EGFR and EGFRvIII undergo stress- and EGFR kinase inhibitor-induced mitochondrial translocalization: A potential mechanism of EGFR-driven antagonism of apoptosis
© Cao et al; licensee BioMed Central Ltd. 2011
Received: 23 September 2010
Accepted: 9 March 2011
Published: 9 March 2011
Epidermal growth factor receptor (EGFR) plays an essential role in normal development, tumorigenesis and malignant biology of human cancers, and is known to undergo intracellular trafficking to subcellular organelles. Although several studies have shown that EGFR translocates into the mitochondria in cancer cells, it remains unclear whether mitochondrially localized EGFR has an impact on the cells and whether EGFRvIII, a constitutively activated variant of EGFR, undergoes mitochondrial transport similar to EGFR.
We report that both receptors translocate into the mitochondria of human glioblastoma and breast cancer cells, following treatments with the apoptosis inducers, staurosporine and anisomycin, and with an EGFR kinase inhibitor. Using mutant EGFR/EGFRvIII receptors engineered to undergo enriched intracellular trafficking into the mitochondria, we showed that glioblastoma cells expressing the mitochondrially enriched EGFRvIII were more resistant to staurosporine- and anisomycin-induced growth suppression and apoptosis and were highly resistant to EGFR kinase inhibitor-mediated growth inhibition.
These findings indicate that apoptosis inducers and EGFR-targeted inhibitors enhance mitochondrial translocalization of both EGFR and EGFRvIII and that mitochondrial accumulation of these receptors contributes to tumor drug resistance. The findings also provide evidence for a potential link between the mitochondrial EGFR pathway and apoptosis.
EGFR is an important mediator of normal cell growth and differentiation [1, 2]. In cancer cells, EGFR is frequently over-expressed and is associated with tumor proliferation, progression and drug resistance [3–5]. EGFRvIII, a constitutively activated EGFR variant, is a product of rearrangement with an in-frame deletion of 801 bp of the coding sequence of the EGFR extracellular domain that results in a deletion of residues 6 through 273 and a glycine insertion as residue 6 [6–9]. EGFR/EGFRvIII gene amplification is frequent in glioblastoma multiforme (GBM), the most common and deadliest brain cancer in adults [9, 10]. Consequently, both EGFR and EGFRvIII are being targeted for cancer therapy [3, 11, 12].
The anticancer efficacy of anti-EGFR small molecule inhibitors and monoclonal antibodies has been evaluated in clinical trials both as single agent and in combination with other chemotherapeutic agents, but to date, have shown only modest effects [13–18]. Much effort is thus being directed at understanding the mechanisms that underlie tumor resistance to anti-EGFR therapy. For example, we have recently shown that nuclear EGFR interacts with STAT3 and that the interaction contributes to tumor resistance to the anti-EGFR agent, Iressa, in human GBM  and breast cancer cells . In addition, it has been recently reported that EGFR and EGFRvIII interacts with apoptotic protein PUMA and inhibits PUMA's apoptotic function . PTEN loss has also been implicated in resistance to EGFR inhibition, although, other studies did not find such a linkage [17, 18, 21, 22]. In lung cancer, gain-of-function EGFR mutations have been shown to be predictive of sensitivity to EGFR-targeted treatments, however, in other tumor types, these mutations are either absent or are very rare. The biology underlying tumor resistance to EGFR-targeted therapy is thus complex and remains not well understood.
An area of EGFR-associated biology in human cancers that is receiving increasing attention is the ability of EGFR to escape lysosome-mediated degradation and recycling to the plasma membranes and, subsequently, to undergo intracellular trafficking to subcellular organelles, such as, nuclei [4, 19, 23–25] and mitochondria [26, 27]. Nuclear EGFR and mitochondrial EGFR are expressed as the full-length proteins, in contrast to HER4/ErbB4 which enters nuclei and mitochondria as its C-terminal fragment. While the cellular functions and role of nuclear EGFR are becoming clearer, those of mitochondrial EGFR are still largely unknown. Also unknown is whether EGFRvIII undergoes mitochondrial translocalization. Nevertheless, it has been shown that EGF stimulation enhances EGFR mitochondrial localization in MDA-MB-231 breast cancer cells  and that mitochondrial EGFR interacts with cytochrome c oxidase subunit II (CoxII) in an EGFR Y845-dependent manner . EGFR Y845 is a specific phosphorylation residue targeted by c-Src and interestingly, c-Src appears to also undergo mitochondrial import with kinetics similar to that of EGFR . In the mitochondria, both EGFR and c-Src can phosphorylate Cox II, albeit the consequence of this phosphorylation remains unclear .
