Expression Of TIP30 In Lung Cancer Tissue And Inhibition Of Tumor Metastasis By TIP30 In Vitro

BackgroudLung cancer is one of the leading causes of death worldwide with increased incidence in China.The overall survival of patients with lung cancer remains poor because of the advanced stage in most patients at the time of diagnosis and lack of effective therapies. Although about 50%of lung cancer patients respond well to initial chemotherapy and external bean radiation therapy with regression of the primary tumor,the survival of lung cancer patients is dismal because of metastases and the development of resistance to chemotherapy.Transformation of a normal phenotype into a malignant phenotype requires accumulation of multiple genetic and epigenetic changes,which result in growth and/or cellular survival advantage.Loss of tumor suppressor genes is one of the critical alterations in this multi-step process.Therefore,identification of novel molecular biomarkers to distinguish tumor cells from normal cells and predict tumor behavior such as pathologic stage,response to chemotherapy and potential to metastasis,is of great importance for improving the outcomes of lung cancer patients.TIP30,also called CC3,is a tumor suppressor with pro-apoptotic and anti-metastasis properties.It was originally identified by a differential display analysis of messenger RNA from the highly metastatic human variant small cell lung cancer(v-SCLC) versus less metastatic classic small cell lung cancer(c-SCLC) cell lines.The expression of TIP30 has been detected in many human normal tissues and was down-regulated in certain tumor cells such as melanoma,breast cancer,neuroblastoma,glioblastoma,colon cancer,and hepatocellular carcinoma.TIP30 was considered to have tumor suppressor activity by inhibiting tumor growth,invasion,and angiogenesis and inducing apoptosis.TIP30 mutants not only abrogated the tumor suppressor potential but also gained oncogenic activities.The depletion of TIP30 predisposed mammary epithelial cells to neoplastic transformation and TIP30-deficient mice spontaneously developed various tumors.It has been reported that v-SCLC lacking endogenous TIP30 expression showed more resistance to death-inducing signals than c-SCLC with high TIP30 expression. Furthermore,over-expression of TIP30 in highly metastatic v-SCLC can significantly inhibited cell proliferation,sensitized cells to cytotoxic drugs,promoted apoptosis and inhibited metastasis. Taken together,these data demonstrated the importance of TIP30 in suppressing the progression and metastasis of SCLC.In this study,we generate the polyclonal antibody against human TIP30 protein with high-purity and high-specificity.The expression profile of TIP30 in clinical specimens is evaluated by immunohistochemistry in 206 lung carcinoma tissues using a high-density tissue microarray.We also assess the effects of TIP30 on tumor cell metastasis potential and proliferation in vitro.Results:1.The gene encoding TIP30 was amplified from the plasmid and inserted into the prokaryotic expression vector pGEX-4T-2 with tag GST(glutathione S-transferase).The E.coli BL21(DE3) transformed with pGEX-TIP30 was induced by IPTG(isopropylbeta-D-thiogalactopyranaside) for expression of TIP30 fusion protein which was recovered in the supernatants and purified via glutathione-Sepharose 4B affinity column.The rabbits were subcutaneously immunized with purified protein and bled.The antibody purification was performed by using Protein A Sepharose^TM CL-4B.2.TIP30 expression was assessed by an immunohistochemic approach on 206 paired lung cancers and adjacent non-tumor tissues,as well as 70 lymph node metastases.The correlation of TIP30 expression with the clinicopathological features of the patients was analysed.In non-tumor lung tissues,TIP30 was preferentially expressed in the cytoplasm of bronchial epithelia cells and alveolar epithelia cells.TIP30 expression was found decreased in all the nine cases of SCLC and in 72 of 197(36.5%) cases of non-small cell lung cancer(NSCLC),with 36.2%(34/94) of adenocarcinoma(AC),36.7%(33/90) of squamous cell carcimona(SCC),and 38.5%(5/13) of adenosquamous carcinoma. Decreased TIP30 expression was correlated with the poor differentiation of tumor and the advanced stage of disease in squamous cell carcinoma(SCC).TIP30 expression in the metastatic lesion was decreased in comparison with that in the primary lung lesions in 22 out of 70(31.4%) NSCLC.There was an inverse association between TIP30 expression and abnormal p53 accumulation in lung cancer(P＜0.001).Importantly,the survival rate of patients who had high TIP30 expression in primary tumor was significantly higher than that of patients with low TIP30 expression in AC(log-rank test,P=0.0112),and in SCC (log-rank test,P=0.0306).3.The expression of TIP30 mRNA and protein in five lung cancer cell lines were analysed. The expression of TIP30 was knockdowned in A549 which contains high level of endogenous TIP30 or introduced TIP30 gene into H1299 which contains low level of endogenous TIP30 by lentivirus.LV-shTIP30 significantly promoted cell growth of A549, inhibited its apoptosis,enhanced its motility and invasion through matrigel as compared with LV-None or mock treatment.However,introduction of TIP30 in H1299 cells dramatically retarded its growth and reduced its invasive and migration properties.Conclusions:1.Obtain the polyclonal antibody against TIP30 protein with high-purity and high-specificity which paves the way for future study.2.Decreased TIP30 expression is correlated with the poor differentiation of tumor and the advanced stage of disease in SCC and the abnormal p53 accumulation in NSCLC,which indicates the importance of down-regulation of TIP30 in the development and progression of non-small cell lung cancer.3.Down-regulation of TIP30 is statistically related to poor prognosis in patients with NSCLC.The determination of the immunohistochemical expression status of TIP30 might be a valuable prognosticator for patients with this disease.4.TIP30 expression in lung cancer shows a sequential decreased pattern from normal tissue,tumor tissue to metastatic carcinoma.The importance of TIP30 in suppression of metastasis is further proved by wound-healing and matrigel invasion assays in vitro.TIP30 serves as a tumor metastasis suppressor gene in lung cancer.