Discoidin Domain Receptor Tyrosine Kinase Research Articles

Low Stability and a ConservedN-Glycosylation Site Are Associated with Regulation of the Discoidin Domain Receptor Family by GlucoseviaPost-TranslationalN-Glycosylation

Cell-surface expression of the discoidin domain receptor (DDR) tyrosine kinase family in high molecular mass form was controlled sensitively by the glucose concentration through a post-translational N-glycosylation process. Cycloheximide time-course experiments revealed that the high-molecular-mass forms of DDR1 and DDR2 were significantly less stable than control receptor tyrosine kinases. Site-directed mutational analysis of the consensus N-glycosylation sites of the DDRs revealed that mutations of asparagine 213 of DDR2 and asparagine 211 of DDR1, a conserved N-glycosylation site among vertebrate DDRs, inhibited the generation of the high-molecular-mass isoform. Taken together, these results suggest a mechanism to control the activity of the DDR family by regulating its cell-surface expression. Due to low stability, the steady-state population of functional DDR proteins in the cell surface depends sensitively on its maturation process via post-translational N-glycosylation, which is controlled by the glucose supply and the presence of a conserved N-glycosylation site.

Detection of DDR2 mutations in malignant melanoma: A potentially targetable feature of advanced stage disease.

9100 Background: The discoidin domain receptor tyrosine kinase 2 (DDR2) gene is mutated in a subset of non-small cell carcinoma of the lung. In lung cancer, targeting of DDR2 with the inhibitor dasatinib has been shown to inhibit DDR2-mutated cell line growth. Responses to dasatinib in DDR2-mutated lung tumors are currently being investigated in clinical trials. We investigated whether somatic DDR2 mutations also occur in melanomas using a set of different clinical subtypes bearing a variety of melanoma-associated molecular alterations. Methods: DNA was extracted from macrodissected FFPE sections of 168 melanomas and subjected to next-generation sequencing using a custom-designed 36-amplicon panel including the coding regions of DDR2 and exons 11 and 15 of BRAF. PCR products were prepared by Access Array, sequenced on the Ion Torrent PGM, and analyzed using Sequence Pilot software. All DDR2 mutations were confirmed by bidirectional Sanger sequencing. Relative expression analysis of the DDR2 gene was performed using RT-PCR on RNA extracted from FFPE sections, with levels normalized to ABL1 and GAPDH. Results: Heterozygous DDR2 point mutations were identified in 3.4% of BRAF-mutated melanoma (n=117) but not in any cases that lacked BRAF mutations (n = 51). All DDR2-mutated melanomas were advanced stage with lymph node and/or extensive soft tissue involvement and included both BRAF V600K and V600E cases. In contrast to the range of mutation sites reported in lung cancers, DDR2 mutations in melanoma (I488S, F574C, S667F and L701F) were at highly evolutionarily conserved positions in the kinase domain. The mutations are expected to be disruptive and may therefore modulate kinase activity. DDR2 transcripts were expressed in tumor cells in all mutated cases. Conclusions: DDR2 mutations in advanced stage melanoma may provide a targetable genetic feature for tyrosine kinase inhibitors such as dasatinib. The invariant association of DDR2 mutation with activating BRAF mutations suggests synergy in transformation between these distinct signaling pathways.

DDR2 mutations in lung cancer in adenocarcinoma and squamous cell carcinoma and association with MET activation.

