The Study Of Ubiquitin Ligase Fbw7 Regulating Cell Proliferation,invation,migration And TMZ Chemosensitivity In Human Glioma Cells

Glioma is one of the highest malignant human tumors. Currently despite the most effective treatment regimen, ie, the maximum extent of surgical resection plus postoperative chemotherapy with radiation and alkylating agents, the median survival period of grade IV glioblastoma multiforme(GBM) is only 14 months, due to tumor invasion, drug resistance and tumor recurrence. Previous research has already proved malignance of glioma is dependent on the network of plural molecules and signal pathways,which leads to the however extremely limited therapeutic effect targeting single molecule or pathway within the complex network. Thus,to find a capable target with interest which can act with and regulate the critical molecules and pathways responsible for malignant transformation of gliomas is the primary aming of this study.By rapidly degrading proteins, Ubiquitin-proteasome system(UPS) regulates a variety of cellular processes, including cell proliferation, growth, differentiation, apoptosis, and senescence. UPS is comprised of three distinct enzymes: ubiquitin-activating enzyme E1, ubiquitin conjugating enzyme E2(UBC) which regulates the formation of ubiquitin chain, and ubiquitin ligase E3 which directly binds to substrates. E3 enzyme thus determines the specificity of UPS regulation, which includes two main types: the APC(anaphase promoting complex) type, and SCF(SKP1-CUL1-F-box complex) type. Fbw7(F-box and WD repeat domain- containing 7) is the substrate binding unit of SCF-type E3 ligase enzyme, which bears the most regulatory abnormality in UPS.A variety of well-known carcinogenic factors are within the substrate spectrum of Fbw7, a majority of which are key transcription factors or regulatory protein molecules of carcinogenic pathways responsible for the malignant transformation of cells,such as cyclin E, MYC, JUN,and Notch. Cyclin E together with cell cycle kinases CDK2 control the transition of cells from G1 to S phase, which plays a key role in cell proliferation.Such an important regulator of cell cycle is precisely regulated by the ubiquitin-proteasome system, with the fact that abnormal over-activation of Cyclin E can cause chromosomal and genomic instability, resulting in carcinogenesis. c-Myc is also a key transcription factor controlling cell cycle, which has been found over-expressed in tumor cells caused by copy number amplification, gene transposition and other mutations. The transcriptional activity determines the biological processes of cell proliferation, protein synthesis, apoptosis, metabolism and cell differentiation; c-Jun is the component of mitogen-activated API transcription factor complex, which has been proved constitutively activated in various tumor cells with the effect of cell proliferation promotion; Notch is another transcription factor located in the cell membrane, which is activated and translocated to the nucleus after cleavage of the intracellular domain by the γ-secretase proteolytic complex. And this activates numerous cellular programs, especially cell proliferation and differentiation inhibition. Fbw7 hence is the most important tumor suppressor gene with its ability to regulate the oncogenes above.Fbw7 frequently mutations in tumors leading to loss of function. With a mutation rate of 6% to 35%, it is considered the most common tumor suppressor right behind p53 and PTEN. In addition, post-transcriptional dysregulation of Fbw7 itself is also commonly observed in tumor cells.p53, SREBP2, NF-k B1, Pin1, RITA, MEK-ERK, as well as micro RNAs regulate the expression and function of Fbw7 either transcriptionaly(e.g. NF-κB1) or post-translationaly(e.g. phosphorylation by ERK kinase). The abnormity of the above regulation together with Fbw7 gene mutations impact its precise ubiquitin ligase function, resulting in excess accumulation of Fbw7 substrates,and which eventually destructs the instinct rhythm of biological processes. For example, in physiological condition,when cells from G1 phase to the M phase of replication, the expression level of Cyclin E begins to increase right after G1 phase and and peaks in early S phase.This expression change of Cyclin E drives cells to complete the G1 / S transition. And then, the transcriptional negative regulation and Fbw7 mediated ubiquitin- proteasome degradation decreases Cyclin E to normal level, while Cyclin A, which transcriptionally activates DNA synthesis enzyme begins to increase. It is apparent that when the expression level of Fbw7 is impacted due to mutations and dysregulation, the activation of cyclin E will be sustained ignorant of cell cycle checkpoint mechanism, leading to abnormal DNA synthesis, and the generation of haploid or polyploid which initially triggers malignant proliferation of