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[Cancer Research 66, 8123-8130, August 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Discoidin Domain Receptor 1 Receptor Tyrosine Kinase Induces Cyclooxygenase-2 and Promotes Chemoresistance through Nuclear Factor-{kappa}B Pathway Activation

Sanjeev Das1, Pat P. Ongusaha1, Yoon Sun Yang2, Jin-Mo Park1, Stuart A. Aaronson3 and Sam W. Lee1

1 Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts; 2 Curonix, Inc., Seoul, South Korea; and 3 Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York

Requests for reprints: Sam W. Lee, Cutaneous Biology Research Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129. Phone: 617-726-6691; Fax: 617-643-2334; E-mail: sam.lee{at}cbrc2.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by various types of collagens and is known to play a role in cell attachment, migration, survival, and proliferation. However, little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. We report here that DDR1 induces cyclooxygenase-2 (Cox-2) expression resulting in enhanced chemoresistance. Depletion of DDR1-mediated Cox-2 induction using short hairpin RNA (shRNA) results in increased chemosensitivity. We also show that DDR1 activates the nuclear factor-{kappa}B (NF-{kappa}B) pathway and blocking this activation by an I{kappa}B superrepressor mutant results in the ablation of DDR1-induced Cox-2, leading to enhanced chemosensitivity, indicating that DDR1-mediated Cox-2 induction is NF-{kappa}B dependent. We identify the upstream activating kinases of the NF-{kappa}B pathway, IKKß and IKK{gamma}, as essential for DDR1-mediated NF-{kappa}B activation, whereas IKK{alpha} seems to be dispensable. Finally, shRNA-mediated inhibition of DDR1 expression significantly enhanced chemosensitivity to genotoxic drugs in breast cancer cells. Thus, DDR1 signaling provides a novel target for therapeutic intervention with the prosurvival/antiapoptotic machinery of tumor cells. (Cancer Res 2006; 66(16): 8123-30)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Discoidin domain receptor 1 (DDR1) belongs to a small family of receptor tyrosine kinases that contain an extracellular domain of 160 amino acids exhibiting strong homology to the Dictyostelium discoideum protein discoidin 1 (1). DDR1 was isolated as a novel member of the receptor tyrosine kinase (RTK) family (2) and was also identified as a gene overexpressed in breast cancer cells (3). Most RTKs are activated by soluble proteins present in the blood or other body fluids. The DDR1 RTK is unusual in that it is activated by the extracellular matrix protein collagen (4, 5), but independent of the major cellular collagen receptor integrins (6). Various types of collagen have been identified as ligands capable of activating DDR1 (4, 5). The activation process is unique in that it is slow but is sustained over long periods with no down-regulation by endocytosis or receptor degradation (4, 5). DDR1 is widely expressed in human and mouse epithelial cells (7). It has been found to be involved in cell interactions with the extracellular matrix and in controlling adhesion and cell motility (8). DDR1 signaling is essential for cerebellar granule differentiation (9), the development of adaptive immune responses (10), arterial wound repair (11), and mammary gland development (12). A number of studies have reported overexpression of DDR1 in lung, esophageal, breast, ovary, and pediatric brain cancers, suggesting a role for DDR1 in tumor progression (2, 7, 13–17). Moreover, the elevated expression of DDR1 in a number of fast-growing invasive tumors has suggested that this matrix-activated RTK may be involved in the proliferation and stromal invasion of tumors.

Accumulating evidence indicates that cyclooxygenase-2 (Cox-2) plays an important role in the promotion of various proliferative diseases, including cancer. Cox-2 catalyzes the synthesis of prostaglandins (18), and Cox-2 up-regulation as well as increased levels of prostanoids have been reported in a high percentage of tumors (19). Prostanoids affect multiple mechanisms that have been implicated in carcinogenesis (19–21). For example, prostaglandin E2 can stimulate cell proliferation and angiogenesis while inhibiting apoptosis and immune surveillance (21). Furthermore, overexpression of Cox-2 is sufficient to induce tumorigenesis in transgenic mice (22). Epidemiologic studies have also shown that the use of nonsteroidal anti-inflammatory drugs, prototypic inhibitors of cyclooxygenases, is associated with a reduced risk of several malignancies (23).

There is as yet relatively little information concerning DDR1 downstream signaling pathways or functions. It was shown that the DDR1 is a direct p53 transcriptional target and that it can function as a survival effector in wild-type (wt) p53-containing cells exposed to genotoxic stress (24), suggesting that inhibition of DDR1 function may provide a novel approach to selectively enhance therapy of such tumors. We undertook our present studies in an effort to identify downstream signaling targets of DDR1, in particular those that may contribute to the DDR1-induced prosurvival pathways. Our results identify a signaling pathway mediated by DDR1 involving activation of nuclear factor-{kappa}B (NF-{kappa}B) and its downstream effectors, including Cox-2 and XIAP. These findings establish the molecular mechanisms underlying DDR1 protection against genotoxic stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. Human breast cancer cell lines MDAMB435 and T47D were cultured in DMEM containing fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C. All mouse embryonic fibroblasts (MEF), including Cox-2–/– MEF, IKK{alpha}–/– MEF, IKKß–/– MEF, and IKK{gamma}–/– MEF (25–27) were also maintained in DMEM containing 10% FBS. For drug treatment, cells were grown to ~50% confluency before exposure to the DNA-damaging agents, mitomycin C at a concentrations of 2.5 to 5.0 µg/mL, etoposide at concentrations of 25 to 80 µmol/L for the indicated time. At the time of drug addition, collagen IV (10 µg/mL, Sigma, St. Louis, MO) was also added to the cells. Adenoviruses expressing DDR1 (Ad-DDR1) or green fluorescent protein (Ad-GFP) were generated, amplified, and titrated as previously reported (24). Cells were grown to ~50% to 70% confluency and infected with recombinant adenovirus at multiplicity of infection (MOI) of 10 to 20 for the indicated time. Ad-GFP was used as control.