Given the pivotal role that mitochondria plays in intrinsic apoptosis, we investigated, in this study, the effects of apoptosis-inducing agents on mitochondrial translocalization of both EGFR and EGFRvIII. We also conducted a series of experiments to address the impact of the mitochondrial accumulation of EGFR and EGFRvIII on the apoptotic response of cancer cells treated with apoptosis-inducing agents and an EGFR kinase inhibitor, Iressa. Our findings demonstrate that both EGFR and EGFRvIII undergo mitochondrial translocalization when cancer cells encounter apoptotic stimuli. Using cells that stably express EGFRvIII and mitochondrially enriched EGFRvIII mutant, we found that mitochondrial accumulation of EGFRvIII rendered the cells highly resistant to apoptosis induced by these agents. These results implicate mitochondrial EGFR/EGFRvIII in the modulation of mitochondria-mediated apoptosis.
EGFR mitochondrial translocalization is enhanced by apoptotic inducers and an EGFR kinase inhibitor
EGFRvIII undergoes mitochondrial translocalization and the degree of transport is enhanced by apoptosis inducers and Iressa
Amino acid substitutions of N-terminal conserved motifs increase mitochondrial import of both EGFR and EGFRvIII
Tumor cells expressing the mitochondrially enriched EGFRvIII are more resistant to mitochondria-mediated apoptosis
Translocation of EGFRvIII to mitochondria renders tumor cells resistant to EGFR inhibition
We report in this study that both EGFR and EGFRvIII translocate into mitochondria upon exposure of GBM and breast cancer cells to apoptosis inducers and EGFR inhibitors and that accumulation of EGFRvIII in mitochondria renders GBM cells more resistant to the EGFR inhibitor, Iressa, and other apoptosis inducers. Two modes of EGFR signaling, cell-surface and nuclear, have been well characterized to play important roles in human cancers [3–5]. The cell-surface EGFR signaling pathway occurs through transduction of mitogenic signals from the cell-surface to PLC-γ-CaMK/PKC, Ras-Raf-MAPK, PI-3K-Akt-GSK, and JAK/STATs, and downstream to targets in the cytoplasm and the cell nucleus. The nuclear EGFR pathway involves EGFR nuclear transport and can be activated by ligand binding, radiation, cisplatin, heat- and oxidative-stress, and inhibited by exposure to vitamin D [4, 32, 33]. Nuclear EGFR functions as a transcription factor and tyrosine kinase, leading to increased proliferation and poor clinical outcomes in cancer [4, 19, 25, 33–36]. Our findings in this study provide for and extend the previously described mitochondrial EGFR signaling by demonstrating the functional consequences of mitochondrial EGFR translocalization, specifically, its impact on apoptosis and drug resistance.
Our findings confirm those in previous reports [26, 27, 37] indicating that the full-length EGFR translocates into the mitochondria. We also demonstrate, for the first time, that EGFRvIII also translocates to mitochondria as a full-length protein. This contrasts with HER2, which has been shown to exist as both full-length (185 kD) and truncated (155 kD) proteins in the mitochondria of normal kidney and breast cells, and in SK-BR-3 breast cancer cells . In contrast to EGFR and HER2, HER4 undergoes γ-secretase processing  and the resulting intracellular domain translocates into the mitochondria . In the case of EGFR, the full-length receptor has been shown to exist in the cytoplasm as free non-membrane-bound form  and this is the consequence of Sec61β translocon-mediated retro-translocation that extract membrane-bound EGFR from the ER to the cytoplasm as a non-membrane-bound receptor . In this form, EGFR interacts with importin β1, a nuclear import protein, crosses through nuclear pore complex into the nucleus, where it functions as a transcription factor and tyrosine kinase [35, 42]. We recently showed that the non-membrane-bound cytoplasmic EGFR/EGFRvIII interacts with the pro-apoptotic protein PUMA and traps PUMA in the cytoplasm . These observations are in line with the notion that full-length EGFR translocates into the mitochondria, as shown in this study and in previous other reports [26, 27, 37].
