Discoidin Domain Receptor 1 Protein Research Articles

Discovery of LLC355 as an Autophagy-Tethering Compound for the Degradation of Discoidin Domain Receptor 1.

Discoidin domain receptor 1 (DDR1) is a potential target for cancer drug discovery. Although several DDR1 kinase inhibitors have been developed, recent studies have revealed the critical roles of the noncatalytic functions of DDR1 in tumor progression, metastasis, and immune exclusion. Degradation of DDR1 presents an opportunity to block its noncatalytic functions. Here, we report the discovery of the DDR1 degrader LLC355 by employing autophagosome-tethering compound technology. Compound LLC355 efficiently degraded DDR1 protein with a DC50 value of 150.8 nM in non-small cell lung cancer NCI-H23 cells. Mechanistic studies revealed compound LLC355 to induce DDR1 degradation via lysosome-mediated autophagy. Importantly, compound LLC355 potently suppressed cancer cell tumorigenicity, migration, and invasion and significantly outperformed the corresponding inhibitor 1. These results underline the therapeutic advantage of targeting the noncatalytic function of DDR1 over inhibition of its kinase activity.

Abstract 2916: Discoidin domain receptor 1 (DDR1) expression is associated with degree of immune exclusion across epithelial tumors

Abstract Background: Discoidin domain receptor 1 (DDR1) is highly expressed in epithelial cancers and has been implicated in tumor growth, invasion, and lack of response to therapy. DDR1 contributes to immune exclusion by promoting tumor collagen alignment in in vivo models. However, it is unclear how DDR1 expression impacts immune cell infiltration in human tumors. A first-in-human trial of PRTH-101, a DDR1-targeted therapeutic antibody, is underway. Establishing a correlation between DDR1 expression and immune infiltration in the tumor microenvironment (TME) will shed light on the role of DDR1 in the TME and inform indication and patient selection strategies for DDR1-targeted therapies. Methods: Adjacent formalin fixed paraffin embedded slides from colorectal (CRC), non-small cell lung (NSCLC), ovarian (OC), pancreatic (PDAC), and triple-negative breast cancers (TNBC) were stained by H&E and a multiplex immunofluorescence (mIF) panel containing DDR1 and immune cell markers CD8 and CD45. Tumor-stromal segmentation and cell identification was done from H&E using AI-powered models developed by PathAI (PathExplore™) or from mIF by image analysis using QuPath. DDR1 mRNA expression was measured by bulk RNA-sequencing, while DDR1 protein expression was measured by mIF. Immune Exclusion Scores (IESs) were calculated for each tumor based on lymphocyte density (from H&E), CD8+ T cell density (from mIF), or CD45+ cell density (from mIF). Each IES is the orthogonal distance between the coordinate of immune cell density in tumor epithelium and stroma, and the regression line representing equal density in epithelium and stroma. Correlations between lymphocyte, CD8, and CD45 IESs and DDR1 mRNA or protein expression levels were evaluated. Results: Both DDR1 mRNA and protein expression levels were significantly correlated with all Immune Exclusion Scores (IESs) measured by the distribution of lymphocytes (H&E), CD8+, or CD45+ cells (mIF) in tumor epithelium and stroma (R: 0.31-0.55, adjusted p: <0.001-0.01) at a pan-cancer level. While the degree of the correlation varied between tumor indications by IES immune cell type, pancreatic cancer exhibited the strongest correlation between the lymphocyte-based IES and DDR1 mRNA and protein expression (R: 0.48-0.54, adjusted p: 0.04-0.07). DDR1 mRNA was also moderately correlated with lymphocyte IES in NSCLC and TNBC (R: 0.37-0.48, adjusted p: 0.12-0.14). Conclusions: We developed a continuous scoring method to quantify the degree of immune exclusion in tumors based on the spatial distributions of lymphocytes, CD8+ T cells, and CD45+ immune cells from H&E and mIF images. DDR1 mRNA and protein expression are correlated with immune exclusion at both the pan-cancer and specific indication level. This adds additional insight into the role of DDR1 in human cancers and may be useful in selecting indications and stratifying patients for DDR1-targeted therapies. Citation Format: Xinwei Sher, Fredrick D. Gootkind, Thomas Schürpf, Florent Peyraud, Jean-Philippe Guégan, Antoine Italiano, G. Travis Clifton, Laura A. Dillon. Discoidin domain receptor 1 (DDR1) expression is associated with degree of immune exclusion across epithelial tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2916.

