Metastasis is a highly inefficient process that requires the co-ordinated regulation of a series of complex processes with only a small percent of tumor cells acquiring the characteristics necessary to escape from the primary tumor and successfully colonize distant sites. We have used LCC6 cells as a model of high-risk metastatic disease. We have previously shown that a dominant negative IGF1R inhibited pulmonary metastases of these cells without affecting growth of the primary tumor in the mammary fat pad of athymic mice (Sachdev et al., 2004
). Herein, we demonstrate that a therapeutic inhibition of IGF1R with an antibody against IGF1R also inhibited pulmonary metastases without affecting primary tumor growth ( and ). EM164 down-regulated IGF1R levels in the tumors () and also inhibited IGF-I stimulated phosphorylation of IRS and downstream Akt, suggesting that EM164 inhibited the biochemical pathways activated by IGF1R but did not affect primary tumor growth. Based on these data, we have shown that IGF1R can affect metastasis without affecting tumor growth in vivo
This regulation of metastasis independent of tumor growth is not isolated to LCC6 cells only. Our in vitro
data with other breast cancer cell lines showed similar findings. In MDA-MB231BO cells (Yoneda et al., 2001
), a metastatic variant of the MDA-MB231 breast cancer cells, we have shown that IGF-I does not stimulate proliferation but enhances motility in vitro
(Jackson et al., 2001
). We have also shown that neutralization of IGF-I with IGF binding protein-1 (IGFBP-1) inhibits IGF-I stimulated motility but not proliferation of MDA-MB-231BO cells (Zhang and Yee, 2002
). Similarly in T47D-YA cells (a variant of T47D breast cancer cells), ectopic expression of the adaptor protein IRS-2 results in IGF-I stimulated motility but not proliferation of the T47D-YA/IRS-2 cells (Byron et al., 2006
). Furthermore in T47D-YA/IRS-2 cells, inhibition of IGF1R with the antibody αIR3 inhibits motility in response to IGF-I (Byron et al., 2006
). In fact, this has also been observed by other groups in other types of cancers such as neuroblastomas (van Golen et al., 2006
). Thus, these results indicate that regulation of metastasis independently of tumor growth may be seen in a wide variety of cancers. This has important clinical implications for the development of therapies against IGF1R for clinical use as discussed later.
Various in vitro
and in vivo
models have suggested that activation of IGF1R regulates various steps in the metastatic cascade. In this study, we found that disruption of IGF1R inhibited invasion across Matrigel in vitro
, suggesting that IGF1R regulates invasion of these cells. When IGF1R was inhibited, we observed no difference in the activity of metalloproteinases or expression of the chemokine receptor CXCR4 (data not shown) which has been shown to be important for organotropism of breast cancer metastasis to organs such as bone and lungs (Muller et al., 2001
). Furthermore, we show here that disruption of IGF1R either with a dominant negative construct or EM164 significantly inhibited the presence of CTC in the blood of mice. Pulmonary metastases were also inhibited when IGF1R was disrupted following direct injection of cells into the circulation. Thus, our data suggest that functional IGF1R is required at multiple steps in the metastatic process including cell invasion and survival in the circulation.