It remains unknown how EGFR or EGFRvIII translocate into mitochondria, particularly, since both receptors lack any of the known mitochondrial targeting signal (MTS). Interestingly, a number of cytoplasmic and nuclear proteins have been shown to undergo mitochondrial translocalization including, HER2 that lacks MTS. Similarly, p53 does not contain a MTS but translocates into the mitochondria in response to DNA damage, leading to apoptosis [43–45]. The transmembrane glycoprotein, MUC1, also lacks a MTS but undergoes proteolytic cleavage in ER, retro-translocated into cytoplasm and subsequently, its C-terminal peptide, MUC1-C localizes to the mitochondria . It has been shown that MUC1-C forms intracellular complexes with chaperone HSP70/HSP90 complex  which enables its mitochondrial trafficking. In light of these observations, future investigations are warranted to explore whether EGFR/EGFRvIII utilizes a similar chaperone mechanism to gain entry into mitochondria.
Emerging evidence indicates that cell-surface and nuclear proteins translocate into mitochondria and that while in the mitochondria, elicit biological effects that are uniquely distinct from their cell-surface and nuclear functions. For example, cell-surface receptors (EGFR, EGFRvIII and HER2), cytoplasmic proteins (c-Src, intracellular domains of HER4 and MUC-1) and nuclear proteins (p53) can be shuttled into mitochondria and some of them have been shown to elicit unique functions [26, 27, 37, 38, 43–48]. We show in this study that mitochondrial transport of EGFR and EGFRvIII can be induced by apoptosis-inducing agents and EGFR inhibitors and that tumor cells with accumulated mitochondrial EGFRvIII are resistant to apoptosis induced by these agents. It has been previously shown that EGF stimulates EGFR mitochondrial import leading to mitochondrial interaction of EGFR with Cox II; the cellular consequence of the interaction, however, remains unclear [26, 27]. The role of HER2 in the mitochondria is unknown. C-terminal region of HER4 binds to and antagonizes anti-apoptotic protein Bcl-2 and thereby promotes apoptosis of breast cancer cells . Mitochondrial MUC1 has been shown to prevent cisplatin-induced apoptosis . Mitochondrial p53 binds to anti-apoptotic protein Bcl-xL and induces apoptosis [43–45]. It is worth noting that a recent study by Yue et al  showed that EGFR mitochondrial translocation can be increased by the mTOR inhibitor, rapamycin, and decreased by the topoisomerase inhibitor, etoposide, and by 3'-methyladenine, an inhibitor of autophagy. The reported link between autophagy and mitochondrial EGFR is particularly interesting and is in line with our observation that apoptotic stress induces EGFR mitochondrial transport. Together, the results of Yue et al  and the current study implicate mitochondrial EGFR in apoptosis and autophagy of therapy-challenged tumor cells. These findings also call for additional efforts to elucidate the exact actions of mitochondrially localized EGFR and EGFRvIII and their impact on tumor response to anti-cancer drugs that often induce apoptosis and/or autophagy.
Our findings in this study indicate that apoptosis inducers and EGFR-targeted inhibitors enhance mitochondrial translocalization of both EGFR and EGFRvIII and that mitochondrial accumulation of these receptors contributes to tumor drug resistance. The findings also provide evidence for a potential link between the mitochondrial EGFR pathway and apoptosis. These results also suggest that tumor cells may re-program their intracellular trafficking of EGFR by increasing its mitochondrial accumulation, as a mechanism for escape from therapy- and stress-induced apoptosis and growth suppression. The exact mechanisms by which mitochondrial EGFR modulates apoptotic response are currently being investigated in our laboratory.
Cell lines and cell culture
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 . 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.
Reagents and chemicals
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).
Determination of EGFR/EGFRvIII mitochondrial translocalization
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 , 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%).
Detection of mitochondrial EGFR/EGFRvIII via immunofluorescence staining and confocal microscopy
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.
