Crucial Role of TSC-22 in Preventing the Proteasomal Degradation of p53 in Cervical Cancer

et al. (2012) Crucial Role of TSC-22 in Preventing the Proteasomal Degradation of p53 in Cervical Cancer. PLoS ONE 7(8): e42006. doi:10.1371/journal.pone.0042006 Crucial Role of TSC-22 in Preventing the Proteasomal Degradation of p53 in Cervical Cancer Cheol-Hee Yoon 0 Seung Bae Rho 0 Seong-Tae Kim 0 Seongho Kho 0 Junsoo Park 0 Ik-Soon Jang 0 Seonock Woo 0 Sung Soon Kim 0 Je-Ho Lee 0 Seung-Hoon Lee 0 Qian Tao, The Chinese University of Hong Kong, Hong Kong 0 1 Department of Life Science, Yongin University , Cheoin-gu, Yongin-si, Gyeonggi-do, Korea , 2 Division of AIDS, Center for Immunology and Pathology, National Institute of Health , Cheongwon-gun, Chungbuk , Korea , 3 Research Institute, National Cancer Center , Ilsandong-gu, Goyang-si Gyeonggi-do, Korea , 4 Division of Biological Sciences and Technology, Yonsei University , Wonju , Korea , 5 Korea Basic Science Institute, Daegeon Center , Daegion , Korea , 6 South Sea Environment Research Department, Korea Ocean Research and Development Institute , Geoje , Korea , 7 School of Medicine, Sungkyunkwan University, Samsung Medical Center , Seoul , Korea The p53 tumor suppressor function can be compromised in many tumors by the cellular antagonist HDM2 and human papillomavirus oncogene E6 that induce p53 degradation. Restoration of p53 activity has strong therapeutic potential. Here, we identified TSC-22 as a novel p53-interacting protein and show its novel function as a positive regulator of p53. We found that TSC-22 level was significantly down-regulated in cervical cancer tissues. Moreover, over-expression of TSC-22 was sufficient to inhibit cell proliferation, promote cellular apoptosis in cervical cancer cells and suppress growth of xenograft tumors in mice. Expression of also TSC-22 enhanced the protein level of p53 by protecting it from poly-ubiquitination. When bound to the motif between amino acids 100 and 200 of p53, TSC-22 inhibited the HDM2- and E6-mediated p53 polyubiquitination and degradation. Consequently, ectopic over-expression of TSC-22 activated the function of p53, followed by increased expression of p21Waf1/Cip1 and PUMA in human cervical cancer cell lines. Interestingly, TSC-22 did not affect the interaction between p53 and HDM2. Knock-down of TSC-22 by small interfering RNA clearly enhanced the polyubiquitination of p53, leading to the degradation of p53. These results suggest that TSC-22 acts as a tumor suppressor by safeguarding p53 from poly-ubiquitination mediated-degradation. - Funding: This research was mainly supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20110026217) and partially assisted by the Korea Basic Science Institute NAP grant (T3278B) and the internal grant from Korea National Institute of Health. The funders had no role in study design, data collection and analysis, dicision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. TGF-b stimulated clone 22 (TSC-22) was first identified as a TGF-b-inducible gene in mouse osteoblastic cells. TSC-22 expression is induced in a variety of cell lines by TGF-b, phorbol ester, serum, and progestin and positively regulates the TGF-b signaling [1,2]. TSC-22 contains a leucine zipper-like motif, but it does not have a DNA-binding motif at the N-terminal region. TSC-22 can homodimerize and heterodimerize with TSC-22 homologous gene-1 (THG-1), and has transcriptional repressor activity [3]. Some researchers have identified the physiological roles of TSC22 in the developmental process. TSC-22 is required for gastrulation during early embryogenesis in Xenopus laevis [4] and for oogenesis in Drosophila [5]. It has been also suggested that TSC22 induces erythroid cell and Cardiac myofibroblast differentiation via activating the transcriptional activity of Smad3 and Smad4, and antagonizing the Smad7 in response to TGF-b-dependent signaling [6,7]. Additionally, several studies have focused on the tumor suppression functions of TSC22. TSC22 is thought to be a potent tumor suppressor in salivary cancer cells [8,9] human gastric carcinoma cells [10], hepatic carcinoma [11], human astrocytic tumors [12], and large granular lymphocyte leukemia [13]. The detailed mechanisms of the tumor suppressor function of TSC22 have been reported along with the hypothesis that TSC-22 represses the expression of the anti-apoptotic genes Gadd45b and Lzts2 [11], negatively regulates Ras/Raf signaling [14] and involved in the TGF-b-mediated gastric carcinoma cell death in a caspase3-dependent manner [10]. On the other hand, TSC22mediated apoptotic activity is inhibited by the interaction between TSC-22 and fortillin, followed by leading to TSC-22 destabilization [15]. p53 is a well-known tumor suppressor gene that acts by activating the transcription of its targeted genes such as p21, PUMA, , PKR and BAX [1618]. p53 functions are regulated by post-translational modifications such as phosphorylation, acetylation, and ubiquitination. It is well-understood that p53 levels are tightly regulated by MDM2-mediated ubiquitination via an auto-regulatory feedback loop [19,20]. Occasionally, the regulation of p53 expression is correlated with tumorigenesis through infection by human papilloma virus expressing E6, which leads to ubiquitin-mediated p53 degradation [21,22]. Even though the regulation of p53 has been examined via multiple Figure 1. Expression of TSC-22 was decreased in human cancer tissue. (A) Total RNA was prepared from patients cancer specimens and the TSC-22 mRNA level was then evaluated with real-time RT-PCR. Cx; serial number of patient tissue samples. (B) HeLa and Caski cells were plated on 6well plates. After 24 h, the cells were infected with Ad-TSC-22 or Ad-LacZ. At the indicated time points, cell numbers were determined by MTT assay to analyze cell proliferation rates. (C) HeLa cells infected with Ad-TSC22 or Ad-LacZ were cultured for the indicated times, and the cells were then stained with propidium iodide (PI). The sub-G1 cell population (dead cell) and cell cycle profile of the PI-stained cells were analyzed by flow cytometry after PI-staining. (D) DNA fragmentation assay was performed by isolating chromosomal DNA from 16106 number of HeLa and Caski cells infected with Ad-TSC22 or Ad-lacZ for 72 hrs (left). After 3 days infection of Ad-TSC-22, p53, Puma, p21, E6 and TSC22 expression were analyzed by Western hybridization (right). doi:10.1371/journal.pone.0042006.g001 routes related to tumorigenesis, many questions remain about the tumor inhibition mechanism of the p53 pathway. Since the mechanism underlying the network of tumor suppressor genes has not yet been explicitly elucidated, we conducted a cDNA microarray analysis of the gene expression profile in cancer tissue to find novel tumor-related genes. We found (...truncated)