Transfections and luciferase assays. Transfections were carried out using LipofectAMINE 2000 (Invitrogen) according to instructions from the manufacturer. In transient transfection experiments, plasmid DNA was kept constant with empty vector. For luciferase assays, cells were harvested 24 hours posttransfection and luciferase activity was determined using Dual Luciferase Reporter assay system (Promega, Madison, WI). The difference in transfection efficiency across samples was normalized by cotransfecting pRL-TK, which expresses Renilla luciferase.

Apoptosis assay. Apoptosis was measured using the Cell Death Detection ELISA kit (Roche, Indianapolis, IN). Briefly, the cells were lysed and the amount of nucleosomes in the cytoplasmic fraction of the cell lysates was measured using antihistone antibody and anti-DNA antibody linked to peroxidase in a sandwich ELISA-based protocol. For calculating relative nucleosome content, the absorbance of all the samples was normalized with respect to that of untreated/untransfected cells.

Short hairpin RNAs. Vectors expressing short hairpin RNA (shRNA) against Cox-2 (5'-GCTCAACACCGGAATTTTT-3'), luciferase (5'-GTGTTGGGCGCGTTATTTC-3'), DDR1 (5'-AATCTCCTTCATCTCTGAT-3'), and GFP (5'-GCAAGCTGACCCTGAAGTC-3') were generated using the pBabe-U6-shRNA plasmid. Cells were transfected with LipofectAMINE 2000 (Invitrogen) according to the protocol of the manufacturer in the presence of shRNA.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assays (EMSA) were done as described previously (28, 29). Briefly, after the indicated drug treatment, nuclear extracts were made using a Nuclear Extraction kit (Panomics, Redwood City, CA). Ten micrograms of nuclear extract were incubated with 32P-labeled NF-{kappa}B consensus oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) in gel shift binding buffer (Promega). The DNA-protein complexes were then resolved by electrophoresis through Novex 6% DNA retardation gel (Invitrogen). Control oligos representing the consensus oligonucleotide unlabeled or unlabeled NF-{kappa}B mutant oligonucleotide (Santa Cruz Biotechnology) were added in 500-fold excess.

Western blot analysis. Western blots were done as described previously (30). The following antibodies were used: DDR1 (Santa Cruz Biotechnology), ß-actin (Sigma), Bcl-2 (BD Transduction Laboratories, San Diego, CA), XIAP (Cell Signaling Technology, Danvers, MA), Cox-2 (BD Transduction Laboratories), and I{kappa}B (Santa Cruz Biotechnology).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DDR1 induces Cox-2 expression. We have previously shown that both DDR1 and Cox-2 promote cell survival upon p53-dependent genotoxic stress (24, 30). To identify the downstream signaling targets of DDR1, in particular those that may contribute to the DDR1-induced prosurvival pathway, we initially investigated the effects of DDR1 overexpression on Cox-2 expression. For this purpose, we infected wt and Cox-2–/– MEF with recombinant Ad-DDR1 or control Ad-GFP and measured levels of Cox-2 expression. As shown in Fig. 1A , ectopic DDR1 expression in wt-MEF resulted in induction of Cox-2 expression in the presence of its ligand collagen, whereas the control virus, Ad-GFP, had no effect. We also generated tetracycline-regulated (tet-off) DDR1 expression system in MDAMB435 cells (MDAMB435-DDR1) and tested whether DDR1 also induced Cox-2 in a different cell type. The results showed that following tet withdrawal, Cox-2 expression increased concomitantly with increasing DDR1 expression and only in the presence of collagen IV (Fig. 1A, middle), suggesting that DDR1-mediated Cox-2 induction was specific for the activated DDR1. In fact, activated induced DDR1 as measured by tyrosine phosphorylation, upon tet-removal, was detected only in the presence of collagen (Fig. 1A, right). These results support the conclusion that activation of the overexpressed DDR1 is required for its induction of Cox-2 expression.


Figure 1
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  		Figure 1. DDR1 induces Cox2 expression, and DDR-mediated chemoresistance is abolished in Cox-2 null cells. A, wt and Cox-2–/– MEFs were infected with Ad-DDR1 or the control Ad-GFP at 20 MOI in the presence of collagen IV (10 µg/mL). Forty-eight hours postinfection, cell extracts were made and Western blotting was carried out for the indicated proteins (left). Right, tetracycline-regulated DDR1 expression (MDAMB435-DDR1 tet-off) induces Cox-2 expression after tet removal for indicated times only in the presence of collagen IV, and cell extracts were made and Western blotting was carried out for DDR1, Cox-2, and ß-actin. At the indicated time after tet removal, cells were lysed and DDR1 proteins were immunoprecipitated. The levels of tyrosine phosphorylation were determined by Western blot (WB) analysis using PY20 phosphotyrosine antibody. The quantity of DDR1 proteins in the immunoprecipitate (IP) was determined by Western blotting using DDR1 antibody. B, DDR1 expression renders chemoresistance in a Cox-2–dependent manner. Wt- and Cox-2–/– MEFs were infected with Ad-DDR1 or the control Ad-GFP at 20 MOI, and 24 hours later, cells were treated with the indicated concentrations of the chemotherapeutic drugs, etoposide and mitomycin C, for 24 and 48 hours. At the same time, collagen IV was also added to the cells. At the indicated time points, cell death detection ELISA was carried out to determine the apoptosis rate as described in Materials and Methods. DMSO was treated as a choice of solvent. Columns, mean of three independent experiments with each sample in triplicate; bars, SD. C, DDR1 expression increases cell survival in response to genotoxic stress in MDAMB435 cells. MDAMB435 cells were infected with Ad-DDR1 or the control virus Ad-GFP at 10 MOI in the presence of collagen IV. Twenty-four hours later, indicated concentrations of etoposide and mitomycin C were added. Twenty-four hours after drug treatment, cell death detection ELISA was carried out. A similar experiment was also carried out with MDAMB435-DDR1 tet-off cells, which were treated with etoposide in the presence of collagen IV with or without tet.

