Identification Of Novel Driver Mutations Of The Discoidin Domain Receptor2(DDR2) Gene In Squamous Cell Lung Cancer Of Chinese Patients

Background and ObjectiveLung cancer incidence and mortality has become the highest which is one of the largest malignant threating to human health. Because of the limited palliation provided by surgery, chemotherapy, and radiation, the improvement in prognosis and survival of patients with lung cancer in the past20years is still unfavorable. Therefore, there is a need for better understanding of the genetic abnormalities in NSCLC cancers to identify and develop novel and effective targeted therapies. Encouraging new treatments have afforded benefits to patients with adenocarcinoma (ie EGFR tyrosine kinase inhibitors, ALK inhibitors) but, unfortunately, the same is not true for Squamous-cell carcinoma (SCC), which is the second most frequent histological subtype in non-small cell lung cancer. Given the burden of disease from lung SCC, identifying new therapeutic targets of mutated kinases is essential for lung SCCs.DDR2, a receptor tyrosine kinase that binds collagen Ⅰ and Ⅲ as its endogenous ligand, is known to increase expression of matrix metalloproteinases and has been previously shown to promote cell proliferation, migration and metastasis by regulating epithelial-mesenchymal transition (EMT). Recently, a3.8%incidence of DDR2mutations after sequencing analysis of290SCC of lung cancer has been reported and showed a gain-of-function phenotype that was abrogated by treatment with dasatinib, a multikinase inhibitor with activity against both DDR1and DDR2.The different incidence of DDR2mutations in the reports between United Stated and European patients suggests that demographics may also affect the rate of observed DDR2mutations. It is not clear whether there are differences between Chinese and Western patients, whether there are novel mutations in Chinese population and the novel mutations are the driver mutation.Materials and Methods1. DDR2was sequenced from DNA obtained from lung SCC patient samples by conventional Sanger sequencing. Mutations were identified using an automated mutation caller online (http://www.ncbj.nlm.njh.gov/blast/Blast.cgj?PAGE TYPE=BlastSearch&BLASTSPEC=OGP96069558) and then verified manually with comparison made to the matched normal sequence.2. To generate a DDR2and its mutated transcript expression vector, the entire sequence of human DDR2and mutatedDDR2was synthesized and subcloned into pEGFP-N1vector with incorporate external Nhel and BamHI sites, respectively. Lung SCC cells and HBE cell cultured on six-well plate were transfected with the pEGFP-DDR2, pEGFP-DDR2-S131C, pEGFP-DDR2-T681I or empty vector using Lipofectamine2000. Cell proliferation assay was performed by both MTT assay and colony formation assay. The change of cell migration ability in DDR2mutation overexpressed cells was observed through scratch assay. Cell migration and invasion in each transfected DDR2mutation cell lines were furthermore observed in Transwell experiment without and with Matrigel matrix respectively.3. Subcutaneous tumor implantation model was conducted by DDR2S131C overexpressed NCI-H1703cells and control cells which were screened by G418injecting in BALB/c-nu mice, with5×106cells in each mouse. Tumor growth was measured by calipers every3days. The tumors were removed from all of the animals after15days, and the subcutaneous growth of each tumor was examined. The tumor volumes were calculated using the equation V=0.5×D×d2(V, volume; D, longitudinal diameter; d, latitudinal diameter). Meanwhile, the expression of Ki67protein in paraffin sections which is a nuclear protein that is associated with and may be necessary for cellular proliferation was detected by immunohistochemical staining.4. The possible mechanism of tumor growth, migration and invasion of DDR2driver mutations was explained by the tested expressions of E-cadherin, N-cadherin and MMP-2/9were examined by qRT-PCR and western blot alone or both.Results1. Identified three novel recurrent somatic mutations of DDR2in86primary lung SCC samples. We performed Sanger sequencing of DDR2gene in an set of86primary lung SCC samples and identified three novel recurrent somatic mutations (G531V, S131C, T681I) in4samples in the tyrosine kinase genes: DDR2. Meanwhile, four synonymous mutations in7samples and one intron mutation in28samples were identified.2. DDR2-S131C overexpression promoted the cell proliferation, migration and invasion. We construct the DDR2wild type and S131C and T681I mutated DDR2expression plasmid vector (pEGFP-DDR2, pEGFP-DDR2-S131C, pEGFP-DDR2-T681I). Furthermore, MTT assay revealed that cell growth was significantly increased in HBE, HI703and SK-MES-1cells transfected with pEGFP-DDR2-S131C compared with cells transfected with empty vector, wildtype pEGFP-DDR2or pEGFP-DDR2-T681I vector. Similarly, the results of colony-formation assay also showed that clonogenic survival was increased following transfection of pEGFP-DDR2-S131C in HBE, H1703and SK-MES-1cells. We evaluated cancer cell invasion through matrigel and migration through wound healing and transwell assays. The results showed that DDR2-S131C overexpression promoted the migration and invasion compared with empty vector, wildtype pEGFP-DDR2or pEGFP-DDR2-T681I vector. These data indicated that only DDR2S131C mutation can promote the migratory and invasive phenotype of lung SCC cells.3. DDR2S131C mutation increased tumor growth in a xenograft mouse model. To further provide in vivo evidence for the oncogenic role of DDR2S131C mutation in lung SCC, we used a xenograft mouse model. Compared to the control treatment, DDR2-S131C over-expression dramatically increased tumor growth, which was demonstrated by significantly increased tumor size and weight. In addition, the HE staining showed the typical characteristics of tumor cells, and the proliferation index Ki67determined by immunohistochemical staining significantly upregulated in the pEGFP-DDR2-S131C transfected tumors. The DDR2-S131C mutation was the gain-of-function mutations.4. DDR2S131C mutation regulated the E-cadherin and MMP-2expression. We investigated the oncogenic role of DDR2S131C mutation by qRT-PCR and western blot analysis. The results showed that DDR2ovexpression could induce the MMP-2expression and decrease E-cadherin expression, while transfection of pEGFP-DDR2-S131C could induce more significantly changes in E-cadherin and MMP-2expression.Conclusion1. The incidence of DDR2mutations in Chinese primary lung SCC patients was4.6%and no difference of DDR2mutations incidence was found between Chinese and Western.Our results showed the in an overall frequency of4.6%(n=4) in86total primary lung SCC samples. The results suggested that there was no difference of DDR2mutations incidence was found between Chinese and Western. The S131C mutation was identified in the exon5, G531V and T681I mutations were found in exonl3and exonl5, respectively. There was no concentrated mutation in DDR2gene. The interesting findings need more efforts to test the implicit meaning. 2. DDR2-S131C mutation is the gain-of-function mutation and potential for the targeted therapy.DDR2-S131C over-expression dramatically increased tumor growth, cell migration and invasion partly via regulating E-cadherin and MMP-2expression. All the functions are the important oncogenic events, which mean the mutation is the gain-of-function mutation and be targeted therapy.3. DDR2-T681I mutation failed to promote the cell proliferation, migration and invasion, which means not all DDR2mutations are the gain-of-function mutations.The MTT, colony-formation, wound healing and transwell assays are proved the DDR2-T681I mutation failed to increase cell proliferation, promote the migration and invasion compared with empty vector, wildtype DDR2. These mean that not all DDR2mutations are the driver mutations, which suggest that targeting the DDR2mutations in some patients may be not a suitably therapeutic strategy.