The high frequency of intra-hepatic and distant metastases in HCC is a critical issue in the management of patients with this disease. Currently, surgical resection remains the only curative option, yet the presence of metastases eliminates this treatment option. Thus, identifying factors that correlate with the development of metastatic disease and tumor recurrence is of great importance. Indeed, recently published work has identified tumor cell-dependent and tumor microenvironment-dependent gene expression changes associated with the progression to a metastatic state in HCC (46
Therefore, the development of experimental model systems, both in vivo
and in vitro
, that allow the dissection of the molecular mechanisms involved in HCC metastasis is of primary importance. We have previously demonstrated the feasibility of modeling metastatic HCC after the somatic and sporadic activation of oncogenes (24
). In this paper, we described the refinement of this system to utilize tissue-specific deletion of tumor suppressor genes. This modification allows us to clearly assess contributions that occur in a cell autonomous manner, and we demonstrate here that the effects of Trp53
deficiency on HCC metastasis occur in a cell autonomous fashion. Further, our data suggest that while loss of p53 enhances the metastatic potential of HCCs, it may not increase the ability of the tumor cells to migrate and invade. A critical caveat for this latter finding is that, to date, we have analyzed only a single cell line derived from a p53 null tumor. Importantly, however, the p53-deficient cell line efficiently colonizes the lung after introduction into the blood stream. This is consistent with the hypothesis that tumor cells develop at an early stage many of the traits required for metastasis (48
). It also suggests that in p53-deficient HCCs, the rate-limiting step may be invasion through the basement membrane and intravasation to enter the circulation, because once in the bloodstream, these cells home effectively to the lungs and establish metastatic lesions.
In HCC patients, p53 function is frequently compromised by point mutation and loss of heterozygosity of the wild-type allele (8
). Olive et al
have recently demonstrated that p53 point mutants commonly found in human tumors display gain of function properties (49
). Therefore, it would be intriguing to determine whether the presence of p53 point mutants will enhance the formation of metastasis in our model system and increase the migration and invasion capabilities of cell lines derived from these tumors.
One advantage of our experimental system is the ability to analyze the role of specific domains of oncoproteins and downstream signaling molecules through the rapid generation and introduction of RCAS viruses encoding mutant proteins. We previously exploited this feature to demonstrate the importance of PI3 kinase signaling in the induction of metastasis by PyMT (24
). Significantly, we have now observed that inhibition of this pathway with rapamycin reduces the invasion of HCC cell lines, suggesting that one potential mechanism by which PI3 kinase signaling contributes to HCC metastasis may be the induction of tumor cell invasion. Importantly, activating mutations in the catalytic subunit of PI3 kinase are common in HCC (50
), and therefore our results suggest that one potential consequence of these activating mutations is the enhancement of cell invasion. Thus, our new results confirm and extend our prior in vivo
observations on the role of PI3 kinase signaling in HCC metastasis, and highlight the utility of our experimental model systems for the elucidation of the mechanisms involved in HCC metastasis.
Our data also point to potential roles for the p16 and p19 tumor suppressors in HCC progression. Inactivation of the locus encoding these proteins is a common finding in HCC, yet the roles of these tumor suppressors in the initiation and progression of this disease remain unknown (9
). Our results indicate that these tumor suppressors may act to enhance both tumor initiation and tumor progression. Indeed, analysis of the TP53
loci in human HCC samples suggested that the loss of these loci might act cooperatively in this disease, although the specific roles of p16INK4A
remain to be elucidated (12
). Our data suggest that p16 and p19 may act to constrain tumor growth. Concomitant loss of Trp53
led to the development of palpable PyMT-induced liver tumors at 4 months of age, a phenotype not observed with Trp53
deletion alone. Our results also suggest that p16 and p19 may influence tumor progression by impeding cell invasion, although the mechanisms by which this occurs remain under investigation.
We also observed increased mRNA levels for the transcription factor snail in p53 and Ink4a/Arf
double deficient tumors relative to p53 wild-type tumors that have low metastatic potential. Intriguingly, snail mRNA levels are elevated in invasive subclones of the non-invasive BL185 cell line (Y-W.C., J.L.T., and B.C.L., unpublished observations), and previous studies have associated snail with tumor metastasis and poor outcome in HCC (40
). It remains to be seen whether snail is required for HCC tumor cell invasion, and whether it is involved in the regulation of Igf2 and cathepsin E, two additional molecules that we have previously associated with metastatic potential in our mouse model (24
Thus, our results describe a new system for the generation and analysis of HCC invasion and metastasis. The examination of these processes using our model should significantly aid our understanding of malignant progression in this disease.