 
DDR1-mediated chemoresistance is Cox-2 dependent. The DDR1 kinase is activated/phosphorylated in response to genotoxic stress in wt-p53-containing cells and confers increased resistance to apoptosis (24). Cox-2 is also up-regulated in wt-p53-containing cells and is protective against genotoxic stress (30). Thus, we next tested whether DDR1-induced Cox-2 played an important role in DDR1-mediated cell survival upon genotoxic stress. For this purpose, we infected wt- and Cox-2–/– MEFs with Ad-DDR1 or control virus Ad-GFP at a MOI of 20, and measured the extent of cell death at different time points following exposure to different chemotherapeutic agents. DDR1 overexpression resulted in a marked decrease in apoptosis in the presence of etoposide at two different concentrations (40 and 80 µmol/L) in wt-MEFs, but had no significant effects in Cox-2-null MEFs under the same conditions (Fig. 1B). Similar results were observed with another therapeutic agent mitomycin C. These findings supported the concept that DDR1-mediated survival or chemoresistance is Cox-2 dependent. To test the role of DDR1 in chemoresistance in human cancer cells, we induced DDR1 expression in tet-off–regulated MDAMB435-DDR1 and measured the extent of apoptosis upon the addition of genotoxic drugs, etoposide or mitomycin C. As shown in Fig. 1C, DDR1 induction by tet removal or Ad-DDR1 infection resulted in ~2- to 3-fold reduction in the extent of apoptosis, upon the addition of etoposide or mitomycin C, compared with similarly treated control cells grown in the presence of tet or infected with Ad-GFP (Fig. 1C). To further validate the effect of Cox-2 induction on DDR1-mediated survival, we used shRNA to suppress the expression of endogenous Cox-2 in human breast cancer cells. Transfection of Cox-2 shRNA resulted in the suppression of Cox-2 expression in Ad-DDR1–infected MDAMB435 cells, compared with cells transfected with control shRNA vector (pBabe-U6-shRNA-luciferase; Fig. 2A, left ). Next, we examined the effects of Cox-2 depletion on DDR1-mediated chemoresistance in DDR1-overexpressing MDAMB435 cells. As shown in Fig. 2A (right), the Ad-DDR1–infected cells were more resistant to etoposide treatment compared with control Ad-GFP–infected cells. However, depletion of Cox-2 induction via Cox-2 shRNA transfection resulted in a dramatic increase of genotoxic stress–induced apoptosis in Ad-DDR1–infected cells (compare columns 3 and 6). The antiapoptotic protein, Bcl-2, was also increased in response to ectopic expression of DDR1, but Bcl-2 induction was drastically reduced by Cox-2 depletion (Fig. 2A), consistent with several previous reports that Bcl-2 expression is regulated through a Cox-2–dependent mechanism (22, 31). To further validate these results, we examined the effect of Cox-2 inhibition in a human breast cancer cell line, T47D, containing high basal expression of both DDR1 and Cox-2 proteins (Fig. 2B, left). Endogenous Cox-2 expression was significantly suppressed by transfection with Cox-2 shRNA but not by luciferase control shRNA (Fig. 2B). Moreover, depletion of Cox-2 resulted in a reproducible decrease in Bcl-2 expression in T47D cells and also caused a marked increase in apoptosis in the presence of etoposide, compared with results with shRNA-transfected or nontransfected T47D cells (Fig. 2B, right). Thus, the high basal expression level of DDR1 in T47D cells seems likely to be a causative factor in the chemoresistant phenotype of these tumor cells. Taken together, these results show that DDR1, through a Cox-2–dependent mechanism, can play an important role in the chemoresistant phenotype of tumor cells.


Figure 2
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  		Figure 2. DDR1-mediated chemoresistance is Cox-2 dependent. A, depletion of Cox-2 induction represses Bcl-2 expression and reduces DDR1-mediated chemoresistance. MDAMB435 cells were transfected with Cox-2 shRNA or luciferase shRNA constructs, then the cells were infected with Ad-GFP or Ad-DDR1 at 10 MOI. Twenty-four hours later, 40 µmol/L etoposide was added in the presence of collagen IV. Twenty-four hours after drug addition, cell extracts were made and Western blotting were carried out for the indicated proteins or subjected to cell death detection ELISA. Left, Western blot analysis using lysates prepared from MDAMB435 cells transfected with Cox-2 shRNA or control shRNA. Right, the pattern of apoptosis analysis by cell death ELISA. B, knockdown of basal Cox-2 expression suppresses Bcl-2 expression and enhances apoptosis in response to etoposide treatment. T47D cells were transfected with Cox-2 shRNA or luciferase shRNA constructs. Twenty-four hours later, 40 µmol/L etoposide was added in the presence of collagen IV. Twenty-four hours after drug treatment, cell extracts were made and Western blots were carried out for the indicated proteins or subjected to cell death detection ELISA.

