The Expression And Significance Of SIAH1 And Bim In Breast Cancer

Breast cancer is the most leading cause of cancer death in the women all over the world. The occurrence of breast cancer experienced several stages:normal, normal ductal hyperplasis, atypical ductal hyperplasis, ductal carcinomain situ and invasive ductal carcinoma. Thus, acquire of new target molecules that play important roles in breast carcinogenesis will be essential for improving therapeutic intervention and prognosis of breast cancers.SIAH (seven in absentia homolog) proteins are homologues of Drosophila seven in absentia (SINA) protein. SINA has two human homologues, SIAH1 and SIAH2. Siahl locates in chromosome 16q12 and encodes a 282-amino-acid protein with 76% amino acid identity to the Drosophila SINA protein. Siah2 maps to chromosome 3q25 and encodes a 324-amino-acid protein that shares 68% identity with Drosophila SINA and 77% identity with human SIAH1.SIAH1 and SIAH2 protein differ significantly at their N termini. SIAH1 has been described to have E3 ubiquitin ligase activity and target some proteins, such asΒ-catenin, c-myb, APC and SIAH1, for ubiquitin-mediate degradation. SIAH1 may function as a tumor suppressor gene. The study showed that SIAH1 expression was lower in HCC cell lines, and overexpression of SIAH1 could induce apoptosis of HCC cells. Some reports showed that SIAH1 overexpression inhibited cell growth and increased apoptosis through major alteration of mitosis. Other showed that SIAH1 was activated during apoptosis and tumor suppression in p53-dependent and-independent cellular models.The BH3-only protein Bim (Bcl-2-interacting mediator of cell death) is a member of Bcl-2 famliy. It is a critical mediator of cell apoptosis in various cell types. It was reported that the activation of Bim by certain sitmulation of apoptosis was interacted with Bcl-2/Bax, inducing mitochondrial pathway of apoptosis.The emerging evidence showed that SIAH1 could induce apoptosis by activating the c-Jun NH2-terminal kinade (JNK) pathway. And JNK pathway played an important role in cell apoptosis. Some studies suggested that Bim was involved in JNK-dependent apoptosis. So we hypothesized that SIAH1 might induce apoptosis by up-regulating the expression of Bim via JNK pathway. It is necessary to confirm this suppose in our further studies.In our present study, we implored the expression of SIAH1 and Bim in breast tissues and analyzed the relationship between SIAH1 expression and clinical pathology parameters. In addition, we examined the overexpression or knockdown of SIAH1 had an effect on the expression of Bim, the activity of MAPK pathway, apoptosis and invasion in breast cancer cells.Materials and Methods1. Patients and specimensA total of 231 cases of breast tissues (including 40 cases of normal breast tissues (NBT),53 cases of atypical ductal hyperplasia (ADH),36 cases of ductal carcinoma in situ (DCIS), and 102 cases of invasive ductal carcinoma (IDC))were obtained from the January 1st,2005 to the December31st,2008 at the First Affiliated Hospital of China Medical University. All of the enrolled patients underwent curative surgical resection without having chemotherapy or radiation therapy. Formalin-fixed paraffin-embedded sections of tissues obtained from surgical samples were stained routinely with hematoxylin and eosin (H&E). Among these samples, some fresh specimens and corresponding normal tissue samples were stored at-70℃immediately after resection until the extraction of protein and RNA.2. Immunohistochemical study and result assessmentFour-micron thick sections were prepared from the paraffin-embedded tissues. Immunostaining was performed by the streptavidin-peroxidase (S-P) method. For negative control, the