8115 Background: The discoidin domain receptor tyrosine kinase 2 (DDR2) gene is mutated in a small subset of squamous cell carcinomas (SQCAs) of the lung. Targeting of DDR2 with the inhibitor dasatinib inhibits DDR2-mutated cell line growth and is being investigated in clinical trials for treatment of DDR2-mutated lung tumors. We investigated the frequency and type of DDR2 mutations and their association with other targetable growth factor pathway alterations in non-small cell lung carcinoma (NSCLC). Methods: DNA was extracted from macro-dissected FFPE sections of 105 advanced stage lung carcinomas. Next-generation sequencing was performed using a 36-amplicon panel including the coding regions of DDR2 and exons 11 and 15 of BRAF. DDR2 mutations were confirmed by bidirectional Sanger sequencing. Lung tumors were classified using a standard immunohistochemistry panel. MET pathway activation was assessed with immunohistochemistry using an activation-specific phospho-antibody (pMET, Try1234/1235). MET amplification and ALK rearrangement were assessed using FISH. Mutational analysis for EGFR (exon 18-21), PIK3CA (exons 9 and 20), and KRAS(exons 1-2) was performed using PCR-based DNA sequencing. Results: Heterozygous DDR2 point mutations were identified in 7/105 (6.7%) NSCLCs, including 3/29 (10.3%) SQCAs and 4/69 (5.8%) ACAs, but not in neuroendocrine/large cell carcinomas (0/7). DDR2 mutations were scattered throughout the gene, with 4 present in the discoidin domain and 3 in the kinase domain. MET pathway activation, seen in 25% of DDR2-unmutated cases, was found in 3/6 (50%) DDR2-mutated cases, including 1 SQCA with high-level MET gene amplification. DDR2 mutations were mutually exclusive with BRAF or EGFR mutation or ALK rearrangement, with 1 KRAS-mutated case. Conclusions: DDR2 mutations occur in lung carcinomas with a range of histologic features and in association with MET activation. Dual targeting of the MET and DDR2 kinases in such cases may warrant further investigation.

Protein Analysis of Atrial Fibrosis via Label-Free Proteomics in Chronic Atrial Fibrillation Patients with Mitral Valve Disease

BackgroundAtrial fibrosis, as a hallmark of atrial structure remodeling, plays an important role in maintenance of chronic atrial fibrillation, but interrelationship of atrial fibrosis and atrial fibrillation is uncertain. Label-free proteomics can implement high throughput screening for finding and analyzing pivotal proteins related to the disease.. Therefore, we used label-free proteomics to explore and analyze differentially proteins in chronic atrial fibrillation patients with mitral valve disease.MethodsLeft and right atrial appendages obtained from patients with mitral valve disease were both in chronic atrial fibrillation (CAF, AF≥6 months, n = 6) and in sinus rhythm (SR, n = 6). One part of the sample was used for histological analysis and fibrosis quantification; other part were analyzed by label-free proteomic combining liquid chromatography with mass spectrometry (LC-MS), we utilized bioinformatics analysis to identify differential proteins.ResultsDegree of atrial fibrosis was higher in CAF patients than that of SR patients. 223 differential proteins were detected between two groups. These proteins mainly had vital functions such as cell proliferation, stress response, focal adhesion apoptosis. We evaluated that serine/threonine protein kinase N2 (PKN2), dermatopontin(DP), S100 calcium binding protein B(S100B), protein tyrosine kinase 2(PTK2) and discoidin domain receptor tyrosine kinase 2(DDR2) played important roles in fibrotic process related to atrial fibrillation.ConclusionThe study presented differential proteins responsible for atrial fibrosis in chronic atrial fibrillation patients through label-free proteomic analysis. We assessed some vital proteins including their characters and roles. These findings may open up new realm for mechanism research of atrial fibrillation.

Potential Association of DDR1 Genetic Variant with FEV1 Decline by Aspirin Provocation in Asthmatics