 
DDR1 activates the NF-{kappa}B prosurvival pathway. It is well established that Cox-2 is a downstream target of the NF-{kappa}B pathway (32, 33). To explore whether DDR1 activates NF-{kappa}B signaling, we first examined its effects on NF-{kappa}B activity by using an NF-{kappa}B promoter–luciferase reporter construct. As shown in Fig. 3A , Ad-DDR1 infection activated NF-{kappa}B activity, whereas Ad-GFP infection of parental MDAMB435 cells had no effect on the NF-{kappa}B promoter. Next, we did EMSA to examine the effects of DDR1 on NF-{kappa}B DNA-binding activity. The DNA-binding activity of NF-{kappa}B was significantly increased in cells infected with Ad-DDR1 to a level comparable with that observed with tumor necrosis factor-{alpha}, activator of this pathway, whereas there was no NF-{kappa}B binding activity in Ad-GFP–infected control cells (Fig. 3B). The increased level of NF-{kappa}B DNA-protein complexes observed in response to DDR1 overexpression was abrogated when an excess of cold wt-NF-{kappa}B binding oligonucleotide was added to compete its binding, whereas the addition of excess of mutant oligo had no effect on NF-{kappa}B DNA-binding activity (Fig. 3B). To further determine the specific role of DDR1 in NF-{kappa}B–mediated Cox-2 induction, we tested whether the I{kappa}B{alpha} superrepressor mutant abolishes Cox-2 expression induced by DDR1. MDAMB435 cells were transfected with the superrepressor expressing plasmid in the presence of Ad-DDR1 or Ad-GFP. Western blot analysis, as shown in Fig. 3C, shows that the superrepresor I{kappa}B markedly inhibited Cox-2 expression induced by DDR1 (Ad-DDR1 infection), whereas transfection of the vector alone (pCDNA3) failed to do so. Taken together, these results show that DDR1 increases NF-{kappa}B activity and that DDR1-mediated Cox-2 induction is NF-{kappa}B dependent.


Figure 3
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  		Figure 3. DDR1 activates NF-{kappa}B signaling. A, modulation of NF-{kappa}B activity by DDR1. MDAMB435 cells were transfected with vector (pGL3-basic) or NF-{kappa}B-Luc. The cells were then infected with Ad-GFP or Ad-DDR1 at 10 MOI in the presence of collagen IV. Forty-eight hours postinfection, luciferase activity was measured as described in Materials and Methods. B, increasing NF-{kappa}B DNA-binding activity by DDR1. MDAMB435 cells were treated with TNF-{alpha} (positive control) or infected with Ad-GFP or Ad-DDR1 at 10 MOI. Twelve hours after TNF-{alpha} addition or 48 hours postinfection, nuclear extracts were isolated. About 5 µg nuclear extracts were incubated with 32P-labeled NF-{kappa}B oligo, cold NF-{kappa}B oligo, or its mutant, followed by gel electrophoresis. C, inhibition of DDR1-mediated Cox-2 induction by an I{kappa}B superrepressor. MDAMB435cells were transfected with I{kappa}B mutant (superrepressor), then infected with Ad-GFP or Ad-DD1 at 10 MOI. Forty-eight hours postinfection, cell extracts were made and Western blots were carried out.

 
Abrogation of NF-{kappa}B activity abolishes DDR1-mediated chemoresistance. Given the ability of DDR1 to enhance NF-{kappa}B activity, we investigated whether DDR1 actually exerts its prosurvival effect via NF-{kappa}B activation. To assess the specificity of this response, we examined whether abrogation of the NF-{kappa}B pathway by the superrepressor I{kappa}B suppresses DDR1-mediated chemoresistance in DDR1-overexpressing breast cancer cells. Ectopic DDR1 expression in MDAMB435 cells with little basal DDR1 expression significantly reduced the apoptotic response to etoposide, compared with that of cells with ectopic GFP expression (Fig. 4A ). However, transfection of the superrepressor I{kappa}B resulted in a significant reduction of DDR1-induced chemoresistance (Fig. 4A, columns 3 and 6). We also examined the effect of NF-{kappa}B inhibition in T47D cells, which have a high basal level of DDR1 expression. As shown in Fig. 4B, transfection of the I{kappa}B superrepressor resulted in a drastic increase in etoposide-induced apoptosis in a dose-dependent manner. In contrast, transfection of vector itself along with increasing doses of etoposide induced only a marginal increase in apoptosis. These results indicate that DDR1 increases NF-{kappa}B activity and its activation plays an important role in DDR1-mediated chemoresistance in some breast cancer cells.


Figure 4
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  		Figure 4. DDR1-mediated chemoresistance is NF-{kappa}B pathway dependent in human breast cancer cells. A, inhibition of DDR1-mediated chemoresistance by an I{kappa}B superrepressor. MDAMB435cells were transfected with an I{kappa}B mutant (superrepressor) or control vector (pcDNA3), then infected with Ad-GFP or Ad-DDR1 at 10 MOI. Twenty-four hours later, 40 µmol/L etoposide were added to cells in the presence of collagen IV. Twenty-four hours after drug addition, cell death detection ELISA was carried out to determine the pattern of apoptosis. B, I{kappa}B superrepressor expression increases apoptosis in T47D cells. T47D cells were transfected with an I{kappa}B superrepressor or vector (pcDNA3). Twenty-four hours later, etoposide was added. Twenty-four hours after drug addition, cell death detection ELISA was carried out. Points, mean of three independent experiments with triplicate samples; bars, SD.

