RESUMO
Didscoidin domain receptor 1 (DDR1) is involved in the progression of prostate cancer metastasis through stimulation of epithelial-mesenchymal transition (EMT). So DDR1 inhibition can be a helpful target for cancer metastasis prevention. So, we studied the effects of DDR1 inhibition on EMT as well as induction of cell-cycle arrest and apoptosis in prostate cancer cell lines. DDR1 expression was evaluated using reverse-transcription polymerase chain reaction and western blot analysis. The EMT-associated protein expression was determined using the western blot analysis and immunocytochemistry following treatment with various concentrations of DDR1 inhibitor. The activation of DDR1 and also downstream-signaling molecules Pyk2 and MKK7 were determined using western blot analysis. Cell survival and proliferation after DDR1 inhibition were evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, and colony formation assays. Flow cytometry analysis was used to determine the effects of DDR1 inhibition on cell-cycle arrest and apoptosis using annexin V/propidium iodide-based flow cytometry. Results showed that the protein expression of N-cadherin and vimentin were decreased whereas protein expression of E-cadherin was increased after DDR1 inhibition. Results of our western blot analysis indicated that DDR1 inhibitor effectively downregulated P-DDR1, P-Pyk2, and P-MKK7 levels. This result also showed that DDR1 inhibition decreased cell survival and proliferation, induced G1 cell-cycle arrest, induced apoptosis by an increase in the Bax/Bcl-2 ratio and depletion of the mitochondrial membrane potential, and also by reactive oxygen species creation in prostate cancer cells. These data show that DDR1 inhibition can result in the EMT prevention via inhibition of Pyk2 and MKK7 signaling pathway and induces cell-cycle arrest and apoptosis in prostate cancer cell lines. Thus, this study identifies DDR1 as an important target for modulating EMT and induction of apoptosis in prostate cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/genética , Receptor com Domínio Discoidina 1/genética , Neoplasias da Próstata/genética , Antineoplásicos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Receptor com Domínio Discoidina 1/antagonistas & inibidores , Transição Epitelial-Mesenquimal/genética , Quinase 2 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MAP Quinase Quinase 7/genética , Masculino , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/genética , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Proteína X Associada a bcl-2/genéticaRESUMO
Background: Circular RNA hsa_circ_0087378 (circ_0087378) has been found to have different functions in different cancer types. However, its function in non-small cell lung cancer (NSCLC) remains unclear. This study revealed the effect of circ_0087378 on the malignant behavior of NSCLC cells in vitro to broaden the options for NSCLC treatment. Methods: This study detected the expression of circ_0087378 in NSCLC cells via real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The discoidin domain receptor 1 (DDR1) protein in NSCLC cells was investigated through western blot. The influence of circ_0087378 on the malignant behavior of NSCLC cells in vitro was investigated by cell counting kit-8 assay, colony formation assay, Transwell assay, and flow cytometry. Dual-luciferase reporter gene assay and RNA pull-down assay were performed to verify the binding between two genes. Results: Circ_0087378 was abundantly expressed in NSCLC cells. The loss of circ_0087378 repressed the proliferation, colony formation, migration, invasion, but enhanced the apoptosis in NSCLC cells in vitro. Circ_0087378 could repress microRNA-199a-5p (miR-199a-5p) by acting as a sponge. The loss of miR-199a-5p abrogated the inhibition of circ_0087378 loss on the malignant phenotype of NSCLC cells in vitro. DDR1 was directly repressed via miR-199a-5p. DDR1 counteracted the repressive role of miR-199a-5p on the malignant behavior of NSCLC cells in vitro. Conclusions: Circ_0087378 promotes the malignant behavior of NSCLC cells in vitro by facilitating DDR1 via sponging miR-199a-5p. It may be a promising target for treatment.
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Background: Hepatocellular carcinoma (HCC) is a malignant cancer with rapid progression, vascular invasion, a high recurrence rate and poor prognosis, so it is necessary to take early measures to halt this process. Accumulating evidence indicates that collagen XV (translated by Col15a1) is a basement membrane molecule related to tumour metastasis in several organs. However, the potential function of collagen XV in the liver associated with HCC remains to be further elucidated. Methods: Col15a1 was overexpressed in HepG2 and HCCLM3 cells. CCK8 and colony formation assays were used to assess the capacity of cell proliferation, and Transwell and wound healing assays were utilized to measure cell migration. Western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) quantified the protein and mRNA expression levels of genes related to the epithelial-mesenchymal transition (EMT). Then, the effect of collagen XV on tumour metastasis was confirmed in vivo. Finally, we inhibited discoidin domain receptor 1 (DDR1) via DDR1-IN-1 to explore whether the collagen XV interacted with DDR1 to regulate EMT. Results: Patients of HCC with higher expression of Col15a1 showed better survival than patients with low expression. Overexpression of collagen XV in HepG2 and HCCLM3 cells suppressed cell proliferation and migration in vitro and inhibited pulmonary and liver metastasis in vivo. In addition, collagen XV downregulated the DDR1 and transcription factor (Snail, Slug), regulated the EMT markers (Vimentin, E-cadherin, N-cadherin, and MMP9). Furthermore, inhibition of the DDR1 receptor by DDR1-IN-1 suppressed the gene promoting the EMT. Conclusions: Collagen XV functioned as a metastasis inhibitor in HCC by regulating the DDR1-Snail/Slug axis to regulate EMT.
