The Role And Mechanism Of Recombinant Ubiquitin Ligases In The Targeted Therapy Of Lung Cancer

Worldwide, lung cancer is the most common cause of cancer-related death inmen and women, the most common type of lung cancer is non-small-cell lungcancer(NSCLC). Epidermal growth factor receptor(EGFR) gene can be detectedin most patients with non-small-cell lung cancer. The epidermal growth factorreceptor is a member of a family of closely related growth factor receptortyrosine kinases that includes EGFR (ErbB1), HER2/neu (ErbB2),HER3,(ErbB3), and HER4(ErbB4). Because EGFR is expressed in a majority ofnon–small cell lung carcinomas (NSCLC), it has been an attractive target for thedevelopment of therapeutic agents. The small-molecule EGFR tyrosine kinaseinhibitors (TKI), including gefitinib and erlotinib, have been evaluated in clinicaltrials for patients with NSCLC. Both agents produce partial responses in10%to20%of all NSCLC patients. But acquired resistance is the major clinical problemfaced with almost all patients responsive to EGFR-TKIs. Mechanisms of acquiredresistance reported in the past few years include secondary mutation of the EGFRgene, amplifcation of the MET gene, and overexpression of HGF; We hope novelpharmaceutical agents can be devised to promote the treatment of lung cancer.Ubiquitylation-induced degradation is a main negative regulating method of protein in cytoplasm. Ubiquitylation is a process that finished byubiquitin-activating enzyme (E1), ubiquitin-conjunction enzyme (E2) andubiquitin-ligase (E3), ubiquitylate the substrate and make it degraded inproteasome. E3plays a key role in this process, it can identify and combine withthe substrate specifically, and transfer ubiquitin to it. In this research, we useU-box domain of CHIP and RING(Y371E) domain of Cbl, both of them have theactivation of E3, catalyze and degrade EGFR to achieve the aim of cancertherapy.As an efficient tool to degrade the special protein, we hoped it can solvemore problem of patients with lung cancer, especially the advanced lung cancer.Bone metastases are a frequent complication of lung cancer, once tumorsmetastasize to bone, they are virtually incurable and result in significantmorbidity prior to a patient’s death.We hoped that we could inhibite the bonedestruction caused by the metastasis of lung cancer. Most in vivo studies indicatethat osteolysis is caused by osteoclast stimulation, not by the direct effects ofcancer cells on bone. Osteolytic metastases are associated with increasedosteoclast activity and reduced osteoblast activity that is uncoupled from boneresorption. The TNF-family molecule RANKL (Receptor Activator ofNF-kappaB Ligand; also known as OPGL, TRANCE, ODF and TNFSF11) andits receptor RANK are key regulators of bone remodeling, and they are essentialfor the development and activation of osteoclasts. RANKL induces thedifferentiation of osteoclast precursor cells and stimulates the resorption functionand survival of mature osteoclasts. The TRAF (TNF Receptor-Associated Factor)adaptor proteins play an important role in the initial event of the signaltransduction pathway induced by RANK. TRAF proteins may act by transmittingthe RANK signal to downstream targets that include NF-kappaB (Nuclear Factor-kappa B) and JNK (c-Jun N-terminal kinase). So TRAF proteins can betreated as the target in our strategy.In the first part of this research, we used recombinant PCR to fuse PTB,SH2,coiled-coiledgene of shc,Grb2and TRIP with U-box gene of CHIP orRING(Y371E) gene of Cbl, then cloned these fusion genes into eukaryoticexpression vector pFLAG-CMV4, structured recombinant ubiquitin ligases. Inthe second part, we selected the most suitable cell lines and investigated thefunctions of recombinant ubiquitin ligases to downregulate the target proteins.And in the third part, we examined the effects of recombinant ubiquitin ligases onproliferation and metastasis in Spc-A1cells. In the fourth part, we investigatedthe the effectiveness and mechanism of recombinant ubiquitin ligases insuppressing osteoclasia.In part1, designed primers, used PCR amplify SH2,PTB,TRIP gene, U-boxgene and RING(Y371E) gene respectively, there are several continuous reservecomplement base pairs between the reverse primer of SH2,PTB,TRIP gene andthe forword primer of U-box gene and RING(Y371E) gene. Fuse SH2,PTB,TRIPand U-box or RING with recombinant PCR. Treated the production withrestriction enzyme and then cloned them into eukaryotic vector pFLAG-CMV4.After identifing and sequencing, recombinant ubiquitin ligases were builtsuccessfully.In part two, the most suitable cell line was selected based on its transfectionefficiency and its expression of endogenous EGFR. Recombinant ubiquitinligases co-transfected into293T cell with EGFR. And the lung cancer cell linewith high-expression of EGFR was tranfected with recombinant ubiquitin ligasestoo. After the transfection the expression of EGFR was detected and thedownregulation of target protein was proved by Western blot. Meanwhile, mRNA abundance of the target protein were detected with real-time PCR, ensure thattheir downregulation occur in post-translational level.In part three, we invetigated the effects of recombinant ubiquitin ligases onproliferation and metastasis in Spc-A1cell line, which was the most suitable onein part2. MTT and Flat cloning formation assey examined the effects of cellproliferation after transfected with recombinant ubiquitin ligases, and transwellassey reflected the metastasis ability of Spc-A1cell lines. And the apoptosis wasdetected by flow cytometry. The results showed that the3recombinant ubiquitinligases could inhibite proliferation and metastasis and promote apoptosis ofSpc-A1cell lines significantly.In Part four, Recombinant ubiquitin ligases were co-transfected into293Tcell with TRAF6. And the target proteins were degraded in cells transfected withTRIP-U-box.The test of co-immunoprecipitation proved the direct interactionbetween recombinant ubiquitin ligases and target proteins.In conclusion, because of the abnormal activation and overexpression ofEGFR in many types of cancer, targeted degradation may be an effective method.EGFR is important in RTK family because it participate in cancer malignanttransformation. Its downregulation can inhibit the development of cancer,therefore, it is an ideal target in cancer therapy. And because the key roles inosteoclasia,TRAF proteins are also the ideal target for bone protecting.Ubiquitylation is a common modification in post-translational level, classicubiquitylation of protein induces ubiquitin-proteasome pathway, lead to thedegradation of ubiquitylated substrates. Using the binding domain of the adaptorswhich can bind to target proteins specifically to achieve the specificity ofrecombinant ubiquitin ligases, while the catalytic domain achieve thefunctionality, thereby reach our goal of degradation of EGFR and TRAF proteins, inhibiting lung cancer cell proliferation, metastasis and the activation ofosteoclasts. It is a novel and effective cancer therapy strategy.