PTTG1 Attenuates Drug-Induced Cellular Senescence Yunguang Tong, Weijiang Zhao, Cuiqi Zhou, Kolja Wawrowsky, Shlomo Melmed* Department of Medicine, Cedars-Sinai Medical Center, University of California Los Angeles, Los Angeles, California, United States of America Abstract As PTTG1 (pituitary tumor transforming gene) abundance correlates with adverse outcomes in cancer treatment, we determined mechanisms underlying this observation by assessing the role of PTTG1 in regulating cell response to anti- neoplastic drugs. HCT116 cells devoid of PTTG1 (PTTG12/2) exhibited enhanced drug sensitivity as assessed by measuring BrdU incorporation in vitro. Apoptosis, mitosis catastrophe or DNA damage were not detected, but features of senescence were observed using low doses of doxorubicin and TSA. The number of drug-induced PTTG12/2 senescent cells increased ,4 fold as compared to WT PTTG1-replete cells (p,0.001). p21, an important regulator of cell senescence, was induced ,3 fold in HCT116 PTTG12/2 cells upon doxorubicin or Trichostatin A treatment. Binding of Sp1, p53 and p300 to the p21 promoter was enhanced in PTTG12/2 cells after treatment, suggesting transcriptional regulation of p21. p21 knock down abrogated the observed senescent effects of these drugs, indicating that PTTG1 likely suppresses p21 to regulate drug- induced senescence. PTTG1 also regulated SW620 colon cancer cells response to doxorubicin and TSA mediated by p21. Subcutaneously xenografted PTTG12/2 HCT116 cells developed smaller tumors and exhibited enhanced responses to doxorubicin. PTTG12/2 tumor tissue derived from excised tumors exhibited increased doxorubicin-induced senescence. As senescence is a determinant of cell responses to anti-neoplastic treatments, these findings suggest PTTG1 as a tumor cell marker to predict anti-neoplastic treatment outcomes. Citation: Tong Y, Zhao W, Zhou C, Wawrowsky K, Melmed S (2011) PTTG1 Attenuates Drug-Induced Cellular Senescence. PLoS ONE 6(8): e23754. doi:10.1371/ journal.pone.0023754 Editor: S.K. Batra, University of Nebraska Medical Center, United States of America Received March 16, 2011; Accepted July 24, 2011; Published August 17, 2011 Copyright: 2011 Tong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The manuscript is supported by NIH Grant CA 75979 (SM), K99CA138914 (YT), and The Doris Factor Molecular Endocrinology Laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Melmed@csmc.edu Introduction PTTG1 (pituitary tumor transforming gene), originally isolated from rat pituitary tumor cells [1], is a securin protein abundantly expressed in hematopoietic, colorectal, thyroid, lung, breast and ovarian malignancies [2]. PTTG1 was proposed as a marker of breast cancer aggressiveness [3,4,5]. Increased PTTG1 expression correlates with higher colorectal tumor grade, invasion of surrounding lymph nodes and increased tumor vascularity [6,7]. PTTG1 is a natively unfolded protein [8] which binds to ,700 gene promoters as indicated by ChIP-on-Chip assay [9], and interacts with ,80 proteins [10]. In addition to its securin function [11], PTTG1 regulates G2/M phase transition by inhibiting separase cleavage and aurora kinase A activity [10,12]. PTTG1 also regulates G1/S transition by inducing cyclin D3 and repressing p21 expression [9]. PTTG1 induces FGF2, stimulates c-myc expression, inhibits p53 DNA binding and transactivation and regulates apoptosis [13,14,15,16,17]. PTTG1 over-expression leads to cell transformation in NIH-3T3 cells, while mouse embryo fibroblast cells devoid of Pttg are resistant to oncogenic transformation [9]. Targeted pituitary PTTG over-expression in Rb+/2 mice resulted in enhanced cellular activity, increased gland volume, and higher prevalence of anterior pituitary tumors [18,19]. Conversely, transgenic pttg deletion abrogated murine pituitary tumor development [20]. Recent research indicated that PTTG1 regulates cancer cell growth responses to treatments. Head and neck squamous cell carcinomas with higher tumor PTTG1 levels exhibit increased recurrences after radiation, 5-fluorouracil (5-FU) and cisplatinum treatment [21]. Breast cancers with higher PTTG1 levels are resistant to tamoxifen and fulvestrant [22]. Reports on gliomas [23], hepatocellular [24], thyroid [25] and esophageal carcinoma patients [26] also indicate that higher tumor PTTG1 levels correlate with adverse clinical outcomes. PTTG1 has been implicated in mediating oncogene-induced pituitary tumor senescence [27]. As senescence also determines cancer therapy outcomes, we examined whether PTTG1 regulates drug-induced cellular senescence. Here we show here that the presence of PTTG1 is a determinant of drug-induced senescence. These findings contribute to understanding the requirement of PTTG1 for regulation of cancer cell response to anti-neoplastic drugs. Materials and Methods Cell lines and reagents HCT116 is a human colon carcinoma cell line, expressing wild- type p53 and PTTG1 genes, and HCT116 p532/2 and HCT116 PTTG12/2 cell lines were derived using homologous recombina- tion to knock out p53 or PTTG1 respectively [28,29]. Cells were cultured in McCoy’s 5A medium containing 15% FBS. SW620 human colon cancer cells were purchased from ATCC (Manassas, VA) and cultured as recommended. PTTG1 and p300 siRNA were purchased from Ambion (Austin, TX), p21 and p53 siRNA from Cell Signaling (Danvers, MA), PTTG antibody from Invitrogen (Carlsbad, CA), p300 and HDAC1 antibodies from Santa Cruz (Santa Cruz, CA), p21 antibody from Cell Signaling, and p53 antibody from CalBioChem (Gibbstown, NJ). PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23754