The adaptation of newly arrived cancer cells to the microenvironment of distal organs is a stringent rate-limiting step in metastasis, and the probability of completing this step varies widely depending on the tumor type and the target organ. The specific stromal interactions that dictate the compatibility of DTCs with a particular organ site have remained largely a mystery. Focusing on VCAM-1 as an overexpressed gene in breast cancer cells that preferentially colonize the lungs, we show that this cell adhesion molecule is engaged by counter-receptor α4 integrins on leukocytes to trigger PI3K/Akt activation and cancer cell survival in the pulmonary parenchyma (). Our findings provide a biochemical explanation for the clinical association of VCAM-1 expression in breast tumors with cancer relapse to the lungs (Minn et al., 2005
). Our findings also show that VCAM-1 expression is high in leukocyte-rich lung metastasis samples from breast cancer patients compared to brain metastasis samples. We propose that these associations are based on a pro-survival advantage provided by VCAM-1 in cancer cells that invade leukocyte-rich sites such as the lungs.
Tumor-associated macrophages and other stromal leukocytes play crucial roles in various aspects of tumorigenesis through the secretion of specific cytokines and proteases (Cheng et al., 2007
; Giraudo et al., 2004
; Gocheva et al., 2010
; Lin et al., 2006
; Sangaletti et al., 2008
; Wyckoff et al., 2004
). The present results reveal an unprecedented role for juxtacrine stimulation of cancer cells by macrophages during metastatic colonization. VCAM-1 on the surface of cancer cells mediates α4 integrin-dependent binding of macrophages, and its oligomerization triggers PI3K/Akt signaling in the cancer cells. High-resolution imaging techniques recently demonstrated physical contacts between macrophages and breast cancer cells during macrophage-facilitated CTC extravasation in mice (Qian et al., 2009
). However, we found no contribution of VCAM-1 to cancer cell extravasation or macrophage recruitment into tumors. Rather, our evidence suggests that the α4 integrin–VCAM-1 interaction promotes cancer cell survival through PI3K/Akt pathway activation by the VCAM-1 cytoplasmic domain. During leukocyte transmigration, VCAM-1 on endothelial cells can communicate with the cytoplasmic proteins ezrin and moesin, and trigger Rac1 activation (Barreiro et al., 2002
; van Wetering et al., 2003
). Our data indicate that VCAM-1 clustering in breast cancer cells mediates Ezrin phosphorylation, Akt binding to Ezrin, and Akt phosphorylation at the TORC2 site S473 to activate pro-survival signaling. The protein kinase(s) responsible for Ezrin phosphorylation at Y353 remains unknown, although Src has been implicated as an indirect mediator (Chuan et al., 2006
; Krieg and Hunter, 1992
). Thus, leukocyte engagement of VCAM-1 in breast cancer cells may serve to amplify the activation of the PI3K/Akt pathway in mitogen-poor microenvironment during the initial stages of seeding and adaptation of DTCs to new sites.
We propose that VCAM-1 expression confers breast cancer cells with a selective survival advantage in leukocyte-rich tissues. As organs exposed to the outside environment, the lungs are rich in resident immune cells (Lipscomb et al., 1995
). In tumor-bearing mice, the lungs accumulate bone marrow-derived mononuclear phagocytes and endothelial progenitors, cell types that express α4 integrins (Gao et al., 2008
; Kaplan et al., 2005
). Our data suggest that α4 integrin expressing leukocytes and endothelial cells in lungs mediate Akt prosurvival signaling by directly interacting with VCAM-1 in metastatic breast cancer cells. The pulmonary parenchyma thereby provides a matching receptive soil for circulating cancer cells that express VCAM-1, explaining the observed link between VCAM-1 expression in primary tumors and selective relapse to the lungs (Minn et al., 2005
Cancer cells departing from a breast tumor with high expression of VCAM-1 would be primed for survival in the leukocyte-rich microenvironment of the lungs. In comparison, the brain has a limited presence of microglial macrophages, which is in line with the lack of an association of VCAM-1 with brain metastasis that we observed. In the case of bone metastasis we found no association between VCAM-1 expression in primary tumors and bone relapse, and no effect of VCAM-1 overexpression on the ability of breast cancer cells to initiate bone marrow colonization in mice. VCAM-1 may not provide an early advantage in the bone marrow because Akt activation in cancer cells that enter this site can be provided by other mechanisms, such as Src-dependent CXCL12 signaling (Zhang et al., 2009
). We do not rule out a role of VCAM-1 later in the osteolytic phase of bone metastasis.
What drives the selection for VCAM-1-expressing cancer cells in breast tumors is unknown, but it is possible that VCAM-1-rich clones are positively selected in breast tumor areas of active infiltration or inflammation. Macrophage-rich areas of a primary tumor would nurture the expansion of VCAM-1-expressing cancer cell clones. Furthermore, breast cancer cells in lung metastatic nodules can reenter the circulation, seed back a mammary tumor, and abundantly proliferate in leukocyte-rich tumor areas (Kim et al., 2009
), and we provide evidence that VCAM-1 favors the outgrowth of CTCs that infiltrate a self-seeding mammary tumor. Accordingly, lung metastatic clones expressing VCAM-1 might undergo recurrent cycles of amplification by shuttling between lung nodules and a leukocyte-rich primary tumor microenvironment.
By disrupting the α4 integrin–VCAM-1 interaction between macrophages and cancer cells with anti-α4 integrin blocking antibodies we were able to cancel the pro-survival action of VCAM-1 in the cancer cells. The interaction between endothelial VCAM1 and leukocyte α4 integrins is a validated target in multiple sclerosis and other diseases that involve rampant recruitment of circulating leukocytes into tissue (Comi, 2009
; Schmidt et al., 2009
). Our results point at a potential application of such targeted drugs to suppress VCAM-1-mediated survival of disseminated cancer cells after the removal of a primary tumor.