The present invention relates to the inhibition of stress-induced receptor tyrosine kinase activity by inhibiting a ligand of said receptor tyrosine kinase, particularly an extracellular ligand.
Exposure of mammalian cells to environmental stress such as hyperosmolarity and oxidative agents, ionizing radiation or UV-light activates a variety of signal transduction cascades. While reactive oxygen species (ROS) have been implicated as second messengers, oxidative stress due to their uncontrolled production and exposure to oxidants has been related to cellular damage and pathophysiological disorders such as cancer (Finkel, 1998; Kamata and Hirata, 1999). Moreover, mammalian cells have to adapt to changes in the extracellular environment including increasing osmolarity, which results in cell shrinkage and increased synthesis of small molecules to equalize the intra- and extracellular conditions (de Nadal et al., 2002).
Osmotic or oxidative stress activate a variety of receptor tyrosine kinases, the most prominent being the epidermal growth factor receptor (EGFR) (King et al., 1989; Knebel et al., 1996; Rao, 1996; Rosette and Karin, 1996). The EGFR controls a plethora of important biological responses including cell proliferation, differentiation, migration or antiapoptotic signals, and has therefore frequently been implicated in diverse human disorders (Prenzel et al., 2001). Ligand-dependent and -independent receptor activation mechanisms have been described for the EGFR. Ligand-mediated receptor activation occurs by binding of an EGF-like ligand, such as EGF, HB-EGF, amphiregulin or TGF-α to receptor ectodomains leading to dimerization of two ligand-receptor heterodimers, subsequent activation of the intrinsic kinase activity and autophosphorylation (Schlessinger, 2002). Ligand-Independent receptor activation has been proposed to occur via inactivation of phosphatases involving oxidation of a critical cysteine residue within their catalytic pocket (Knebel et al., 1996). Hence, the equilibrium of receptor phosphorylation is shifted from the non-phosphorylated to the phosphorylated state. Another mechanism for ligand-independent receptor activation has been suggested to involve non-specific clustering and internalization of the EGFR (Rosette and Karin, 1996). Furthermore, cytoplasmic non-receptor tyrosine kinases such as c-Src have been shown to phosphorylate the EGFR (Biscardi et al., 1999; Tice et al., 1999).
Tyrosine phosphorylation of the EGFR can also be induced by G protein-coupled receptor (GPCR) stimulation, a process that has been termed EGFR transactivation (Daub et al., 1996). The mechanism has originally been attributed to an exclusively ligand-independent process. In many cell systems however, EGFR transactivation occurs via metalloprotease-mediated shedding of transmembrane EGF-like ligands that have to be processed to become active (Prenzel et al., 1999). Very recently members of the ADAM family of metalloproteases were identified as the sheddases required for GPCR-induced proHB-EGF and pro-AR processing (Gschwind et al., 2003; Yan et al., 2002). Above all, ADAM17, also named TNF-α converting enzyme (TACE), but also ADAM10 and ADAM12 have been shown to be involved (Asakura et al., 2002; Gschwind et al., 2003; Yan et al., 2002). Aberrant signalling processes involving ligand-dependent EGFR transactivation have been related to different human disorders, such as head and neck squamous cell carcinoma, cardiac and gastrointestinal hypertrophy and cystic fibrosis (Asakura et al., 2002; Gschwind et al., 2002; Keates et al., 2001; Lemjabbar and Basbaum, 2002). Apart from EGFR transactivation ADAM9 has been shown to process proHB-EGF in response to TPA stimulation (Izumi et al., 1998), while also cleavage of proTGF-α, proHB-EGF and proamphiregulin by ADAM17 has been reported (Merlos-Suarez et al., 2001; Peschon et al., 1998; Sunnarborg et al., 2002) and implicated in tumourigenesis in the case of TGF-α (Borrell-Pages et al., 2003).
