Roles For Clusterin In Progression, Chemoresistance And Metastasis Of Human Ovarian Cancer And The Underlying Mechanisms

Background:Ovarian cancer, the leading cause of death from gynecological malignancy. With the limitations in the timely screening methods, in more than two-thirds of the cases, patients present with widespread metastatic dissemination at time of initial presentation. Rencently, as the result of advances in surgical management and cisplatin-based combination chemotherapy, initially, 80% patients achieved a clinical complete remission. However, following preliminary success in tumor regression, recurrence and resistance to further chemotherapeutic treatment often ensues, which lead to no significant alteration in overall survival. Therefore, shortage of suitable biologic marker, limitations in the timely screening methods, early metastasis and resistance to anticancer chemotherapeutic drugs become the major obstacles for ovarian cancer therapy. Thus, to identify the specific molecular that promote chemoresistance and metastasis, investigate the mechanisms involved in the modulation is critical in the search for effective therapeutic strategies.Clusterin (CLU) is a fascinating heterodimeric multivalent glycoprotein with nearly ubiquitous tissue distribution. It is implicated in quite a lot of diverse physiological processes such as immune regulation, cell adhesion, cell-cell/cell-substratum interactions, cell differentiation and transformation, cell cycle regulation, lipid transportation and DNA repair. With the regulation of pre-mRNA alternative splicing, there exist two different but related Clusterin protein isoforms in human cells, depending on the type of stimulus and cell context. That is, the secretory form (sCLU), which function as a heat shock protein with its chaperone activity, was suggest to be a cytoprotective protein; and the nuclear form of Clusterin protein (nCLU), acts as the proapoptotic role. Clusterin expression has been associated with tumorigenesis of various malignancies, including tumours of prostate, colon, and breast. What's more, sCLU upregulation appears to be a general molecular stress response in these tumors. Conclusively, it is suggested that important functions of sCLU overexpression in the pathogenesis, progression and acquisition of therapy-resistant phenotype of cancer. In 2005, it was reported that the overexpression of cytoplasmic Clusterin may represent an acquired malignant phenotypic feature of ovarian carcinoma and may be one of the important factors in determining its aggressive nature. Thus, Clusterin gene is a promising target for cancer therapy. A better understanding of its physiologic and pathophysiologic roles in ovarian carcinoma may open new opportunities for effective therapeutic intervention and better outcomes.Objective:1) To observe the differential expression of the sCLU and nCLU isoform, and each roles in the phenotype of human ovarian carcinoma cells;2) To explore the possible roles and mechanisms of different Clusterin isoforms plays in the pathogenesis, progression, acquisition of therapy-resistant phenotype, metastasis and apoptosis of ovarian cancer;3) To investigate the feasibility of RNAi targeting sCLU in gene therapy for ovarian carcinoma.Methods:1) The mRNAs of different Clusterin isoforms (CLUF, CLUT, CLUN) were cloned from the pcDNA3.1(+)/CLU plasmids by RT-PCR. Sense eukaryotic expression vectors of pIRES2-EGFP/CLUF,pIRES2-EGFP/CLUT,pIRES2-EGFP/CLUN were constructed by cloning the mRNAs of CLUF, CLUT, CLUN into pIRES2-EGFP;2) Differential expression of Clusterin was examined by RT-PCR and Immunohistochemistry analysis in different ovarian carcinoma cell lines;3) Ovarian carcinoma SKOV3 cells was transfected with sense vectors by Lipofectin, the expression level and localization of Clusterin protein in each transfected cells were examined by Western blot;4) Indirect immunofluorescence was used to observe subcellular localization of Clusterin isforms in transfected SKOV3 cells and their phenotypic effects;5) Apoptosis of the transfeceted