Downregulation of ER60 Protease Inhibits Cellular Proliferation by Inducing G1S Arrest in Breast Cancer Cells In Vitro.код для вставкиСкачать
THE ANATOMICAL RECORD 295:410–416 (2012) Downregulation of ER60 Protease Inhibits Cellular Proliferation by Inducing G1/S Arrest in Breast Cancer Cells In Vitro ZIN-MAR LWIN,1 GEORGE WAI-CHEONG YIP,1 FOOK-TIM CHEW,2 1 AND BOON-HUAT BAY * 1 Department of Anatomy, National University of Singapore, Singapore 2 Department of Biological Sciences, National University of Singapore, Singapore ABSTRACT ER60 protease, a 58-kDa molecular chaperone in the endoplasmic reticulum, is involved in glycoprotein synthesis. ER60 protease has been reported to be differentially expressed in various cancers including breast carcinoma. This study explored the relationship of ER60 protease with cell proliferation in breast cancer in vitro. ER60 protease expression was first determined in a panel of breast cell lines by real-time RT-PCR and Western blot analysis and found to be most abundantly expressed in T47D breast cancer cells. The ER60 protease gene was then successfully knocked down in T47D breast cancer cells using two different sequences of small-interfering RNA. The silencing efficiencies of siER-1 and siER-2 at 48-hr post-transfection were found to be >80% at the mRNA level with concomitant downregulation of the ER60 protease protein by >60% when compared with control T47D breast cancer cells. Downregulation of ER60 protease was also associated with inhibition of cell proliferation when assessed by the AlamarBlue assay. Cell cycle analysis performed on the siER-1- and siER-2-transfected cells, revealed an increase in G1 phase population and a decrease in the S and G2/M phase populations compared with control cells, implicating G1/S cell cycle arrest. It would appear that ER60 protease is involved in breast tumorigenesis and could therefore be a prospective target for cancer therapeutics. Anat Rec, 295:410–416, C 2012 Wiley Periodicals, Inc. 2012. V Key words: ER60; endoplasmic reticulum; breast cancer; gene silencing INTRODUCTION Breast cancer, the most prevalent female cancer in many parts of the world (Hery et al., 2008; Matsuda et al., 2010; Vuong et al., 2010), is also the leading cause of cancer death among women in both developed and developing countries (Jemal et al., 2011). The incidence rate of breast cancer is considered low in Asia [with an age-standardized rate (ASR) 24.0–32.5 per 100,000 depending on which part of Asia] when compared with Europe, Australia/New Zealand, and North America (ASR 76.7–89.9 per 100,000) (Jemal et al., 2011). However, the incidence of breast cancer in Singapore is much higher than that reported in the other parts of Asia. According to the Singapore Cancer Registry interim C 2012 WILEY PERIODICALS, INC. V report, the ASR for breast cancer in Singapore is 58.5 per 100,000 accounting for 17.1% of all female cancer deaths between 2004 and 2008 (National Registry of Diseases Office of Singapore). As a heterogeneous disease Grant sponsor: Singapore National Medical Research Council; Grant number: NMRC/1081/2006. *Correspondence to: Boon-Huat Bay, MBBS, PhD, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, MD10, Singapore 117 597, Singapore. E-mail: email@example.com Received 6 June 2011; Accepted 24 December 2011. DOI 10.1002/ar.22413 Published online 20 January 2012 in Wiley Online Library (wileyonlinelibrary.com). ER60 PROTEASE AND BREAST CANCER PROLIFERATION with distinct molecular profiles (Hsiao et al., 2010), breast cancer presents a great challenge to healthcare professionals in terms of diagnosis, management, and prognostication. Currently, clinical classification of breast cancer subtypes for deciding treatment regimes are based on the expression profile of common biomarkers including estrogen receptor, progesterone receptor, human epidermal growth factor receptor 1/2, and cytokeratin 5/6. ER60 protease, a protein with high sequence similarity to phosphoinositide-specific phospholipase C (PI-PLC) (Urade and Kito, 1992; Otsu et al., 1995; Urade et al., 2000), was first isolated from guinea pig uterus and was mistakenly identified as an isoenzyme of PI-PLC (Bennett et al., 1988). It was later purified as a novel cysteine protease from the endoplasmic reticulum of rat liver (Urade and Kito, 1992). ER60 protease is also known by several names including: ER60, ERp60, ERp57, GRp58, or 1,25D3-MARRS (Membrane Associated, Rapid Response Steroid binding) receptor (Khanal and Nemere, 2007a,b). ER60 has been studied extensively as an endoplasmic reticulum luminal chaperone protein involved in the quality