Prognostic significance of TM4SF1 and DDR1 expression in epithelial ovarian cancer.

Transmembrane 4 L6 family member 1 (TM4SF1) and discoidin domain receptor 1 (DDR1) are expressed in numerous types of cancer, but their expression in epithelial ovarian cancer and the association between their expression and patient prognosis are unclear. The present study aimed to explore the expression of TM4SF1 and DDR1 and their relationship with prognosis in epithelial ovarian cancer. Firstly, the Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) platforms were used to compare the expression levels of TM4SF1 and DDR1 in ovarian cancer and normal ovarian tissue, and Kaplan-Meier plotter was used to analyze the association between gene expression and patient prognosis. The proteins interacting with TM4SF1 and DDR1 were analyzed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways was conducted for the interacting proteins. Furthermore, immunohistochemical staining was performed to detect the expression of TM4SF1 and DDR1 protein in epithelial ovarian cancer tissue and to analyze the association between expression and prognosis. The Oncomine and GEPIA analyses showed that the expression levels of TM4SF1 and DDR1 were significantly higher in epithelial ovarian cancer than in normal ovarian tissue, and the analysis of clinical samples revealed that TM4SF1 and DDR1 were coexpressed in some cases. STRING analysis indicated that the TM4SF1 and DDR1 proteins interact with each other. The overall survival and progression-free survival of patients whose epithelial ovarian cancer coexpressed TM4SF1 and DDR1 were significantly shorter than those of patients lacking TM4SF1 and DDR1 coexpression. Multivariate analysis indicated that TM4SF1 and DDR1 protein coexpression was an independent prognostic factor. In summary, TM4SF1 and DDR1 proteins were coexpressed in some epithelial ovarian cancer tissues and appear to be adverse prognostic factors for epithelial ovarian cancer. In addition, TM4SF1 and DDR1 may have an interactive or mutual regulatory mechanism.

Discoidin domain receptor 1 promotes lung adenocarcinoma migration via the AKT/snail signaling axis.

Discoidin domain receptor 1 (DDR1), a member of receptor tyrosine kinase, has been implicated in tumor progression. However, the function and underlying mechanism of DDR1 in lung adenocarcinoma (LUAD) progression is unclear. Thus, we explored the molecular regulatory mechanism of DDR1 in the migration of LUAD. Transwell assays, wound healing assays and xenograft tumor assays were performed to study the function of DDR1 in the progression of LUAD. Immunoblotting and quantitative real-time polymerase chain reaction (RT-qPCR) were used to detect the expression levels of genes. Co-immunoprecipitation (co-IP) assays were performed to detect the interaction between DDR1 and AKT. Immunofluorescence and immunohistochemistry assays were used to determine the expression level of proteins in cells and tissues, respectively. DDR1 expression was significantly higher in LUAD tissues than in normal lung tissues, and the level of DDR1 was inversely correlated with prognosis in patients. We found that DDR1 promoted the migration and invasion of LUAD cells in vitro. Furthermore, ectopic expression of DDR1 in LUAD cells altered EMT-related markers expression. Importantly, the DDR1 protein interacted with AKT and phosphorylated AKT. The AKT inhibitor MK2206 interrupted Snail upregulation in DDR1-overexpressing LUAD cells. Finally, our study revealed that depletion of DDR1 attenuated LUAD cell migration in a tumor xenograft mouse model. Our findings uncovered that a high abundance of DDR1 increased the migration and invasion capability of LUAD cells via the AKT/Snail signaling axis and indicated that DDR1 could be a potential target for treating LUAD.

