IGF-induced signaling has been implicated in the pathogenesis of several human malignancies [2
]. In particular, induction of IGF signaling, by a variety of mechanisms, has been implicated in HCC. Indeed, the induction of IGF2 gene expression is a common event in HCC, and serum levels of IGF2 mRNA correlate well with the presence of extrahepatic metastases in this disease [17,29
]. In agreement with these findings, we have previously shown that elevated IGF2 mRNA levels identify tumors with enhanced metastatic potential in a novel HCC mouse model [21
We therefore sought to determine the role of IGF2-induced signaling in the metastatic capability of HCC cells. Although exogenous IGF2 was insufficient to stimulate HCC cell migration and invasion, we observed that knockdown of IGF2, as well as blockade with anti-IGF2 antibodies, impaired the ability of HCC cells to migrate and invade in transwell assays, indicating that IGF2 is required for these phenotypes and that IGF2 may act in an autocrine manner to induce these effects.
Using an shRNA-mediated inhibition strategy, we found that IGF1R-mediated signaling is required for cell migration and invasion in vitro
, indicating that IGF2 signals through this receptor to stimulate these phenotypes. These findings are consistent with a recent report showing that IGF1R inhibition impaired the migration of HCC cells in a wound healing assay [41
]. Importantly, we also observed that IGF1R-mediated signaling is required for lung colonization in vivo
, providing experimental support for the connection between IGF2 expression and extrahepatic metastasis in HCC patients. Our results are consistent with previous findings indicating roles for IGF1R-mediated signaling in the metastasis of other tumor types [4
Interestingly, whereas IGF1R knockdown impaired tumor lung colonization, knockdown of IGF2 had only a modest effect, perhaps suggesting that in vivo circulating IGFs produced by the recipient animals may stimulate the IGF1R signaling cascade in the injected HCC cells, circumventing the need for the cells to produce IGF2. Another possibility is that IGF2 is important at an earlier step in metastasis, such as the migration and invasion of tumor cells away from the primary site and into the circulation, a step that is circumvented in this particular assay. This hypothesis is consistent with our in vitro migration and invasion data.
In contrast to the importance of IGF1R-stimulated signaling in lung colonization by HCC cells, we observed that IGF1R-mediated signaling was not required for tumor growth after the SC injection of tumor cells. The different results obtained in the SC tumor assay and the lung colonization assay also suggest the possibility that IGF1R signaling is specifically required for tumor progression but not for tumor development per se
. Further experiments are required to test this hypothesis and to provide mechanistic insights into how this might occur. However, these observations are consistent with findings reported in mammary tumorigenesis [42,43
Our results contrast with those of Nussbaum et al. [41
] who found that inhibition of IGF1R signaling using small molecules impaired tumor growth. A potential explanation for this discrepancy is that in addition to impairing IGF1R signaling in tumor cells, treatment of mice with small-molecule antagonists also impairs IGF1R signaling in stromal cells such as fibroblasts and vascular endothelial cells that are critical for tumor growth. Our use of shRNA specifically targets IGF1R signaling within the tumor cells and therefore allows more precise evaluation of the requirement for IGF1R-stimulated signaling cascades in tumor development.
Previous studies have suggested that in addition to IGF1R, IGFs may signal through IGF1R/IR heterodimers and IR-A homodimers [6
]. Of significance, our results demonstrate that IGF1R-mediated, but not IR-mediated signaling, is required for many of the phenotypes assayed. These results are consistent with recent findings on the effects of IGF1R, but not IR, on the viability, proliferation, and migration of human HCC cells [41
]. These results suggest, that in HCC cells, IGF ligands signal through IGF1R holoreceptors, not IGF1R/IR heterodimers, and that insulin signaling is not important for transformation phenotypes in HCC, despite the fact that the liver is a critical site of insulin action. Further, these results suggest that although the IGF1R and IR share common downstream IRS adapter proteins, the signals stimulated downstream of these receptors are different.
Consistent with this idea, knockdown of IRS2, but not IRS1, partially recapitulated the migration and invasion defect observed in IGF1R depleted cells. The involvement of IRS2 in the invasion phenotype is consistent with its role in tumor metastasis in mouse models of human cancer [35
]. However, IRS2 knockdown does not induce as severe a phenotype as IGF1R depletion (nor does concomitant knockdown of IRS1 and IRS2). This suggests that signals propagated downstream of IGF1R independently of the IRS proteins play critical roles in these phenotypes.
Indeed, the identities of the downstream signaling pathways important for the metastasis-related phenotypes induced by IGF1R-stimulated signaling remain unknown. Direct pharmacologic inhibition of these pathways in our HCC cell lines is an unfeasible approach because these inhibitors block the transforming activity of PyMT, the initiating oncoprotein in these cell lines. We have previously demonstrated that treatment of these HCC cell lines with the mTOR antagonist rapamycin impairs invasion [31
], and our unpublished results show that blockade of MAP kinase or PI3 kinase signaling potently impairs tumor cell migration and invasion (Y.-W. Chen and B. Lewis, unpublished data). Thus, rescue experiments in which IGF1R mutants defective in stimulating specific downstream signaling pathways are used to reverse the invasion defect caused by IGF1R knockdown will be needed to address this issue.
What also remain unclear are the identities of the terminal effectors that mediate IGF-induced cell migration and invasion. Using a candidate gene approach, we showed that MMP2 is involved in IGF1R-mediated invasion, yet MMP2 inhibition does not produce as severe an impact on cell invasion as IGF1R inhibition, indicating that other critical players remain to be identified. It is likely that a more global approach may identify important players in this process. Indeed, Nussbaum et al. [41
] recently reported that IGF signaling induces the expression of several genes involved in motility. An intriguing possibility, yet to be tested experimentally, is that the metastatic phenotype induced by IGF signaling is dependent on the inhibition of FoxO transcription factors, recently shown to act as tumor suppressors in vivo
In summary, we have demonstrated a critical role for IGF-stimulated signaling in metastasis-associated phenotypes in HCC cells. These data provide further support for the pathologic significance of IGF2 gene reactivation in HCCs and suggest that interfering with this signaling pathway may be a critical strategy in treating HCC patients with disseminated disease.