Hallmarks of the metastatic process include increased motility of tumor cells and decreased adhesion to surrounding cells and tissues. Both of these processes are associated with increased and decreased expression of specific integrins (Hood and Cheresh, 2002
). The expression of β4 integrin is maintained, and often upregulated, in various types of invasive and metastatic tumors, correlating with poor prognosis (Mercurio and Rabinovitz, 2001
; Tagliabue et al., 1998
; Grossman et al., 2000
; Lu et al., 2008
). Consistent with these findings, we found that β4 integrin is highly expressed in osteosarcoma cell lines and patient tumors, in both metastatic sites and primary tumors. The latter is not surprising, since historical data shows that 80% of patients who undergo surgical resection only, will eventually develop metastatic disease, demonstrating the high metastatic potential of this disease (Gaffney et al., 2006
). Furthermore, β4 integrin is upregulated in highly metastatic MNNG-HOS cells, compared to non-metastatic parental HOS cells. We tested whether the metastasic behavior of MNNG-HOS may be a reflection of changes in β4 integrin.
We examined the consequences β4 integrin knockdown in MNNG-HOS cells in vitro and found an inability to grow in anchorage independent conditions. Our in vivo data demonstrated that expression of β4 integrin is crucial for the metastatic phenotype in the MNNG-HOS human osteosarcoma model, since knockdown of β4 integrin markedly suppressed experimental pulmonary metastasis. This was strengthened by our findings that sh-RNA mediated knockdown also led to an increase in survival using the more stringent spontaneous model of metastasis.
Integrins influence cell behavior not only by providing a docking site for ECM proteins at the cell surface, but also by acting to relay molecular cues regarding the cellular environment that influence cell shape, growth, survival and migration (Hood and Cheresh, 2002
; Gao and Giancotti, 2004). β4 integrin contains a large unique cytoplasmic domain (1017 amino acid), in which a critical tyrosine residue (Y1494) has been identified in the third fibronectin type III repeat of this domain. This residue has been shown to be essential for β4 integrin-mediated activation of PI3K (Shaw, 2001
). Introduction of mutant β4 (Y1494F) into MNNG-HOS (Luc) cells resulted in reduced phosphorylation of Akt and S6, both of which are downstream targets of PI3K and mTOR. Furthermore, expression of mutant β4 integrin (Y1494F) resulted in significant reduction of experimental pulmonary metastasis of MNNH-HOS cells, but only a minimal effect on the inhibition of primary tumor growth. Thus, these data suggest that β4 integrin plays a more important role in promoting tumor metastases.
Our previous studies have shown that high expression of ezrin, a membrane-cytoskeletal linker protein, is associated with the metastatic phenotype and activation of Akt/mTOR signaling in osteosarcoma (Khanna et al., 2004
; Wan et al., 2005
). However, the mechanism by which ezrin mediates metastases remains unclear. In this study, we found that ezrin can interact with β4 integrin both in vitro
and in vivo
(). Moreover, a key finding in the present study is that the association between β4 integrin and ezrin is essential for maintenance of β4 integrin protein expression in osteosarcoma cell lines. We have demonstrated that this occurs at the level of β4 interin transcription but the mechanism by which this occurs is currently unclear.Thus, these data provide a new link in understanding the mechanism of ezrin on the regulation of tumor metastases. Integrins lack enzymatic activity, and are thus dependent on recruitment of adaptor and signaling proteins for effector functions (Liu et al., 2002
; Geiger et al., 2001
). These proteins include integrin-binding proteins such as talin, adaptors and/or scaffolding proteins that lack intrinsic enzymatic activity such as vinculin, paxillin, tensin and α-actin, and enzymes that modify integrin downstream effectors such as the non-receptor tyrosine kinases FAK (focal adhesion kinase) and Src. Ligand binding to the extracellular integrin domain induces conformational changes and lead to integrin clustering and recruitment of actin-associated proteins and signaling proteins, which link the integrin to the cytoskeleton (Hynes, 2002
; Giancotti, 2003
). Talin has been demonstrated to be a major cytoskeletal actin-binding protein that binds to the tails of integrins β1, β2, β3, β5 and β7, linking them to the cytoskeleton (Galderwood et al., 1999
; Calderwood et al., 2001
; Calderwood et al., 2003
;Pfaff et al., 1999
). In addition, at least two other β integrin tail-binding proteins, β3-endonexin and cytohesin, have also been identified (Kashiwagi et al., 1997
; Kolanus et al., 1996
). Our study identifies a previously uncharacterized interaction between ezrin and β4 integrin, but not β1 integrin. The interaction between ezrin and β4 integrin appears to be essential for maintenance of β4 integrin expression levels. In this regard, we do not yet know how ezrin regulates β4 integrin but this will be another interesting avenue to investigate in the future.