 
IKKß and IKK{gamma} are required for DDR1-mediated NF-{kappa}B activation and chemoresistance. The IKK complex (I{kappa}B kinase) is the upstream activating kinase of the NF-{kappa}B pathway. It is composed of two catalytic subunits, IKK{alpha} and IKKß, which can directly phosphorylate I{kappa}B, whereas IKK{gamma} acts as the regulatory subunit. Activation of the canonical NF-{kappa}B pathway based on I{kappa}B degradation is mostly dependent on IKKß. To investigate whether these NF-{kappa}B upstream regulators affect DDR1-mediated NF-{kappa}B activation, we examined the ability of DDR1 to activate NF-{kappa}B and its targets in IKK knockout MEFs (i.e., IKK{alpha}–/–, IKKß–/–, and IKK{gamma}–/– MEFs). Each MEF was transfected with an NF-{kappa}B luciferase reporter construct (NF-{kappa}B-Luc) or control luciferase construct (pGL3) along with Ad-DDR1 or Ad-GFP. DDR1 enhanced NF-{kappa}B activity in wt-MEF and IKK{alpha}–/– MEF (Fig. 5A ), but the activation of NF-{kappa}B by DDR1 was significantly diminished in both IKKß–/– and IKK{gamma}–/– MEFs (Fig. 5A). Ectopic DDR1 induced increased expression of NF-{kappa}B targets, Cox-2 and XIAP, in wt-MEFs, and transfection of the superrepressor I{kappa}B mutant into wt-MEFs significantly inhibited the DDR1-mediated increase of Cox-2 and XIAP expression (Fig. 5B, left). Moreover, the DDR1-mediated activation of NF-{kappa}B targets, Cox-2 and XIAP, was abolished in IKKß–/– MEFs (Fig. 5B, right). Taken together, these data show that IKKß and IKK{gamma} are essential for DDR1-mediated NF-{kappa}B activation, but that IKK{alpha} is dispensable. To determine whether DDR1-mediated survival or chemoresistance also requires the IKK subunits, we examined the antiapoptotic role of DDR1 in response to etoposide in IKK knockout MEFs. As seen in other cell types, DDR1 overexpression suppressed etoposide-induced apoptosis in wt-MEFs and IKK{alpha}–/– MEFs (Fig. 5C and D). Moreover, abrogation of NF-{kappa}B pathway via the superrepressor I{kappa}B mutant further enhanced the apoptotic response to etoposide in wt-MEFs. Of note, the DDR1-mediated antiapoptotic effect was not observed in IKKß–/– or IKK{gamma}–/– MEFs (Fig. 5D). These results strongly imply that DDR1 promotes cell survival via the activation of NF-{kappa}B and its target genes and that IKKß and IKK{gamma} subunits of the IKK complex are required for mediating the prosurvival signal of DDR1 upon genotoxic stress.


Figure 5
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  		Figure 5. DDR1-mediated NF-{kappa}B activation requires IKKß and IKK{gamma}. A, IKKß and IKK{gamma} are required for DDR1-mediated NF-{kappa}B activation. Wt–, IKK{alpha}–/–, IKKß–/–, and IKK{gamma}–/– MEFs were transfected with pGL3Basic or NF-{kappa}B-Luc along with 0.1 µg tk-Renilla-Luc for normalization. Cells were then infected with Ad-GFP or Ad-DDR1 at 20 MOI. Forty-eight hours postinfection, luciferase activity was measured as described in Materials and Methods. B, DDR1-mediated induction of the NF-{kappa}B target genes, Cox-2 and XIAP, is suppressed by the superrepressor I{kappa}B mutant (left). Wt-MEFs were infected with Ad-GFP or Ad-DDR1 at 20 MOI. Twenty-four hours later, 40 µmol/L etoposide were treated. After additional 24 hours, cell extracts were made and Western blots were carried out for the indicated proteins. DDR1 does not induce Cox-2 and XIAP expression in IKKß-null MEFs (right). IKKß-null MEFs were infected with Ad-GFP or Ad-DDR1 at 20 MOI. Twenty-four hours later, 40 µmol/L etoposide were treated. C and D, IKKß and IKK{gamma} are required for DDR1-dependent, NF-{kappa}B–mediated prosurvival. C, wt-MEFs were transfected with I{kappa}B superrepressor, then infected with Ad-GFP or Ad-DDR1 at 20 MOI. Twenty-four hours later, MEFs were treated with 40 µmol/L etoposide. Twenty-four hours after drug addition, cell death detection ELISA was carried out. D, Wt–, IKK{alpha}–/–, IKKß–/–, and IKK{gamma}–/– MEFs were infected with Ad-GFP or Ad-DDR1 at 20 MOI. Twenty-four hours later, cells were treated with 40 µmol/L etoposide. Twenty-four hours after drug addition, apoptosis was measured by cell death detection ELISA. Columns (A, C, and D), mean of three independent experiments with each sample in triplicate; bars, SD.