primary antibodies were replaced by non-immune serum. Brown particles appearing in cytoplasm was as regarded as positive cells. The intensity of immunostaining (1=weak,2=moderate, and 3=intense) and the percentage of positive cells (0%=negative,1-50%=1,51-75%=2,≥76%=3) were assessed in at least 5 high power fields (×400 magnification). The scores of each sample were multiplied to give a final score of 0,1,2,3,4,6 or 9, and the tissues were finally determined as negative if score< 4; and positive expression if score≥4.3. Cell cultureHuman breast cancer cell lines MCF-7, MDA-MB-231, and human breast epithelial MCF-10A cell line were maintained in Dulbcco's Modifed Eagle Medium (DMEM) supplemented 10% fetal bovine serum in a humidified atmosphere with 5% of CO2.4. Western blotThe protein was extracted with lysis buffer (150 mM NaCl,1%NP-40,0.1%SDS, 2μg/ml aprotinin,1 mM PMSF) for 1 hour at 4℃. The supernatants were centrifuged at 12000 rpm for 30 minutes at 4℃. The supernatants containing total protein were harvested. Aliquots containing 60μg of proteins were separated on a 12% SDS-polyacrylamide gel and transferred to Polyvinylidene Fluoride (PVDF) membranes. After blocking, the blots were respectively incubated with primary antibody directed against SIAH1 (1:400), Bim (1:1000), JNK (1:1000), p-JNK (1:1000), ERK (1:400), p-ERK (1:400), p38 (1:400), p-p38 (1:400) or p-actin (1:200) overnight at 4℃and followed by each corresponding second antibody at room temperature for 2 hour at 37℃. Then the results developed by ECL. The protein bands were then analyzed using the Bioimaging System. The grayscale values of the SIAH1 and Bim bands were normalized to the values of the correspondingβ-actin band to determine the expression of the protein. 5. RT-PCRTotal RNA was isolated using Trizol Reagent according to the manufacturer's instructions. SIAH1 and Bim RT-PCRs were performed using a TaKaRa RNA PCR Kit (AMV) Ver.3.0 according to the manufacturer's protocol.β-actin served as an internal control. After electrophoresis, the PCR products were stained with ethidium bromide and analyzed using a Bioimaging system. Relative band intensities were determined using NIH image software.6. Small RNA InterferenceThe small interfering RNA (siRNA) duplexes were synthesized and purified by Ji Kai. SIAH1 target sequence was as follows:1:5'-AACTCCTGCCTCCTTATGTA TTT-3'; 2:5'-GAUAGGAACACGCAAGCAA-3'; 3:5'-GUUGCAUGUAGUAACA CUA-3'. The nonsilencing siRNA 1 (control siRNA 1) sequence was as follows: 5'-AAGAGCCGTCAGACTGCTACA-3'. Bim target sequence was as follows: 1:5'-ACCGAGAAGGUAGACAAUU-3'; 2:5'-CUACCUCCCUACAGACAGA-3'; 3: 5'-CUACCUCCCUACAGACAGA-3'. The nonsilencing siRNA 2 (control siRNA 2) sequence was as follows:5'-CGUACGCGGAAUACUUCGA-3'. Considering the relative effectiveness and stability, SIAH1 siRNA 1 and Bim siRNA 1 were selected by comparing our pilot experiments.7. Cell transfectionThe SIAH1 expression vector pcDNA3-myc-SIAH1 and the empty vector pcDNA3-myc were kindly provided by Dr. Matsuzawa Shu-ichi (Burnham Institute, La Jolla, California, USA). The cells were transfected with the SIAH1/Bim siRNA or pcDNA3-myc-SIAH1 using Lipofectamine 2000, following the manufacturer's instructions. The control siRNA and pcDNA3-myc were used as negative controls.8.3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay The transfected cells were seeded in 96-well plates (1×104 cells/well). Cell proliferation was evaluated each day for 4 days after transfection using MTT method. The absorbance, which was directly proportional to the mumber of living cells in culture, was measured at 490 nm using a microplate reader. A blank with dimethyl sulfoxide (DMSO) alone was measured and subtracted from all values. All the assays were