Title Nuclear-targeted deleted in liver cancer 1 ( DLC 1 ) is less efficient in exerting its tumor suppressive activity both in vitro and in vivo

Background: Deleted in liver cancer 1 (DLC1) serves as an important RhoGTPase activating protein (RhoGAP) protein that terminates active RhoA signaling in human cancers. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends not only on RhoGAP activity, but also relies on proper focal adhesion localization through its interaction with tensin family proteins. Recently, there are reports showing that DLC1 can also be found in the nucleus; however, the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed. Methodology and Principal Findings: We herein provide new evidence that DLC1 protein, which predominantly associated with focal adhesions and localized in cytosol, dynamically shuttled between cytoplasm and nucleus. Treatment of cells with nuclear export blocker, Leptomycin B (LMB), retained DLC1 in the nucleus. To understand the nuclear entry of DLC1, we identified amino acids 600–700 of DLC1 as a novel region that is important for its nuclear localization. The tumor suppressive activity of nuclear DLC1 was directly assessed by employing a nuclear localization signal (NLS) fusion variant of DLC1 (NLS-DLC1) with preferential nuclear localization. In SMMC-7721 HCC cells, expression of NLS-DLC1 failed to suppress colony formation and actin stress fiber formation in vitro. The abrogated tumor suppressive activity of nuclear DLC1 was demonstrated for the first time in vivo by subcutaneously injecting p53 RasV12 hepatoblasts with stable NLS-DLC1 expression in nude mice. The injected hepatoblasts with NLS-DLC1 expression effectively formed tumors when compared with the non-nuclear targeted DLC1. Conclusions/Significance: Our study identified a novel region responsible for the nuclear entry of DLC1 and demonstrated the functional difference of DLC1 in different cellular compartments both in vitro and in vivo. Citation: Chan L-K, Ko FCF, Sze KM-F, Ng IO-L, Yam JWP (2011) Nuclear-Targeted Deleted in Liver Cancer 1 (DLC1) Is Less Efficient in Exerting Its Tumor Suppressive Activity Both In Vitro and In Vivo. PLoS ONE 6(9): e25547. doi:10.1371/journal.pone. 0025547 Editor: Chad Creighton, Baylor College of Medicine, United States of America Received June 7, 2011; Accepted September 6, 2011; Published September 26, 2011 Copyright: 2011 Chan 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 Hong Kong Research Grants Council (HKU7798/07M), Hong Kong Research Grants Council Collaborative Research Fund (HKU 1/06C and HKU 7/CRG/09) and Outstanding Young Researcher Award (to J.W.P. Yam). I.O. L. Ng is Loke Yew Professor in Pathology. 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: judyyam@pathology.hku.hk (JWPY); iolng@hku.hk (IO-LN)