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Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells

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Tumor Biology

Abstract

Recently, we demonstrated the association of sperm-associated antigen 9 (SPAG9) expression with breast cancer. Among breast cancer, 15 % of the cancers are diagnosed as triple-negative breast cancers (TNBC) based on hormone receptor status and represent an important clinical challenge because of lack of effective available targeted therapy. Therefore, in the present investigation, plasmid-based small hairpin (small hairpin RNA (shRNA)) approach was used to ablate SPAG9 in aggressive breast cancer cell line model (MDA-MB-231) in order to understand the role of SPAG9 at molecular level in apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT) signaling. Our data in MDA-MB-231 cells showed that ablation of SPAG9 resulted in membrane blebbing, increased mitochondrial membrane potential, DNA fragmentation, phosphatidyl serine surface expression, and caspase activation. SPAG9 depletion also resulted in cell cycle arrest in G0–G1 phase and induced cellular senescence. In addition, in in vitro and in vivo xenograft studies, ablation of SPAG9 resulted in upregulation of p21 along with pro-apoptotic molecules such as BAK, BAX, BIM, BID, NOXA, AIF, Cyto-C, PARP1, APAF1, Caspase 3, and Caspase 9 and epithelial marker, E-cadherin. Also, SPAG9-depleted cells showed downregulation of cyclin B1, cyclin D1, cyclin E, CDK1, CDK4, CDK6, BCL2, Bcl-xL, XIAP, cIAP2, MCL1, GRP78, SLUG, SNAIL, TWIST, vimentin, N-cadherin, MMP2, MMP3, MMP9, SMA, and β-catenin. Collectively, our data suggests that SPAG9 promotes tumor growth by inhibiting apoptosis, altering cell cycle, and enhancing EMT signaling in in vitro cells and in vivo mouse model. Hence, SPAG9 may be a potential novel target for therapeutic use in TNBC treatment.

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Acknowledgments

This work is supported by grants from Indo-UK Cancer Research Program (Grant No. BT/IN/UK/NII/2006), Centre for Molecular Medicine (Grant No. BT/PR/14549/MED/14/1291), NII-core funding, Department of Biotechnology, Government of India. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. We acknowledge Dr V. Kumar, Senior Staff Scientist, International Centre for Genetic Engineering and Biotechnology, New Delhi, India, for critical reading and editing of this manuscript. We also thank technical support by Mrs. Rekha Rani, National Institute of Immunology, New Delhi, India, for SEM imaging.

Authors’ contributions

NJ, NG, SA, APT, DP, RF, AS, and VK carried out all the experiments, prepared the figures, and drafted the manuscript. NJ participated in data analysis and interpretation of results. AS designed the study and participated in data analysis and interpretation of results. All authors read and approved the manuscript.

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    Authors and Affiliations

    1. Cancer Research Program, Cancer Microarray, Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India

      Nirmala Jagadish, Namita Gupta, Sumit Agarwal, Deepak Parashar, Aditi Sharma, Rukhsar Fatima, Amos Prashant Topno, Vikash Kumar & Anil Suri

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    1. Nirmala Jagadish
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    Correspondence to Anil Suri.

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    Namita Gupta and Sumit Agarwal contributed equally to this work.

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    Supplementary Fig. 1

    Western blot analysis demonstrates up-regulation of apoptotic moleclues, BAD, BAX, cyto-C, Caspase 7 and down-regulation of anti-apoptotic molecule, BCL-XL in SPAG9 ablated MCF7, BT-474 and SK-BR-3 breast cancer cells. β-actin was used as a loading control. Western Blotting was repeated in three independent experiments. (PPTX 805 kb)

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    Jagadish, N., Gupta, N., Agarwal, S. et al. Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells. Tumor Biol. 37, 13101–13110 (2016). https://doi.org/10.1007/s13277-016-5240-6

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    • DOI: https://doi.org/10.1007/s13277-016-5240-6

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