The Investigation Of Clinical Significance And Function Of Tumor Suppressor Numb In Malignant Pleural Mesothelioma

Background and ObjectiveMalignant pleural mesothelioma (MPM) is a rare and highly aggressive tumor with dismal prognosis. Although the asbestos exposure, which is regarded as the main risk for MPM, has been regulated since the1870s, the incidence is increasing continuedly because of the long latent period between exposure and clinical disease. Its mortality has been reported increasing over the past two decades in industrialized countries and is expected to peak in2010-2020. The histologic subtypes of mesothelioma include epithelioid (most common), sarcomatoid and mixed. MPM is poorly responsive to current oncological therapies with a median survival of around10-12months. In recent years, the success of gene therapy in tumor area lay a foundation for finding effective treatment for MPM.Numb is one of the most important cell fate determinants and has been implicated as a tumor suppressor in organism. It is a membrane-associated protein which contains an amino-terminal phosphotyrosine-binding domain (PTB) and a C-terminal proline-rich site (PRR). The PTB domain is critical for Numb to interact with the E3-ligases Mdm2, Lnx, Siah1and Itch, which leads to poly-ubiquitination and proteolytic degradation of proteins. Numb has also been proven to participate in regulating the cell polarity and asymmetric cell division, epithelial-mesenchymal transition (KMT), ubiquitination and degradation, and down-regulating Notch and Hedgehog-Gli signaling while up-regulating TP53activity. Thus, Numb can inhibit the tumor formation through controlling the balance of proliferation and differentiation as well as apoptosis and regeneration of cells. Loss of Numb expression has been reported in some human cancers, such as breast cancers, non-small cell lung carcinomas, salivary gland carcinomas and medulloblastomas. A role for Numb in preventing tumorigenesis appears to involve the suppression of the oncogenic Hedgehog-Gli signaling. However, the expression and clinical significance of Numb in MPM and its effect on cancer development have not been reported.To explore the effect of Numb in the development of MPM and lay a foundation for gene therapy, we investigated from the following three aspects:1. The expression and clinical significance of Numb in MPM.2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Gli1.3. The effect of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells.Methods1. The expression of Numb and Gli1in MPM and their clinical significanceNumb and Gli1expression were evaluated by immunohistochemistry in61MPM tissues and22normal pleura, and Chi-square test was used to compare the results; Correlations of protein expression (grade scores) with clinical and pathological parameters of the tumors were evaluated with Kruskal-Wallis or Mann-Whitney test for ordinal variables. Correlations between Numb and Gli1expression were evaluated with the Spearman rank order correlation test. The univariate analysis of overall survival (OS) was carried out by the Kaplan-Meier method using the log-rank test. Multivariate Cox proportional hazards regression model was used to test for independency.2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Glil(1) First, the expression of Numb in NCI-H2452cells was detected by qRT-PCR and Western blot. Then we detected the protein expression of Numb in the presence of proteasome inhibitor MG132treatment.(2) First, we transfected the pcDNA3.1Numb (Numb) or empty vector (mock) with LipofectaminTM2000into NCI-2452cells transiently and detected the protein expression of Numb and Glil by Western blot. Then we detected the protein expression of Gli1in Numb-transfected cells with the treatment of proteasome inhibitor MG132.3.The effects of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells(1) To explore the effect of Numb overexpression on the proliferation in NCI-H2452cells, MTT was used to detect survival rate at48h and72h after transfection.(2) To explore the effect of Numb overexpression on the apoptosis in NCI-H2452cells, Hoechst33258staining and Annexin V-FITC/PI assay using flow cytometry were used to detect the change of cellular morphology and apoptosis rate at72h after transfection.(3) To explore the mechanism of Numb overexpression enhancing apoptosis, the apoptosis related proteins caspase-3, caspase-9, cytochrome c, XIAP, survivin were detected by Western blot after transfection.