Background. The discoidin domain receptor tyrosine kinase 1 (DDR1) is positioned within the major histocompatibility complex (MHC) region which plays an important role in the immune system. In addition, DDR1 has been elucidated to be downregulated during the epithelial–mesenchymal transition of bronchial epithelium. Objective. To investigate the potential genetic associations between DDR1 and aspirin-exacerbated respiratory disease (AERD), this study conducted association studies of DDR1 single nucleotide polymorphisms (SNPs) with AERD and the obstructive symptom of forced expiratory volume in 1 s (FEV1) decline after aspirin provocation. Methods. Nine common SNPs were genotyped in 93 AERD patients and 96 aspirin-tolerant asthma (ATA) controls. The genotype distributions of all loci were in Hardy–Weinberg equilibrium (HWE; p > .05). Results. In the results of logistic analyses using age, sex, smoking status, and atopy as covariates, DDR1 rs1264320 in the intronic region showed a potent association signal with FEV1 decline by aspirin provocation in asthmatics of this study even after corrections for multiple testing (p = .003 and corrected p = .01). However, the variants of DDR1 were not significantly associated with the AERD development (corrected p > .05). On further comparison of FEV1 decline by aspirin provocation between AERD and ATA, the variant rs1264320 was found to be associated with the FEV1 decline of ATA rather than AERD. Conclusion. Despite the need for further functional evaluations and replications, we conclude that DDR1 polymorphisms are not likely to contribute to predispositions of AERD, but may be potentially associated with FEV1 decline by aspirin provocation in asthmatics.

DDR1 (discoidin domain receptor tyrosine kinase 1)

Review on DDR1 (discoidin domain receptor tyrosine kinase 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Pancreatic islet and progenitor cell surface markers with cell sorting potential

The aim of the study was to identify surface bio-markers and corresponding antibody tools that can be used for the imaging and immunoisolation of the pancreatic beta cell and its progenitors. This may prove essential to obtain therapeutic grade human beta cells via stem cell differentiation. Using bioinformatics-driven data mining, we generated a gene list encoding putative plasma membrane proteins specifically expressed at distinct stages of the developing pancreas and islet beta cells. In situ hybridisation and immunohistochemistry were used to further prioritise and identify candidates. In the developing pancreas seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor-related protein 11 (LRP11), dispatched homologue 2 (Drosophila) (DISP2) and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) were found to be expressed in early islet cells, whereas discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch-like EGF repeat containing (DNER) were expressed in early pancreatic progenitors. The expression pattern of DDR1 overlaps with the early pancreatic and duodenal homeobox 1 (PDX1)⁺/NK6 homeobox 1 (NKX6-1)⁺ multipotent progenitor cells from embryonic day 11, whereas DNER expression in part overlaps with neurogenin 3 (NEUROG3)⁺ cells. In the adult pancreas SEZ6L2, LRP11, DISP2 and SLC30A8, but also FXYD domain containing ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7) and transmembrane protein 27 (TMEM27), retain an islet-specific expression, whereas DDR1 is undetectable. In contrast, DNER is expressed at low levels in peripheral mouse and human islet cells. Re-expression of DDR1 and upregulation of DNER is observed in duct-ligated pancreas. Antibodies to DNER and DISP2 have been successfully used in cell sorting. Extracellular epitopes of SEZ6L2, LRP11, DISP2, DDR1 and DNER have been identified as useful tags by applying specific antibodies to visualise pancreatic cell types at specific stages of development. Furthermore, antibodies recognising DISP2 and DNER are suitable for FACS-mediated cell purification.

Microarray Profiling Analysis Uncovers Common Molecular Mechanisms of Rubella Virus, Human Cytomegalovirus, and Herpes Simplex Virus Type 2 Infections in ECV304 Cells

