2 Although recent evidence suggest that hepatocellular EMT plays a pivotal role in the dissemination of malignant hepatocytes during HCC progression, the underlying molecular mechanisms remain to be characterized.3, 4 Ras homolog (Rho) GTPases, including RhoA, Rac1, and cell division cycle 42 (Cdc42), are the main regulators of the actin cytoskeleton and therefore general modulators of cellular processes important for tumor biology.

Moreover, deregulated Rho GTPase signaling was reported to play an important role in the initiation and the progression of HCC.5, 6 Rnd3/RhoE is an atypical member of the Rho GTPase family because it is devoid of GTPase activity. The best-characterized function of Rnd3 is the inhibition of RhoA activity and the subsequent down-regulation of ROCK-mediated actomyosin contractility.7, 8 Through GSK1120212 mouse its role as a negative regulator of the Rho/ROCK pathway, Rnd3 was involved in tumor cell migration and invasion9-11 and myoblast MAPK inhibitor fusion.12 More recently, Rnd3 was shown to inhibit cell-cycle progression, apparently independently of cytoskeleton remodeling.8 Thus, Rnd3 has been implicated in different steps of cancer development, such as regulation of cell proliferation and apoptosis,13-15 cell transformation,13 or cell migration and invasion. Our reanalysis of five transcriptomic studies revealed an alteration

of Rnd3 messenger RNA (mRNA) expression in HCC, compared to nontumor liver tissues,5 with four of five showing a down-expression16-19 and a single one, based on only four cases, an overexpression.20 Here, we confirm that Rnd3 is down-regulated in most human HCC and HCC-related cell lines, and we provide evidence that Rnd3 down-regulation increases HCC invasion and thus may favor HCC progression. 3D, three-dimensional; ANOVA, analysis of variance; Cdc42, cell division cycle HAS1 42; DMEM,

Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; IF, immunofluorescence; IHC, immunohistochemistry; miRNA, microRNA; MMPs, matrix metalloproteinases; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; Rho, Ras homology; SIP1, Smad-interacting protein 1; siRNA, short interfering RNA; UTRs, untranslated regions; ZEB1, zinc finger E-box binding homeobox 1. Samples came from resected or explanted livers with HCC of patients treated in Bordeaux from 1992 to 2005. Fragments of fresh tumor and nontumor liver tissues (taken at a distance of at least 2 cm from the tumor) were immediately snap-frozen in liquid nitrogen and stored at −80°C. RNA or proteins were extracted as previously described.21 HCCs used as the Affymetrix hybridization set (57 HCCs) and the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) validation set (63 HCCs) were described.