SKOV3-CLUN cell lines was observed by cell cycle distribution with FCM and transmission electron microscopy study.6) By G418 screening, stable transfectants of SKOV3-CLUF,SKOV3-CLUT, SKOV3-Vec were obtained. MTT assays were performed to evaluate the effects of sCLU and pnCLU overexpression on cell proliferation;7) MTT assays were performed to determine the in vitro drug sensitivity of the transfected SKOV3-CLUF,SKOV3-CLUT,SKOV3-Vec and SKOV3 cells, the IC50 values were calculated;8) The inducible expression of Clusterin was examined by RT-PCR and Western blot in cisplatin treated A2780 cells;9) Acorrding to the design rules for siRNA, three hairpin siRNA templates were designed based on Clusterin gene sequence, cloned into eukaryotic expression plasmid pSuppressorNeo, and confirmed by restrict endonuclease digestion and DNA sequencing;10) Ovarian carcinoma CP70 cells were transfected with shRNA vectors by Lipofectin. Stable transfectants were obtained by G418 screening, mRNA and the corresponding protein in the transfected cells were examined;11) Cell proliferation and clonogenic assays were carried out to evaluate the effect of Clusterin depletion on cell proliferation. Phenotypic effects by Clusterin-silenced in CP70 cells were also studied by recording both cellular morphology and phenotype of the transfected cells;12) MTT assays were performed to determine the in vitro drug sensitivity of the CP70-shRNAI, CP70-Sc and CP70 cells. The IC50 values were calculated to evaluate the effects of sustained silencing of Clusterin expression on the chemosensitivity of CP70 cells to cisplatin;13) Apoptosis of the cisplatin-treated CP70-shRNAI cells was examined by AnnexinⅤ/PI analysis and Western blot, to study the underlying mechanisms for sensitization of CP70 cells to chemotherapy by Clusterin gene silencing;14) To study the effect of silencing of the Clusterin gene expression in CP70 cells on cell metastasis, the wound migration and cell invasive assay were performed.Results:1) By RT-PCR and Western blot, it was confirmed that Clusterin gene were expressed in all the five observed ovarian carcinoma cell lines. Among which, the expression level in CP70 cells is most significant;2) Successfully constructed the sense eukaryotic expression vectors of pIRES2-EGFP/CLUF, pIRES2-EGFP/CLUT, pIRES2-EGFP/CLUN, and sequencing verified the proper encoding of the mRNAs; Transfection sense vectors of each Clusterin isoforms into the SKOV3 significantly increased their expression. Western blot study suggested SKOV3-CLUF cells showed the 60 kDa and 40kDa sCLU isoforms expression in the cytoplasmic extract; SKOV3-CLUT cells exhibited pnCLU expression in the cytoplasm as ~50 kDa; while, SKOV3-CLUN cells not only showed the ~50 kDa pnCLU isoforms in the cytoplasmic extract, but also expressed 55 kDa mature nCLU isoforms in the nuclear extract.3) The expression of Clusterin protein in SKOV3-CLUF and SKOV3-CLUT cells were predominantly cytoplasmic, which showed no significant effect on cell phenotype. While, Clusterin protein expressed in SKOV3-CLUN cells primarily localized in the nuclear, which lead to apoptotic morphological alteration observed by Hoechst nuclear staining and electron microscopy study, as well as the G2-M arrest and appearance of sub G1-G0 fraction in cell cycle analysis with FCM;4) Followed by selection with G418, stabilized transfected SKOV3-CLUF,SKOV3-CLUT cell lines were constructed successfully. MTT assay showed overexpression of sCLU elicited proliferative effect on cell growth, whereas pnCLU induced no obvious change, as compared with mock controls;5) MTT assay suggested sCLU overexpression considerably inhibited SKOV3-CLUF cells'chemosensitivity to cisplatin in a dose-dependent manner, enhancing the IC50 of cisplatin to ~2.4 fold. Whereas for SKOV3-CLUT cells, pnCLU overexpression boosted chemosensitivity and reduced the IC50 by >40%;6) With cisplatin treatment, the expression of Clusterin RNA and protein in A2780 cells were both upregulated in a time dependent manner;7) Successfully constructed the shRNA eukaryotic