Detection of mitochondrial EGFR by transmission electron microscopy (EM)
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.
Generation of mutant EGFR and EGFRvIII with enriched mitochondrial presence using site-directed mutagenesis
This was performed using QuikChange Mutagenesis Kit (Stratagene) and PCR, according to the manufacturer's instructions . The plasmids carrying EGFR and EGFRvIII cDNAs, namely, pCMV-Tag5A-EGFR and pCMV-Tag5A-EGFRvIII , 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.
Nuclear fractionation and western blotting
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.
Determination of half-life of EGFRvIII and its MTS mutants
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).
TUNEL assay for apoptosis
This was performed using a TUNEL assay kit (Invitrogen), according to the manufacturer's instructions . 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).
Cell survival and clonogenic growth assays
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 . 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.
Student t-test was performed using STATISTICA (StatSoft Inc., Tulsa, OK) and Microsoft Excel.
This work was supported by NIH grant 5K01-CA118423, DoD grant W81XWH-07-1-0390, Pediatric Brain Tumor Foundation and Beez Foundation (to HWL).
- Cohen S, Fava RA, Sawyer ST: Purification and characterization of epidermal growth factor receptor/protein kinase from normal mouse liver. Proc Natl Acad Sci USA. 1982, 79: 6237-6241. 10.1073/pnas.79.20.6237PubMed CentralView ArticlePubMedGoogle Scholar
- Sato JD, Kawamoto T, Le AD, Mendelsohn J, Polikoff J, Sato GH: Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med. 1983, 1: 511-529.PubMedGoogle Scholar
- Lo HW: EGFR-targeted therapy in malignant glioma: novel aspects and mechanisms of drug resistance. Curr Mol Pharmacol. 2010, 3: 37-52.PubMed CentralView ArticlePubMedGoogle Scholar
- Lo HW: Nuclear mode of the EGFR signaling network: biology, prognostic value, and therapeutic implications. Discov Med. 2010, 10: 44-51.PubMed CentralPubMedGoogle Scholar
- Lo HW, Hung MC: Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br J Cancer. 2006, 94: 184-188. 10.1038/sj.bjc.6602941PubMed CentralView ArticlePubMedGoogle Scholar
- Yamazaki H, Ohba Y, Tamaoki N, Shibuya M: A deletion mutation within the ligand binding domain is responsible for activation of epidermal growth factor receptor gene in human brain tumors. Jpn J Cancer Res. 1990, 81: 773-779.View ArticlePubMedGoogle Scholar
- Ekstrand AJ, Sugawa N, James CD, Collins VP: Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails. Proc Natl Acad Sci USA. 1992, 89: 4309-4313. 10.1073/pnas.89.10.4309PubMed CentralView ArticlePubMedGoogle Scholar
- Wong AJ, Ruppert JM, Bigner SH, Grzeschik CH, Humphrey PA, Bigner DS, Vogelstein B: Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl Acad Sci USA. 1992, 89: 2965-2969. 10.1073/pnas.89.7.2965PubMed CentralView ArticlePubMedGoogle Scholar
- Sugawa N, Ekstrand AJ, James CD, Collins VP: Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc Natl Acad Sci USA. 1990, 87: 8602-8606. 10.1073/pnas.87.21.8602PubMed CentralView ArticlePubMedGoogle Scholar
- Wong AJ, Bigner SH, Bigner DD, Kinzler KW, Hamilton SR, Vogelstein B: Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA. 1987, 84: 6899-6903. 10.1073/pnas.84.19.6899PubMed CentralView ArticlePubMedGoogle Scholar