Expression and subcellular localization of Discoidin Domain Receptor 1 (DDR1) define prostate cancer aggressiveness

BackgroundThe Discoidin Domain Receptor 1 (DDR1) is one of the two members of a unique family of receptor tyrosine kinase receptors that signal in response to collagen, which has been implicated in cancer progression. Here, we examined the expression of DDR1 in prostate cancer (PCa), and assessed its potential value as a prognostic marker, as a function of grade, stage and other clinicopathologic parameters.MethodsWe investigated the association between the expression level and subcellular localization of DDR1 protein and PCa aggressiveness by immunohistochemistry, using tissue microarrays (TMAs) encompassing 200 cases of PCa with various Gleason scores (GS) and pathologic stages with matched normal tissue, and a highly specific monoclonal antibody.ResultsDDR1 was found to be localized in the membrane, cytoplasm, and nuclear compartments of both normal and cancerous prostate epithelial cells. Analyses of DDR1 expression in low GS (≤ 7[3 + 4]) vs high GS (≥ 7[4 + 3]) tissues showed no differences in nuclear or cytoplasmic DDR1in either cancerous or adjacent normal tissue cores. However, relative to normal-matched tissue, the percentage of cases with higher membranous DDR1 expression was significantly lower in high vs. low GS cancers. Although nuclear localization of DDR1 was consistently detected in our tissue samples and also in cultured human PCa and normal prostate-derived cell lines, its presence in that site could not be associated with disease aggressiveness. No associations between DDR1 expression and overall survival or biochemical recurrence were found in this cohort of patients.ConclusionThe data obtained through multivariate logistic regression model analysis suggest that the level of membranous DDR1 expression status may represent a potential biomarker of utility for better determination of PCa aggressiveness.

Epithelial polarization in 3D matrix requires DDR1 signaling to regulate actomyosin contractility.

Epithelial cells form sheets and tubules in various epithelial organs and establish apicobasal polarity and asymmetric vesicle transport to provide functionality in these structures. However, the molecular mechanisms that allow epithelial cells to establish polarity are not clearly understood. Here, we present evidence that the kinase activity of the receptor tyrosine kinase for collagen, discoidin domain receptor 1 (DDR1), is required for efficient establishment of epithelial polarity, proper asymmetric protein secretion, and execution of morphogenic programs. Lack of DDR1 protein or inhibition of DDR1 kinase activity disturbed tubulogenesis, cystogenesis, and the establishment of epithelial polarity and caused defects in the polarized localization of membrane-type 1 matrix metalloproteinase (MT1-MMP), GP135, primary cilia, laminin, and the Golgi apparatus. Disturbed epithelial polarity and cystogenesis upon DDR1 inhibition was caused by excess ROCK (rho-associated, coiled-coil-containing protein kinase)-driven actomyosin contractility, and pharmacological inhibition of ROCK was sufficient to correct these defects. Our data indicate that a DDR1-ROCK signaling axis is essential for the efficient establishment of epithelial polarity.

Prognostic Impact of Phosphorylated Discoidin Domain Receptor-1 in Esophageal Cancer

