3-Deazaadenosine mg DDR is characterized by an N terminal di
DDR2 is characterized by an N-terminal discoidin domain and a C-terminal kinase domain . Characteristics of DDR1, which is a homologue of DDR2, have been extensively investigated in terms of expression level in cancer tissues and biological functions in normal and cancer cell conditions, but characteristics of DDR2 remain unclear , , . Since DDR2 mutations in lung SQCC were discovered, DDR2 has commanded considerable attention as a new potential molecular target . So far, fifteen kinds of mutations of DDR2 have been reported in the discoidin domain, juxtamembrane region, and catalytic kinase domain. The frequency of each mutation is only a few percent, and hot spots of mutation do not exist so far , , . The T681I mutation in DDR2 was observed in a sample of lung SQCC and a lung SQCC cell line (PCT/JP2014/084755) in our study. Miao et al. similarly reported that T681I was detected in one among 54 lung SQCC patients . Some DDR2 mutations (L63V, S131C, and I638F) were likely to be activating mutations, and the others had no promoting biological effects compared to wild type. We found that DDR2 protein with T681I mutation was not phosphorylated by collagen I treatment and did not increase tumor metastasis in animal model, suggesting T681I mutation produces loss of function. Metastasis consists of multiple steps, such as increased cell mobility, disruption of basement membrane, circulation in vessels, and colonization in other tissues , . During this process, MMPs are involved in degradation of the basement membrane, one of the first steps of metastasis . Overexpression of MMPs is related with poor prognosis in patients with various cancers, and inhibition of MMPs leads to inhibition of cancer invasion and metastasis , . MMP-1 is transcriptionally regulated by the AP-1 complex in osteosarcoma 3-Deazaadenosine mg . As for our results on the time-course of phosphorylation status and protein changes induced by collagen I treatment, phosphorylation of DDR2 and c-Jun was observed in cells overexpressing DDR2. However, the phenomenon was not observed in cells expressing T681I. These results indicate that activation of DDR2 induces MMP-1 gene expression through phosphorylation of c-Jun, a component of AP-1, resulting in tumor metastasis in animal model. Huang TL et al. reported DDR2 − AP-1 − CYR61 − MMP-1 signaling pathway promoted bone erosion in rheumatoid arthritis through regulating migration and invasion of fibroblast-like synoviocytes . We did not observe significant overexpression of MMP-1 protein in whole cell lysates of H226B-WT cells (data not shown), and it is probably because most MMP-1 protein is secreted into the surrounding extracellular matrix , . The phosphorylation of DDR2 has been reported to regulate not only signal transduction but also protein stability. Yu, J. et al. showed that phosphorylated DDR2 underwent proteasome degradation through Cbl-b-mediated ubiquitination . We found Collagen I treatment induced DDR2 degradation and phosphorylation in wild type but not in T681I (Fig. 6B), suggesting inactive T681I mutant protein might be accumulated in tumor. In fact, we found DDR2 protein was overexpressed in 29% of SQCC clinical samples. Threonine at 681 of DDR2 might be a key amino acid to determine the amount of DDR2 protein, so in future research it is necessary to verify the correlation between DDR2 protein overexpression and the T681I mutation. DDR1 has been reported to promote invasion in a variety of human cancer cell lines, including prostate, breast, and lung , , . DDR1 is thought to upregulate expression of MMP components such as MMP-2 and MMP-9, resulting in degradation of matrix components . Although there exists scant evidence concerning DDR2, the DDR2 mutation S131C promoted proliferation, migration, and invasion by lung SQCC cells, and these processes were mediated by induction of MMP-2 . Reduction of DDR2 by siRNA inhibited the induction of Epithelial-Mesenchymal Transition (EMT) by TGF-β1 . In breast cancer, DDR2 stabilizes SNAIL1, which is transcriptionally regulated by TGF-β, resulting in induction of EMT . In our results, EMT phenotypes including SNAIL1 expression, down-regulation of E-cadherin, and up-regulation of Vimentin, were not induced by ectopic expression of DDR2 (data not shown). These results suggest that the DDR family, including DDR2, is involved in tumor progression, although the mechanism varies according to cancer cell type.