tumor cells. Inadditio to excess proliferation, carcinogenic biological behaviors also include invading to the surrounding tissues or even growing to distant tissues(metastasis), differentiation abnormalities such as epithelial- mesenchymal transition(EMT) and cell stemness. Like Cyclin E, Molecules(pathways) behind these biological behaviors will be dysregulated with impacted Fbw7 function, and this is the theoretical basis of our research to study the involvement of Fbw7 in malignant biological behaviors of Gliomas.The clinical therapeutics of gliomas is somehow another issue with our more attention.Since the highly malignancy of glioblastomas, postoperative concurrent chemoradiotherapy and adjuvant chemotherapy has become the gold standard for the comprehensive treatment of glioma. Temozolomide(TMZ) is an second-generation alkylating agent, with the most common application in clinical treatment of Gliomas. TMZ can cause DNA double-strand breaks(DSB), followed by apoptosis, autophagy or senescence like cell death. Unfortunately, s significant population of glioma patients are either not sensitive to TMZ treatment or experiencing chemoresistance which leads to tumor progression and recurrence. Our previous studies have found a great deal of molecules which paticipate in tumor cell proliferation, invasion, and apoptosis are also involved in the mechanism of tumor chemoresitance, which include but not limited to detoxification of membrane transporter proteins, regulation of apoptosis and autophagy, DNA damage repair pathways. There also exists crosstalks between these TMZ chemoresistance-related pathways and The regulation network of Fbw7 guided proteasome system.And this is the theoretical basis of our research into the involvement of Fbw7 in the regulation of TMZ chemosensitivity.The effect of Fbw7 expression on the biological behavior and TMZ chemosensitivity of glioma cells have not yet been profoundly studied. This project aimed to study the regulatory effect of Fbw7 on the proliferation, invasion and migration and chemosensitivity to TMZ of glioma cells, as well as the mechanisms behind these biological phenotypes. This study are divided into four parts.In the first part, we studied Fbw7 expression in clinical glioma samples, and its relationship with the pathological grades as well as the survival prognosis, by means of bio-information inquiry and immunohistochemical analysis. In the second part, we constructed Fbw7 overexpression vector, to study the effect of Fbw7 overexpression on cell proliferation, invasion and migration in U251 and U373 glioma cell lines. In the third part, we observed the expression of Fbw7 in TMZ-induced U251 resistant cell line,and its effect on the chemoresistance of U251/TMZ cells. We also studied the therapeutic effect of Fbw7 overexpression together with TMZ in vitro.In the last part, the regulation of Fbw7 on cell cycle and apoptosis as well as the expression of downstream substrates Auroa B, Mcl-1 and Notch1, were further studied for the mechanism of timorous behaviors.Part I Expressional and prognostic analysis of Fbw7 in Glioma samplesObjective: To study the m RNA and protein expression of Fbw7 in different grades of gliomas tissues, and analyze the relationship between Fbw7 expression and the prognosis of patients.Methods: The m RNA expressions and sample-matching survival information of Glioma patients in Cancer Genome Atlas(TCGA) based on microarray and sequencing data were analyzed to compare Fbw7 level in high grade(GBM) and low grade gliomas and study the prognostic significance of Fbw7 using Kaplan-Meier survival analysis and log-rank test. Immunohistological staining of Fbw7 on 50 paraffin samples(grade II 15 cases, grade III 10 cases,grade IV 30 cases) to analyze the protein level after quantifying the staining intensity using rank-sum test and the subcellular localization. The expressional correlation between Fbw7 and proliferation marker Ki-67 was also analyzed.Results: m RNA levels of Fbw7 in high grade gliomas reduced significantly, compared to the low grade Gliomas. KM survival analysis showed a significant longer overall survival time in Glioma patients with lower Fbw7 expression level compared to those in which Fbw7 expression was below medium level. Immunohistologi cal analysis showed Fbw7 was expressed in both the nucleus and cytoplasm, and that the staining index of Fbw7 increased obviously with the pathological grade decreased. Moreover, the staining index of Fbw7 and Ki-67 were significantly negatively associated.Conclusion: The m RNA and protein expressions were upregulated with the pathological grades decreased in Glioma samples,and were negatively correlated with the patient’s survival time as well as the proliferation marker Ki-67.Part II To study the effect of Fbw7 over-expression on cell proliferation, invasion and migrationObjective: By