control of newly synthesized glycoproteins (Khanal and Nemere, 2007b) as well as in the assembly of major histocompatibility complex class-I (MHC-I) molecules (Vigneron et al., 2009; Chapman and Williams, 2010). Studies involving various types of tissues have shown differential expression of ER60 protease in normal tissues as compared with cancerous tissues such as breast, lung, gastric, and ovarian carcinomas (Celli and Jaiswal, 2003; Leys et al., 2007; Cicchillitti et al., 2009). However, much remains to be elucidated to fully understand the role of ER60 protease in cancer. In this study, we elucidated the relationship of ER60 protease with cell proliferation (a hall mark of cancer) in breast cancer cells in vitro. MATERIALS AND METHODS Cell Culture Three human breast cancer cell lines, viz., MCF-7, T47D, and MDA-MB-231 breast cancer cells, as well as immortalized normal breast epithelial MCF-12A cells, were purchased from American Type Culture Collection (ATCC). Breast cancer cell lines were cultured in either Dulbecco’s-Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI 1640) medium supplemented with 10% fetal bovine serum. The normal breast epithelial MCF-12A cell line was cultured in DMEM-F12 (Invitrogen, Carlsbad, CA) medium with additional 20 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO), 0.01 mg/mL insulin (Sigma-Aldrich), 100 ng/ mL cholera toxin (Sigma-Aldrich), 500 ng/mL hydrocortisone (Sigma-Aldrich), and 5% fetal bovine serum (Hyclone, Thermo Fisher Scientific, Rockford, IL). All the breast cell lines were cultured in a 5% CO2 incubator at 37 C. 411 following the manufacturer’s protocol. The quantitative real-time polymerase chain reaction was performed using hot-start QuantiTect SYBR Green (Qiagen) and LightCycler 2.0 System (Roche Applied Science, Penzberg, Germany). The cycling conditions used were one cycle of 95 C for 15 min, 45 cycles of 94 C for 15 sec, 60 C for 25 sec, and 72 C for 12 sec. The primer sequences were 30 -AAG-CTC-AGC-AAA-GAC-CCA-AA-50 and 50 CAC-TTA-ATT-CAC-GGC-CAC-CT-30 for ER60 protease, and 30 -ATG-ACA-TCA-AGA-AGG-TGG-TG-50 and 50 CAT-ACC-AGG-AAA-TGA-GCT-TG-30 for GAPDH. The latter was the housekeeping gene used as the internal control for normalizing the expression level of ER60. Relative fold change was based on the formula 2DDCT (Livak and Schmittgen, 2001) with the MCF-12A breast epithelial cell line as the calibrator. Western Blot Total protein was extracted from cells using M-PERV Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) with added HaltTM protease inhibitor cocktail (Pierce). The extract was quantified by the Bradford method (Bradford, 1976), and 20 lg was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene difluoride membrane using the Semi-Dry Transfer Cell unit (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked overnight at 4 C with 5% nonfat milk dissolved in tris-buffered saline containing 0.1% Tween. For detection of targeted proteins, the membrane was incubated with the following antibodies for 1 hr at room temperature: rabbit anti-ER60 (1:1,000; Abcam, Cambridge, UK) and mouse anti-b-actin (1:6,000; SigmaAldrich), horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:6,000; Pierce ECLTM, GE Healthcare, Piscataway, NJ), and HRP-conjugated donkey antimouse antibody (1:6,000; Pierce ECLTM). The protein bands were visualized by incubation with chemiluminescence reagent (Pierce) and subsequent exposure of the X-ray film (Kodak, Rochester, NY). For quantification, the film was scanned and densitometric analysis performed to calculate the optical density (OD) ratio. R Immunocytochemical Staining Cells were fixed with 4% paraformaldehyde for 30 min at room temperature followed by quenching of endogenous peroxidase activity by incubation with 0.5% hydrogen peroxide dissolved in methanol for 30 min. The cells were then blocked with 5% goat serum for 1 hr at room temperature before incubation with rabbit polyclonal anti-ER60 primary antibody at 1:200 dilution for 1 hr at room temperature. The VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) was used following the manufacturer’s protocol and immunostaining was demonstrated using 3,-30 -diaminobenzidine (DAB) as the substrate before visualization of the slides under a light microscope. Quantitative Real-Time RT-PCR Total RNA was extracted using RNeasy Mini kit (Qiagen, Venlo, Netherlands). One microgram of total RNA was converted to cDNA using random hexamers with Superscript III 1st Strand Synthesis System (Invitrogen) Immunofluorescence Staining Cells were fixed as described above and blocked with 1% BSA for 1 hr at room temperature. Cells