BackgroundEsophageal squamous cell carcinoma (ESCC) is common in East Asia and also is often deadly. We sought to determine whether measuring the discoidin domain receptor-1 (DDR1)—both total and phosphorylated proteins—could improve our ability to predict recurrence in ESCC. Materials and MethodsTotal DDR1 and phosphorylated DDR1 (pDDR1) were measured using semiquantitative immunohistochemistry in a cohort of 60 patients with ESCC. Association between these immunohistochemical measurements and standard clinical-pathological variables such as patient recurrence-free survival was examined using univariate and multivariate analyses. ResultsSix patients (10.0%) had regional recurrence and eight patients (13.3%) had distant recurrence. In univariate analysis, early disease recurrence correlated with intense staining of total DDR1 (P = 0.03) as well as intense staining of pDDR1 (P < 0.001). On multivariate analysis, only regional lymph node metastasis (P = 0.04, HR = 4.20) and intensity of pDDR1 immunohistochemistry (P = 0.03, HR = 4.27) emerged as significant independent prognostic factors for recurrence. ConclusionsThis study suggests that immunohistochemical measurements of both the DDR1 protein and pDDR1 can provide prognostic value in ESCC, even when other clinical and pathological factors are also being considered.

DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in a Genetic Mouse Model of Alport Syndrome.

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3–/– mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3–/– mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population.

Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors.

Acute lung injury (ALI) is a deadly symptom for serious lung inflammation. Discoidin Domain Receptor 1 (DDR1) is a new potential target for anti-inflammatory drug discovery. A new selective tetrahydroisoquinoline-7-carboxamide based DDR1 inhibitor 7ae was discovered to tightly bind the DDR1 protein and potently inhibit its kinase function with a Kd value of 2.2 nM and an IC50 value of 6.6 nM, respectively. The compound dose-dependently inhibited lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) release in mouse primary peritoneal macrophages (MPMs). In addition, 7ae also exhibited promising in vivo anti-inflammatory effects in a LPS-induced mouse ALI model. To the best of our knowledge, this is the first "proof of concept" investigation on the potential application of a small molecule DDR1 inhibitor to treat ALI.

IGF-I induces upregulation of DDR1 collagen receptor in breast cancer cells by suppressing MIR-199a-5p through the PI3K/AKT pathway.

Discoidin Domain Receptor 1 (DDR1) is a collagen receptor tyrosine-kinase that contributes to epithelial-to-mesenchymal transition and enhances cancer progression. Our previous data indicate that, in breast cancer cells, DDR1 interacts with IGF-1R and positively modulates IGF-1R expression and biological responses, suggesting that the DDR1-IGF-IR cross-talk may play an important role in cancer.In this study, we set out to evaluate whether IGF-I stimulation may affect DDR1 expression. Indeed, in breast cancer cells (MCF-7 and MDA-MB-231) IGF-I induced significant increase of DDR1 protein expression, in a time and dose dependent manner. However, we did not observe parallel changes in DDR1 mRNA. DDR1 upregulation required the activation of the PI3K/AKT pathway while the ERK1/2, the p70/mTOR and the PKC pathways were not involved. Moreover, we observed that DDR1 protein upregulation was induced by translational mechanisms involving miR-199a-5p suppression through PI3K/AKT activation. This effect was confirmed by both IGF-II produced by cancer-associated fibroblasts from human breast cancer and by stable transfection of breast cancer cells with a human IGF-II expression construct. Transfection with a constitutively active form of AKT was sufficient to decrease miR-199a-5p and upregulate DDR1. Accordingly, IGF-I-induced DDR1 upregulation was inhibited by transfection with pre-miR-199a-5p, which also impaired AKT activation and cell migration and proliferation in response to IGF-I.These results demonstrate that, in breast cancer cells, a novel pathway involving AKT/miR-199a-5p/DDR1 plays a role in modulating IGFs biological responses. Therefore, this signaling pathway may represent an important target for breast cancers with over-activation of the IGF-IR axis.

