Our results establish an important role for IRS-2 in mammary tumor metastasis. Using the PyV-MT mouse model of mammary tumorigenesis, we showed that mice lacking expression of the IRS-2 adaptor protein develop mammary tumors with a latency and growth rate similar to those of their WT littermates and that IRS-2 is not required for tumor angiogenesis. Importantly, however, the ability of PyV-MT-derived mammary tumors to metastasize to the lungs is significantly impaired in the absence of IRS-2 expression. This deficiency in metastasis is also observed when IRS-2−/− mammary tumor cells are grown orthotopically in the mammary fat pads of WT mice, supporting an intrinsic function for IRS-2 in the mammary tumor cells. We have also established a mechanism for IRS-2 function in mammary tumor metastasis. Specifically, IRS-2 promotes mammary tumor cell invasion and resistance to stress-induced apoptosis. Taken together, our findings highlight an important contribution of IRS-2 to breast cancer and identify a novel metastasis-associated signaling pathway.
Our study is the first to establish a specific role for IRS-2 in tumor metastasis and to identify the mechanistic basis for this function. Hallmarks of the metastatic phenotype are the ability to invade from the primary tumor site into the stromal microenvironment and to survive in foreign environments (15
). Our data reveal that IRS-2 contributes to both of these essential metastatic steps. Mammary tumor cells that lack IRS-2 expression are less invasive, whereas IRS-1−/−
cells that express only IRS-2 are more invasive, than WT cells that express both isoforms. These findings are in agreement with previous studies demonstrating a role for IRS-2 in the ability of the α6β4 integrin to promote invasion (40
), as well as with more recent studies that have associated increased motility with IRS-2 function in variants of MDA-MB-231 and MCF-7 breast carcinoma cells (19
). Our results also support that IRS-2 contributes to tumor metastasis through the regulation of cell survival signaling pathways. A higher incidence of apoptosis was observed in IRS-2−/−
tumors than in WT tumors. Furthermore, IRS-2−/−
tumor cells undergo apoptosis at a higher rate in the absence of growth factor stimulation than tumor cells that express IRS-2. In contrast, loss of IRS-1 expression did not increase apoptosis. Rather, cells expressing only IRS-2 (IRS-1−/−
) were more resistant to stress-induced apoptosis. Presumably, tumor cells lacking IRS-2 are more sensitive to apoptotic stimuli and are less likely to survive outside of their normal microenvironment, where intrinsic survival mechanisms are required. A hypothesis that emerges from the present data is that IRS-2 functions in an autocrine signaling pathway that is essential for invasion and survival when exogenous stimuli are not present, such as during metastasis to foreign tissues.
Our data on the importance of IRS-2 for mammary tumor metastasis and the contrasting behavior of the IRS-1−/−
mammary tumor cells indicate that IRS-1 and IRS-2 are not functionally redundant in breast cancer. IRS-2-null tumors express IRS-1, but this isoform is not sufficient to support the metastasis of mammary tumor cells to the lungs. Moreover, IRS-2, but not IRS-1, promotes tumor cell invasion and survival in vitro. These results suggest that IRS-2 is capable of activating a unique signaling pathway that is essential for tumor progression and that this pathway is not activated sufficiently, or at all, through IRS-1. The importance of IRS-2 for mammary tumor cell invasion and survival suggests an involvement of a PI3K-dependent signaling pathway in the function of this adaptor protein (6
). IRS-2 was previously implicated in the activation of PI3K by the α6β4 integrin, and the importance of this signaling pathway for promoting breast carcinoma cell invasion and survival has previously been demonstrated (3
). Although IRS-1 and IRS-2 each contain multiple PI3K binding motifs and can recruit and activate PI3K, the distinct intracellular compartmentalization of IRS-1 and IRS-2 could influence the functional outcomes of their activation of downstream signaling pathways (52
). This mechanism for differential IRS function has been described for IRS-2-dependent insulin-stimulated Glut4 translocation and glucose uptake in brown adipocytes (11
). In addition to PI3K, other downstream effectors might play a role in the metastasis-promoting functions of IRS-2, because this adaptor protein contains potential binding motifs for many intracellular signaling molecules, including the tyrosine phosphatase SHP-2, the adapters Nck, Grb-2, and Crk, and the tyrosine kinase fyn (52
). Many of these effectors function in signaling pathways that could regulate metastasis.
Although few studies have examined IRS expression in human breast cancer, one study did observe a decrease in IRS-1 expression during tumor progression, with IRS-1 expressed at low levels in poorly differentiated breast cancers (37
). It is intriguing to speculate that IRS-2 is the predominant IRS isoform expressed in these later stages of tumor progression. The fact that IRS-1−/−
mammary tumor cells that express only IRS-2 are highly invasive and are resistant to apoptotic stress suggests that these tumors would be more aggressive. With regard to this possibility, IRS-2 expression is maintained in ER−
breast carcinoma cells, while IRS-1 expression is decreased or lost (40
). Loss of ER expression is considered a poor prognostic marker, and ER−
tumors tend to be more aggressive than tumors that retain ER expression (44
Our findings that IRS-1 and IRS-2 play distinct roles in mammary tumor progression raise the question of whether these adaptor proteins contribute to the progression of other types of cancers. IGF-1 signaling has been implicated in the behavior of many other carcinomas, including prostate, colon, thyroid, and pancreatic (17
). It is likely that the IRS proteins function downstream of the IGF-1R in these cancers as well and contribute to tumor development and progression. Strong evidence to support a role for the IRS proteins in progression comes from recent studies on the tumor-suppressor tuberous schlerosis complex (TSC1-2) (31
). This complex consists of two gene products, tuberin and hamartin, which negatively regulate the activity of the growth-regulatory kinase S6K1 (31
). S6K1 regulates insulin and IGF-1-dependent activation of PI3K by suppressing IRS gene expression and also by phosphorylating the IRS proteins and targeting them for degradation in a negative feedback loop (16
). In tumors that arise from TSC1-2 mutations, which are termed harmartomas, S6K activity is elevated and IRS expression is downregulated. These tumors can grow quite large, but they have a low malignancy potential, as was observed in IRS-2−/−
mammary tumors (31
In summary, we have identified a novel role for the IRS-2 adaptor protein in mammary tumor metastasis. Although IRS-2 is not required for tumor initiation or primary tumor growth, signaling through this adaptor protein is important for promoting tumor cell invasion and survival, hallmarks of the metastatic phenotype. Our data reveal that the relative expression levels of IRS-2 in tumors could significantly affect disease progression for breast cancer patients. Therefore, pathways that regulate IRS-2 expression, as well as downstream pathways activated through IRS-2, represent potential novel therapeutic targets.