To study the common molecular mechanisms of various viruses infections that might result in congential cardiovascular diseases in perinatal period, changes in mRNA expression levels of ECV304 cells infected by rubella virus (RUBV), human cytomegalovirus (HCMV), and herpes simplex virus type 2 (HSV-2) were analyzed using a microarray system representing 18,716 human genes. 99 genes were found to exhibit differential expression (80 up-regulated and 19 down-regulated). Biological process analysis showed that 33 signaling pathways including 22 genes were relevant significantly to RV, HCMV and HSV-II infections. Of these 33 biological processes, 28 belong to one-gene biological processes and 5 belong to multiple-gene biological processes. Gene annotation indicated that the 5 multiple-gene biological processes including regulation of cell growth, collagen fibril organization, mRNA transport, cell adhesion and regulation of cell shape, and seven down- or up-regulated genes [CRIM1 (cysteine rich transmembrane BMP regulator 1), WISP2 (WNT1 inducible signaling pathway protein 2), COL12A1 (collagen, type XII, alpha 1), COL11A2 (collagen, type XI, alpha 2), CNTN5 (contactin 5), DDR1 (discoidin domain receptor tyrosine kinase 1), VEGF (vascular endothelial growth factor precursor)], are significantly correlated to RUBV, HCMV and HSV-2 infections in ECV304 cells. The results obtained in this study suggested the common molecular mechanisms of viruses infections that might result in congential cardiovascular diseases.

DDR1 Receptor Tyrosine Kinase Promotes Prosurvival Pathway through Notch1 Activation

DDR1 (discoidin domain receptor tyrosine kinase 1) kinase s highly expressed in a variety of human cancers and occasionally mutated in lung cancer and leukemia. It is now clear that aberrant signaling through the DDR1 receptor is closely associated with various steps of tumorigenesis, although little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. Besides the role of DDR1 in tumorigenesis, we previously identified DDR1 kinase as a transcriptional target of tumor suppressor p53. DDR1 is functionally activated as determined by its tyrosine phosphorylation, in response to p53-dependent DNA damage. In this study, we report the characterization of the Notch1 protein as an interacting partner of DDR1 receptor, as determined by tandem affinity protein purification. Upon ligand-mediated DDR1 kinase activation, Notch1 was activated, bound to DDR1, and activated canonical Notch1 targets, including Hes1 and Hey2. Moreover, DDR1 ligand (collagen I) treatment significantly increased the active form of Notch1 receptor in the nuclear fraction, whereas DDR1 knockdown cells show little or no increase of the active form of Notch1 in the nuclear fraction, suggesting a novel intracellular mechanism underlying autocrine activation of wild-type Notch signaling through DDR1. DDR1 activation suppressed genotoxic-mediated cell death, whereas Notch1 inhibition by a γ-secretase inhibitor, DAPT, enhanced cell death in response to stress. Moreover, the DDR1 knockdown cancer cells showed the reduced transformed phenotypes in vitro and in vivo xenograft studies. The results suggest that DDR1 exerts prosurvival effect, at least in part, through the functional interaction with Notch1.

Abstract P3-09-03: microRNAs and Target Gene Networks in Breast Cancer Cell Lines

Abstract Background: MicroRNAs (miRNAs) are a class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Several miRNAs have been involved in the regulation of several cellular processes during oncogenesis. Objective: We simultaneously analyzed 18 different breast cancer cell lines (BCC) for miRNA and mRNA expression profiles in order to identify regulatory miRNA target gene networks and novel miRNA-directed therapeutic targets. Materials and Methods: We preprocessed the mRNA and miRNA expression data using R software (justRMA) and performed quantile normalization using background-subtracted log ratio distributions across arrays. We identified 39 hsa-miRNAs with the largest variation across samples and examined the Spearman correlations of miRNAs with the Affymetrix probesets. We calculated false discovery rates for probe sets with stringent correlation coefficients. We performed hierarchical clustering using the 39 miRNAs and correlated dendograms branches with basal/luminal classes. Potential miRNA target genes were identified. We performed pathway analyses by using all 39 miRNA target genes. Western blot analyses were performed to confirm regulation of specific proteins. Results: We demonstrated 39 deregulated miRNAs and 10612 mRNA targets genes in BCC. Twenty-three miRNAs were upregulated in basal and downregulated in the luminal groups targeting 6790 genes, and 16 miRNAs downregulated in basal and upregulated in luminal cells, targeting 3822 genes. The first direct comparison screening for the 39 miRNAs and all mRNAs identified 29 direct pairs where the correlation magnitude was above 0.9. MiR-221/222, -31, -143/145, -34a, -200 were the most and consistently deregulated miRNAs in all luminal and basal like BCC. We identified genes, such as CD59, PDGF-β and discoidin domain receptor tyrosine kinase 1-DDR1 and different cancer network pathways, such as cell cycle and cell surface markers, potentially regulated by a subset of these miRNAs. Interestingly, we found that the expression of single genes might be regulated by several miRNAs. Conclusions: We found a specific miRNA signature and respective genome targets that well defined different clusters of BCC. Breast cancer cell lines are associated with changes in expression of multiple miRNAs that, in turn, disrupt a network of genes that either activate or inhibit important cellular functions. A better understanding of the network of genes and cellular pathways regulated by these miRNAs will enable to understand breast cancer pathogenesis and miRNA based breast cancer therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-03.