Abstract 5070: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis

Abstract Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by regulating the interactions of tumor cells with their surrounding collagen matrix. We investigated the role of DDR1 in the tumor progression of colorectal cancer. We examined the DDR1 mRNA and protein expression levels in a series of human colorectal cancer cell lines by RT-PCR and Western blotting. We also analyzed the DDR1 protein expression in 27 surgical specimens of colorectal cancer by immunohistochemistry. Migration, invasion, and proliferation assays were conducted with a high-content time-lapse assay system in a series of colon cancer cell lines. Nilotinib, a well-known DDR1 inhibitor, and DDR1-silenced colon cancer cells were used for inhibition of DDR1 tyrosine kinase activity. In an in vivo study, we orthotopically implanted KM12SM human colon cancer cells into the cecum of nude mice and administered nilotinib (100 mg/kg/day) by daily oral gavage. After 4 weeks of treatment, primary tumors and metastatic lesions were resected, and specimens were analyzed histologically. DDR1 mRNA and protein expression was observed in the human colon cancer cell lines and in cancer cells of most of the surgical specimens. Colon cancer cell lines KM12SM, KM12c, LoVo, and HT29 exposed to nilotinib (20 μM) showed reduced migration. When incubated with type I collagen, however, these cells showed increased migratory activity. Nilotinib treatment significantly inhibited this effect. The migratory activity of DDR1-silenced cells was not affected by nilotinib treatment. The orthotopic xenografts grew invasively as aggressive tumors with a stromal reaction, but under nilotinib treatment, the stromal reaction was reduced, and tumors grew expansively. In addition, the numbers of lymph node metastases were significantly reduced. Disruption of DDR1 attenuated colon cancer cell migration and led to altered primary tumor morphology and to impaired lymph node metastasis. DDR1 may be a promising therapeutic target in patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2015-5070

Abstract 5357: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) tyrosine kinase enhances cytotoxicity of anti-mesothelin immunotoxin for cancer therapy

Abstract Discoidin domain receptor 1 (DDR1) is an emerging anti-cancer target that belongs to the family of tyrosine kinases that are activated by collagen, the most abundant component of extracellular matrix (ECM). Collagen mediated induction of DDR1 facilitates cell adhesion, migration, proliferation and matrix remodeling. The collagen/DDR1 axis is also thought to modulate tumor-stromal interaction and potentially can affect tumor response to therapy. Mesothelin is a cell-surface tumor-associated antigen over-expressed in several human cancers including mesothelioma, ovarian, lung, breast, and pancreatic cancers with limited expression on normal cells. RG7787 is a clinically optimized recombinant immunotoxin (RIT) being developed in collaboration with Roche that should enter clinical trials late this year. It consists of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A in which immunogenic B cell epitopes are silenced. Activation of tyrosine kinase mediated signaling in tumor cells has been shown to modulate the activity of RITs. Therefore, we hypothesized that DDR1 regulates RIT activity and its inhibition might enhance the activity of RG7787. Knockdown of DDR1 by siRNA or its inhibition with a novel and specific inhibitor, ‘7rh’, synergistically enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed the knockdown of DDR1 increased mesothelin expression, internalization of RG7787, and enhanced inhibition of protein synthesis. Furthermore, stimulation of DDR1 activity by collagen treatment protected cancer cells from killing by RG7787. However, this collagen mediated resistance of cancer cells to RIT therapy can be overcome by addition of 7rh. Combination of 7rh with RG7787 synergistically inhibited growth of A431/H9 tumors in nude mice. In conclusion, we report that collagen protects cells from killing by RITs through DDR1 and that lowering DDR1 protein or inhibiting DDR1 kinase activity enhances the cytotoxic activity of RITs. Our data suggest that the combination of ‘7rh’ and RG7787 represents a novel therapeutic strategy to target mesothelin-expressing cancers. These data also provide insight into how tumor stroma might be protecting cancer cells from therapy. Citation Format: Fatima G. Ali-Rahmani, David Fitzgerald, Scott Martin, Paresma Patel, Craig Thomas, Ira Pastan. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) tyrosine kinase enhances cytotoxicity of anti-mesothelin immunotoxin for cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2015-5357

Abstract 5428: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) tyrosine kinase enhances cytotoxicity of anti-mesothelin immunotoxin