expression vectors of pSup/CLU-shRNAⅠ,pSup/CLU-shRNAⅡ,pSup/CLU-shRNAⅢ. Established stable transfected cell line: CP70-shRNAI, CP70-shRNAⅡ, CP70-shRNAⅢ, CP70-Sc;8) Efficient silencing of the Clusterin gene expression in CP70 cells using shRNA vectors were verified by Western blot and RT-PCR assay. Among those, the shRNA I vector appeared to be most effective than shRNA II and shRNAⅢ, which reduced sCLU RNA and protein levels by 91.7% and 88.5%;9) The inhibition of Clusterin depletion on cell proliferation was appraised by cell proliferation and clonogenic assays;10) MTT assays displayed Clusterin depletion extensively sensitized CP70 cells to cisplatin in a dose-dependent manner, reducing the IC50 of cisplatin by>80%(55.57-10.26μM for 48hrs; 37.24-7.06μM for 72hrs), whereas scrambled control showed no effect;11) AnnexinⅤ/PI and Western blot indicated that CP70 cells sensitization to chemotherapy owing to Clusterin gene silencing is related to activation of the cellular apoptotic machinery;12) As shown by wound migration and cell invasive assay, silencing of the Clusterin gene expression in CP70 cells inhibited cell metastasis. Comparing with the wild type CP70 cells, CP70-shRNAI exhibited a marked fold inhibition on cell invasion to 38.8% at 24 hrs (P<0.01), cellular motility was also inhibited with down to 58.2% and 55.0% at 24 hrs and 48 hrs respectively (P < 0.01). Conclusions:1) Different Clusterin mRNA yield different isoforms of Clusterin protein in SKOV3 cells with each special subcellular localization. sCLU and pnCLU were predominantly cytoplasmic, while nCLU primarily localized in the nuclear.2) sCLU is cytoprotective and functions as a central molecule in cell homeostasis. sCLU overexpression extensively inhibited the chemosensitivity of human ovarian carcinoma cells.3) nCLU plays a proapoptisis role in ovarian cancer cells, With nCLU overexpression, SKOV3-CLUN cells were provoked to apoptosis through G2-M arrest.4) Nuclear localization of the truncated Clusterin in SKOV3 cells is NLS dependent, without which pnCLU isoform was sequestrated in cytoplasm to prevent cytotoxicity. Thus,the proapoptisis roles of nCLU is greatly depended on its nuclear localization.5) The expression of sCLU in ovarian carcinoma cells was closely related with drug-resistance. With cisplatin treatment, the expression of sCLU in the cytoplasm was upregulated. Together with its stress-associated cytoprotective functions, we proposed that the overexpression of sCLU plays an important role in the acquisition of chemoresistant phenotype of ovarian cancer.6) Compared with A2780 cells, high level cytoplasmic expression of sCLU in CP70 cells is associated with its resistance to cisplatin.7) With specific shRNA vectors used, the expression of sCLU protein and RNA can be effectively silenced, sensitized ovarian carcinoma cells to chemotherapy, inhibited cancer progression and suppressed cell migration and tumor metastasis. 8) Albeit the mechanistic basis for the regulation of cell apoptotic activity by sCLU is not fully understood at present, our preliminary analysis has revealed Clusterin silencing potently promote the activation of caspase-3, -9 and PARP in cisplatin-treated CP70-shRNA cells, suggesting that perhapes the caspase9-caspase3-dependent intrinsic apoptotic pathway is the key target of sCLU actions in ovarian cancer cells. By suppressing the activiton of this pathway, sCLU plays its cytoprotective role and functions in drug-resistance.9) The anti-apoptotic properties of sCLU are presumably mediated by interference with Bax activation in mitochondria. With the intracellular localization to the membranes of the endoplasmic reticulum or mitochondria, sCLU can bind to Bax, inhibit Bax oligomerization, then suppress the caspase9-caspase3-dependent intrinsic apoptotic pathway, as well as Bax-depended c-Myc-mediated apoptosis. Meanwhile, promotes c-Myc-mediated transformation and tumour progression. Which once more give an elucidation for the causal relation between sCLU depletion and inhibited cell proliferation, decreased tumor progression.