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RASSF5 Inhibits Growth and Invasion and Induces Apoptosis in Osteosarcoma Cells Through Activation of MST1/LATS1 Signaling

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RASSF5 Inhibits Growth and Invasion and Induces Apoptosis in Osteosarcoma Cells Through Activation of MST1/LATS1 Signaling ONCOLOGY REPORTS 32: 1505-1512, 2014 RASSF5 inhibits growth and invasion and induces apoptosis in osteosarcoma cells through activation of MST1/LATS1 signaling XU-HUI ZHOU1, CHAO-QUN YANG1, CHENG-LIN ZHANG1, YANG GAO1, HONG-BIN YUAN2 and CE WANG1 Departments of 1Orthopedic Surgery and 2Anesthesiology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China Received May 13, 2014; Accepted July 9, 2014 DOI: 10.3892/or.2014.3387 Abstract. Ras association (RalGDS/AF-6) domain family tumor suppressor in OS cells through activation of the MST1/ member RASSF5 has been implicated in a variety of key LATS1 pathway. biological processes, including cell proliferation, cell cycle regulation and apoptosis. It is believed to play an important Introduction role in tumorigenesis as a tumor suppressor in a number of malignancies. Yet, little is known concerning the function and Osteosarcoma (OS), originating from bone as an aggres- underlying mechanisms of RASSF5 in human osteosarcoma sive bone tumor, is an infrequent but the most common and (OS). The expression of RASSF5 was examined by immu- destructive primary bone tumor in children and adolescents. nohistochemical assay using a tissue microarray in 45 cases It is the second highest cause of cancer-related mortality, of OS tissues. A gain-of-function approach was used to mainly due to the development of often fatal metastasis (1). In observe the effects of lentiviral vector-mediated overexpres- the past few decades, surgical resection therapy has resulted sion of RASSF5 (Lv-RASSF5) on cell growth, invasion and in the poor prognosis of OS patients. With the application apoptosis, respectively, as indicated by MTT, Transwell and of neoadjuvant chemotherapy in OS, the five-year survival flow cytometry assays, and the expression levels of mamma- rate has significantly increased (2,3). To date, however, the lian sterile 20-like (MST1) kinase, large tumor suppressor 1 molecular pathogenesis and etiology of OS are still not clearly (LATS1), proliferating cell nuclear antigen (PCNA), matrix elucidated. metallopeptidase-9 (MMP-9) and p53 were detected by real- Members of the RASSF family (RASSF1-10) have been time PCR and western blot assays in OS cells (MG-63 and reported to participate in a variety of important biological U-2 OS). The results indicated that the expression of RASSF5 processes, and in particular have been identified as candidate protein was significantly downregulated in OS tissues tumor suppressors in cancers (4), of which RASSF5 (also compared to that in adjacent non-cancerous tissues (ANCT) called NORE1) is expressed in most normal tissues but is (40.0 vs. 73.3%, P=0.002), and had a negative correlation with downregulated in several cancer cell lines (5). The protein distant metastasis of the tumor (P=0.01). Overexpression of expression of RASSF5 is frequently downregulated in lung RASSF5 markedly suppressed cell proliferation and invasion, tumor cell lines and primary lung tumors, impairs cell growth and induced cell apoptosis in the OS cell lines with increased and mediates Ras-dependent apoptosis (6). RASSF5 has also expression of MST1, LATS1 and p53 and decreased expres- been identified as a breakpoint-spanning gene downregulated sion of PCNA and MMP-9. Taken together, our findings in clear cell renal cell carcinoma (CCRCC), and inhibits cell demonstrate that RASSF5 expression is negatively correlated proliferation, representing a new candidate tumor suppressor with distant metastasis of OS, and RASSF5 may function as a for CCRCC (7). Some studies show that no inactivating somatic mutation for RASSF5 is found in lung tumor lines, but the RASSF5 promotor region is hypermethylated in primary tumors and tumor cell lines (8). It is known that methylation of Correspondence to: Dr Ce Wang, Department of Orthopedic promotor CpG islands is a common mechanism inactivating Surgery, Changzheng Hospital, Second Military Medical University, tumor-suppressor genes in cancer. RASSF family genes 415 Fengyang Road, Shanghai 200003, P.R. China including RASSF5 to various degrees are methylated in E-mail: [email protected] neuroblastoma cell lines and primary tumors (9). Methylation Dr Hong-Bin Yuan, Department of Anesthesiology, Changzheng of RASSF5 frequently occurs in squamous cell carcinomas of Hospital, Second Military Medical University, 415 Fengyang Road, the head and neck (10), and even has the potential for serving Shanghai 200003, P.R. China as a recurrence biomarker for bladder cancer (11). E-mail: [email protected] However, few studies indicate that no promotor methylation of RASSF5 is detected in thyroid tumor (12) and methylation is Key words: RASSF5, osteosarcoma, growth, invasion, apoptosis an uncommon event in primary thyroid tumors (13). Moreover, promotor methylation is not the molecular mechanism respon- sible for RASSF5 suppression in neuroblastic tumors (14). 