Screening And Identification Of Lung Cancer Functional Genes By Functional Monoclonal Antibody Library

Lung cancer is one of the most common malignancies worldwide with highest incidence and mortality rate.However,the traditional curative methods have only limited effect on reducing the mortality of lung cancer.Presently molecular target therapy shows great promise in clinical treatment of cancer and became one of the hot research fields of cancer. To develop a effective targeting therapy,the initial and key step is to identify the tumor specially expressed molecules.To this end,a novel high-capacity functional monoclonal antibody library screening technique was employed,and then the functional genes was isolated by the functional antibodies.We then studied the functional relevance of these genes in the development of lung cancer and the underlying molecular mechanism.Part1.Screening and identification of lung cancer related functional genes by functional monoclonal antibody libraryBALB/c mice were immunized with cancerous cells isolated from fresh human lung cancer tissue.The spleen cells were fused with SP2/0 cells to establish a lung cancer functional monoclonal antibody library containing 1440 mAbs.By ICC 377 clones were identified which stably secrete mAbs against human lung tumor cells,271 of these clones were abandoned because of immunohistochemical reacting with normal lung epithelium. Then the remaining 106 clones were further assayed by tissue arrays and 67 of these clones showed predominantly tumor tissues reactivity.106 clones were screened by MTT method and 57 clones displayed ability of proliferation suppression to cancer cells,including inhibitory rate of 24 clones over 20%and 22 clones over 15%.RT-CES^TM system was also employed to estimate effects on cancer cells growth,the results showed that 49 clones can significantly inhibit cell growth of lung cancer,including inhibitory rate of 24 clones over 20%,20clones clones over 15%,The rest clones showed inhibitory rate by about 10-15%. Subclass of 106 clones identified,including IgM,IgA,IgG1,IgG2a and IgG2b and light/heavy chain includingκ,λ.Those result demonstrated that monoclonal antibodies of this library were diverse.27 mAbs were chosen from the antibody library for further estimating the inhibitory effect on carcinoma xenograft in nude mice.23 clones could significantly inhibit tumor growth and tumor weight.Among them,the inhibitory rates of six clones were above 70%,and eight were about 50-70%,6 of these clones were 30-50%,3 clones were under 30%.The result indicated that the antigens recognized by these mAbs might contribute to growth of lung cancer.5 mAbs were finally produced for antigen identification by immuno-affinity and MALDI-TOP-PMF.At last we acquired 3 functional genes including(CPS1) identified by1E2,RyR1 by 5B8,LGALS3BP by 13H3.Part2.Studies on the gene functions of Mac2-BPSeveral studies have reported that Mac2-BP was overexpressed in carcinoma tissues, however,so far few studies focus on the functional role of Mac2-BP in cancer progression. Therefore,we designed functional studies as follows to reveal the role of Mac2-BP in lung cancer progression as well as its potential application in clinic.We firstly applied RNA interference and immunoprecipitation to confirm the results of mass spectrometry.Then,cell immunofluorescence and cell immunocytochemisry showed that Mac2-BP was high expressed in most lung cancer cell lines.Immunohistochemistry assay on 105 case of lung cancers paired with normal lung tissues revealed that Mac2-BP was significantly overexpressed in cancer tissue,and the overexpression of Mac2-BP was correlated with lung cancer differentiation.13H3 mAb had no effects tumor cell growth,adhesion,migration and invasion in vitro,which might due to Mac2-BP was barely secreted by lung tumor cells in vitro.However,when purified 13H3 mAb was to used to treat lung cancer xenograft in vivo a significant tumor growth inhibition was observed by a dose-dependent manner,with the inhibition rates of 60.14%,54.86%and 43.84%for the high,middle and low dosage of 13H3 respectively.These results showed that 13H3 was a functional mAb and Mac2-BP was a lung cancer associated gene that involved in tumor growth.We then applied RNAi to further revealed the role of Mac-2BP in lung cancer development.Transient RNAi,revealed that downregulating Mac-2BP could suppress cell proliferation,cell adhesion with Matrix of CollagenⅠ,Matrigel or FN,cell migration and invasion by about 38.5%,40%,50%,35%, 27%and 29%respectively.Stable RNAi transfection also certified these result in vitro.We also applied ex vivo to study the role of Mac2-BP on xenograte tumor growth.Mac2-BP knockdown could inhibit 80%xenograft tumor burden.The Mac2-BP knockdown study once again showed that Mac2-BP was a functional gene associated with lung cancer cell proliferation,matrix adhesion,migration and invasion.To further study the mechanism of Mac2-BP on lung cancer cell proliferation,we assayed the cell cycle after RNAi and revealed that Mac2-BP knockdown lead to G1 arrest.Further studies revealed that downregulation of Mac2-BP was correlated with P27 upexpression.These results indicated that Mac2-BP promotes lung cancer cell proliferation in vitro and in vivo through down-regulation P27 to regulate cell cycle.Together,these results demonstrated that Mac2-BP gene was a novel potential functional target for lung cancer therapy.