- Lo HW, Hsu SC, Hung MC: EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization. Breast Cancer Research and Treatment. 2006, 95: 211-218. 10.1007/s10549-005-9011-0View ArticlePubMedGoogle Scholar
- Lo HW, Cao X, Zhu H, Ali-Osman F: Constitutively activated STAT3 frequently coexpresses with epidermal growth factor receptor in high-grade gliomas and targeting STAT3 sensitizes them to Iressa and alkylators. Clin Cancer Res. 2008, 14: 6042-6054. 10.1158/1078-0432.CCR-07-4923PubMed CentralView ArticlePubMedGoogle Scholar
- Omuro AMP, Faivre S, Raymond E: Lessons learned in the development of targeted therapy for malignant gliomas. Mol Cancer Ther. 2007, 6: 1909-1919. 10.1158/1535-7163.MCT-07-0047View ArticlePubMedGoogle Scholar
- Reardon DA, Quinn JA, Vredenburgh JJ, Gururangan S, Friedman AH, Desjardins A, Sathornsumetee S, Herndon JE, Dowell JM, McLendon RE: Phase 1 trial of gefitinib plus sirolimus in adults with recurrent malignant glioma. Clin Cancer Res. 2006, 12: 860-868. 10.1158/1078-0432.CCR-05-2215View ArticlePubMedGoogle Scholar
- Friedman HS, Bigner DD: Glioblastoma multiforme and the epidermal growth factor receptor. N Engl J Med. 2005, 353: 1997-1999. 10.1056/NEJMp058186View ArticlePubMedGoogle Scholar
- van den Bent MJ, Brandes AA, Rampling R, Kouwenhoven MC, Kros JM, Carpentier AF, Clement PM, Frenay M, Campone M, Baurain JF: Randomized phase II trial of erlotinib versus temozolomide or carmustine in recurrent glioblastoma: EORTC brain tumor group study 26034. J Clin Oncol. 2009, 27: 1268-1274. 10.1200/JCO.2008.17.5984PubMed CentralView ArticlePubMedGoogle Scholar
- Thiessen B, Stewart C, Tsao M, Kamel-Reid S, Schaiquevich P, Mason W, Easaw J, Belanger K, Forsyth P, McIntosh L, Eisenhauer E: A phase I/II trial of GW572016 (lapatinib) in recurrent glioblastoma multiforme: clinical outcomes, pharmacokinetics and molecular correlation. Cancer Chemother Pharmacol. 2009, 65: 353-361. 10.1007/s00280-009-1041-6View ArticleGoogle Scholar
- Reardon DA, Desjardins A, Vredenburgh JJ, Gururangan S, Friedman AH, Herndon JE, Marcello J, Norfleet JA, McLendon RE, Sampson JH, Friedman HS: Phase 2 trial of erlotinib plus sirolimus in adults with recurrent glioblastoma. J Neurooncol. 2010, 96: 219-230. 10.1007/s11060-009-9950-0PubMed CentralView ArticlePubMedGoogle Scholar
- Lo HW, Hsu SC, Ali-Seyed M, Gunduz M, Xia W, Wei Y, Bartholomeusz G, Shih JY, Hung MC: Nuclear Interaction of EGFR and STAT3 in the Activation of iNOS/NO Pathway. Cancer Cell. 2005, 7: 575-589. 10.1016/j.ccr.2005.05.007View ArticlePubMedGoogle Scholar
- Zhu H, Cao X, Ali-Osman F, Keir S, Lo HW: EGFR and EGFRvIII interact with PUMA to inhibit mitochondrial translocalization of PUMA and PUMA-mediated apoptosis independent of EGFR kinase activity. Cancer Lett. 2010, 294: 101-110. 10.1016/j.canlet.2010.01.028PubMed CentralView ArticlePubMedGoogle Scholar
- Guillamo JS, de Bouard S, Valable S, Marteau L, Leuraud P, Marie Y, Poupon MF, Parienti JJ, Raymond E, Peschanski M: Molecular mechanisms underlying effects of epidermal growth factor receptor inhibition on invasion, proliferation, and angiogenesis in experimental glioma. Clin Cancer Res. 2009, 15: 3697-3704. 10.1158/1078-0432.CCR-08-2042View ArticlePubMedGoogle Scholar
- Mellinghoff IK, Cloughesy TF, Mischel PS: PTEN-Mediated Resistance to Epidermal Growth Factor Receptor Kinase Inhibitors. Clin Cancer Res. 2007, 13: 378-381. 10.1158/1078-0432.CCR-06-1992View ArticlePubMedGoogle Scholar
- Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY, Bourguignon L, Hung MC: Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol. 2001, 3: 802-808. 10.1038/ncb0901-802View ArticlePubMedGoogle Scholar
- Lo HW, Cao X, Zhu H, Ali-Osman F: Cyclooxygenase-2 is a novel transcriptional target of the nuclear EGFR-STAT3 and EGFRvIII-STAT3 signaling axes. Mol Cancer Res. 2010, 8: 232-245. 10.1158/1541-7786.MCR-09-0391PubMed CentralView ArticlePubMedGoogle Scholar
- Lo HW, Xia W, Wei Y, Ali-Seyed M, Huang SF, Hung MC: Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer. Cancer Res. 2005, 65: 338-348.PubMedGoogle Scholar
- Boerner JL, Demory ML, Silva C, Parsons SJ: Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol Cell Biol. 2004, 24: 7059-7071. 10.1128/MCB.24.16.7059-7071.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Demory ML, Boerner JL, Davidson R, Faust W, Miyake T, Lee I, Hüttemann M, Douglas R, Haddad G, Parsons SJ: Epidermal Growth Factor Receptor Translocation to the Mitochondria. Journal of Biological Chemistry. 2009, 284: 36592-36604. 10.1074/jbc.M109.000760PubMed CentralView ArticlePubMedGoogle Scholar
- Bigner SH, Humphrey PA, Wong AJ, Vogelstein B, Mark J, Friedman HS, Bigner DD: Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res. 1990, 50: 8017-8022.PubMedGoogle Scholar
- Wang Y, Lyu YL, Wang JC: Dual localization of human DNA topoisomerase IIIalpha to mitochondria and nucleus. Proc Natl Acad Sci USA. 2002, 99: 12114-12119. 10.1073/pnas.192449499PubMed CentralView ArticlePubMedGoogle Scholar
- Kutay U, Guttinger S: Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol. 2005, 15: 121-124. 10.1016/j.tcb.2005.01.005View ArticlePubMedGoogle Scholar
- la Cour T, Gupta R, Rapacki K, Skriver K, Poulsen FM, Brunak S: NESbase version 1.0: a database of nuclear export signals. Nucleic Acids Res. 2003, 31: 393-396. 10.1093/nar/gkg101PubMed CentralView ArticlePubMedGoogle Scholar
- Cordero JB, Cozzolino M, Lu Y, Vidal M, Slatopolsky E, Stahl PD, Barbieri MA, Dusso A: 1, 25-Dihydroxyvitamin D down-regulates cell membrane growth- and nuclear growth-promoting signals by the epidermal growth factor receptor. J Biol Chem. 2002, 277: 38965-38971. 10.1074/jbc.M203736200View ArticlePubMedGoogle Scholar
- Wang SC, Hung MC: Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors. Clin Cancer Res. 2009, 15: 6484-6489. 10.1158/1078-0432.CCR-08-2813View ArticlePubMedGoogle Scholar
- Psyrri A, Yu Z, Weinberger PM, Sasaki C, Haffty B, Camp R, Rimm D, Burtness BA: Quantitative determination of nuclear and cytoplasmic epidermal growth factor receptor expression in oropharyngeal squamous cell cancer by using automated quantitative analysis. Clin Cancer Res. 2005, 11: 5856-5862. 10.1158/1078-0432.CCR-05-0420View ArticlePubMedGoogle Scholar
- Wang SC, Nakajima Y, Yu YL, Xia W, Chen CT, Yang CC, McIntush EW, Li LY, Hawke DH, Kobayashi R, Hung MC: Tyrosine phosphorylation controls PCNA function through protein stability. Nat Cell Biol. 2006, 8: 1359-1368. 10.1038/ncb1501View ArticlePubMedGoogle Scholar
- Xia W, Wei Y, Du Y, Liu J, Chang B, Yu YL, Huo LF, Miller S, Hung MC: Nuclear expression of epidermal growth factor receptor is a novel prognostic value in patients with ovarian cancer. Mol Carcinog. 2009, 48: 610-617. 10.1002/mc.20504PubMed CentralView ArticlePubMedGoogle Scholar
- Yue X, Song W, Zhang W, Chen L, Xi Z, Xin Z, Jiang X: Mitochondrially localized EGFR is subjected to autophagic regulation and implicated in cell survival. Autophagy. 2008, 4: 641-649.View ArticlePubMedGoogle Scholar