Abstract Discoidin domain receptor 1 (DDR1) is an emerging anti-cancer target that belongs to the family of tyrosine kinases that are activated by collagen, the most abundant component of extracellular matrix (ECM). Collagen mediated induction of DDR1 facilitates cell adhesion, migration, proliferation and matrix remodeling. The collagen/DDR1 axis is also thought to modulate tumor-stromal interaction and potentially can affect tumor response to therapy. Mesothelin is a cell-surface tumor-associated antigen over-expressed in several human cancers including mesothelioma, ovarian, lung, breast, and pancreatic cancers with limited expression on normal cells. RG7787 is a second-generation less immunogenic recombinant immunotoxin (RIT) consisting of an Fv portion of an anti-mesothelin antibody attached to a portion of Pseudomonas exotoxin A in which immunogenic B cell epitopes were silenced. Activation of tyrosine kinase mediated signaling in tumor cells has been shown to modulate the activity of RITs. Therefore, we hypothesized that DDR1 regulates RIT action and its inhibition might enhance the activity of RG7787. Knockdown of DDR1 by siRNA or its inhibition with a novel and specific inhibitor, ‘7rh’, synergistically enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed increased inhibition of protein synthesis in cells treated with combination of ‘7rh’ and RG7787. Furthermore, stimulation of DDR1 activity by collagen treatment protected cancer cells from killing by RG7787. In conclusion, we report that collagen protects cells from killing by RITs through DDR1 and that lowering DDR1 protein or inhibiting DDR1 kinase activity enhances the cytotoxic activity of RITs. Our data suggest that the combination of ‘7rh’ and RG7787 represents a novel therapeutic strategy to target mesothelin-expressing cancers. Citation Format: Fatima Ali-Rahmani, David Fitzgerald, Scott Martin, Paresma Patel, Craig Thomas, Ira Pastan. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) tyrosine kinase enhances cytotoxicity of anti-mesothelin immunotoxin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5428. doi:10.1158/1538-7445.AM2014-5428

