Studies Of Expression And Role Of ATP Citrate Lyase In Epithelial Ovarian Cancer

BackgroudOvarian cancer is the third most common neoplasm of the female reproductive tract and the leading cause of death related to a gynecological malignancy. Because early-stage tumors commonly show no obvious and often only unspecific signs or symptoms, nearly70%of the patients is suffered in advanced or has been transferred at the time of diagnosis. Epithelial ovarian cancer (EOC) accounts for more than90%of all ovarian cancers. EOC is always more aggressive, and local invasion or distant metastasis occurs early. Cytoreductive surgery with concomitant platinum and paclitaxel-based chemotherapy is the main treatment for ovarian cancer, but chemoresistant and side-effects make the tumor recurrence. The5-year survival rate remains approximately15-30%only. Therefore, it is essential to explore mechanisms of carcinogenesis of ovarian cancer and find reliable prognostic and/or predictive factors.Fatty acids play an essential role in a variety of cellular processes, but now it is identified as the critical step in tumor cell transformation and proliferation. Fatty acids are obtained from the diet or can be synthesized de novo from carbohydrate precursors. In most normal tissues, fatty acid synthesis occurs at low rates since fatty acids are acquired via the circulation to support the needs of nonproliferating cells, in contrast, de novo fatty acid synthesis occurs at very high rates in tumor tissues. Therefore, abnormal metabolism has become one of most common properties of cancer cells. The increased de novo fatty acid synthesis is caused by multiple mechanisms, including increased expression or activation of lipogenic enzymes. Thus, further research will be required to understand the mechanisms of lipogenic enzymes dysregulation and ovarian cancer progression, so as to develop novel therapeutic molecular targets to overcome the disease.ATP citrate lyase (ACL), a key enzyme of de novo lipid synthesis, is involved in the generation of cytosolic acetyl-CoA and oxaloacetate from citrate and thus contributes to the translocation of acetyl-CoA from mitochondria to cytosol. Acetyl-CoA is a building block for cholesterol and fatty acid synthesis, is pivotal in energy and material metabolism. As a first step in lipid synthesis, ACL integrates glucose and lipid metabolism. ACL overexpression or activation has been reported in bladder, breast, liver, stomach and lung tumors. However, the role of ACL in human epithelial ovarian cancer has yet to be determined.In this study, to identify associations with clinicopathologic parameters and prognosis, the expression of ACL in epithelial ovarian cancer and normal ovarian tissues was detected. To show the therapeutic potential and mechanism of ACL inhibition for ovarian cancer treatment, the effect of ACL gene silencing on biological behavior of ovarian cancer cells were assessed.PART I:Expression of ATP citrate lyase in epithelial ovarian cancer tissues and their clinical significanceObjective:To analyze the expression of ACL in epithelial ovarian cancer and normal ovarian tissues, and their associations with clinicopathologic parameters and prognosis. SREBP-1is a basic-helix-loop-helix-leucine zipper protein that regulates the transcription of lipogenic enzymes. To dissect the relationship of SREBP-1with ACL in EOC, the SREBP-1expression was detected.Clinical samples and methods:1. Clinical samplesTumor samples were obtained from patients who underwent surgical resection at the Qilu Hospital of Shandong University. The age of the patients ranged from30to73years. All tumors were pathologically diagnosed based on the World Health Organization criteria and staged according to the classification of the International Federation of Gynecology and Obstetrics. Clinicopathologic parameters and patient prognosis data were also obtained.18epithelial ovarian cancer tissues were collected, while12normal ovarian tissues removed during surgery for benign conditions were also collected as controls. These tissues were frozen immediately after surgical resection and stored in liquid nitrogen until use for RNA or protein extraction. This study was approved by the Ethics Committee of Qilu Hospital, and signed informed consent was obtained from each patient.2. MethodsQuantitative real-time PCR and Western blot was used to detect the mRNA and protein expression of ACL in18epithelial ovarian cancer tissues and12normal ovarian tissues. To identify associations with clinicopathologic parameters and prognosis, ACL and phosphorylated ACL (p-ACL) expression in82epithelial ovarian cancer tissues were detected by immunohistochemistry.Results:1. ACL mRNA expression in EOC tissues and normal ovarian tissuesACL mRNA expression was detected by quantitative real-time PCR. The result showed that the relative expression (2-ACT) of ACL mRNA in18EOC tissues was0.0154±0.00500, which was obviously higher than that in12normal ovarian tissues (0.0042±0.00085, p<0.05).2. ACL protein expression in EOC tissues and normal ovarian tissuesACL protein expression was detected by Western blot. The result showed that the expression of ACL and phosphorylated ACL (p-ACL) protein in EOC tissues were significantly increased compare to normal ovarian tissues (p<0.05). SREBP-1is a basic-helix-loop-helix-leucine zipper protein that regulates the transcription of lipogenic enzymes. To dissect the relationship of SREBP-1with ACL in EOC, we detected SREBP-1expression by western blot. Our data showed that ACL and p-ACL expression were independent of either