- De Potter CR, Quatacker J, Maertens G, Van Daele S, Pauwels C, Verhofstede C, Eechaute W, Roels H: The subcellular localization of the neu protein in human normal and neoplastic cells. Int J Cancer. 1989, 44: 969-974. 10.1002/ijc.2910440604View ArticlePubMedGoogle Scholar
- Vidal GA, Naresh A, Marrero L, Jones FE: Presenilin-dependent gamma-secretase processing regulates multiple ERBB4/HER4 activities. J Biol Chem. 2005, 280: 19777-19783. 10.1074/jbc.M412457200View ArticlePubMedGoogle Scholar
- Naresh A, Long W, Vidal GA, Wimley WC, Marrero L, Sartor CI, Tovey S, Cooke TG, Bartlett JMS, Jones FE: The ERBB4/HER4 Intracellular Domain 4ICD Is a BH3-Only Protein Promoting Apoptosis of Breast Cancer Cells. Cancer Res. 2006, 66: 6412-6420. 10.1158/0008-5472.CAN-05-2368View ArticlePubMedGoogle Scholar
- Liao HJ, Carpenter G: Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression. Mol Biol Cell. 2007, 18: 1064-1072. 10.1091/mbc.E06-09-0802PubMed CentralView ArticlePubMedGoogle Scholar
- Lo HW, Ali-Seyed M, Wu Y, Bartholomeusz G, Hsu SC, Hung MC: Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1. J Cell Biochem. 2006, 98: 1570-1583. 10.1002/jcb.20876View ArticlePubMedGoogle Scholar
- Marchenko ND, Zaika A, Moll UM: Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem. 2000, 275: 16202-16212. 10.1074/jbc.275.21.16202View ArticlePubMedGoogle Scholar
- Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, Moll UM: p53 has a direct apoptogenic role at the mitochondria. Mol Cell. 2003, 11: 577-590. 10.1016/S1097-2765(03)00050-9View ArticlePubMedGoogle Scholar
- Sansome C, Zaika A, Marchenko ND, Moll UM: Hypoxia death stimulus induces translocation of p53 protein to mitochondria. Detection by immunofluorescence on whole cells. FEBS Lett. 2001, 488: 110-115. 10.1016/S0014-5793(00)02368-1View ArticlePubMedGoogle Scholar
- Ren J, Agata N, Chen D, Li Y, Yu WH, Huang L, Raina D, Chen W, Kharbanda S, Kufe D: Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents. Cancer Cell. 2004, 5: 163-175. 10.1016/S1535-6108(04)00020-0PubMed CentralView ArticlePubMedGoogle Scholar
- Ren J, Bharti A, Raina D, Chen W, Ahmad R, Kufe D: MUC1 oncoprotein is targeted to mitochondria by heregulin-induced activation of c-Src and the molecular chaperone HSP90. Oncogene. 2006, 25: 20-31.PubMedGoogle Scholar
- Zhao Y, Chaiswing L, Velez JM, Batinic-Haberle I, Colburn NH, Oberley TD, St Clair DK: p53 translocation to mitochondria precedes its nuclear translocation and targets mitochondrial oxidative defense protein-manganese superoxide dismutase. Cancer Res. 2005, 65: 3745-3750. 10.1158/0008-5472.CAN-04-3835View ArticlePubMedGoogle Scholar
- Lo H-W, Hsu S-C, Xia W, Cao X, Shih J-Y, Wei Y, Abbruzzese JL, Hortobagyi GN, Hung M-C: Epidermal Growth Factor Receptor Cooperates with Signal Transducer and Activator of Transcription 3 to Induce Epithelial-Mesenchymal Transition in Cancer Cells via Up-regulation of TWIST Gene Expression. Cancer Res. 2007, 67: 9066-9076. 10.1158/0008-5472.CAN-07-0575PubMed CentralView ArticlePubMedGoogle Scholar
- Lo HW, Zhu H, Cao X, Aldrich A, Ali-Osman F: A novel splice variant of GLI1 that promotes glioblastoma cell migration and invasion. Cancer Res. 2009, 69: 6790-6798. 10.1158/0008-5472.CAN-09-0886PubMed CentralView ArticlePubMedGoogle Scholar
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