1506 ZHOU et al: RASSF5 for cancer therapy Thus, to further clarify the function and molecular mecha- IHC staining. Tissue microarray sections were processed nism of RASSF5 in cancer, we examined the expression of for IHC analysis of RASSF5 protein as follows. RASSF5 in OS tissues by immunohistochemical (IHC) assay, Immunohistochemical examinations were carried out on and investigated the effects of RASSF5 overexpression on cell 3-mm sections. For anti-RASSF5 IHC, unmasking was growth and invasion in vitro. We hypothesized that RASSF5 performed with 10 mM sodium citrate buffer, pH 6.0, at 90˚C may function as a tumor suppressor implicated in the develop- for 30 min. For anti-RASSF5 IHC, antigen unmasking was ment of OS. not necessary. Sections were incubated in 0.03% hydrogen peroxide for 10 min at room temperature, to remove endog- Materials and methods enous peroxidase activity, and then in blocking serum [0.04% bovine serum albumin, A2153; Sigma-Aldrich, Shanghai, Materials. The OS cell lines (MG-63 and U-2 OS) used for the China and 0.5% normal goat serum X0907; Dako Corporation, experiments were obtained from the Institute of Biochemistry Carpinteria, CA, USA, in phosphate-buffered saline (PBS)] for and Cell Biology (Shanghai, China). Lentiviral-mediated 30 min at room temperature. Anti-RASSF5 antibody was used RASSF5 vector (Lv-RASSF5), negative control vector, and at a dilution of 1:200. The antibody was incubated overnight virion-packaging elements were purchased from GeneChem at 4˚C. Sections were then washed three times for 5 min in (Shanghai, China). Human OS tissues and the corresponding PBS. Non-specific staining was blocked with 0.5% casein and ANCT were collected from the Department of Orthopedic 5% normal serum for 30 min at room temperature. Finally, Surgery, Changzheng Hospital. OS tissue microarray was staining was developed using diaminobenzidine substrate, constructed by Shanghai Outdo Biotech Co., Ltd. (Shanghai, and sections were counterstained with hematoxylin. Normal China). All the antibodies were purchased from Cell Signaling serum or PBS was used to replace the anti-RASSF5 antibody Technology (Boston, MA, USA). RASSF5 primer was synthe- in negative controls. sized by ABI (Framingham, MA, USA). Quantification of protein expression. The expression of Drugs and reagents. Dulbecco's modified Eagle's medium RASSF5 was semi-quantitatively estimated as total immu- (DMEM) and fetal bovine serum (FBS) were purchased nostaining scores, which were calculated as the product of a from Thermo Fisher Scientific Inc. (Waltham, MA, USA); proportion score and an intensity score. The proportion and TRIzol reagent and Lipofectamine 2000 were obtained from intensity of the staining were evaluated independently by two Invitrogen (Carlsbad, CA, USA); M-MLV reverse transcrip- observers. The proportion score reflected the fraction of posi- tase was purchased from Promega (Madison, WI, USA); tive staining cells (0, none; 1, <10%; 2, 10 to ≥25%; 3, >25 SYBR-Green Master Mix was obtained from Takara (Otsu, to 50%; and 4, >50%), and the intensity score represented the Japan); and the ECL Plus kit was obtained from GE Healthcare staining intensity (0, no staining; 1, weak; 2, intermediate; (Piscataway, NJ, USA). Cell apoptosis kit [propidium iodide and 3, strong). Finally, a total expression score was obtained (PI), RNase A, Annexin V-FITC] was from KeyGen Biology ranging from 0 to 12. Based on the analysis in advance, (Nanjing, China). RASSF5 expression was categorized into two groups: low- level RASSF5 expression (score 0-3) and high-level RASSF5 Clinical samples and data. The tissue microarray was expression (score 4-12). The scoring was independently prepared for IHC test. Human OS tissues and the corre- assessed by two pathologists. sponding ANCT were obtained from biopsies in a total of 45 consecutive OS cases admitted to our hospital from January Cell culture and transfection. OS cells were cultured in 2005 to December 2011. The baseline characteristics of the DMEM supplemented with 10% heat-inactivated FBS, 100 U/ patients before neo-adjuvant chemotherapy are summarized ml of penicillin, and 100 µg/ml of streptomycin. Cells in this (Table II). The study was approved by the Medical Ethics medium were placed in a humidified atmosphere containing Committee of the Second Military Medical University, and 5% CO2 at 37˚C. Cells were subcultured at a 1:5 dilution in written informed consent was obtained from the patients medium containing 300 µg/ml G418 (an aminoglycoside or their parents before sample collection. Two pathologists antibody; a commonly used stable transfection reagent in respectively reviewed all of the cases. molecular genetic testing). On the day of transduction, OS cells were replated at 5x104 cells/well in 24-well plates containing Tissue microarray. The Advanced Tissue Arrayer (ATA-100; serum-free growth medium with Polybrene (5 mg/ml). When Chemicon International, Temecula, CA, USA) was used to reaching 50% confluency, the cells were transfected with create holes in a recipient paraffin block and to acquire cylin- recombinant experimental virus or control virus at the optimal drical core tissue biopsies with a diameter of 1 mm from the multiplicity of infection (MOI) of 50, and cultured at 37˚C specific areas of the ‘donor’ block.
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