(4) To explore the effect of Numb overexpression on the sensibility to chemotherapy in NCI-H2452cells, MTT was used to detect survival rate with the treatment of cisplatin, pemetrexed or carboplatin at different concentrations.Result1.The expression of Numb and Glil in MPM and their clinical significanceThe immunoreactivity of Numb in MPM was significantly lower than that of normal control group(P<0.05), and Numb has an inverse correlation with Ki-67labeling index (P<0.05). The immunoreactivity of Gli1in MPM was significantly higher than that of normal control group(P<0.05),and nuclear Gli1was found in associated with the tumor IMIG-stage (P<0.05).The expression levels of Numb with nuclear Glil exhibited a significant inverse correlation (r=-0.361P<0.05). Univariate analysis indicated that the OS was influenced by expression of Numb (P<0.05) and histological subtype (P<0.05), and further Cox regression analysis showed that only histological subtype was an independent prognostic factor for OS (P<0.05).2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Gli1(1) The expression of Numb in NCI-H2452cellsBefore transfection, we detected comparable levels of NumbmRNA in NCI-112452vs. 293T cells, but the protein level of Numb in NCI-H2452was lower than that of293T cells. Numb protein was restored to a high level by treatment with the proteasome inhibitor MG132, which meaned that loss of Numb expression in NCI-H2452cells was determined at the post-translational level, through enhanced protein degradation.(2) Numb overexpression in NCI-H2452cells inhibited Gli1protein expression The NCI-H2452cells transfected with pcDNA3.1Numb showed higher Numb protein expression compared with that of mock-transfected, While the Gli1protein expresssion was weakened. The Gli1protein in Numb-transfected cells restored to a high level with the treatment of proteasome inhibitor MG132, which meaned that Numb lost the inhibition effect on Glil in the presence of MG132.3. The effects of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells(1) Overexpression of Numb inhibited the proliferation in NCI-H2452cells The absorbance of Numb-transfected cells, as indicated by MTT, was markedly lower at48h and72h compared with that of mock-transfected cells or blank controls (P<0.05,<0.01), which meaned that Numb overexpression inhibited growth of NCI-H2452cells(2) Overexpression of Numb promoted the apoptosis in NCI-H2452cells. Staining with Hoechst33258showed that Numb overexpression increased the number of apoptotic cells characterized by chromatin condensation and nuclear fragmentation, comparing with that of mock-transfected cells or blank controls; The apoptosis rate was determined by two-color analysis of Annexin V/FITC binding and propidium iodide (PI) uptake using flow cytometiy at72h after transfection.The percentages of early and late apoptotic cells ware notably higher in Numb-transfected than that of mock-transfected cells or blank controls (P<0.01).(3) Numb induced apoptosis in NCI-H2452cells through the intrinsic caspase-9pathway We analyzed some proteins related to apoptosis in NCI-H2452cells by Western blot after transfection. Numb-transfected cells showed increased active caspase-9(35kDa) and caspase-3(17kDa) bands compared with that of mock-transfected cells. In addition, we found release of cytochrome c to cytoplasm as well as down-regulation of XIAP and survivin in Numb-transfected cells.(4) Overexpression of Numb sensitized NCI-H2452cells to cisplatin and carboplatin The survival rate of Numb-transfected cells was significantly lower than that of mock-transfected cells and blank controls with the treatment of cisplatin and carboplatin at the same concentrations, which meaned that overexpression of Numb sensitized NCI-H2452cells to cisplatin and carboplatin.But the survival rates of the three groups had no statistically differences with the treatment of pemetrexed.Conclusions1. Numb expression was significantly decreased or complete absent in MPM tissues, which correlated with high Ki-67labeling index and poor prognosis.2. Glil expression was significantly increased and correlated with IMIG stages in MPM tissues; Numb had an inverse correlation with Gli1and Numb overexpression inhibited Gli1protein through the ubiquitin-proteasome pathway.3. Overexpression of Numb inhibited the proliferation and induced the apoptosis through the intrinsic caspase-9pathway in MPM cells.4. Overexpression of Numb sensitized MPM cells to cisplatin and carboplatin.Originality1. We demonstrated, for the first time,that the frequent loss of Numb expression in MPM and it is a poor predictor of survival.2. We investigated, for the first time, the correlation of Numb and Gli1as well as the regulation of Numb on Gli1.3. We investigated, for the first time, the effect of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in MPM cells, which could lay a foundation for the gene therapy targeted Numb for MPM.