Besides RTK phosphorylatlon, environmental stress leads to the activation of mitogen-activated protein kinases (MAPKs) which are part of major intracellular signal transduction cascades coupling extracellular stimuli to the nucleus by activating transcription factors. Thus, MAPK pathways control important processes such as proliferation, migration, differentiation and stress responses (Chen et al., 2001b; Johnson and Lapadat, 2002). However, apart from activating transcription factors the MAPKs extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 have been implicated in controlling processing of transmembrane proteins (Fan and Derynck, 1999) by phosphorylating the intracellular domain of ADAM17 (Diaz-Rodriguez et al., 2002; Fan et al., 2003). Intensive research has focused on mechanisms of MAPK activation by osmotic and oxidative stress (Kyriakis and Avruch, 2001) as the cell's fate is determined by cross-talk between these signalling pathways. Previous reports proposed the inactivation or downregulation of phosphatases in MAPK activation due to stress agents as well as the modification of scaffolding protein function, leading to association or dissociation of signalling complexes (Benhar et al., 2002; de Nadal et al., 2002). Moreover, small G-proteins have been demonstrated to play a role in the activation of MAPK by osmotic stress (de Nadal et al., 2002). The activation of MAPK by stress stimuli has severe consequences for the development and progression of human cancer as increasing evidence implicates particularly ROS, the stress-activated kinases p38 and JNK and stress signalling in the susceptibility of cancer cells to apoptosis and in proliferative responses. Thereby, stress signalling via the EGFR can affect cancer therapy, as recent studies indicate that anti-cancer drugs activate stress signalling cascades (reviewed in Benhar et al., 2002).
The mechanisms of RTK and MAPK activation in response to oxidative and osmotic stress have been intensively studied, but so far, these regulatory pathways were generally described as ligand-independent processes in human carcinoma cells. Recent advances in understanding the regulation of EGFR activation by ADAM metalloproteases prompted us to investigate the mechanisms of stress-induced EGFR and MAPK stimulation with respect to a potential involvement of EGF-like ligand processing.
Fischer et al. (Poster PS01-0916, Eur. J. Biochem.) describe that p38 and metalloproteases of the ADAM family control stress-induced ligand-dependent EGFR activation in downstream signalling in human carcinoma cells. Specific metalloproteases are, however, not identified.
Takenobu et al. (J. Biol. Chem. 278, (2003), 1725-1762) disclose stress- and inflammatory cytokine-induced ectodomain shedding of HB-EGF-like growth factors mediated by p38 MAPK. It was found that the metalloprotease ADAM9 is not required for stress-induced pro HB-EGF shedding in VeroH cells.
Herrlich et al. (FASEB J. 16 (2002), A56) and Tschumperlin et al. (FASEB J. 16 (2002), A1150) disclose stimulation of HER2/HER3 or EGFR activation respectively by heregulin shedding mediated by osmotic or mechanical stress.
In the present application it is now demonstrated that EGFR activation in response to osmotic or oxidative stress involves metalloprotease-mediated cleavage of proHB-EGF. The responsible metalloproteases comprise members of the ADAM family processing proHB-EGF, particularly ADAM9, ADAM10 and ADAM17. Furthermore, stress-induced ligand-dependent EGFR activation can be linked to the MAPKs ERK1/2 and JNK. We provide evidence that stress-activated EGFR phosphorylation depends on p38 activity, implicating p38 as an upstream activator of ADAM metalloproteases in the stress response of human carcinoma cells.
Further a combination treatment of tumour cells with a chemotherapeutic agent, e.g. with doxorubicin, which induces p38 activation, and blockade of HB-EGF effect strongly enhanced cell death when compared to doxorubicin treatment alone. This result suggests a role of this signalling mechanism for tumour cells to escape chemotherapy-induced cell death.
Thus, a method is provided which allows modulating of stress-induced activation of a receptor tyrosine kinase or an RTK-mediated signalling pathway in a cell, preferably in a mammalian cell, more preferably in a human cell, e.g. in a tumour cell, in particular in a hyperproliferative or apoptosis-resistant cell comprising inhibiting the activity of a ligand of said receptor tyrosine kinase.
A first aspect of the invention relates to the use of an inhibitor of a receptor tyrosine kinase ligand for the manufacture of a medicament for the prevention or treatment of an at least partially therapy-resistant hyperproliferative disorder.
A further aspect of the present invention relates to the use of an inhibitor of a is receptor tyrosine kinase ligand for increasing the efficacy of therapies against hyperproliferative disorders.
A further aspect of the present invention relates to the use of an inhibitor of a receptor tyrosine kinase ligand for the manufacture of a medicament for increasing the sensitivity of hyperproliferative disorders against irradiation and/or medicament treatment.
Still a further aspect of the present invention relates to the use of an inhibitor of a receptor tyrosine kinase ligand for the manufacture of a medicament for the prevention or treatment of a disorder which is caused by or associated with stress-induced activation of a receptor tyrosine kinase.