Transcriptional upregulation of DDR2 by ATF4 facilitates osteoblastic differentiation through p38 MAPK-mediated Runx2 activation

Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at -1150 bp in the DDR2 promoter was required for ATF4-mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast-specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2-induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation.

GRB7-dependent pathways are potential therapeutic targets in triple-negative breast cancer.

Abstract Abstract #25 Background: Breast cancer lacking expression of the estrogen and progesterone receptor and overexpression of HER2/neu (ie, "triple-negative” disease) accounts for about 10-15% of all breast cancer and is characterized by a higher risk of recurrence, early recurrence, resistance to cytotoxic therapy, and lack of any specific targeted therapy.
 Methods: We extracted RNA from primary tumor samples of 246 patients with stage I-III triple-negative breast cancer (confirmed in a central lab) treated with 4 cycles of adjuvant doxorubicin (60 mg/m2) plus cyclophosphamide (600 mg/m2) or docetaxel (60 mg/m2) who were enrolled on trial E2197, and correlated RNA expression (by quantitative RT-PCR using a panel of 371 rationally selected genes) with recurrence. There was no difference in recurrence between the two treatment arms in the entire study population, nor in the 246 patients in this analysis (of whom 59 recurred) after a median followup of 76 months.
 Results: Higher expression of GRB7 was the only gene significantly associated with an increased risk of recurrence (nominal p value 0.0000853, Korn's adjusted p value controlling false discovery at 10% (KP10) p=0.0359), but did not correlate with any clinicopathologic features except age (low expression associated with age > 65 years, p=0.03). In a Cox proportional hazards model adjusted for age, nodal status, tumor size, and grade, higher GRB7 expression was associated with an increased risk of recurrence when evaluated as a continuous variable (hazard ratio 3.41; p = 0.001) or as a dichotomous variable (hazard ratio 2.24 above vs. below median; p=0.006). The 5-year recurrence rates were 10.5% (95% C.I.7.8%, 14.1%) in the low and 20.4% (95% C.I. 16.5%, 25.0%) in the high GRB7 groups. There were only six genes whose expression correlated with GRB7 (r> 0.4), including ERBB2 (r=0.70), DDR1 (discoidin domain receptor tyrosine kinase 1; r=0.53), KRT19 (keratin 19; r=0.49), ERBB3 (r=0.48), GPR56 (G protein-coupled receptor 56; r=0.48) and PHB (prohibitin; r=0.42).
 Conclusions: GRB7 is a calmodulin-binding protein which has an SH2 (Src homology 2) domain that binds to phosphorylated tyrosine residues and other specific protein targets, and which plays a critical role in signaling (EGFR, HER2), motility (eprhins), migration (focal adhesion kinase), and cell-matrix/cell-cell interactions (integrins). Higher GRB7 RNA expression is associated with a significantly higher risk of recurrence in triple-negative breast cancer, indicating that GRB7 or GRB7-dependent pathways are potential therapeutic targets in triple-negative disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 25.