Tumor and Stem Cell Biology Microtubule-Associated Histone Deacetylase 6 Supports the Calcium Store Sensor STIM1 in Mediating Malignant Cell Behaviors

Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca2þ storage sensor that promotes cell growth, migration, and angiogenesis in breast and cervical cancers. Here, we report that the microtubuleassociated histone deacetylase 6 (HDAC6) differentially regulates activation of STIM1-mediated store-operated Ca2þ entry (SOCE) between cervical cancer cells andnormal cervical epithelial cells. Confocalmicroscopy of living cells indicated that microtubule integrity was necessary for STIM1 trafficking to the plasma membrane and interaction with Orai1, an essential pore subunit of SOCE. Cancer cells overexpressed both STIM1 and Orai1 compared with normal cervical epithelial cells. HDAC6 upregulation in cancer cells was accompanied by hypoacetylated a-tubulin. Tubastatin-A, a specific HDAC6 inhibitor, inhibited STIM1 translocation to plasma membrane and blocked SOCE activation in cancer cells but not normal epithelial cells. Genetic or pharmacologic inhibition of HDAC6 blocked STIM1 membrane trafficking and downstream Ca2þ influx, as evidenced by total internal reflection fluorescent images and intracellular Ca2þ determination. In contrast, HDAC6 inhibition did not affect interactions between STIM1 and the microtubule plus end-binding protein EB1. Analysis of surgical specimens confirmed that most cervical cancer tissues overexpressed STIM1 and Orai1, accompanied by hypoacetylated a-tubulin. Together, our results identify HDAC6 as a candidate target to disrupt STIM1-mediated SOCE as a general strategy to block malignant cell behavior. Cancer Res; 73(14); 4500–9. 2013 AACR. Introduction Modulation of cytosolic Ca2þ levels provides versatile and dynamic signaling that mediates fundamental cellular functions, such as proliferation, migration, gene regulation, and apoptosis (1). Increase in cytosolic Ca2þ concentrations occurs as a result of Ca2þ entry from the extracellular space and Ca2þ release from intracellular stores, mainly from the endoplasmic reticulum. Store-operated Ca2þ entry (SOCE) is a major Ca2þ entry pathway in nonexcitable cells (2, 3). The SOCE activation includes several steps: (i) stimulation of G proteins or protein tyrosine kinases activates phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate to release the second messenger inositol-1, 4, 5-trisphosphate (IP3). (ii) Binding of IP3 to IP3 receptor in the endoplasmic reticulum membrane causes rapid and transient Ca2þ release from endoplasmic reticulum lumen. (iii) The decrease of endoplasmic reticulum luminal Ca2þ activates store-operated Ca2þ (SOC) channels in the plasma membrane, leading to a sustained influx of extracellular Ca2þ across the plasma membrane (2, 4). Two genes, STIM1 (stromal-interaction molecule 1) and Orai1, are responsible for SOCE activation (2). STIM1 functions as an endoplasmic reticulum Ca2þ sensor that detects store depletion. Once endoplasmic reticulum Ca2þ is depleted, STIM1 aggregates into multiple punctate that translocates to the close proximity of plasma membranes. Orai1, an essential pore-forming component of SOC channel, translocates to the same STIM1containing structures during endoplasmic reticulum Ca2þ depletion and opens to mediate Ca2þ entry. STIM1 is required for the development and function of regulatory T cells (5, 6) and STIM1 deficiency causes several autoimmune diseases and myopathy in human subjects and mouse models (7). The emerging importance of STIM1 in tumor biology has been highlighted in breast and cervical cancer (8–10). Inhibiting STIM1-mediating Ca2þ influx impaired focal adhesion turnover of breast cancer cells, which could be rescued by the small GTPases Ras and Rac (8). The reduction of Orai1 or STIM1 by RNA interference in highly Authors' Affiliations: Departments of Biomedical Engineering, and Pharmacology; Institute of Basic Medical Sciences; Medical Device Innovation Center; Department of Obstetrics andGynecology; Advanced Optoelectronic Technology Center; Infectious Diseases and Signaling Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University; and National Institute of Cancer Research, National Health Research Institutes, Taiwan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Y.T. Chen and Y.F. Chen contributed equally to this work. Corresponding Author: Meng-Ru Shen, Department of Obstetrics & Gynecology, National Cheng Kung University Hospital, Tainan 704, Taiwan. Phone: 886-6-2353535 ext 5505; Fax: 886-6-2766185; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-4127 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(14) July 15, 2013 4500 on April 15, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 22, 2013; DOI: 10.1158/0008-5472.CAN-12-4127