OP1-05 INTEGRATIVE DNA METHYLATION AND GENE EXPRESSION ANALYSES IDENTIFIES DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) ASSOCIATION WITH IDIOPATHIC NONOBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyOutstanding Posters: Research1 Apr 2014OP1-05 INTEGRATIVE DNA METHYLATION AND GENE EXPRESSION ANALYSES IDENTIFIES DISCOIDIN DOMAIN RECEPTOR 1 (DDR1) ASSOCIATION WITH IDIOPATHIC NONOBSTRUCTIVE AZOOSPERMIA Ranjith Ramasamy, Alex Ridgeway, Josephine Addai, Larry Lipshultz, and Dolores Lamb Ranjith RamasamyRanjith Ramasamy More articles by this author , Alex RidgewayAlex Ridgeway More articles by this author , Josephine AddaiJosephine Addai More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.613AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogenesis is a complex process that involves proliferation, differentiation, and cell adhesion. Spermatogenic failure or non−obstructive azoospermia (NOA) results from mechanisms involved are incompletely understood. DDR1 is a member of a small subfamily of receptor tyrosine kinases that is involved in adhesion, migration, proliferation, apoptosis, cell morphogenesis and differentiation. Since, DDR1 is expressed in human post−meiotic germ cells of testis, we hypothesized that abnormal DDR1 expression could be a possible mechanism that can compromise spermatogenesis in a subset of men with idiopathic NOA. METHODS We used the high resolution Infinium 450K methylation array and compared fibrobalsts cultured from testicular biopsies of 19 NOA men and 4 fertile controls. Microarray data was analyzed using Minfi (R software package) utilizing subset−quantile within array normalization. We investigated the functional role of abnormal promoter DNA methylation for selected genes using mRNA expression by quantitative RT−real time PCR. Immunohistochemistry was used to confirm testicular expression and potential importance in spermatogenesis RESULTS Differentially methylated CpG sites (∼20K) were identified using an F−Test (p<0.05) in the NOA samples. We identified 24 genes with the >30% difference in methylation within promoter region of men with NOA and fertile controls. Of the aberrantly methylated CpGs, 13 were hypomethylated and 11 were hypermethylated groups. From the top 11 hypermethylated genes, six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, NPHS2) were selected for validation since they were shown to be expressed in testis. Of the 6 genes validated with qPCR, DDR1 showed aberrant gene expression pattern. Four (21%) patients out of the 19 NOA men had lower expression levels (1.8x) of DDR1, whereas two (10.5%) men had higher expression levels (2.5x) of DDR1compared to fertile men (p<0.05). Immunohistochemical analysis suggests presence of DDR1 within cytoplasm of germ cells in fertile men and men with maturation arrest histology. DDR1 protein is absent in men with Sertoli−cell only or germ cell aplasia. CONCLUSIONS Aberrant expression of DDR1 is associated with NOA. The functional relevance of abnormal methylation of DDR1 to NOA warrants further investigation © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e163-e164 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ranjith Ramasamy More articles by this author Alex Ridgeway More articles by this author Josephine Addai More articles by this author Larry Lipshultz More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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Introduction: Integrins are crucial for migration of leukocytes through endothelium in systemic inflammatory response syndrome (SIRS). Some reports suggested integrins independent mechanisms appeared to regulate the migration of leukocytes through inflammatory extracellular matrix. We identified the up-regulation of discoidin domain receptor 1 (DDR1) in the genes profile of activated neutrophils by cDNA arrray. DDR1 has two major isoforms, DDR1a and DDR1b, which are a receptor tyrosine kinase activated by collagen. Methods: We extracted human leukocytes from bloods of healthy donors for in-vitro assay. The expression of DDR1 mRNAs and proteins in leukocytes was examined by Northern blotting, RT-PCR, IP-Western Blotting, In-situ hybridization in a various condition. DDR1a- or DDR1b -overexpressing THP-1 cells were analyzed the migration and adhesion with or without collagen. Results: Isolated neutrophils and PBMC expressed DDR1 mRNA and protein that was increased by LPS, TNF alpha or IL-1 beta, but not by IFN gamma. Infiltrating leukocytes expressed DDR1 mRNA in human stromal tissues by in-situ hybridization. DDR-1a and -1b expressing THP-1 cells resulted in increased adherence to collagen-coated plates in a beta1-integrin independent manner. DDR1a-, but not DDR1b-, overexpressing cells exhibited marked pseudopod extension and migrated successfully through three-dimensional collagen lattices. Conclusions: The interaction of DDR1a with stromal collagen enhanced leukocytes migration in inflammatory tissue microenvironment. Regulation of DDR1a might contribute to host defense in SIRS.

Involvement of discoidin domain 1 receptor in recurrence of hepatocellular carcinoma by genome-wide analysis

Hepatocellular carcinoma (HCC) is highly invasive with a high frequency of recurrence following surgery and poor prognosis. The underlying molecular mechanisms for HCC recurrence are not well understood. Here, we used microarray technology for genome-wide analysis to identify genes who may be involved in tumor recurrence. cDNA from HCC tumor tissues of patients with early recurrence (ER; n = 10) and patients whose HCC had not recurred ≥ 2 years postsurgery (nER; n = 10) was hybridized to the Affymetrix Human Geome U133 plus 2.0 whole-genome microarray. Gene clusters were identified and used for hierarchial clustering and principal component analysis. Genes with more than twofold change in expression between ER and nER groups were further analyzed. Expression levels of a subset of genes were validated using RT-PCR and immunohistochemistry. A total of 1,646 genes had significantly different expression between the ER and nER groups (P < 0.05) with 61 and 49 genes in the ER upregulated and downregulated for more than twofold in comparison with the nER group, respectively. The cellular functions of differentially expressed genes included cell adhesion, motility, cytoskeleton, transcription, metabolism, signal transduction, and apoptosis. The discoidin domain receptor 1 (DDR1) mRNA expression was significantly higher in the ER (3.36 ± 0.39) compared with the nER group (3.01 ± 0.49; P = 0.020). A greater proportion of liver tissue samples from ER versus nER patients had DDR1 protein expression (80.0 vs. 40.0 %, P = 0.022). Using microarray technology, we identified a number of genes whose expression differed between patients with recurrent HCC compared to those without. DD1 mRNA and protein levels were higher in patients with recurrent HCC, suggesting this gene maybe involved in tumor invasion and metastasis.

