Functional Study On Two Lung Cancer Related Novel Genes, DENND2D And OLC1

Lung cancer is the leading cause of cancer death worldwide.There are still no effective methods for early detection and therapy of the malignant disease,and the 5 year survival rate of lung cancer patients is below 15%,due to poor understanding of the mechanisms involved in lung cancer development and progerssion. Identification and research on the oncogenes and the tumor suppressor genes will provide new targets of prevention and treatment to lung cancer.Two lung cancer-related novel genes had been obtained from the different expression cDNA libraries previously established in this lab,one is the candidate oncogene OLC1,the other is candidate tumor suppressor DENND2D.This project was mainly focused on the functional study for the two genes,especially on DENND2D.To understand expression status of DENND2D gene in human normal and pathological tissues,using Northern blotting analysis we detected the natural transcript of DENND2D with the predicted molecular size in lung,testis and placenta tissues,but there was no detectable expression of this gene in heart,brain and muscle tissues.Then we tested the expression levels of DENND2D with Real-Time PCR,in 16 pairs of tumor and normal lung tissue samples derived from patients of lung squamous cell carcinoma.There was 50%of the tumor samples showing decreased expression of DENND2D,setting 2-fold as a cutoff ratio.To explore the gene function,we construct a Tet-on inducible expression system with adenovirus as the vector,and successfully get the gene expression under control. Induced expression of DENND2D can directly induce apoptosis in lung cancer cells H1299,and the apoptosis incidence presented in manner of the time-dose dependent of DENND2D expression level.With induced DENND2D expression,the loss of mitochondria membrane potential,and the activity of Caspase 3 and Caspase 9 which is key regulator of mitochondria-apoptosis pathway were increased;whereas when treated the cells with general Caspase inhibitor and specific Caspase 9 inhibitor,the DENND2D induced apoptosis was significantly down-regulated.It suggests that DENND2D could induce Caspase dependent mitochondrial apoptosis.However, there was no experimental evidence support the DENND2D as a mitochondria protein, using fusion tagged protein to show this protein location.Moreover,we found that DENND2D could activate the JNK pathway,but the DENND2D induced apoptosis is in JNK independent manner because it was not affected by the JNK dominant negative mutant and the JNK inhibitor SP600125.Combine the results in lung cancer cell line A549 and H520,we could summary up that the DENND2D induced apoptosis was not affected by the endogenous DENND2D expression.With xenograft of H1299 cells in nude mice,we confirmed the tumor suppressor activity of DENND2D in vivo,and it also seemed that DENND2D could induce apoptosis in vivo.For study on OLC1 gene,an inducible adenovirus expression system of OLC1 was also successfully constructed.Several monoclonal antibodies against OLC1 protein were prepared in hope of measuring OLC1 protein levels in the circulating plasma.To identify the OLC1 gene fuction,the gene engineering mouse model were established,and both the general expression transgenic and the lung specific expression transgenic mouse showed the hyperplasia of typeâ…¡alveolar cells,giving the physiology function clue to this gene.Further investigations are still undertaking.