Still a further aspect of the present invention is a pharmaceutical composition or kit comprising as active ingredients
The term “receptor tyrosine kinase” is well understood in the art and preferably relates to membrane-bound molecules having tyrosine kinase activity which are comprised of an extracellular domain, a transmembrane domain and an intracellular domain. Examples of suitable receptor tyrosine kinases are EGFR and other members of the EGFR family such as HER2, HER3 or HER4 as well as other receptor tyrosine kinases such as PDGFR, the vascular endothelial growth factor receptor KDR/FLK-1, the TRK receptor, FGFR-1 or IGF-1 receptor but also other types of growth factor receptors such as TNF receptor 1, TNF receptor 2, CD30 and IL6 receptor. Preferably, the receptor tyrosine kinase is EGFR.
The present invention comprises the inhibition of the activity of a receptor tyrosine kinase ligand. The inhibition is preferably a “specific” inhibition, wherein the activity of a specific receptor tyrosine kinase ligand is selectively inhibited, i.e. the activity of other receptor tyrosine kinase ligands is not significantly inhibited. By means of selective inhibition of specific receptor tyrosine kinase ligands a highly specific disruption of receptor tyrosine kinase activity may be achieved which is important for pharmaceutical applications in that the occurrence of undesired side effects may be reduced. It should be noted, however, that the method of the present invention also comprises a non-specific inhibition of receptor tyrosine kinase ligands.
Further, the invention preferably relates to an inhibition, wherein an inhibitor acts directly on the receptor tyrosine kinase ligand itself or on a metalloprotease capable of cleaving the receptor tyrosine kinase ligand, e.g. by binding. The invention, however, also encompasses an inhibition wherein the inhibitor does not directly act on the metalloprotease and/or the receptor tyrosine kinase ligand but to a precursor or metabolite thereof, particularly a precursor of the receptor tyrosine kinase ligand. Furthermore, the invention also relates to an inhibition of p38 for the modulation of stress-induced receptor tyrosine kinase activity.
The receptor tyrosine kinase ligand is preferably a molecule binding to the extracellular receptor tyrosine kinase domain. Examples of suitable receptor tyrosine kinase ligands are HB-EGF, EGF, amphiregulin, betacellulin, epiregulin, TGF-α, neuregulin or heregulin. More preferably, the receptor tyrosine kinase ligand is HB-EGF.
The inhibition of the activity of a receptor tyrosine kinase ligand preferably relates to an inhibition of the cleavage of a precursor, particularly a membrane-bound precursor of the ligand by a metalloprotease and/or the activation of a receptor tyrosine kinase, e.g. EGFR by the ligand. A metalloprotease inhibitor of the present invention is preferably capable of inhibiting the cleavage of the ligand precursor and its release. Examples of suitable metalloproteases are ADAM9, 10 and 17, particularly ADAM17 which are critical mediators of the cellular stress response. Alternatively, the inhibitor of the present invention may be capable of inhibiting the biological activity of the receptor tyrosine kinase ligand, particularly EGFR tyrosine phosphorylation, downstream mitogenic signalling events, e.g. activation of mitogen-activated protein kinases (MAPKs) such as ERK1/2 and/or JNK, cell proliferation and/or migration.
Inhibitors of a receptor tyrosine kinase ligand may be used for the prevention or treatment of disorders, particularly hyperproliferative disorders. Preferably the disorder is caused by or associated with stress-induced activation of a receptor tyrosine kinase. The stress is preferably an oxidative and/or osmotic stress. More preferably, the stress is a p38-mediated stress. The presence of such a type of disorder may be determined by measuring p38 expression, e.g. on mRNA level (cDNA array analysis, SAGE, Northern Blot, etc) and/or on the protein level (Western Blot analysis, immunofluoresence microscopy, in situ hybridization techniques, etc). The presence of such type of disorder may also be determined by examining the occurrence of activating mutations In genomic and/or mRNA molecules encoding p38. Further, elevated levels of p38 agonists in serum and/or disease-affected tissues may be determined. It should be pointed out that this type of disorder need not be associated with enhanced receptor tyrosine kinase expression.
For example, the disorder may be a hyperproliferative disorder such as a cancer, e.g. breast, stomach, prostate, bladder, ovarial, lung, liver, kidney or pancreas cancer, glioma, melanoma, leukemia, etc or another disorder such as a hyperproliferative skin disease, e.g. psoriasis or inflammatory diseases.
The activity of receptor tyrosine kinase ligands (preferably either directly or via metalloprotease inhibition) may be inhibited on the nucleic acid level, e.g. on the gene level or on the transcription level.
