TIG3 Tumor Suppressor-Dependent Organelle Redistribution and Apoptosis in Skin Cancer Cells Tiffany M. Scharadin1, Haibing Jiang 1 , Ralph Jans1, Ellen A. Rorke4, Richard L. Eckert1,2,3* 1 Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America, 2 Department of Dermatology, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America, 3 Department of Obstetrics and Gynecology and Reproductive Sciences, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America, 4 Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland, United States of America Abstract TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis. Citation: Scharadin TM, Jiang H, Jans R, Rorke EA, Eckert RL (2011) TIG3 Tumor Suppressor-Dependent Organelle Redistribution and Apoptosis in Skin Cancer Cells. PLoS ONE 6(8): e23230. doi:10.1371/journal.pone.0023230 Editor: Janine Santos, University of Medicine and Dentistry of New Jersey, United States of America Received March 25, 2011; Accepted July 12, 2011; Published August 17, 2011 Copyright: 2011 Scharadin 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: This work was supported by the National Institutes of Health grants to RE (NIH R01 AR094713 and NIH R01 AR04694). 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: reckert@umaryland.edu Introduction TIG3 (Tazarotene-induced gene 3), which is also called retinoic acid receptor responder 3 (RARRES3) and retinoid-inducible gene 1 (RIG1) [1–5], is a one hundred sixty-four amino acid protein [6]. TIG3 was originally identified as increased following treatment of cultured epidermal keratinocytes or psoriatic epidermis with the synthetic retinoid, Tazarotene [6]. It is expressed at low levels in hyperproliferative epidermis (e.g., squamous cell carcinoma and psoriasis) and expression is restored by retinoid treatment [7–9]. In retinoid-treated psoriatic epidermis, increased TIG3 expression is associated with restoration of normal differentiation [6,10]. The association of increased TIG3 expression with normal epidermal phenotype suggests that TIG3 may act as a pro-differentiation regulator. To examine the mechanism of action, we studied TIG3 function in normal human keratinocytes [10–12]. These studies show that TIG3 is present at vanishingly low levels in keratinocytes in monolayer culture, but is increased in differentiated raft cultures [12]. Vector-mediated expression of TIG3 in keratinocytes results in reduced proliferation and increased cornified envelope formation, suggesting that TIG3 regulates keratinocyte differentiation [10–12]. Ongoing studies show that TIG3 operates via several mechanisms, but a prominent mechanism of action is regulation of transgluta- minase activity [10,11]. Type I transglutaminase (TG1) is a key enzyme in keratinocytes and other surface epithelia that is expressed in suprabasal differentiated cells [13–20]. Transglutaminase catalyzes formation of M-(c-glutamyl)lysine protein-protein cross- links to assemble the cornified envelope, an essential component of the epidermal barrier [21,22]. Our studies suggest that TIG3 co- localizes with TG1 leading to increased transglutaminase activity [10,11]. Additional studies show that TIG3 reduces keratinocyte proliferation, but does not cause apoptosis [10,11]. TIG3 consists of an amino terminal hydrophilic segment and a c-terminal membrane anchoring domain [6,23]. Mutagenesis studies indicate that mutants lacking the c-terminal membrane-anchoring domain are not active [10,11,23]. In contrast, N-terminal truncation converts TIG3 into a protein that causes apoptosis in keratinocytes [12]. TIG3 is expressed at reduced levels in skin tumors [7]. Thus, a major goal of the present study is to characterize the impact of TIG3 expression in skin cancer cells. We show that restoring TIG3 expression reduces survival of epidermal squamous cell carcino- ma cells via a mechanism that involves pericentrosomal TIG3 localization leading to altered microtubule organization and organelle distribution. This is associated with changes in the level of cell cycle and apoptosis regulators. Results TIG3 expression decreases cell number We began by examining the impact of TIG3 on SCC-13 cell survival. TIG3 was delivered by adenovirus infection. Fig. 1A shows that empty vector-infected cells increase in number over PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23230