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Dysregulation of the discoidin domain receptor (DDR1), a collagen-activated receptor tyrosine kinase, has been linked to several human cancer diseases including non-small cell lung carcinoma (NSCLC), ovarian cancer, glioblastoma, and breast cancer, in addition to several inflammatory and neurological conditions. Although there are some selective DDR1 inhibitors that have been discovered during the last two decades, a combination of elevated cytotoxicity, kinome selectivity and/or poor DMPK profile has prevented more in-depth studies from being performed. As such, no DDR1 inhibitor has reached clinical investigation to date, forming an urgent need to develop specific DDR1 inhibitor(s) using various drug discovery means. However, the recent discovery of VU6015929, a potent and selective DDR1 kinase inhibitor, with enhanced physiochemical and DMPK properties in addition to its clean kinome profile marked a milestone in the development of DDR1 inhibitors. Herein, VU6015929 was used to construct a 3D e-pharmacophore model which was validated via calculating the difference of score between the active compounds and decoys. The validated e-pharmacophore model was then utilized to screen 20 million drug-like compounds obtained from the freely accessible Zinc database. The generated hits were ranked using high throughput virtual screening technique (HTVS), and the top 8 small molecules were subjected to a molecular docking study and MM-GBSA calculations. Protein-ligand complexes of compounds 1, 2, 3 and the standard compound (VU6015929) were performed for 100 ns and compared with the DDR1 unbound protein state and the DDR1 bound to a co-crystallized ligand. The molecular docking, MD and MM-GBSA outputs revealed compounds 1-3 as potential DDR1 inhibitors, with compound 2 displaying superior binding affinity, comparable binding stability and average binding free energy for the ligand-enzyme complex compared to VU6015929.
Assuntos
Receptor com Domínio Discoidina 1 , Simulação de Dinâmica Molecular , Receptor com Domínio Discoidina 1/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Neoplasias/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The insulin receptor isoform A (IR-A), a dual receptor for insulin and IGF2, plays a role in breast cancer (BC) progression and metabolic reprogramming. Notably, discoidin domain receptor 1 (DDR1), a collagen receptor often dysregulated in cancer, is involved in a functional crosstalk and feed forward loop with both the IR-A and the insulin like growth factor receptor 1 (IGF1R). Here, we aimed at investigating whether DDR1 might affect BC cell metabolism by modulating the IGF1R and/or the IR. To this aim, we generated MCF7 BC cells engineered to stably overexpress either IGF2 (MCF7/IGF2) or the IR-A (MCF7/IR-A). In both cell models, we observed that DDR1 silencing induced a significant decrease of total ATP production, particularly affecting the rate of mitochondrial ATP production. We also observed the downregulation of key molecules implicated in both glycolysis and oxidative phosphorylation. These metabolic changes were not modulated by DDR1 binding to collagen and occurred in part in the absence of IR/IGF1R phosphorylation. DDR1 silencing was ineffective in MCF7 knocked out for DDR1. Taken together, these results indicate that DDR1, acting in part independently of IR/IGF1R stimulation, might work as a novel regulator of BC metabolism and should be considered as putative target for therapy in BC.
Assuntos
Neoplasias da Mama/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Feminino , Humanos , Células MCF-7RESUMO
The discoidin domain receptor-1 (DDR1) is a non-integrin collagen receptor recently implicated in the collective cell migration of other cancer types. Previously, we identified an elevated expression of DDR1 in oral squamous cell carcinoma (OSCC) cells. Through the data mining of a microarray dataset composed of matched tumor-normal tissues from forty OSCC patients, we distilled overexpressed genes statistically associated with angiolymphatic invasion, including DDR1, COL4A5, COL4A6 and PDPN. Dual immunohistochemical staining further confirmed the spatial locations of DDR1 and PDPN in OSCC tissues indicative of collective cancer cell invasion. An elevated DDR1 expression at both the transcription and protein level was observed by treating keratinocytes with collagen of fibrillar or basement membrane types. In addition, inhibition of DDR1 kinase activity in OSCC TW2.6 cells disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues. Taken together, these results suggest that collagen deposition in the affected tissues followed by DDR1 overexpression could be central to OSCC tumor growth and angiolymphatic invasion. Thus, DDR1 inhibitors are potential therapeutic compounds in restraining oral cancer, which has not been previously explored.