Inhibition on the gene level may comprise a partial or complete gene inactivation, i.e. by gene disruption. On the other hand, inhibition may occur on the transcript level, e.g. by application of anti-sense molecules, e.g. DNA molecules, RNA molecules or nucleic acid analogues, ribozymes, e.g. RNA molecules or nucleic acid analogues or small RNA molecules capable of RNA interference (RNAi), e.g. RNA molecules or nucleic acid analogues, directed against metalloprotease and/or ligand mRNA.
Further, the activity may be inhibited on the protein level, e.g. by application of compounds which result in a specific inhibition of a metalloprotease and/or ligand activity. The inhibition on the protein level may comprise, for example, the application of antibodies or antibody fragments directed against a metalloprotease such as ADAM9, 10 or 17 or a ligand or ligand precursor such as HB-EGF or PreHB-EGF. The antibodies may be polyclonal antibodies or monoclonal antibodies, recombinant antibodies, e.g. single chain antibodies or fragments of such antibodies which contain at least one antigen-binding site. e.g. proteolytic antibody fragments such as Fab, Fab′ or F(ab′)2 fragments or recombinant antibody fragments such as scFv fragments. For therapeutic purposes, particularly for the treatment of humans, the application of chimeric antibodies, humanized antibodies or human antibodies is especially preferred.
Furthermore, proteinaceous or low-molecular weight inhibitors of metalloproteases and/or ligands may be used. Examples of such inhibitors are CRM197, batimastat, marimastat, heparin or blocking antibodies against the ligands. Further inhibitors may be identified by screening procedures as outlined in detail below.
For therapeutic purposes, the medicament is administered in the form of a pharmaceutical composition which additionally comprises pharmaceutically acceptable carriers, diluents and/or adjuvants.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e. the concentration of the test compound which achieves a half-maximal inhibition of the growth-factor receptor activity). Such information can be used to more accurately determine useful doses in humans. The dose ration between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g. Fingi et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1, p. 1). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the receptor modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data, e.g. the concentration necessary to achieve a 50-90% inhibition of the receptor using the assays described herein. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. Dosages necessary to achieve the MEC will depend on individual characteristics and route administration. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
The actual amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgement of the prescribing physician. For antibodies or therapeutically active nucleic acid molecules, and other compounds e.g. a daily dosage of 0.001 to 100 mg/kg, particuarly 0.01 to 10 mg/kg per day is suitable.
Suitable routes of administration may, for example, include oral, rectal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections.
Alternatively, one may administer the compound in a local rather than a systematic manner, for example, via injection of the compound directly into a solid tumour, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example in a liposome coated with a tumour-specific antibody, The liposomes will be targeted to and taken up selectively by the tumour.
As outlined above, the present invention is particuarly suitable for the treatment or prevention of therapy-resistant hyperproliferative disorders, preferably therapy-resistant types of cancer, e.g. irradiation and/or medicament resistant types of cancer.
Treatment of cancer with irradiation and/or cytostatic and/or cytotoxic agents has been shown to activate stress kinase p38. Surprisingly, it was found that inhibition of receptor tyrosine kinase ligands such as HB-EGF strongly enhances the therapeutic activity of irradiation and/or chemotherapeutics, particuarly the apoptosis-inducing activity thereof. Thus, co-application of a direct receptor tyrosine kinase ligand inhibitor or inhibitors which prevent ligand precursor shedding with a further therapeutic procedure and/or medicament results in a substantive increase in sensitivity of the disorder against application of said further procedure and/or medicament and thus enhancement of the efficacy of said further therapeutic procedure and/or medicament. The administration if ligand inhibitors as described above is particularly suitable for the treatment or prevention of disorders which are at least partially resistant against irradiation therapy and/or administration of cytostatic and/or cytotoxic medicaments, particularly which are at least partially resistant against apoptosis-inducing procedures and medicaments.
In a preferred embodiment of the present invention the receptor tyrosine kinase ligand inhibitor is co-applied with an irradiation therapy, particuarly a gamma irradiation therapy. In a further preferrred embodiment the receptor tyrosine kinase ligand inhibitor is co-applied with a further anti-cancer medicament, particuarly an apoptosis-inducing medicament. Preferred examples of suitable anti-cancer medicaments are doxorubicin, taxanes, cis/trans-platin or derivatives thereof, 5-fluorouracil, mitomycin D, paclitaxel, etoposide, cyclophosphoamide, docetaxel or other apoptosis-inducing drugs or proteins, such as antibodies.
