Abstract
BackgroundDeleted 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 FindingsWe 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/SignificanceOur 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.
Highlights
Deleted in Liver Cancer 1 (DLC1) was first cloned by subtractive hybridization as a gene fragment that was frequently deleted in human hepatocellular carcinoma (HCC) [1]
DLC1 shuttled between cytoplasm and nucleus Expression of Myc-tagged DLC1 (Myc-DLC1) in SMMC-7721 cells revealed its existence in focal adhesions, cytoplasm and nucleus (Fig. 1A)
We extended our investigation by examining the localization of endogenous DLC1 in various cell lines
Summary
Deleted in Liver Cancer 1 (DLC1) was first cloned by subtractive hybridization as a gene fragment that was frequently deleted in human hepatocellular carcinoma (HCC) [1]. Focal adhesion localization through interaction with tensin family protein is one of the characteristics of DLC1 and is functionally associated with its tumor suppressive activity. This was supported by the evidence that DLC1 mutant with intact RhoGAP domain but failed to be targeted to the focal adhesions exhibited reduced growth suppressive activity in vitro [10,14]. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends on RhoGAP activity, and relies on proper focal adhesion localization through its interaction with tensin family proteins. There are reports showing that DLC1 can be found in the nucleus; the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed
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