How does IGF1R regulate metastasis independently of tumor growth? One possibility is that functional IGF1R is essential for survival in the circulation as discussed earlier. This is supported by evidence showing that indeed IGF1R is essential for anchorage-independent survival once cells are free of adhesion to the extracellular matrix (ECM) in vitro
; Valentinis et al., 1999
). Further, activation of IGF1R has been shown to mediate resistance to anoikis or enhance survival in the absence of attachment to ECM in cancer cells (Ravid et al., 2005
). Thus, surviving in the circulation or at a secondary site is one of the critical steps in the metastatic cascade as tumor cells are subjected to some of the harshest environment in the host in the circulation (Townson et al., 2003
). Additionally, our data do not rule out the possibility that functional IGF1R is a critical growth regulator at a secondary site or is required to co-opt other signals promoting tumor cell survival in the lungs. Indeed, a key regulator of metastasis is the regulation of growth of tumor cells at the secondary site (Chambers et al., 2002
Work from several groups has demonstrated that there is a mutually sustaining reciprocity between tumor epithelial cells and their surrounding stroma (Gupta and Massague, 2006
). Many of the genes that comprise metastasis signatures have included genes expressed by the stroma (Allinen et al., 2004
; Minn et al., 2005b
). As tumor cells invade across the basement membrane, they can initiate development of the reactive stroma, further propelling metastasis (Radisky and Radisky, 2007
). Thus, it has become increasingly clear that the microenvironment influences the invasion and metastasis of tumor cells and that the local microenvironment impacts tumor cells as they escape. Our data suggest that disruption of IGF1R does not affect tumor growth of LCC6 cells in the mammary fat pad of mice. Perhaps this is due to the fact that in the mammary fat pad of the mouse, the microenvironment provides the necessary signals for growth, and IGF1R is not needed in this pathway.
In contrast, LCC6 cells need functional IGF1R for survival in the circulation and colonization of metastatic sites. Thus, it is probable that the microenvironment at the metastatic site in the lungs is insufficient to provide all the cues needed for successful establishment of metastatic growth in the lungs. Although we have only studied metastatic growth in the lungs, it is possible that IGF1R also regulates growth in the bone microenvironment. However, it has been shown that only six genes are shared in common between the bone metastasis signature (Kang et al., 2003
; Minn et al., 2005b
) and lung metastasis signature (LMS) of breast cancer cells (Gupta et al., 2005
; Minn et al., 2005a
). Therefore, it is possible that our results are specific for metastasis to the lungs. Ongoing studies are examining differential expression of genes in LCC6 cells without and with disruption of IGF1R that may further provide a better insight into how IGF1R regulates metastasis independent of tumor growth.
Recently, the notion of self-seeding has been postulated to explain metastasis where circulating tumor cells return to the primary site to enhance growth (Norton and Massague, 2006
). As a result, increased growth of the primary tumor is accompanied by decreased metastases at distant sites. Our data argue against this model; disruption of IGF1R did not affect “primary” tumor growth but effectively reduced metastases. Thus, it seems unlikely that self-seeding was responsible for the growth of these cells in the mammary fat pad.
Our results have important implications for the clinical development of IGF1R targeted strategies. First, this metastatic phenotype regulated by IGF1R will be difficult to measure in clinical trials. Second, biochemical inhibition of the target does not automatically lead to inhibition of tumor growth. Inhibition of IGF1R kinase activity is an important biodynamic marker, but may not be linked to a clinical outcome measured in a phase 2 drug study. Thus, suitable biomarkers need to be developed to identify tumors responsive to anti-IGF1R therapy. We have previously shown that IRS-1 activation is associated with proliferation of cancer cells (Jackson et al., 1998
), while IRS-2 is associated with motility of breast cancer cells (Jackson et al., 2001
). These observations have also been confirmed in vivo
by studies showing that IRS-2 null animals, but not IRS-1 null animals, have significantly decreased incidence of metastasis compared to wild-type mice expressing polyoma virus middle T antigen (PyV-MT) in the mammary gland (Gibson et al., 2007
; Nagle et al., 2004
). This suggests that one biomarker could be expression of phosphorylated IRS-1 compared to IRS-2. However, it has recently also been shown that both IRS-1 and IRS-2 overexpression can lead to metastasis (Dearth et al., 2006
). Perhaps the identification of an “IGF1R driven metastasis signature” would be useful in identifying and monitoring patients who could benefit from IGF1R targeted therapy for inhibition of metastatic disease and we are currently investigating this.
In the present study, we show that IGF1R regulates metastasis independently of tumor growth. As such, our study has several important implications for clinical trials with agents targeting IGF1R. Clinical trials of this therapeutic strategy may need to adjust their endpoints for assessing benefit by taking into account that inhibition of IGF1R signaling may inhibit metastasis but not primary tumor growth.