Co-application of the ligand inhibitor and the further procedure and/or medicament may be carried out simultaneously and/or sequentially. Application of the ligand inhibitor leads to an increased sensitivity of the disorder to be treated against application of other therapies. Particularly, tumour resistance against irradiation and/or chemotherapeutics is reduced.
Thus, a further aspect of the present invention is a pharmaceutical composition or kit comprising as active ingredients
Preferably, the composition or kit additionally comprises pharmaceutically acceptable carriers, diluents and/or adjuvants.
Furthermore, a method is provided which allows for identifying modulators of stress-induced receptor tyrosine kinase activity e.g. p38-induced activity, comprising determining if a test compound is capable of inhibiting the activity of a ligand of said receptor tyrosine kinase. This method is suitable as a screening procedure, e.g. a high-through put screening procedure for identifying novel compounds or classes of compounds which are capable of modulating stress induced signal transduction. Further, the method is suitable as a validation procedure for characterizing the pharmaceutical efficacy and/or the side effects of compounds. The method may comprise the use of isolated proteins, cell extracts, recombinant cells or transgenic non-human animals. The recombinant cells or transgenic non-human animals preferably exhibit an altered metalloprotease and/or ligand expression compared to a corresponding wild-type cell or animal.
Furthermore, the invention shall be explained by the following figures and examples.
1. Materials and Methods
1.1 Cell Culture, Plasmids and Transfections
All cell lines (American Type Culture Collection, Manassas, Va.) were routinely grown according to the supplier's instructions. Transfections of Cos-7 cells were carried out using Lipofectamine (Invitrogen) according to the manufacturer's protocol. Briefly, for transient transfections in 6 cm dishes cells were incubated for 4 h in 2 mL of serum-free medium containing 20 μL Lipofectamine and 2 μg of total plasmid DNA per dish. The transfection mixture was then supplemented with an equal volume of 20% fetal bovine serum and, 20 h later, cells were washed and cultured in serum-free medium for another 24 h prior to stimulation. The inhibitors AG1478 (Alexis Biochemicals), Batimastat (BB94, British Biotech, Oxford, UK), Crm197 (Quadratech Ltd., UK), SB202190 (Calbiochem) and PD98059 (Alexis Biochemicals) were added to serum-starved cells before the respective stimulation.
Dominant negative protease constructs of ADAM10, 12, 15 and 17 lacking the pro- and metalloprotease domains were previously described (Gschwind et al., 2003). The plasmids pcDNA3-HA-ERK2 (Daub et al., 1997) and pcDNA3-proHB-EGF-VSV (Prenzel et al., 1999) were used in this study.
1.2 Protein Analysis
Cells were lysed and proteins immunoprecipitated as described before (Daub et al., 1997). Prior to lysis, cells grown to 80% confluence were treated with inhibitors and agonists as indicated in the figure legends and then lysed for 10 min on ice in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/mL aprotinin. Lysates were precleared by centrifugation at 13,000 rpm for 10 min at 4° C. Precleared lysates were immunoprecipitated using the respective antibodies and 20 μL of protein A-Sepharose for 4 h at 4° C. Precipitates were washed three times with 0.5 mL of HNTG buffer, suspended in 2×SDS sample buffer, boiled for 3 min, and subjected to gel electrophoresis. Following SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose membrane. Western blots were performed according to standard methods. The antibodies against human EGFR (108.1) and SHC have been characterized before (Prenzel et al., 1999). Phosphotyrosine was detected with the 4G10 monoclonal antibody (UBI, Lake Placid, N.Y.). Polyclonal anti-phospho-p44/p42 (Thr202/Tyr204) MAPK antibody, and anti-phospho-JNK (Thr183/Tyr185) and anti-phospho-p38 (Thr180/Tyr182) antibody were purchased from New England Biolabs (Beverly, Mass.). Polyclonal anti-ERK2, anti-JNK1 and anti-p38 antibody was from Santa Cruz Biotechnology (Santa Cruz, Calif.).
