locus is regarded as one of the most important anti-tumoral defenses in mammalian systems, and the two protein-coding genes at this locus are frequently independently inactivated in HCC. Studies of human tumors and mouse models have suggested a role for the Arf tumor suppressor in constraining tumor initiation (22
). We have recently shown that deletion of the Ink4a/Arf
locus enhances the progression of liver tumors induced by polyoma middle T antigen and Trp53
deletion, consistent with findings in a skin carcinogenesis mouse model that indicated that Arf may additionally play a role in tumor progression (31
). Further, we showed that mouse HCC cell lines that lacked both Trp53
displayed enhanced migration and invasion capabilities in cell culture assays compared to a cell line that lacks Trp53
). We have now shown that p19Arf
specifically impairs tumor cell invasion without affecting either proliferation or soft agar colony formation, pointing to a potential role for Arf in regulating tumor metastasis.
induces cell cycle arrest in a p53-dependent manner, and relays signals to p53 after oncogene-induced stress that stimulates the onset of cellular senescence, a potent anti-tumorigenic checkpoint. However, accumulating data indicate that Arf may additionally have p53-independent tumor suppressor functions (32
). Our data show that Arf-mediated inhibition of tumor cell invasion is independent of its interaction with Mdm2, suggesting that regulation of tumor invasion may be another p53-independent tumor suppressor function of Arf. Importantly, this function is also independent of Arf’s ability to bind to other interacting proteins such as E2F1, c-Myc, and Foxm1b, as deletion mutants that interfere with the ability of Arf to bind to these proteins still effectively block invasion.
Instead, our data demonstrate that the effect of p19Arf
on tumor cell invasion occurs via a p53-independent mechanism involving CtBP. Mutant Arf proteins that fail to bind to CtBP do not inhibit cell invasion, and shRNA-mediated ablation of CtBP reduces invasion in HCC cells expressing CtBP-binding-deficient Arf proteins. Guo et al. have previously shown that genetic disruption of Arf
in mouse embryonic fibroblasts (MEFs) enhances cell motility in a p53-dependent manner through the stimulation of Rac1 activity (33
). Our data indicate that Arf expression in HCC cells does not alter cell morphology or the actin cytoskeleton, as assessed by phalloidin staining. This suggests that the activity of Rac1, or other Rho family GTPases involved in cytoskeletal remodeling, may not be affected by Arf expression, although this has not be formally tested.
Additional studies demonstrated that activated Rho GTPases could stimulate invasion by Trp53
null, but not Arf
null, MEFs suggesting that p19Arf
regulates cell migration but not invasion (34
). Thus, our experiments are the first to show that Arf inhibits tumor cell invasion via a p53-indpendent pathway. The differences between the previous studies and ours may reflect the different cell types utilized - epithelial-derived tumor cell lines versus transformed mouse fibroblasts. Importantly, in a related study, we have also shown that p14Arf
inhibits the hypoxia-induced migration of H1299 human lung carcinoma cells in a p53-independent and CtBP-dependent manner (35
), and previous studies have indicated that CtBP can regulate the migration of this cell line, although cell invasion was not assayed in these studies (19
). Significantly, the leucine 46 residue that is critical for the Arf-CtBP interaction is one of six invariant residues conserved across several species including human, mouse, chicken, pig and opossum (32
). Thus, the Arf-CtBP interaction likely mediates a highly conserved tumor suppressor function of Arf, and may have implications for the dissemination of human tumors.
CtBPs play a critical role in cellular regulation by binding to a variety of transcriptional repressors important for development and tumorigenesis (36
). CtBPs have also been suggested to play a role in inhibiting anoikis (18
). However, the mechanisms by which Arf inhibits CtBP function remain unclear. Our findings suggest that Arf may impede CtBP function, yet the mechanisms by which this might occur remain unclear, although potential links are beginning to emerge. While we have observed that Arf expression does not influence the steady state levels of CtBP, Arf, but not a mutant defective for CtBP binding, stimulates the degradation of CtBP in response to cellular stresses such as UV irradiation [Supplemental Figure 2
)]. Therefore, it is possible that Arf stimulates the degradation of CtBP under stress conditions relevant during cell migration and invasion, such as detachment from the basement membrane. In addition, recent findings from the Grossman lab indicate that Arf can inhibit CtBP-mediated transcriptional repression of a target promoter (R. Kovi and S.R.G., unpublished observations).
CtBP has also been previously shown to induce an epithelial to mesenchymal transition (EMT) (17
). EMT, first identified as a critical process during normal embryonic development, involves the downregulation of epithelial cell markers such as E-cadherin and the induction of mesenchymal markers such as vimentin, and is postulated to be involved in tumor cell invasion and metastasis (27
). However, our data indicate that our HCC cell lines invade without undergoing EMT, and E-cadherin protein levels are not significantly altered in cells by reintroduction of p19Arf
. A similar phenomenon has been recently described by Christofori and colleagues in connection to tumor cell invasion stimulated by the mucin-like protein podoplanin (37
). EMT has been postulated to a transient process. Thus, an alternate possibility is that Arf may prevent the transient induction of CtBP, and the consequent transient reduction of E-cadherin levels, in response to pro-invasion stimuli that would allow a tumor cell to initiate invasion. A similar model has been postulated for metastasis of colorectal carcinomas (38
Thus, our findings indicate a potential new role for Arf in tumor invasion. Further exploration of the pathways involved in this process may yield promising new targets for therapeutic intervention.