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Calcium Sensor, NCS-1, Promotes Tumor Aggressiveness and Predicts Patient Survival Lauren M

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Calcium Sensor, NCS-1, Promotes Tumor Aggressiveness and Predicts Patient Survival Lauren M Published OnlineFirst March 8, 2017; DOI: 10.1158/1541-7786.MCR-16-0408 Signal Transduction Molecular Cancer Research Calcium Sensor, NCS-1, Promotes Tumor Aggressiveness and Predicts Patient Survival Lauren M. Moore1, Allison England1, Barbara E. Ehrlich2, and David L. Rimm1 Abstract Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional pendent breast cancer cohorts by the Automated Quantitative þ Ca2 -binding protein that affects a range of cellular processes Analysis method of quantitative immunofluorescence. Elevated beyond those related to neurons. Functional characterization of levels of NCS-1 were significantly correlated with shorter survival NCS-1 in neuronal model systems suggests that NCS-1 may rates. Furthermore, multivariate analysis demonstrated that influence oncogenic processes. To this end, the biological role NCS-1 status was prognostic, independent of estrogen receptor, of NCS-1 was investigated by altering its endogenous expression progesterone receptor, HER2, and lymph node status. These in MCF-7 and MB-231 breast cancer cells. Overexpression of findings indicate that NCS-1 plays a role in the aggressive behavior NCS-1 resulted in a more aggressive tumor phenotype demon- of a subset of breast cancers and has therapeutic or biomarker strated by a marked increase in invasion and motility, and a potential. decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitax- Implications: NCS-1, a calcium-binding protein, is associated el-induced cell death in a manner that was independent of cellular with clinicopathologic features of aggressiveness in breast cancer proliferation. To determine the association between NCS-1 and cells and worse outcome in two breast cancer patient cohorts. Mol clinical outcome, NCS-1 expression was measured in two inde- Cancer Res; 1–11. Ó2017 AACR. Introduction In addition to neurons, NCS-1 (14) has been identified in non- þ neuronal tissues (34–36). The widespread expression of this Calcium (Ca2 ), a ubiquitous second messenger molecule, is protein suggests that it may have other functions in addition to involved in numerous physiologic processes associated with its characterized role in neuronal models. For example, the tumor progression (e.g., proliferation, adhesion, migration, and þ functional properties of NCS-1 indicate that it may influence invasion; refs. 1–4). The dysregulation of Ca2 homeostasis has oncogenic processes. Recently, NCS-1 has been shown to act as been implicated as an important driver in the development of a survival factor through the indirect activation of the PI3K/AKT metastatic phenotypes (5). Numerous studies demonstrate that þ signaling pathway (24), a pathway known to be perturbed in Ca2 -binding proteins [e.g., S100 proteins (6–9), calmodulin many forms of cancer (37). Based upon its potential to influence (10, 11), calcineurin (12, 13)] have the potential to promote oncogenic processes and its established relationship with taxanes tumor progression. þ (30, 38), we hypothesize that increased expression of NCS-1 may Neuronal Calcium Sensor-1 (NCS-1) is a Ca2 -binding protein be associated with tumor aggressiveness, taxane response, and that is expressed at high concentrations in neurons (14). The poor clinical outcome in breast cancer patients. In this study, we characterization of NCS-1 in neuronal models demonstrates its þ assess the role of NCS-1 in breast cancer biology and drug involvement in Ca2 -signaling transduction (15–18), exocytosis response by altering the endogenous expression of this protein (19–21), membrane trafficking (22, 23), cell survival (24), and in MCF-7 and MB-231 cell lines. In addition, we examined the functional regulation of a diverse group of proteins (Supplemen- clinical association between NCS-1 expression and survival in tary Fig. S1; refs. 25–29). NCS-1 has also been identified as a novel breast cancer patients. binding partner of the chemotherapeutic drug, paclitaxel (30), and this binding interaction was shown to alter the biological activity of NCS-1 in vitro and in vivo (31–33). Materials and Methods Validation and reproducibility assessment of the NCS-1 antibody 1 Department of Experimental Pathology, Yale School of Medicine, New Haven, A rabbit monoclonal antibody targeting NCS-1 (Abcam, cata- 2 Connecticut. Department of Pharmacology, Yale School of Medicine, New log number: ab129166) was validated for specificity, selectivity, Haven, Connecticut. and reproducibility using in-house techniques, previously Note: Supplementary data for this article are available at Molecular Cancer described (39). The optimized signal-to-noise