1.3 JNK Activity Assay
JNK activity was assayed as described previously (Sudo and Karin, 2000). Cultured cells were lysed in lysis buffer containing 20 mM Tris (pH7.6), 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 1 mM dithiotreitol, 0.5 mM phenylmethylsulfonylfluoride, 20 mM β-gylcerophosphate, 1 mM sodium orthovanadate and 1 μg/mL leupeptin. JNK was immunoprecipitated from lysates obtained from 6-well dishes using polyclonal anti-JNK antibody. Immunoprecipitates were washed twice using lysis buffer and twice using kinase assay buffer (25 mM HEPES (pH 7.5), 20 mM β-gylcerophosphate, 20 mM PNPP, 20 mM MgCl2, 2 mM dithiotreitol and 0.1 mM sodium orthovanadate). Kinase reactions were performed in 30 μL of kinase buffer supplemented with 1 μg GST-c-Jun (aa1-79), 20 μM cold ATP and 5 μCi of [y-32P]ATP at 30° C. for 30 minutes. Reactions were stopped by addition of 30 μL of Laemmli buffer and subjected to gel electrophoresis on 12.5% gels. Labeled GST-c-Jun was quantitated using a Phosphoimager (Fuji).
1.4 Flow Cytometric Analysis
FACS analysis was performed as described before (Prenzel et al., 1999). In brief, cells were seeded, grown for 20 h and serum-starved for 24 h. Cells were treated with inhibitors and stimulated as indicated. After collection, cells were stained with an ectodomain-specific antibody against proHB-EGF for 45 min. After washing with PBS, cells were incubated with FITC-conjugated secondary antibodies for 15 min and washed again with PBS. Cells were analysed on a Becton Dickinson FACScalibur flow cytometer.
1.5 TCA Precipitation of HB-EGF
Cos-7 transiently transfected with pcDNA3-proHB-EGF-VSV were serum-starved for 24 h. Prior to stimulation cells were washed, preincubated with BB94 (10 μM) and stimulated as indicated. After stimulation the supernatant was collected, sodium-desoxycholate was added (100 μg/mL) and following incubation on ice for 10 minutes the solution was supplemented with trichloro acetic acid (TCA) to a final concentration of 10% TCA. After incubation on ice for 30 minutes samples were centrifuged, the supernatant was discarded and the precipitates were resuspended in Schägger-Jargow sample-buffer. TCA was neutralized using Tris-HCl (pH 8.8), and samples were separated using the tricine-SDS gel electrophoresis protocol (Schägger and von Jargow, 1987).
1.6 RNA Interference and RT-PCR Analysis
Transfection of 21-nucleotide siRNA duplexes (Dharmacon Research, Lafayette, Colo., USA) for targeting endogenous genes was carried out using Oligofectamine (Invitrogen) for NCI-H292 cells and 4.2 μg siRNA duplex per 6-well plate as previously described (Elbashir et al., 2001). Cos-7 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, 8.4 μg siRNA duplex per 6 cm dish were incubated with 10 μL Lipofectamine 2000 in 1 mL serum-free medium for 20 minutes. The transfection mixture was added to the cell culture medium containing serum and, after 6 h, cells were washed and incubated in medium containing serum overnight. NCI-H292 and Cos-7 cells were serum-starved and assayed 2 d after transfection. Highest efficiencies in silencing target genes were obtained by using mixtures of siRNA duplexes targeting different regions of the gene of interest. Sequences of siRNA used were
The siRNA-duplexes against ADAM12 and ADAM17 have been described earlier (Gschwind et al., 2003).
Specific silencing of targeted genes was confirmed by RT-PCR analysis. RNA isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) was reverse transcribed using AMV Reverse Transcriptase (Roche, Mannheim, Germany). PuReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, N.J.) were used for PCR amplification. Primers (Sigma Ark, Steinheim, Germany) were
PCR products were subjected to electrophoresis on a 2.5% agarose gel and DNA was visualized by ethidium bromide staining.
1.7 Apoptosis Assay
TCC-Sup bladder carcinoma cells were seeded, grown for 20 h and treated with 10 μM doxorubicin and Crm197 as indicated for 72 h. Cells were collected in assay buffer containing propidium iodide (PI), and incubated at 4° C. for 3 h. Nuclear PI staining was analysed on a Becton Dickinson FACScalibur flow cytometer.
2. Results
2.1 Distinct Kinetics of EGFR and MAPK Activation in Cos-7 and Human Carcinoma Cell Lines
Osmotic and oxidative stress lead to phosphorylation of the EGFR and MAPKs in a wide variety of cell systems (Carpenter, 1999; de Nadal et al., 2002; King et al., 1989). To investigate the underlying mechanisms we performed time course experiments in Cos-7 and TCC-Sup bladder carcinoma cell lines by immunoblot analysis. As shown in
2.2 p38 Controls EGFR Activation by Osmotic and Oxidative Stress.