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BACKGROUND: In cancer cells, re-activation of Epithelial-Mesenchymal Transition (EMT) program through Discoidin Domain Receptor1 (DDR1) leads to metastasis. DDR1-targeted therapy with siRNA might be a promising strategy for EMT inhibition. Therefore, the aim of this study was to investigate the effect of DDR1 knockdown in the EMT, migration, and apoptosis of prostate cancer cells. For this purpose, the expression of DDR1 was down regulated by the siRNA approach in LNcap-FGC and DU-145 prostate cancer cells. METHODS: Immunocytochemistry was carried out for the assessment of EMT. E-cadherin, N-cadherin, Bax, Bcl2, and the phosphorylation level of Proline-rich tyrosine kinase 2 (Pyk2) and Map Kinase Kinase 7 (MKK7) was determined using the western blot. Wound healing assay was used to evaluate cell migration. Flow cytometry was employed to determine the apoptosis rate in siRNA-transfected cancer cells. RESULTS: Our findings showed that the stimulation of DDR1 with collagen-I caused increased phosphorylation of Pyk2 and MKK7 signaling molecules that led to the induction of EMT and migration in DU-145 and LNcap- FGC cells. In contrast, DDR1 knockdown led to significant attenuation of EMT, migration, and phosphorylation levels of Pyk2 and MKK7. Moreover, DDR1 knockdown via induction of Bax expression and suppression of Bcl-2 expression induces apoptosis. CONCLUSION: Collectively, our results indicate that the DDR1 targeting with siRNA may be beneficial for the inhibition of EMT and the induction of apoptosis in prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Receptor com Domínio Discoidina 1/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Quinase 2 de Adesão Focal/antagonistas & inibidores , MAP Quinase Quinase 7/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Apoptose/efeitos dos fármacos , Receptor com Domínio Discoidina 1/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quinase 2 de Adesão Focal/metabolismo , Humanos , MAP Quinase Quinase 7/metabolismo , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase which utilizes collagen as a ligand to regulate the interaction between cancer cells and tumor stroma. However, the clinical relevance of DDR1 expression in bladder cancer as well as its molecular regulation have not been previously investigated. Here, we assessed the role of DDR1 in bladder cancer. The DDR1 levels in bladder cancer specimens were examined by Western blot, compared to the paired adhesive normal controls. The effects of DDR1 were explored on both cell migration in bladder cancer cells and tumor growth as xenograft. We detected significant higher levels of DDR1 in bladder cancer tissues. Moreover, high levels of DDR1 were correlated with poor prognosis of corresponding patients. Both the in vitro cell invasiveness and in vivo tumor xenograft growth could be promoted by the overexpressed DDR1, while both of which could be inhibited after the depletion of DDR1. Furthermore, DDR1 increased the levels of ZEB1 and Slug, based on its effects on tumor invasion. In conclusion, DDR1 may promote the aggressiveness of bladder cancer cells and drive an aggressive phenotype in bladder cancer.
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BACKGROUND: Discoidin Domain Receptor 1 (DDR1) belongs to the family of collagen receptor tyrosine kinases that confers the progression of various cancers. Aberrant expression of DDR1 was detected in several human cancers including ovarian cancer, which had been shown to increase the migration and invasion of tumor cells. However, the precise mechanisms underlying the abnormal expression of DDR1 in ovarian cancer has not been well investigated in previous studies. RESULTS: In this work, a negative correlation between DDR1 and a tumor suppressor miRNA, miR-199a-3p, was observed in ovarian cancer tissues. Furthermore, in vitro experimental results confirmed that miR-199a-3p decreased the expression of DDR1 via targeting the 3'UTR of DDR1 mRNA. To explore the mechanisms for miR-199a-3p silence in ovarian cancer, the methylation status of the miR-199a promoter was analyzed in ovarian epithelial or cancer cells by methylation-specific PCR and bisulphite sequencing. As expected, the miR-199a promoter was hypermethylated in ovarian cancer cells but not in normal ovarianepithelial cells. Interestingly, knockdown of DNA methyltransferase 3A (DNMT3A) notably increased miR-199a-3p level and then attenuated the expression of DDR1 in ovarian cancer cells, which suggested that DNMT3A was responsible for the miR-199a promoter hypermethylation. Phenotype experiments showed that overexpression of miR-199a-3p significantly impaired the migratory, invasive, and tumorigenic capabilities of ovarian cancer cells as well as enhanced cisplatin resistance through inhibiting DDR1 expression. CONCLUSION: These findings demonstrate a critical role of miR-199a-3p/DDR1 pathway in ovarian cancer development.