Piwil2 Suppresses P53 by Inducing Phosphorylation of Signal Transducer and Activator of Transcription 3 in Tumor Cells Yilu Lu . , Kun Zhang . , Chao Li., Youlin Yao, Dachang Tao, Yunqiang Liu, Sizong Zhang, Yongxin Ma* State Key Laboratory of Biotherapy, Division of Morbid Genomics, Department of Medical Genetics, West China Hospital, Sichuan University, Chengdu, China Abstract Piwi proteins have been implicated in germ cell proliferation, differentiation, germline stem cell maintenance and transposon control in germline from Drosophila to mammals. The Piwi-like2 (piwil2) gene is mainly expressed in testis or embryonic cells among normal tissues but widely expressed in tumors. However, it remains to be fully determined through which mechanism piwil2 is involved in tumorigenesis. Here we report that Human piwil2, or Hili represses the tumor suppressor P53 in human cancer cells. Immunoprecipitation analysis shows that Piwil2 can directly associate with Signal Transducer and Activator of Transcription 3 (STAT3) protein via its PAZ domain and form a Piwil2/STAT3/c-Src triple protein- protein complex. Furthermore, STAT3 is phosphorylated by c-Src and translocated to nucleus, then binds to P53 promoter and represses its transcription. The present study demonstrated that Piwil2 plays a role in anti-apoptosis in tumor cells possessing P53 as a positive regulator of STAT3 signaling pathway, providing novel sights into roles of Piwil2 in tumorigenesis. Citation: Lu Y, Zhang K, Li C, Yao Y, Tao D, et al. (2012) Piwil2 Suppresses P53 by Inducing Phosphorylation of Signal Transducer and Activator of Transcription 3 in Tumor Cells. PLoS ONE 7(1): e30999. doi:10.1371/journal.pone.0030999 Editor: Kevin D. Bunting, Emory University, United States of America Received September 29, 2011; Accepted December 29, 2011; Published January 27, 2012 Copyright: 2012 Lu 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 is supported by the National Natural Science Foundation of China (90919006 and 31070676), Trans-Century Training Programme Foundation for the Talents by the Ministry of Education of China (grant no. NCET-07-0580) and Spring Sunshine Programme by the Ministry of Education of China (grant no. Z2009-1-61003). 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: mayongxin@gmail.com . These authors contributed equally to this work. Introduction The Argonaute gene family which encode basic proteins that contain both PAZ and Piwi conserved domains, have been reported to induce histone and DNA methylation, mRNA breakdown and inhibition of translation [1,2]. As a predominantly germline specific clade of Argonaute gene family, Piwi subfamily are found in all animals tested so far and play essential roles in stem-cell self-renewal, gametogenesis and RNA silencing in diverse organisms [3–8]. Mutations of three Piwi homologs in mice (miwi, mili and miwi2) respectively cause arrestment of spermatogenesis and male sterility [9–11]. The piwil2 gene, alias mili in mouse or Hili in human, is mainly expressed in testis or embryonic cells among normal tissues but widely expressed in tumors, suggesting that Piwil2 may disturb cell division, inhibit apoptosis and play a role as dose-dependent oncogenic fate determinants. However, the underlying mechanism through which Piwil2 involved in tumorigenesis remains largely unknown yet [12,13]. The tumor suppressor p53 which involved in a number of cellular signaling pathways is known to play an essential role in regulating apoptosis. Loss of P53 function is a common feature of many human cancers [14–16]. Though deletions or mutations of P53 have been observed in a great number of tumors, the HeLa cell line possesses wild-type P53 alleles detectable in both mRNA and protein level [17]. Interestingly, Lin et al. reported that in prostate cancer cell lines wild-type but not mutant P53 can significantly inhibit STAT3 activity, whereas it has also been reported that STAT3 can bind to P53 promoter and inhibit the P53 gene transcription rate [18,19]. STAT signaling pathways activated in response to cytokines and growth factors have been reported to constitutively express in varied tumor-derived cell lines and tumor tissues [20]. STAT3 activation can resist apoptotic machinery relied anti-tumor therapies, and also enhance the growth of tumor cell [18]. Because its activation can mediate oncogenic transformation in cultured cells and tumor formation in nude mice, Stat3 has been classified as an oncogene [21]. Here we present that human piwil2 gene suppresses apoptosis by phosphorylating STAT3 along with c-Src and initiating transcriptional silencing of P53. Results Piwil2 inhibits P53 involved apoptosis in HeLa Cells Previous studies have reported that Piwil2 is expressed in various tumors and inhibits apoptosis when transfected into embryo fibroblast cells [13]. To investigate the role which human piwil2 protein, alias HILI plays in tumorigenesis and apoptosis in cancer cells, expression vectors and siRNAs were transfected into HeLa Cells. Real-time qPCR and Western blot analysis revealed that there was a significant decrease in P53 expression following the over-expression of Piwil2 in both mRNA and protein level, while its counterpart, Piwil2-knockdowned HeLa cells express a higher level of P53 (Fig. 1A). We also examined several other PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30999