done at least 3 times independently. The cell growth curve was analyzed.9. Matrigel invasion assayCell invasive ability was examined using a 24-well Transwell with 8μm pore polycarbonate memberance inserts according to the manufacturer's protocol. The Matrigel (100μg/ml 1:7) which was precooled with serum-free DMEM was applied to the upper surface of the membranes. After transfection for 24 hours, cells were seeded on the upper chamber (5×104 cells/well) and incubated for 32 hours. Nontransfected cells served as the control. Cells that had invaded the surface of the membrane were fixed with methanol and stained with hematoxylin. Five random high-magnification microscope fields per filter were counted.10. Flow cytometry (FCM)After 48 hours of culture, cells from each experimental group were collected and digested with trypsin and fixed with 75% ice-cold ethanol at 4℃overnight. Cells (1×107) were centrifuged at 1000 rpm 5 minutes, and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis. The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.11. Statistical analysisAll statistical calculations were performed by SPSS 13.0 for Windows software. P values less than 0.05 were considered statistically significant.Results1,SIAH1 was down-regulated in breast cancer tissues and cell, and inducing apoptosis of breast cancer cells by up-regulating the level of Bim(1)The expression of SIAH1 and Bim significantly decreased concurrently in the process of breast carcinogenesis and were correlated with well to moderately-differentiated and early-stage breast cancer. The immunohistochemistry results showed there was a decreasing tendency in positive rate of SIAH1 and Bim expression from normal breast tissues (NBT) (85.0% and 87.5%, respectively), atypical ductal hyperplasia (ADH) (66.0% and 67.9%, respectively), ductal carcinoma in situ (DCIS) (52.8% and 63.9%, respectively) to invasive ductal carcinoma (IDC) (28.4% and 34.3%, respectively). There were statistically significant difference of SIAH1 (P=0.002 and 0.008, respectively) and Bim (P= 0.016 and 0.002, respectively) expression between the NBT and DCIS and between the DCIS and IDC. The Spearman's correlation test revealed that the SIAH1 expression was significantly positively associated with Bim expression in the 231 breast specimens (P< 0.001, rs=0.395). The clinicopathological data analysis showed that the expression of SIAH1 (P=0.025) and Bim (P=0.045) were significantly lower in poorly-differentiated tumors (gradeⅢ) than in well to moderately-differentiated tumors (gradeⅠandⅡ). The SIAH1 (P=0.018) and Bim (P=0.032) expression were significantly lower in advanced-stage tumors (stageⅢandⅣ) compared to early-stage tumors (stageⅠandⅡ).(2) SIAH1 mRNA and protein expression were significantly lower in breast cancer tissues and cell linesThe RT-PCR and Western blot results showed that SIAH1 mRNA (P<0.05) and protein (P<0.05) expression were significantly lower in breast tumor tissues in comparison with the non-tumorous counterparts, and in human breast cancer MDA-MB-231, MCF-7 cell lines in comparison with human normal breast epithelial MCF-10A cell line (P<0.05).(3) Overexpression of SIAH1 inducing apoptosis of breast cancer cells by up-regulating the level of Bim:Our study showed a moderate increase in apoptosis after MDA-MB-231 and MCF-7 cells transfected with pcDNA3-myc-SIAH1 (approximately 18.06%±3.35% and 19.27%±4.32%, respectively, P< 0.05), compared with the cells transfected with the empty vector (approximately 1.11%±0.21% and 0.05%±0.008%, respectively). MDA-MB-231 and MCF-7 cells co-transfected pcDNA3-myc-SIAHl and Bim siRNA 1 (approximately 0.96%±0.18% and 0.01%±0.002%, respectively) showed lower apoptosis rate than cells transfected with pcDNA3-myc-SIAH1 (approximately 18.06%±3.35% and 19.27%±4.32%, respectively).