employing Lentiviral vectors over-expressing Fbw7, to study the effect of Fbw7 on cell proliferation,invasion and migration in U251 and U373 Glima cell lines.Methods: Fbw7 over-expressing lentivirus vectors were constructed to steadily transfect U251 and U373 Glioma cells. Cell growth at different time points(24,48,72,96,120h) were determined by means of MTT assays to compare the effect of Fbw7 on cell proliferation. In the same time, colony formation assays were performed to compare the ability of U251/U373 cells to grow confluently. Transwell experiments and wounding healing assays were conducted to study the effect of Fbw7 over-expression on cell invasion and migration.Results: Fbw7 overexpression lentivirus vector was successfully constructed. By MTT assays, cell viability started to be significantly inhibited in LV-Fbw7 transfected cells compared to blank/negative control cells at 48 hours from the beginning of assays. After 3 weeks of confluent culture, LV-Fbw7 transfected cells formed significantly reduced colonies(>20 cells) compared with t blank/negative control cells in both U251 and U373 cell lines. Transwell results showed that, after 72 hours after LV-Fbw7 transfection, U251 and U373 cells expressed notably impacted invasive ability compared to controls. In wound healing assays, reduced migration distance of two Glioma cell lines was observed after 18 h and 36 h of LV-Fbw7 transfection compared with controls.Conclusion: Over-expression of Fbw7 significantly inhibited the growth and colony formation; and significantly impacted the ablity of invasion and migration of glioma cells.Part III To study the effect of Fbw7 over-expression on TMZ chemosensitivity of glioma cellsObjective: To study Fbw7 expression levels and its role in the chemoresistance in U251/TMZ resistant cell line, and the effect of Fbw7 on TMZ chemosensitivity in vitro.Methods: U251/TMZ cell line which was resistant to TMZ was induced by incrementally increasing TMZ concentrations. Proteic expression of Fbw7 was assessed by Western Blot assay. The chemoresistant ability(IC 50) was compared before and after LV-Fbw7 transfection by MTT assays. The inhibitory effect of Fbw7 over-expression in combination with TMZ was studied in vitro by MTT assays too.Results: Western Blot results showed that Fbw7 expression in U251 / TMZ cell line was significantly lower than the parent U251 cell line. The IC50 of U251/TMZ was lowered after Fbw7 over-expression. In vitro inhibition experiments cell vitality of U251 Glioma cells was significantly reduced in LV-Fbw7 transfection + TMZ group compared with either TMZ alone or LV-Fbw7 alone group, at both 36 and 72 hours of treatment.Conclusion: The enhaunced chemoesistance of U251/TMZ cell line was Fbw7 relevant. Moreover, Fbw7 over-expression can significantly increase the chemosensitivity of TMZ in vitro.Part IV To study the mechanisms of how Fbw7 regulates malignant phenotype and TMZ sensitivity of glioma cellsObjective: To study specific mechanisms of Fbw7 over-expression on glioma cell proliferation, invasion and migrationas well as the regulation on TMZ chemosensitivity.Methods: Flow cytometry was used to analyze the cell cycle and apoptosis changes of U251 and U373 glioma cells with Fbw7 over-expression. The expression of cycle kinase Aurora B, apoptotic proteins Caspase 3、Mcl-1 and transcription factor Notch1 were assessed by Western Blot assay,after LV-Fbw7 transfection alone or combined LV-Fbw7 and TMZ treatment.Results: Flow cytometry results showed significantly higher fraction of G2/M cells together with lower fraction of heteroploid cells, and higher apoptosis rate in Fbw7 over-expression and TMZ combined group compared with control, LV-Fbw7 alone, and TMZ alone groups. WB results showed that the expression cycle kinase Aurora B in 24 points and 72 hours transfection group were lower than control groups with a greater decrease at 72 h.The expressional level of Caspase 3 increased compared to controls after 24 and 72 hours of Fbw7 transfection whereas Mcl-1,which is representative of anti-apoptotic factor,decreased with persistent Fbw7 over-expression. Moreover, proteic expression of Notch1 was reduced, especially at 24 h after Fbw7 over-expression,which is obviously lower than that of Aurora B or Mcl-1. Combined treatment of Fbw7 plus TMZ significantly down-regulated Aurora B level, increased Caspase 3 whereas reduced Mcl-1 and Notch1 level compared with TMZ alone treatment and vehicle treatment. Noticeably there was an increase of Mcl-1 expression after TMZ treatment compared to control.Conclusion: Fbw7 over-expression down-regulated the expression of Aurora B, Mcl-1 and Notch1, increased Caspase 3 level, furthermore increased G2 / M arrest, reduced heteroploidy division and enhanced apoptosis in U251 cells, which were likely to be the mechanisms of Fbw7 inhibiting malignancy of Gliomas.