeded per well as previously described [19]. Seeded cells were treated with 1% DMSO or 12.5 μM Iressa in 1% DMSO for 24 hrs, medium removed and replaced with fresh drug-free growth medium for 10-14 days. Colonies were stained with crystal violet blue solution (Sigma) for 1 hr, washed with water, dried and counted. Triplicate wells were used for each treatment and three independent experiments were performed to derive means and standard deviations.