were then incubated with rabbit anti-ER60 primary antibody at 412 LWIN ET AL. Fig. 1. ER60 mRNA expression in breast cell lines. A: ER60 mRNA expression was measured using real-time RT-PCR. Relative fold change was calculated using 2DDCT using GAPDH as internal control and MCF-12A normal breast epithelial cell line as the calibrator cell line. Error bar ¼ SEM. **P < 0.01. B: Single bands with the correct product size (160 bp for ER60 and 177 bp for GAPDH) as verified by 1.2% agarose gel electrophoresis. Lanes 1 and 5 ¼ MCF-12A, lanes 2 and 6 ¼ MCF-7, lanes 3 and 7 ¼ MDA-MB-231, and lanes 4 and 8 ¼ T47D. For verification of the specificity of the PCR products, a standardized 45 PCR cycle was performed and the band intensities do not reflect the expression level. C: Representative melting curves of primers for ER60 and GAPDH. Single peak for each primer further validates the absence of primer dimers and nonspecific amplifications. 1:200 dilution for 1 hr at room temperature followed by Cy3-conjugated goat anti-rabbit secondary antibody (1:200; Sigma-Aldrich). The nuclei of the breast cancer cells were stained with DAPI (40 -6-diamidino-2-phenylindole) dye. The slides were subsequently visualized by confocal microscopy. mg/mL RNase A (Roche Applied Science), and 20 lg/mL propidium iodide (Sigma-Aldrich) dissolved in phosphate-buffered saline before analysis by the Dako Cytomation Cyan LX flow cytometer (Dako, Glostrup, Denmark). Silencing of ER60 Protease Gene by Small-Interfering RNA Two ER60 specific small-interfering RNAs (siRNAs; labeled siER60-1 and siER60-2) and one sequence of nontargeting siRNA (siNeg) were purchased from Ambion (Applied Biosystems, Austin, TX). DharmaFECT 1 transfection reagent was purchased from Dharmacon (Chicago, IL). Briefly, T47D cells were seeded into 6-well plates at 2.5 105 cells per well and allowed to stabilize for 24 hr before transfection with 2 mL of RPMI medium containing 10% FBS, 5 nM siRNA, and 4 lL transfection reagent per well. Statistical Analysis Comparisons between groups were analyzed with the one-way ANOVA and post-hoc Tukey’s test using the PASW Statistics 18 (SPSS, Chicago, IL). P-value < 0.05 was considered to be statistically significant. RESULTS ER60 Protease Expression in Breast Cancer Cell Lines Cell proliferation was assessed using the AlamarBlue assay (Invitrogen) by measuring the fluorescence reading at 570-nm excitation wavelength and 585-nm emission wavelength using a fluorescence spectrometer. After 24-hr post-transfection, cells were synchronized by serum starvation for 24 hr. The medium was then replaced with fresh medium containing 10% FBS and cells were allowed to grow for 1 day before subjecting them to cell viability assay. Cells were incubated in 2 mL of complete culture medium containing 10% AlamarBlue solution for 90 min before transferring 120-lL solution from each well into a 96-well plate for measurement of fluorescence reading. ER60 protease was expressed in all the four breast cell lines examined (MCF-7, MDA-MB-231, T47D, and MCF-12A cells) with T47D cells exhibiting the highest expression. As shown in Fig. 1A, ER60 protease mRNA transcript in T47D cells was almost 10 folds higher compared with the other three cell lines. The specificity of the primers was validated by DNA gel electrophoresis (Fig. 1B) and melting curve analysis (Fig. 1C). Western blotting also revealed that T47D cells had the highest ER60 protease protein expression (Fig. 2A), which was verified by quantification of the intensity of the protein bands using optical densitometry (Fig. 2B). The ER60 protease protein was observed to be localized primarily in the cytoplasm of the cells particularly at the perinuclear region by immunocytochemistry (Fig. 3A) and confirmed by immunofluorescence staining (Fig. 3B), corroborating the notion that ER60 is an endoplasmic reticulum luminal protein. Cell Cycle Analysis Silencing the ER60 Protease Gene siRNA-transfected cells were synchronized as described above before harvesting by trypsinization and fixation with ice-cold 70% ethanol overnight. Cells were stained using a cocktail containing 0.1% Triton-X, 0.2 T47D breast cancer cells were subsequently selected for ER60 protease knockdown experiments in view of the high expression of the protein in this breast cancer cell line. The ER60 protease gene was successfully Cell Proliferation Assay ER60 PROTEASE AND BREAST CANCER PROLIFERATION knocked down using two sequences of siRNA, labeled siER60-1 and siER60-2 with silencing efficiencies of 88% for both sequences at 48-hr post-transfection (Fig. 4A). There was concomitant downregulation of the ER60 protease protein (Fig. 4B), which was also demonstrated qualitatively by immunocytochemistry at 72-hr posttransfection, where a markedly lower staining intensity was observed in the siER60-1- and siER60-2-transfected cells (Fig. 4C). 