 
Chemosensitization of breast cancer cells by depletion of DDR1 expression. Given that DDR1 is involved in NF-{kappa}B prosurvival signaling and chemoresistance, we sought to determine whether DDR1 inhibition might be sufficient to promote chemosensitization in human breast cancer cells exhibiting high basal levels of DDR1. Inhibition of DDR1 expression by DDR1 shRNA in T47D cells in which DDR1 and Cox-2 are expressed at high levels significantly suppressed the levels of endogenous DDR1 and Cox-2 as measured by immunoblotting (Fig. 6A ). Moreover, depletion of DDR1 modulated total I{kappa}B protein level (Fig. 6A). Although transfection of control shRNA (luciferase shRNA) as well as vector alone (pBabe-U6-shRNA) had no effect on the levels of Cox-2, Bcl-2, and I{kappa}B, cotransfection of the superrepressor I{kappa}B mutant with either luciferase-shRNA or vector alone inhibited both Cox-2 and Bcl-2 expression (Fig. 6A). These data further argue that DDR1 is an upstream regulator of the NF-{kappa}B pathway. To directly assess the involvement of DDR1 in survival or chemoresistance of T47D tumor cells, we compared the levels of etoposide-induced apoptosis observed in these breast cancer cells transfected with a control shRNA construct (pBabe-luciferase-shRNA), vector alone (pBabe), or DDR1 shRNA (pBabe-DDR1-shRNA). As shown in Fig. 6B, depletion of DDR1 expression resulted in a significant increase in apoptosis with increasing etoposide concentration, whereas there was only a marginal increase in apoptosis with empty vector or luciferase shRNA-transfected cells. These findings establish that the reduced levels of endogenous DDR1 contributed directly to etoposide-induced sensitization of these tumor cells to apoptosis. Finally, we examined whether DDR1-mediated chemoresistance in T47D cells was NF-{kappa}B dependent. Suppression of Cox-2 expression or NF-{kappa}B activity by the superrepressor I{kappa}B mutant significantly reduced apoptosis induced by etoposide in control shRNA or vector-transfected cells. Of note, DDR1 depletion by DDR1 shRNA failed to further increase the level of etoposide-induced apoptosis in the presence of the superrepressor (Fig. 6C). These results indicate that DDR1-mediated chemoresistance in breast cancer cells, which exhibit high basal levels of this receptor, is dependent upon activation of NF-{kappa}B pathway and consequent Cox-2 induction.


Figure 6
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  		Figure 6. Knockdown of endogenous DDR1 expression enhances chemosensitivity. A, modulation of NF-{kappa}B pathway proteins, Cox-2, Bcl-2, and I{kappa}B. T47D cells were stably transfected with pBabe (vector), luciferase shRNA (pBabe-luciferase shRNA), or DDR1 shRNA construct (pBabe-DDR1 shRNA), and selected for stable clones under puromycin. The resistant clones were pooled and determined for reduction of DDR1 expression. DDR1 knockdown cells (T47-DDR1 shRNA) as well as control shRNA lines (T47D-luciferase shRNA and T47D-pBabe shRNA) were then transiently transfected with I{kappa}B superrepressor mutant or vector (pcDNA3), and total cell lysates were isolated for Western blot analysis using anti-DDR1, anti-Cox-2, anti-I{kappa}B, anti-Bcl-2, or anti-ß-actin antibodies. Note that depletion of DDR1 significantly reduced Cox-2 expression along with an increase of total I{kappa}B level and that expression of I{kappa}B mutant superrepressor inhibited Cox-2 expression. DDR1 knockdown or control T47D cells were also transfected with Cox-2 shRNA or GFP-shRNA. Forty-eight hours posttransfection, cell extracts were made and Western blots were carried out for the indicated proteins. B, effect of DDR1 depletion on genotoxic agent (etoposide)–mediated apoptosis. DDR1 knockdown T47D cells and control T47D cells (pBabe-shRNA and Luciferase shRNA) were treated with indicated concentrations of etoposide. Twenty-four hours after drug addition, apoptosis was measured by cell death detection ELISA. C, depletion of Cox-2 or I{kappa}B superrepressor mutant antagonizes DDR1-mediated prosurvival. The same double-knockdown cells from (A) were also treated with etoposide (40 µmol/L) for 24 hours, then analyzed to determine the patterns of apoptosis by cell death detection ELISA.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well documented that Cox-2 can be induced by growth factors and cytokines, inflammatory stimuli, and tumor promoters (34). Some studies have provided evidence that oxidative and DNA damage stresses can induce Cox-2 expression (35, 36). However, the precise mechanisms of induction of Cox-2 in response to these stresses are unknown. Our previous studies showed that tumor suppressor p53 or DNA-damaging agents acting through p53 can induce Cox-2, which is able to counteract p53-mediated cell death (30). These studies indicated that Cox-2 is a p53 response gene that is induced to alleviate the adverse effects arising from DNA damage/genotoxic stress. A number of recent reports strongly imply that enhanced Cox-2 expression plays a significant role in resistance to chemotherapy-induced apoptosis, by protecting cancer cells against apoptosis (37). Moreover, increased Cox-2 levels are commonly detected in a variety of premalignant and malignant tissues (19). Expression of Cox-2 in several types of cancer, including breast cancer, correlates with poor prognosis, and Cox-2 enzyme inhibitors have been reported to reduce breast cancer incidence in humans (38, 39). Recent studies have shown that overexpression of Cox-2 in the mammary gland of CD1 transgenic mice results in tumorigenic transformation as well as angiogenic stimulation (22, 40). Cox-2 is expressed by the tumor endothelium in human colon, breast, prostate, lung, and other cancers (41), whereas normal vascular endothelial cells express only Cox-1 (42). In addition, several studies have shown that selective inhibition of Cox-2 activity suppresses angiogenesis in vitro and in vivo (43). Thus, it has been strongly suggested that Cox-2 is a promising molecular target for cancer prognosis and treatment (44).