ratio for the Research Online (http://mcr.aacrjournals.org/). NCS-1 antibody was 0.339 mg/mL (Fig. 1A). Antibody specificity Corresponding Author: David L. Rimm, Yale University School of Medicine, 310 was assessed by staining for NCS-1 expression in formalin-fixed, Cedar Street P.O. Box 208023, New Haven, CT 06520-8023. Phone: 203-737- paraffin-embedded brain tissues from NCS-1 knockout mice and 4204; Fax: 203-737-5089; E-mail: [email protected] wild-type controls (Fig. 1B). Specificity was further evaluated by doi: 10.1158/1541-7786.MCR-16-0408 examining the expression of NCS-1 in a panel of cancer cell lines Ó2017 American Association for Cancer Research. (Fig. 1C). Reproducibility was assessed by comparing NCS-1 www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst March 8, 2017; DOI: 10.1158/1541-7786.MCR-16-0408 Moore et al. NCS-1+/+ brain -/- B NCS-1 brain A NCS-1 Antibody titration curve 8,000 2.5 2.0 6,000 1.5 4,000 1.0 2,000 0.5 0 0.0 3.39 0.679 0.339 0.0679 NCS-1 in Tumor mask AQUA_Norm mask NCS-1 in Tumor Antibody concentrations (mg/mL) Bottom 10% Top 10% Ratio C D kDa 1234567891011 12,000 250 Lane 1: BT-20 150 Lane 2: MB-453 10,000 Lane 3: MB-231 100 8,000 Lane 4: MB-468 75 Lane 5: SKBR3 6,000 Lane 6: A431 50 4,000 Lane 7: ZR75 37 Lane 8: UACC812 2,000 R2 = 0.9818 Lane 9: BT747 Scores (Run 2) AQUA NCS-1 25 0 NCS-1 Lane 10: MCF-7 21 kDa 20 0 5,000 10,000 15,000 15 Lane 11: T47D NCS-1 AQUA Scores (Run 1) Figure 1. The NCS-1 antibody is specific and reproducible. A, Antibody titration curve of commercially available NCS-1 antibody reveals optimal antibody concentration of 0.339 mg/mL. B, Photomicrograph of cerebellum tissue from wild-type and NCS-1 knockout mice stained for NCS-1 and DAPI reveals specificity of NCS-1 antibody. C, Immunoblot analysis of NCS-1 in multiple cancer cell lines shows NCS-1 at predicted 21 kDa. D, Linear regression of control TMAs stained on two different days. Pearson's coefficient, R2 values > 0.90 denotes assay reproducibility. staining from serial sections run on different days. The regression trol (catalog no.: sc-108080) and NCS-1 shRNA lentiviral particles of these control tissue microarrays (TMA) showed a high linear (catalog no.: sc-36019-V) were purchased from Santa Cruz Bio- regression coefficient (R2 ¼ 0.98; Fig. 1D). technology Inc. (Paso Robles, CA, USA). For the hNCS-1 plasmid, lentiviral particles were produced through the transfection of Breast cancer cell line culture maintenance HEK-293T cells. Briefly, HEK-293T cells were seeded and trans- Breast cancer cell lines (MCF-7, ATCC HTB-22 and MB-231, fected with a mixture of hNCS-1 lentiviral plasmid and second- ATCC HTB-26) were obtained from the American Type Culture generation packaging plasmids using Lipofectamine 2000 per the Collection (ATCC). The ATCC validates all cell lines by short manufacturer's instructions. After 18 to 24 hours, transfection tandem repeat analysis. Upon receipt, cell lines were used within 6 media were removed and replaced with fresh media. Cells were months. MCF-7 cell lines were maintained at 37 C, 5% CO2 in then incubated for additional 24 hours. After this period, lenti- Eagle's Minimum Essential Medium supplemented with 10% viral-containing supernatant was collected, centrifuged, and fil- FBS, 1% penicillin/streptomycin, 1% MEM nonessential amino tered through a 0.45-mm filter. The virus was stored at –80 C until acids, 1% sodium pyruvate, and 0.01 mg/mL human recombi- the day of lentiviral transduction. nant insulin. MB-231 cell lines were maintained at 37 C, 0% CO2 in Leibovitz's L-15 Medium (L-15) supplemented with 10% FBS Lentiviral transduction and 1% penicillin/streptomycin. Cells were seeded in a 6-well plate at a density of 5 Â 105 cells/ well 24 hours prior to transduction. The following day, media Lentiviral plasmid, particles, and production were replaced with fresh media supplemented with polybrene at a A Human NCS-1 (hNCS-1) lentiviral plasmid was purchased concentration of 8 mg/mL. For hNCS-1 lentiviral particles, 0.5 mL from Applied Biological Materials (catalog no.: LV800661). Con- of virus was directly added to each well. For the more concentrated OF2 Mol Cancer Res; 2017 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst March 8, 2017; DOI: 10.1158/1541-7786.MCR-16-0408 Functional Role of NCS-1 in Breast Cancer control and NCS-1 shRNA lentiviral particles, 10 mL of virus was upper chamber, which contained SFM. Cells were allowed to added to each well. Following transduction, cells were incubated migrate toward the chemoattractant (media supplemented with at 37C. After 24 hours, media were replaced with fresh media. 10% FBS) in the lower chamber. At the endpoints indicated, 72 After 48 hours, puromycin was added at a predetermined optimal hours (MCF-7) or 24 hours (MB-231), cells were removed from concentration.
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