the125kDa precursor form of SREBP-1or the68kDa mature and cleaved nuclear form.3. ACL/p-ACL protein expression in82EOC tissues and the relationships with clinicopathologic parameters 3.1ACL and p-ACL expression scores and localizationACL and p-ACL expression in82EOC tissues were detected by immunohistochemistry. Immunohistochemical results showed that ACL and p-ACL expression was detected in both the cytoplasm and the nucleus of tumor cells in some samples. The staining intensity of ACL or p-ACL was scored on a scale of0-4(0, no staining;1, weak;2, moderate;3, moderate to strong;4, strong). The patients were classified into two groups:low ACL/p-ACL (scores of0,1, or2) and high ACL/p-ACL (scores of3or4). SREBP-1expression was also divided into two groups according to the ACL classification.3.2p-ACL protein expression and the relationships with clinicopathologic parametersIn82EOC tissues, high p-ACL and low p-ACL rates were64.6%(53/82cases) and35.4%(29/82cases), respectively. The high p-ACL rates were30%(3/10cases),63.2%(12/19cases),71.7%(38/53cases) in tumor grade1,2,3, respectively; and were42.9%(9/21cases) and72.1%(44/61cases) in stage Ⅰ-Ⅱ and Ⅲ-Ⅳ, respectively. High p-ACL expression was significantly correlated with poor differentiation (p=0.044) and advanced FIGO stage (p=0.020). p-ACL expression was not correlated with age, tumor size, histological type or SREBP-1expression (p>0.05).3.3ACL protein expression and the relationships with clinicopathologic parametersIn82EOC tissues, high ACL and low ACL rates were62.2%(51/82cases) and37.8%(31/82cases), respectively. ACL expression was not correlated with any of the clinicopathologic parameters studied (p>0.05).4. The relationship between ACL/p-ACL expression and survival in patients with EOC4.1Univariate survival analysis of clinicopathologic parametersUnivariate survival analysis showed that p-ACL expression, age, tumor grade and FIGO stage were significantly associated with survival (p<0.05).4.2Cox regression modelIn a multivariate Cox regression model with these significant covariates, age and FIGO stage were significant factors for prediction of a poor prognosis (p=0.001and p=0.031, respectively), the risk ratio (RR) were3.189(95%CI,1.658-6.134) and9.089(95%CI,1.224-67.500)Conclusion:1. ACL is overexpressed and activated in EOC, and p-ACL expression was significantly correlated with tumor grade and FIGO stage, which suggests that ACL could promote ovarian cancer tumorigenesis and progression.2. p-ACL expression was significantly correlated with overall survival. ACL may be a useful marker for EOCs with especially poor prognoses. PART II:Effect of ACL gene silencing on biological characteristics of ovarian cancer cell linesObjective:To investigate the effect of ACL-siRNA-mediated gene silencing on biological characteristics of ovarian cancer cell lines A2780and OVCAR3. To explore the therapeutic potential of ACL.Methods:Design and chemically synthesize siRNAs based on the sequence of ACL mRNA. They were transfected into ovarian cancer cell lines A2780and OVCAR3with LipofectamineTM2000. The ACL protein level was detected by Western blot. The ACL-siRNA which has the more powerful inhibition was selected. Cell growth inhibition was observed with MTT; cell cycle distribution was observed with flow cytometry; the invasive ability was observed with transwell matrigel invasion assays.Results:1. ACL-siRNAs transfection and ACL protein expression. ACL-siRNAs were transfected into ovarian cancer cell lines A2780and OVCAR3with LipofectamineTM2000. After transfection with siRNA for72h, protein expression in A2780and OVCAR3cells were detected by Western blot. The result showed that the expression of ACL protein was reduced after ACL-siRNAs transfection (p<0.05). Between the two ACL-siRNAs (siACL-1and siACL-2), siACL-1is more effective.2. Cell growth inhibition by ACL-siRNA (siACL-1) transfection. Cell growth inhibition rates in A2780and OVCAR3cells were detected by MTT after siACL-1transfected for24,48,72,96h. The result showed that the ACL knockdown cells proliferated more slowly than did the control cells, and significant differences between the ACL knockdown cells and control cells were observed at48,72and96h (p<0.05), the cell growth inhibition rates were (19.3±1.8)%,(21.5±2.2)%and (19.1±2.1)%in A2780cells, and (13.7±1.1)%,(14.3±1.3)%and (7.1±0.8)%in OVCAR3cells.3. The effect of ACL-siRNA (siACL-1) on cell cycle progression. Cell cycle distribution in A2780and OVCAR3cells were detected by flow cytometry (FCM) after siACL-1were transfected. The result showed that after transfection for72h, ACL knockdown induced a progressive reduction in the S-phase population that was associated with a progressive parallel accumulation of G1-phase cells in both A2780and OVCAR3(p<0.05). The percentages of A2780cells in the G1, G2/M, and S phases were G1,66.86%; S,29.01%; and G2-M,4.13%(negative control siRNA); G1,89.11%; S,6.76%; and G2-M,4.14%(siACL-1); The percentages of OVCAR3cells were G1,64.53%; S,25.54%; and G2-M,9.92%(negative control siRNA); G1,84.01%; S,10.12%; and G2-M,5.86%(siACL-1).4. The effect of ACL-siRNA (siACL-1) on invision. Invision ability of A2780and OVCAR3cells were detected by transwell assay after siACL-1were transfected. The result showed that after ACL knockdown, there was no significant change in invasion ability in A2780or OVCAR3cells (p>0.05).Conclusion:ACL-siRNAs can significantly reduced ACL expression. ACL knockdown inhibits cellular proliferation and induces cell cycle arrest in vitro. ACL may be a novel therapeutic target for epithelial ovarian cancer.