The wide range of genetic, environmental, and metabolic stimuli that activate p53 clearly distinguishes it from other known tumor suppressor genes. Our previous studies have shown that DDR1 is a direct p53 target gene and can be functionally activated/phosphorylated in a p53-dependent manner (24). We also found that inhibition of DDR1 function resulted in strikingly increased apoptosis of wt-p53–containing cells in response to genotoxic stress (24). Hence, DDR1 seems to play a critical role in determining the cellular outcome in response to genotoxic/therapeutic stress in wt-p53–containing cells. Our present studies show that DDR1 acts as a novel upstream regulator for Cox-2, and that Cox-2 functions as an important mediator for DDR1-mediated survival in the p53-dependent response to genotoxic stress. Moreover, we showed that the mechanism by which DDR1 causes increased Cox-2 expression is mediated through DDR1 activation of the NF-{kappa}B pathway, which induces increased levels of its known targets, including Cox-2 and XIAP. DDR1 up-regulation of Cox-2 as well as XIAP was shown to be dependent on functional components of the NF-{kappa}B signaling. DDR1 activation led to strong induction of NF-{kappa}B–mediated transcription, and MEFs null for either IKKß or IKK{gamma} failed to induce Cox-2 in response to DDR1 signaling. One well-known characteristic of cancer cytotoxic treatment is the activation of the transcription factor NF-{kappa}B, which regulates cell survival (45). NF-{kappa}B activation suppresses the apoptotic potential of chemotherapeutic agents and contributes to chemotherapy resistance. It is also known that many human tumors display constitutive NF-{kappa}B activation that allows malignant cells escape apoptosis (46). Our present findings show that DDR1 induces NF-{kappa}B activation, establishing this RTK as an important upstream regulator for the NF-{kappa}B prosurvival pathway.

Numerous studies have reported overexpression of DDR1 in lung, esophageal, breast, ovary, and pediatric brain cancers (2, 7, 13–17). Moreover, the elevated expression of DDR1 in a number of fast-growing invasive tumors has suggested an important role of this matrix-activated RTK in the proliferation and stromal invasion of tumors (13, 15). In the present studies, we showed that a human breast tumor line, T47D, which overexpressed DDR1, exhibited high constitutive levels of Cox-2. Moreover, inhibition of NF-{kappa}B by the I{kappa}B superrepressor as well as shRNA inhibition of the expression of Cox-2 or DDR1 itself all dramatically increased the apoptotic response of these tumor cells to genotoxic agents. These findings imply that in DDR1-overexpressing tumors, prosurvial signaling by this newly worked out pathway may offer new tumor-specific therapeutic targets.


   Acknowledgments
 
Grant support: NIH grants CA097216, CA085214, and CA080058.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the members of the Cutaneous Biology Research Center for helpful discussion and Sarah Boswell for critical reading of the manuscript.