(4) SIAH1 inhibiting the invasive ability of breast cancer cells was independent of Bim:Invasion assays showed that both MDA-MB-231 and MCF-7 cells transfected with SIAH1 siRNA 1 had more cells (55.21±5.23 and 43.17±4.12, respectively, P< 0.05) invaded onto the lower surfaces of the Transwell filters than the cells transfected with control siRNA 1 (13.26±2.18 and 11.43±1.83, respectively) or untreated (15.45±2.36 and 11.69±1.62, respectively). In contrast, both MDA-MB-231 and MCF-7 cells transfected with pcDNA3-myc-SIAH1 had fewer cells (3.21±0.96 and 2.93±0.73, respectively, P<0.05) invaded onto the lower surfaces of the Transwell filters than the cells transfected with pcDNA3-myc (14.36±2.32 and 11.73±1.76, respectively) or untreated (15.45±2.36 and 11.69±1.62, respectively). MDA-MB-231 and MCF-7 cells co-transfected with pcDNA3-myc-SIAH1 and Bim siRNA 1 (3.06±0.89 and 2.09±0.68, respectively) had no significant difference with cells transfected with pcDNA3-myc-SIAH1 (3.21±0.96 and 2.93±0.73, respectively).2,SIAH1 induced apoptosis by activation of JNK/Bim pathway and inhibited cell invasion by inactivation of ERK pathway in breast cancer cells(1) SIAH1 regulated the expression of MAPKs in MDA-MB-231 and MCF-7 cell lines:Western blot results showed that overexpression of SIAH1 up-regulated the expression of P-JNK but down-regulated the expression of P-ERK. Meanwhile, SIAH1 siRNAs down-regulated the expression of P-JNK but up-regulated the expression of P-ERK, and SIAH1 siRNA 1 was the most effective in the three SIAH1 siRNAs. The expression of P-P38 had no change after the cells were transfected with pcDNA3-myc-SIAH1 or SIAH1 siRNAs(2) Overexpression of SIAH1 induced apoptosis of MDA-MB-231 and MCF-7 cells by JNK/Bim pathway:Western blot results showed that overexpression of SIAH1 significantly up-regulated the expression of Bim and P-JNK, but SP600125 significantly inhibited overexpression of SIAH1-regulated the expression of Bim and P-JNK. The flow cytometry results showed that overexpression of SIAH1 significantly increased the apoptosis in MDA-MB-231 and MCF-7 cell lines (17.96% or 19.31%, respectively), but SP600125 significantly inhibited overexpression of SIAH1-induced apoptosis (0.05% or 0.02%, respectively).(3) SIAH1 inhibited cell invasion through ERK pathway:Western blot results confirmed that the expression of P-ERK up-regulated by SIAH1 siRNA 1 was suppressed by ERK inhibitor PD98059. And invasion assays showed that both MDA-MB-231 and MCF-7 cells transfected with SIAH1 siRNA 1 (47.78±6.47 or 34.11±5.64, respectively, P<0.05) had greater number of cells which invaded onto the lower surfaces of the Transwell filters than cells transfected with SIAH1 siRNA 1 and meanwhile incubated with PD98059 (6.32±1.01 or 7.14±1.12, respectively, P<0.05).(4) Both JNK and ERK pathway effected on SIAH1-associated cell growth inhibition:MTT assay results showed that the suppressed proliferation of the cancer cells transfected with pcDNA3-myc-SIAH1 was significantly reversed by SP600125. We also observed that the level of proliferation in cells transfected with SIAH1 siRNA 1 was significantly higher than those cells transfected with SIAH1 siRNA 1 and meanwhile incubation with PD98059.Conclusion1. SIAH1 functions as a tumor suppressor gene in the breast carcinogenesis and SIAH1 is significantly positively correlated with the expression of Bim. SIAH1 and Bim expression are significantly correlated with well to moderately-differentiated tumors and early-stage tumors.2. SIAH1 induces apoptosis of MDA-MB-231 and MCF-7 cells by JNK/Bim pathway.3. SIAH1 inhibiting the invasive ability of breast cancer cells is independent of Bim.