The finding that p38 activation preceded EGFR tyrosine phosphorylation (
Preincubation of Cos-7 cells with the selective EGFR-kinase inhibitor AG1478 did not affect p38 phosphorylation in response to stress agents (
2.3 Stress-Induced EGFR Phosphorylation Depends on Metalloprotease Activity and HB-EGF Function.
Recent investigations underlined the importance of EGF-like ligand proteolysis in EGFR signalling, especially in signal transduction events that were previously thought to be ligand-independent such as EGFR signal transactivation by GPCR (Prenzel et al., 1999). Based on these findings we adressed the question whether EGFR activation by stress stimuli can also involve a ligand-dependent mechanism. Therefore, cells were preincubated with the metalloprotease inhibitor batimastat (BB94) that has been shown to inhibit EGF-like ligand processing and subsequent EGFR transactivation (Prenzel et al., 1999). EGFR tyrosine phosphorylation was monitored after stimulation with stress agents using immunoblot analysis. As shown in
Next, we investigated the effect of the diphtheria toxin mutant Crm197 which specifically blocks HB-EGF function on stress-activated EGFR tyrosine phosphorylation. Indeed, Crm197 inhibited EGFR phosphorylation to the same extent as BB94, suggesting that in these three cell lines HB-EGF is critically involved in stress-induced EGFR activation. As a positive control receptor activation by LPA was completely prevented by these inhibitors while direct stimulation with EGF was unaffected.
Furthermore, it was of special interest whether this ligand-dependency can be followed to downstream signalling partners of the EGFR. Shc adaptor proteins are prominent signalling adaptors of the EGFR linking the receptor to activation of the Ras/Raf/ERK-MAPK cascade. Indeed, the data shown in
2.4 Ectodomain Shedding of proHB-EGF is Induced in Response to Osmotic and Oxidative Stress in Cos-7 Cells
To further substantiate the role of HB-EGF in this ligand-dependent EGFR stimulation mechanism we investigated shedding of proHB-EGF in response to hyperosmolarity and hydrogen peroxide treatment on the level of the ligand itself. Therefore, we analyzed the amount of proHB-EGF present on the cell surface of Cos-7 cells prior to and after stimulation with sorbitol or hydrogen peroxide by flow cytometric analysis. As shown in
2.5 Metalloproteases of the ADAM Family Mediate EGFR Activation by Osmotic and Oxidative Stress.
The finding that metalloprotease-dependent mechanisms significantly contribute to stress-induced EGFR and Shc activation raised the question which metalloprotease(s) are involved. We used the RNA interference technique to inhibit the endogenous expression of the ADAM proteases ADAM9, -10, -12, -15 and ADAM17 that have already been implicated in EGF-like ligand cleavage.
2.6 Activation of the MAPKs ERK1/2 and JNK in Response to Hyperosmolarity and Oxidative Stress is Mediated by HB-EGF-Dependent EGFR Activation.
Since the MAPKS ERK1/2 and JNK are activated by hypertonicity and reactive oxygen species (de Nadal et al., 2002; Kyriakis and Avruch, 2001), we asked whether the ligand-dependent EGFR phosphorylation contributes to the induction of MAPK activation by stress stimuli. Therefore, we used the selective EGFR tyrphostin AG1478 to investigate the overall dependence of stress-induced MAPK activation on the EGFR kinase activity. Furthermore, we compared this effect with inhibition of the ligand-dependent EGFR activation process by BB94 and Crm197. As shown in
Apart from ERK1/2, we were interested in the signalling mechanisms leading to activation of JNK.
Treatment of tumour cells with chemotherapeutics has been shown to activate the stress kinases p38 and JNK leading to cell death. However, as p38 activation leads to HB-EGF-dependent EGFR- and Erk1/2 activation, we investigated the effect of blocking HB-EGF function on the doxorubicin-induced apoptosis of TCC-Sup bladder carcinoma cells.
Together, these data demonstrate that ligand-dependent EGFR tyrosine phosphorylation by stress agents plays a critical role in the activation of the MAPKs ERK1/2 and JNK in response to osmotic and oxidative stress in human cancer cells. Moreover, these results implicate the herein presented signalling mechanism as a pathway employed by tumour cells to evade chemotherapy-induced cell death.
3. Discussion
Intensive investigations addressed the question of how mammalian cells deal with stress agents. Our present study provides new insights in growth factor-dependent mechanisms leading to EGFR and subsequent MAPK activation in response to osmotic and oxidative stress in human carcinoma cells.