Received 4/ 3/06. Revised 5/30/06. Accepted 6/13/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Di Marco E, Cutuli N, Guerra L, Cancedda R, De Luca M. Molecular cloning of trkE, a novel trk-related putative tyrosine kinase receptor isolated from normal human keratinocytes and widely expressed by normal human tissues. J Biol Chem 1993;268:24290–5.[Abstract/Free Full Text]
  2. Zerlin M, Julius MA, Goldfarb M. NEP: a novel receptor-like tyrosine kinase expressed in proliferating neuroepithelia. Oncogene 1993;8:2731–9.[Medline]
  3. Johnson JD, Edman JC, Rutter WJ. A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain. Proc Natl Acad Sci U S A 1993;90:5677–81.[Abstract/Free Full Text]
  4. Vogel W, Gish GD, Alves F, Pawson T. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol Cell 1997;1:13–23.[CrossRef][Medline]
  5. Shrivastava A, Radziejewski C, Campbell E, et al. An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors. Mol Cell 1997;1:25–34.[CrossRef][Medline]
  6. Vogel W, Brakebusch C, Fassler R, Alves F, Ruggiero F, Pawson T. Discoidin domain receptor 1 is activated independently of ß(1) integrin. J Biol Chem 2000;275:5779–84.[Abstract/Free Full Text]
  7. Alves F, Vogel W, Mossie K, Millauer B, Hafler H, Ullrich A. Distinct structural characteristics of discoidin I subfamily receptor tyrosine kinases and complementary expression in human cancer. Oncogene 1995;10:609–18.[Medline]
  8. Bhatt RS, Tomoda T, Fang Y, Hatten ME. Discoidin domain receptor 1 functions in axon extension of cerebellar granule neurons. Genes Dev 2000;14:2216–28.[Abstract/Free Full Text]
  9. Ram R, Lorente G, Nikolich K, Urfer R, Foehr E, Nagavarapu U. Discoidin domain receptor-1a (DDR1a) promotes glioma cell invasion and adhesion in association with matrix metalloproteinase-2. J Neurooncol 2006;76:239–48.[CrossRef][Medline]
  10. Matsuyama W, Faure M, Yoshimura T. Activation of discoidin domain receptor 1 facilitates the maturation of human monocyte-derived dendritic cells through the TNF receptor associated factor 6/TGF-ß-activated protein kinase 1 binding protein 1ß/p38{alpha} mitogen-activated protein kinase signaling cascade. J Immunol 2003;171:3520–32.[Abstract/Free Full Text]
  11. Hou G, Vogel W, Bendeck MP. The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair. J Clin Invest 2001;107:727–35.[Medline]
  12. Vogel WF, Aszadi A, Alves F, Pawson T. Discoidin domain receptor 1 tyrosine kinase has an essential role in mammary gland development. Mol Cell Biol 2001;21:2906–17.[Abstract/Free Full Text]
  13. Barker KT, Martindale JE, Mitchell PJ, et al. Expression patterns of the novel receptor-like tyrosine kinase, DDR, in human breast tumours. Oncogene 1995;10:569–75.[Medline]
  14. Laval S, Butler R, Shelling AN, Hanby AM, Poulsom R, Ganesan TS. Isolation and characterization of an epithelial-specific receptor tyrosine kinase from an ovarian cancer cell line. Cell Growth Differ 1994;5:1173–83.[Abstract]
  15. Perez JL, Jing SQ, Wong TW. Identification of two isoforms of the Cak receptor kinase that are coexpressed in breast tumor cell lines. Oncogene 1996;12:1469–77.[Medline]
  16. Nemoto T, Ohashi K, Akashi T, Johnson JD, Hirokawa K. Overexpression of protein tyrosine kinases in human esophageal cancer. Pathobiology 1997;65:195–203.[Medline]
  17. Weiner HL, Rothman M, Miller DC, Ziff EB. Pediatric brain tumors express multiple receptor tyrosine kinases including novel cell adhesion kinases. Pediatr Neurosurg 1996;25:64–71.[Medline]
  18. Dannenberg AJ, Subbaramaiah K. Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 2003;6:431–43.[CrossRef]
  19. Dannenberg AJ, Altorki NK, Boyle JO, et al. Cyclooxygenase 2: a pharmacological target for the prevention of cancer. Lancet Oncol 2001;2:544–51.[CrossRef][Medline]
  20. Gupta RA, Dubois RN. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2. Nat Rev Cancer 2001;1:11–21.[CrossRef][Medline]
  21. Gasparini G, Longo R, Sarmiento R, Morabito A. Inhibitors of cyclo-oxygenase 2: a new class of anticancer agents? Lancet Oncol 2003;4:605–15.[CrossRef][Medline]
  22. Liu CH, Chang SH, Narko K, et al. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001;276:18563–9.[Abstract/Free Full Text]
  23. Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 2002;94:252–26.[Abstract/Free Full Text]
  24. Ongusaha PP, Kim JI, Fang L, et al. p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop. EMBO J 2003;22:1289–301.[CrossRef][Medline]
  25. Hu Y, Baud V, Delhase M, et al. Abnormal morphogenesis but intact IKK activation in mice lacking the IKK{alpha} subunit of the I{kappa}B kinase. Science 1999;284:316–20.[Abstract/Free Full Text]
  26. Li Z-W, Chu W, Hu Y, et al. The IKK{alpha} subunit of I{kappa}B kinase (IKK) is essential for nuclear factor-{kappa}B activation and prevention of apoptosis. J Exp Med 1999;189:1839–45.[Abstract/Free Full Text]
  27. Makris C, Godfrey VL, Krähn-Senftleben G, et al. Female mice heterozygous for IKK{gamma}/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol Cell 2000;5:969–79.[CrossRef][Medline]
  28. Yamaoka S, Courtois G, Bessia C, et al. Complementation cloning of NEMO, a component of the I{kappa}B kinase complex essential for NF-{kappa}B activation. Cell 1998;93:1231–40.[CrossRef][Medline]
  29. Ryo A, Suizu F, Yoshida Y, et al. Regulation of NF-{kappa}B signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 2003;12:1413–26.[CrossRef][Medline]
  30. Han JA, Kim JI, Ongusaha PP, et al. p53-mediated induction of Cox-2 counteracts p53- or genotoxic stress-induced apoptosis. EMBO J 2002;21:5635–44.[CrossRef][Medline]
  31. Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493–501.[CrossRef][Medline]
  32. Schmedtje JF, Ji YS, Liu WL, DuBois RN, Runge MS. Hypoxia induces cyclooxygenase-2 via the NF-{kappa}B p65 transcription factor in human vascular endothelial cells. J Biol Chem 1997;272:601–8.[Abstract/Free Full Text]
  33. Yamamoto K, Arakawa T, Ueda N, Yamamoto S. Transcriptional roles of nuclear factor {kappa} B and nuclear factor-interleukin-6 in the tumor necrosis factor {alpha}-dependent induction of cyclooxygenase-2 in MC3T3-1 cells. J Biol Chem 1995;270:31315–20.[Abstract/Free Full Text]
  34. Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 2000;69:145–82.[CrossRef][Medline]
  35. Steinauer KK, Gibbs I, Ning S, French JN, Armstrong J, Knox SJ. Radiation induces upregulation of cyclooxygenase-2 (COX-2) protein in PC-3 cells. Int J Radiat Oncol Biol Phys 2000;48:325–8.[Medline]
  36. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem 1999;274:5038–46.[Abstract/Free Full Text]
  37. Cao Y, Prescott SM. Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J Cell Physiol 2002;190:279–86.[CrossRef][Medline]
  38. Denkert C, Winzer KJ, Hauptmann S. Prognostic impact of cyclooxygenase-2 in breast cancer. Clin Breast Cancer 2004;4:428–33.[Medline]
  39. Arun B, Goss P. The role of COX-2 inhibition in breast cancer treatment and prevention. Semin Oncol 2004;31:22–9.[Medline]
  40. Chang SH, Liu CH, Conway R, et al. Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proc Natl Acad Sci U S A 2004;101:591–6.[Abstract/Free Full Text]
  41. Masferrer JL, Leahy KM, Koki AT, et al. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res 2000;60:1306–11.[Abstract/Free Full Text]
  42. Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki A, Masferrer L. Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Res 2002;2:625–31.
  43. Gately S, Li WW. Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin Oncol 2004;31:2–11.[Medline]
  44. Subbaramaiah K, Dannenberg AJ. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol Sci 2003;24:96–102.[CrossRef][Medline]
  45. Wang CY, Mayo MW, Baldwin AS, Jr. TNF-{alpha} and cancer therapy-induced apoptosis: potentiation by inhibition of NF-{kappa}B. Science 1996;274:784–7.[Abstract/Free Full Text]
  46. Rayet B, Gelinas C. Aberrant rel/nfkb genes and activity in human cancer. Oncogene 1999;18:6938–47.[CrossRef][Medline]



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