413 Effects on Cell Proliferation and Cell Cycle Cell proliferation was measured by the AlamarBlue assay 72 hr after transfection (including 24 hr of serum starvation). Knocking down ER60 protease expression by siRNA significantly decreased cell proliferation in T47D breast cancer cells (Fig. 5) with the fluorescence readings decreasing by 38.5 6 2.5% for siER60-1-transfected cells and 11.2 6 3.1% for siER60-2-transfected cells (P < 0.001 and P < 0.01, respectively). Cell cycle analysis was carried out at 72-hr post-transfection, after 24-hr serum starvation to synchronize the growth phase of cells. Downregulation of ER60 protease in T47D breast cancer cells was accompanied by a significant increase in G1 population (P < 0.001), while the Sphase and G2/M populations were significantly reduced in siER60-1- and siER60-2-transfected cells (Fig. 6), indicating that ER60 protease downregulation induced G1/S arrest. The sub-G1 population, which represented apoptotic cells, was observed to be marginally increased. The S þ G2M phases which denote cell proliferation were significantly lower in the siER60-1- (26.05 6 0.58%) and siER60-2-transfected cells (22.76 6 0.56%) when compared with the siNeg controls (P < 0.01 in both instances). DISCUSSION Fig. 2. ER60 protein expression in breast cancer cell lines. A: Western blots of ER60 protease protein in breast cancer cell lines with bactin as the loading control. B: Protein bands were quantified by densitometric analysis. Relative expression was calculated by OD ratio of ER60 protease to b-actin and normalized with the MCF-7 cell line. Error bar ¼ SEM. Although the ER60 protease protein has been investigated for more than two decades (Bennett et al., 1988), the main areas of study were focused primarily on its function as a molecular chaperone with relatively little information available regarding its role in carcinogenesis. In fact, ER60 protease has recently attracted the attention of researchers in the field of cancer biology. In our study, we observed expression of ER60 protease at both mRNA and protein levels in breast cancer cells in vitro. MCF-12A cells, which represent normal breast epithelium, had comparable expression with MCF-7 cells but lower than MDA-MB-231 cells and T47D cells at the mRNA transcript level. This is in line with a Fig. 3. Localization of the ER60 protease protein in T47D breast cancer cells. A: Immunocytochemical staining of ER60 protease protein. Brown DAB staining in the cytoplasm indicates specific ER60 protease staining while the nuclei are stained blue by hematoxylin. Scale bar ¼ 50 lm. B: Immunofluorescence staining of ER60 protease protein. Red Cy3 immunofluorescence represents specific ER60 protease staining and DAPI dye stains the nucleus blue. Scale bar ¼ 25 lm. 414 LWIN ET AL. Fig. 4. Silencing of the ER60 protease gene in T47D breast cancer cells. A: Silencing efficiency of siRNAs targeting the ER60 protease gene. Relative mRNA expression of ER60 protease at 48-hr posttransfection was calculated using 2DDCT. Error bar ¼ SEM. B: Western blots of ER60 protease and b-actin (the housekeeping protein) and bar chart showing relative ER60 protease protein expression. Error bar ¼ SEM. C: Qualitative expression of ER60 protease protein as analyzed by immunocytochemistry. ER60 protease was visualized by the brown DAB staining. Nucleus was counterstained with hematoxylin. Scale bar ¼ 100 lm. previous finding reporting an upregulation of ER60 protease in some cancers including breast, uterus, lung, and stomach cancers (Celli and Jaiswal, 2003). It has been proposed that ER60 protease, an endoplasmic reticulum chaperone protein (Coe and Michalak, 2010), is involved in endoplasmic reticulum stress response (Corazzari et al., 2007; Ni and Lee, 2007) and promotes tumor cell survival in hypoxic microenvironment brought about by poor vascularization (Koumenis, 2006). However, ER60 protease expression has also been reported to be partially downregulated or even absent in cancers such as gastric adenocarcinoma (Leys et al., 2007), squamous cell esophageal carcinoma (Qi et al., 2008; Ayshamgul et al., 2011), cervical carcinoma (Mehta et al., 2008), and renal cell carcinoma (Tanaka et al., 2008). Some investigators have attributed this observation to ER60 protease being a key component for antigen presentation by MHC-I molecules during immune surveillance (Garbi et al., 