Discoidin Discoveries

In this issue of Structure, Carafoli et al. investigate the mode of antibody-mediated inhibition of the discoidin domain receptor 1 (DDR1). These studies also provide new insight into activation of the DDRs, which are unique among receptor tyrosine kinases in the composition of their extracellular regions.

The Discoidin domain receptor 1 gene has a functional A2RE sequence

Discoidin domain receptor 1 (DDR1) is expressed in myelin oligodendrocytes and co-localizes with myelin basic protein (MBP). Alternative splicing of DDR1 generates five isoforms designated DDR1a-e. The MBP mRNA contains an hnRNP A2 response element (A2RE) sequence that is recognized by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, which is responsible for transport of the MBP mRNA to oligodendrocyte processes. We hypothesized that DDR1 could have a functional A2RE sequence. By in silico analysis, we identified an A2RE-like sequence in the human DDR1 mRNA. We observed nuclear and dendrite cytoplasmic immunofluorescence, indicating that DDR1 and hnRNP A2/B1 co-localize in human oligodendrocytes and in differentiated HOG16 cells. The A2RE-like sequence of DDR1 contains the single nucleotide polymorphism rs2267641, and we found that in the human brain, the minor allele is associated with lower and higher levels DDR1b and DDR1c mRNA expression, respectively. Moreover, a positive correlation between DDR1c and the myelin genes myelin-associated glycoprotein and oligodendrocyte lineage transcription factor 2 was found. Differentiated HOG16 cells transfected with an hnRNP A2/B1 siRNA simultaneously show a decrease and an increase in the DDR1c and DDR1b mRNA expression levels, respectively, which was accompanied by a decrease in DDR1 protein levels at the cytoplasmic edges. These results suggest that the DDR1 A2RE sequence is functionally involved in the hnRNP A2/B1-mediated splicing and transport of the DDR1c mRNA.

Discoidin Domain Receptor 1: Isoform Expression and Potential Functions in Cirrhotic Human Liver

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

Identification of Receptor Tyrosine Kinase, Discoidin Domain Receptor 1 (DDR1), as a Potential Biomarker for Serous Ovarian Cancer

Ovarian cancer, one of the most common gynecological malignancies, has an aggressive phenotype. It is necessary to develop novel and more effective treatment strategies against advanced disease. Protein tyrosine kinases (PTKs) play an important role in the signal transduction pathways involved in tumorigenesis, and represent potential targets for anticancer therapies. In this study, we performed cDNA subtraction following polymerase chain reaction (PCR) using degenerate oligonucleotide primers to identify specifically overexpressed PTKs in ovarian cancer. Three PTKs, janus kinase 1, insulin-like growth factor 1 receptor, and discoidin domain receptor 1 (DDR1), were identified and only DDR1 was overexpressed in all ovarian cancer tissues examined for the validation by quantitative real-time PCR. The DDR1 protein was expressed in 63% (42/67) of serous ovarian cancer tissue, whereas it was undetectable in normal ovarian surface epithelium. DDR1 was expressed significantly more frequently in high-grade (79%) and advanced stage (77%) tumors compared to low-grade (50%) and early stage (43%) tumors. The expression of the DDR1 protein significantly correlated with poor disease-free survival. Although its functional role and clinical utility remain to be examined in future studies, our results suggest that the expression of DDR1 may serve as both a potential biomarker and a molecular target for advanced ovarian cancer.