We have demonstrated here that EGFR phosphorylation induced by both osmotic and oxidative stress requires a metalloprotease activity and release of HB-EGF in Cos-7 cells and human carcinoma cell lines. This result was obtained both on the level of the EGFR (
Increasing evidence has implicated the ADAM family of zinc-dependent proteases as crucial mediators of EGF-like ligand processing. In accordance with these reports our results demonstrate that ADAM proteases are responsible for shedding of proHB-EGF induced by stress stimuli. Interestingly, while in the context of EGFR transactivation single distinct ADAM proteases are activated (Gschwind et al., 2003; Yan et al., 2002), we found that depending on the cellular system two or more ADAM proteases become active (
How are the metalloproteases of the ADAM family activated, finally leading to EGFR phosphorylation and downstream signalling responses? Previous reports demonstrated regulation of metalloprotease-mediated ectodomain cleavage of transmembrane proteins in response to growth factors and TPA by the MAPK ERK1/2, while the basal level of ectodomain shedding has been attributed to p38 activity (Fan and Derynck, 1999; Gechtman et al., 1999; Rizoli et al., 1999). Moreover, p38 has been implicated as an upstream mediator of the EGFR in the sorbitol-induced EGFR activation in human non-transformed keratinocytes (Cheng et al., 2002). In contrast to our results, the authors excluded a ligand-dependent mechanism based on medium transfer experiments. As the released EGF-like ligand may be retained in the extracellular matrix binding to heparan sulfate glycans involvement of ligand-dependent EGFR activation cannot be ruled out. These reports and the finding that p38 activity in our systems is independent of the EGFR phosphorylation state (
This crucial role of p38 in the stress response is well conserved throughout evolution. In Saccharomyces cerevisae the response to hyperosmolarity has been intensively studied. A central role plays the kinase HOG, the yeast homolog of human p38. HOG is activated by sensor molecules for changing osmolarity that have no human counterparts. Activation of HOG by these proteins leads to adaptive responses in yeast to deal with hyperosmolarity (de Nadal et al., 2002).
Activation of ERK1/2 and JNK in response to oxidative and osmotic stress represents an important step in the cellular stress response (reviewed in Kyriakis and Avruch, 2001). While the phosphorylation of different receptor tyrosine kinases as potential mediators of MAPK signalling is stimulated by stress agents numerous reports reveal an outstanding role for the EGFR in MAPK activation by stress stimuli (reviewed in Carpenter, 1999). Recent investigations provide evidence for the severe consequences of stress signalling via MAPKs in anticancer therapy, as cancer cells frequently produce high levels of ROS (Burdon, 1995; Szatrowski and Nathan, 1991). Moreover, anticancer drugs or radiation therapy lead to activation of stress signalling cascades (Benhar et al., 2002), which has been attributed to the production of ROS caused by these agents. Furthermore, EGFR-dependent MAPK signalling has been reported to affect the expression levels of apoptosis regulators such as the Bcl-2 family (Jost et al., 2001). For these reasons elucidating stress signalling mechanisms and their complex interplay has gained increased attention due to the therapeutic implications.
Here, we show that the stress-induced ligand-dependent EGFR activation leads to downstream signalling events which depend in the case of ERK1/2 critically and in the case of JNK to a large degree on EGFR activation (
To our knowledge this is the first report demonstrating the ligand-dependent activation of the EGFR and subsequent downstream signalling by osmotic and oxidative stress agents regulated by the MAPK p38 within human carcinoma cells. Increasing evidence implicates particularly oxidative stress caused by the excessive production of ROS in a variety of human disorders as diverse as cardiovascular, neurodegenerative or hyperproliferative diseases such as cancer. Therefore, our results are of special importance for pathophysiological disorders and the respective therapeutic approaches involving cellular damage caused by stress agents.
The data presented here extend previous results on the signalling mechanisms of stress stimuli in mammalian cells, particularly in human carcinomas. Our findings substantiate the importance of ADAM family proteases and HB-EGF as critical mediators of the stress response in human cancer cells. Furthermore, our data suggest that cross-communication between different groups of MAPK employs ADAM proteases and the EGFR as signalling intermediates. Within this context the balance between ERK1/2 and JNK activity is of special significance for the cell's fate. Future investigations will further have to focus on other EGFR downstream signalling events and possible pathobiological responses such as enhanced proliferation or migration of cancer cells in response to stress agents.
Number | Date | Country | Kind |
---|---|---|---|
03015209.4 | Jul 2003 | EP | regional |
Filing Document | Filing Date | Country | Kind | 371c Date |
---|---|---|---|---|
PCT/EP04/07329 | 7/5/2004 | WO | 1/4/2006 |