Suppression Of Non-Small Cell Lung Cancer Proliferation And Tumorigenicity By DENND2D A Study On Transcriptional Regulatory Mechanism Of Brk1in Non-small Cell Lung Cancer

Background:DENND2D was identified as being down-regulated in lung cancer using a lung cancer low-expression suppression subtractive hybridization (SSH) library in our previous study. To date, there is no report as to the biological function of DENND2D in NSCLC.Objective:In this study, we sought to assess the expression pattern in lung tumorigenesis and the functional role of DENND2D in non-small cell lung cancer.Methods:DENND2D mRNA expression level of15primary cultured bronchial epithelial cell strains (PBEs), immortalized human bronchial epithelial (IHBE) cell lines and27lung cancer tissues were measured by PCR. DNA copy number, mRNA and protein expression level of9non-small cell lung cancer (NSCLC) cell lines were measured. DENND2D protein expression levels of lung cancer tissues, precancerous lesions and adjacent normal bronchial epithelial tissues were all measured in primary lung cancer samples from97patients by immunohistochemistry (IHC) and compared using Fisher’s exact test. Cell proliferation assay and foci formation assay were used to test the proliferation inhibition property of DENND2D. Tumor formation was assayed in athymic mice (six mice) to test the tumor formation property of HI299cells that stably overexpressed DENND2D. To test whether DENND2D overexpression induces apoptosis and the apoptosis rate, western blot and fluorescence-activated cell sorting (FACS) were used. CCK-8assay was used to test the proliferation of different concentration of cis-platinum treated and DENND2D over-expressed H1299cells, then IC50was calculated. At last, PARP1and PAR protein expression was detected by using western blot.Results:DENND2D mRNA was present in PBEs (15/15) but absent in IHBE cell lines (3/3) and NSCLC cell lines (8/9). Relative DNA copy number was diminished in NSCLC cell lines (6/9). Compared to adjacent normal tissues, DENND2D mRNA (13/27) and protein expression (23.6%vs81.5%, p<0.001) were both significantly down-regulated in tumor tissues. IHC data showed that the protein expression levels of DENND2D were gradually decreased in tumor tissues (IRS=0.90), precancerous lesions (IRS=1.38) compared to adjacent normal bronchial tissues (IRS=1.97). Functionally, overexpression of DENND2D inhibited lung cancer cell proliferation, foci formation and anchorage-independent growth in vitro. It also significantly inhibited tumorigenicity in vivo. Moreover, overexpression of DENND2D induced cleaved PARP, one of the apoptosis markers, significantly down regulated. FACS data showed that the apoptosis rate was related with the protein expression level of DENND2D. The CCK-8data showed that IC50of DENND2D group was remarkably lower than the control group. PARP1and PAR were significantly down regulated in DENND2D over expressed H1299cells.Conclusion:The mechanism of DENND2D down-regulation is very complex. The down-regulation of DENND2D is an early event in lung tumorigenesis, and DENND2D may play an important role in the proliferation and tumorigenicity of NSCLC. The two main functions f DENND2D in NSCLC cells is apoptosis inducing and chemotherapy sensitivity enhancement of cis-platinum. Background:Brkl is one of the most important genes in Rac-Arp2/3pathway, and its abnormal expression may be an important factor causing non-small cell lung cancer metastatic potential.Objective:In this study, we attempt to reveal the mechanism of high expression of Brkl in non-small cell lung cancer on transcriptional level.Methods:We used transcription factor prediction tools to calculate the compact district of transcript factor from5’flanking region of Brkl gene, and then we amplified series of DNA fragments. These fragments were cloned into the luciferase reporter gene plasmids. These recombinant plasmids were transfected into non-small cell lung cancer cells and the luciferase activity was tested using Dual-Luciferase Reporter Assay System (DLR). Fragments which made high luciferase activity were short-cutted for truncated fragments. Use this method, the core promoter region of Brkl was found. Microarray data was used to screen out metastasis related transcription factors, the expression of which was positively correlated with the expression of Brkl in non-small cell lung cancer. These transcription factors were co-transfected into non-small cell lung cancer cells with recombinant luciferase plasmid to test the luciferase activity. Transcription factor prediction tools were used to calculate the transcript factor binding sites. These sites were mutated and luciferase activities were tested.Results:Fragment F1、F3、F5and F8were amplified and identified by DNA sequencing. Luciferase activities of fragments F3and F8were significantly higher than empty vector pGL4.26. Candidate weak promoter region (-3661—3212), candidate enhancer region (-1314—934) and core promoter region (-94—1, S831) were found by testing the luciferase activity of truncated fragments of F3and F8. Eight metastasis related transcription factors were screened out from138non-small cell lung cancer tissues microarray data. In8transcription factors, only jun can improve the luciferase activity of S831in A549and HI299, but not in H520. The mutation of Spl binding site caused the transcriptional activity is significantly down regulated (>90%).Conclusion:The position-78—74upstream of Brkl gene transcriptional initiation site where Sp1binded may be the core promoter region of Brkl gene.