2007; Chapman and Williams, 2010), and therefore a lower expression would facilitate immune evasion by cancer cells (Seliger et al., 2001; Dunn et al., 2006; Seliger et al., 2010). ER60 protein has also been associated with resistance to the chemotherapeutic drug Paclitaxel in A2780 ovarian cancer cells in vitro (Cicchillitti et al., 2009). However, overexpression of ER60 protease in Chinese Hamster Ovary stable transfectants has been reported to increase mitomycin C-induced DNA crosslinking leading to enhanced cytotoxicity on exposure to this anticancer drug (Celli and Jaiswal, 2003). Hence, more work needs to be done before one can ascertain the exact role played by ER60 protease in carcinogenesis. In this study, we observed that T47D cells had the highest ER60 protease gene expression compared with MCF-7 and MDA-MB-231 breast cancer cells. T47D and ER60 PROTEASE AND BREAST CANCER PROLIFERATION Fig. 5. Effect of downregulating ER60 protease expression on cell proliferation in T47D breast cancer cells. Relative fluorescence emitted represents the relative number of cells in each sample normalized against the control (siNeg) group. *P < 0.05, **P < 0.01, and ***P < 0.001. 415 cytokines and mitogens direct cells to enter the mitotic cycle and activate a cascade of signaling pathways including MAP kinase (MAPK) pathway (Tartaglia and Gelb, 2010), phosphatidyl inositol-3 kinase (PI3K)/AKT pathway (Castaneda et al., 2010) and JAK/STAT pathway (Spano et al., 2006; Borges et al., 2008). Dysregulation of these signaling pathways could result in aberrant cell proliferation as in the case of cancer (Kim and Choi, 2010). For instance, vitamin D is known to perturb regulation of the cell cycle and impede cell proliferation by inhibiting the MAPK–extracellular signal-regulated kinase (ERK) 1 and 2 signaling pathways (Deeb et al., 2007). Richard et al. (2010) have also recently demonstrated that the growth inhibition potential of vitamin Ds in MCF-7 breast cancer cells is mediated by a novel receptor identified as 1,25D3MARRS protein. A study by Apati et al. (2003) has shown that calcium induces cell survival and proliferation in TF-1 leukemia cell line by MAPK pathway activation and a known function of ER60 protease is maintenance of homeostasis of intracellular calcium. There is also evidence that ER60 protease can modulate STAT3 signaling (Coe et al., 2010). In conclusion, silencing the ER60 protease gene inhibited cell proliferation and induced G1/S cell cycle arrest in breast cancer in vitro. Several different mechanisms could be involved stemming from the multifunctional roles of ER60 protease. It would be worthwhile to elucidate the mechanistic pathway(s) responsible for regulating the proliferative effect exerted by ER60 protease in breast cancer cells. ER60 protease could therefore be a potential molecular target in breast cancer therapeutics. ACKNOWLEDGEMENTS The authors thank Ms. Saw Marlar from Flow Cytometry Laboratory (National University Medical Institute) and Ms. Bay Song Lin, Multimedia Unit (Department of Anatomy, National University of Singapore) for their technical assistance. Fig. 6. Effect of downregulating ER60 protease expression on regulation of the cell cycle in T47D breast cancer cells. Cell cycle was analyzed by flow cytometry using propidium iodide staining. A representative histogram depicting the different phases of the cell cycle in T47D breast cancer cells is displayed above the bar chart showing the subG1, G1, S, and G2/M cell populations. The y-axis represents the percentage of cells in each phase of the cell cycle from a sample population of 10,000 cells. Error bar ¼ SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. MCF-7 breast cancer cells share similar distinctive characteristics as both cell lines exhibit the luminal epithelial ERþ/PRþ/Her2 phenotype while MDA-MB-231 breast cancer are highly invasive and ER/PR/Her2þ (Lacroix and Leclerc, 2004). It is still unclear at this point in time the reason for T47D breast cancer cells displaying the highest ER 60 protease expression among the three cancer cell lines examined. We also demonstrated that siRNA-mediated silencing of the ER60 protease gene inhibited cell proliferation by inducing cell cycle arrest at the G1/S phase in T47D breast cancer cells. In eukaryotic cells, cell division or proliferation is a tightly controlled process driven by stimulators with fine tuning by inhibitors and feedback mechanisms (Lord, 1986; Kurzawa and Morris, 2010). Growth factors, LITERATURE CITED Apati A, Janossy J, Brozik A, Bauer PI, Magocsi M. 2003. 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