Many advanced human tumors markedly overexpress TGF-β, which allows the tumor cell to generate a more permissive stromal environment through paracrine mechanisms such as enhanced angiogenesis and reduced immune surveillance. However, due to the accumulation of genetic and epigenetic defects, in the later stages of the carcinogenic process the tumor cell itself can also respond to TGF-β in a manner that would promote progression, by reactivating developmentally inappropriate migratory and invasive programs. Therapeutic application of TGF-β antagonists could potentially suppress metastasis by interfering with either or both of these types of mechanisms. While it is clear that bulky TGF-β antagonists such as antibodies could readily interfere with the longer-range effects of tumor-derived TGF-β, it is less clear whether these agents would be able to impact on very short-range effects of TGF-β on the tumor cell itself. To address this question, we recovered metastatic cells from the lungs of tumor-bearing mice treated with an anti-TGF-β antibody, and looked for direct effects of the therapeutic intervention on the metastatic tumor cell.
Applying this approach to the 4T1 mouse model of metastatic breast cancer, we have shown for the first time that integrin-binding sialoprotein (Bsp) is an important mediator of the direct pro-metastatic effects of TGF-β on the tumor cell. In vivo, treatment with TGF-β antibody was associated with reduced expression of Bsp in lung metastases, and the functional signficance of this observation was confirmed when we showed that experimental knockdown of Bsp in the 4T1 cells significantly reduced their metastatic efficiency. Furthermore, Bsp and TGF-β expression were tightly correlated with each other, and with metastatic ability in a series of related breast cancer cell lines of differing metastatic potential, suggesting that TGF-β is a key regulator of Bsp expression during progression.
BSP is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of glycoproteins that also includes osteopontin (OPN). Both BSP and OPN are major constitutents of the non-collagenous matrix in skeletal tissues, where they play important roles in bone turnover (23
), and both are regulated by TGF-β (26
). In addition to its role in bone physiology, OPN has recently been implicated as an important player in metastasis to multiple sites (32
), but BSP is less well studied in this regard. However, a large body of clinical data shows that BSP protein is overexpressed by many malignant tissues, including breast (34
). Serum levels of BSP are increased in colon, breast and prostate cancer patients (16
), and elevated serum BSP in primary breast cancer patients is prognostic for bony metastasis (40
). Moreover, by in silico
analysis of large clinical microarray studies of breast cancer (www.oncomine.org
), we have found that BSP mRNA expression in the primary tumor increases with increasing tumor grade, as had been previously been shown in a smaller scale study (39
), and furthermore that BSP mRNA levels correlate significantly with the presence of metastatic disease ((41
): Supplemental Fig. S1). These correlative clinical studies are all consistent with an important role for BSP in breast cancer metastasis. Indeed, forced overexpression of BSP in the MDA-MB231 human breast cancer cell line can enhance invasion and migration in vitro
, and promote metastasis in vivo
), and anti-BSP antibody treatment can suppress the formation of osteolytic metastases by these cells (44
). Our study is the first to demonstrate a role for endogenous Bsp in metastasis to visceral organs, and to place Bsp as a downstream mediator of TGF-β in this process.
Metastasis is a multi-step process, and Bsp appears to contribute to the prometastatic effects of TGF-β at more than one of these steps. Members of the SIBLING family bind to integrins on the cell surface and mediate formation of tertiary complexes with other effector molecules that then modulate cell behaviour. BSP, OPN and dentin matrix protein 1 can form tertiary complexes on the cell surface with αvβ3 integrin and specific metalloproteinases, thereby enhancing MMP activation (29
). BSP specifically binds to and activates MMP-2, and was the only SIBLING that could enhance invasion in multiple cancer cell lines (27
). TGF-βs are also thought to enhance invasiveness through a mechanism that is dependent on MMPs (28
). Here we have shown that the ability of TGF-β to promote invasion is completely dependent on the presence of Bsp. Knockdown of Bsp had no effect on the ability of TGF-β to enhance the synthesis or secretion of pro-MMPs, and it did not cause detectable activation of MMPs in cell culture medium. However, it did significantly reduce basal and TGF-β stimulated activation of the MMPs locally at the cell surface, as evidenced by a reduction in the degradation of type I or type IV collagen in the immediate vicinity of the cell. Thus the presence of Bsp is critical for TGF-β to cause local matrix degradation, a key step for invasion and metastasis.
BSP can also form a tertiary complex with αvβ3 integrin and complement Factor H (30
). Metastasizing cells as they travel through the blood stream are exposed to the complement system, and must control complement activity on their surfaces so as to avoid direct lysis, opsonization or macrophage activation. The BSP-mediated sequestration of Factor H on the tumor cell surface can inhibit the anti-tumor effects of alternate complement pathway for several cell types (19
), by activating Factor I and inhibiting the lytic pathway of complement (30
). We showed that treatment with BSP can also protect 4T1 cells against complement-mediated cell lysis. Since Bsp must interact with the integrin prior to binding Factor H in order to be effective, this action of Bsp, like its effect on invasion, is likely to be very local (30
). Thus there are at least two distinct mechanisms whereby locally increased levels of Bsp on the cell surface could mediate the promotion of metastasis by TGF-β.
BSP and OPN are thought to play particularly important roles in osteotropic metastasis. In the 4T1 model in the format in which we have used it, the predominant metastatic site is the lung, and the tumor burden in the bone is relatively low (data not shown), but our data clearly show that Bsp can significantly impact on metastatic efficiency to visceral sites in this model. We had hoped that our experimental approach might lead to identification of a circulating biomarker that would aid in the selection of patients for TGF-β antibody treatment, and allow monitoring of the efficacy of TGF-β antagonism. However, when we determined serum levels of Bsp in this model, they were not significantly increased by the presence of tumor (Supplemental Fig. S2). Others have found that BSP expression is lower in visceral metastases than in skeletal ones (46
) and osteolytic bone metastases are likely to liberate additional BSP from the bone matrix, which may explain why increases in circulating BSP in breast cancer patients are only prognostic for bone metastases (40
). In preliminary experiments using intracardiac injection of 4T1 cells, we have found that 1D11 is efficacious in reducing the burden of osteolytic bone metastases (JP and SL, unpublished data), and work is in progress to assess BSP status in this experimental setting. Thus it remains possible that TGF-β antibody treatment might impact on circulating BSP levels for patients with bone metastases, but nevertheless, it is clear that therapeutic benefit can be obtained even in settings where BSP is only locally increased in the tumor. Since BSP mRNA is elevated in metastatic compared with non-metastatic breast cancers (Supplemental Fig. S1B), BSP mRNA levels in the tumor might form a useful component of a patient stratification scheme even if circulating protein levels do not.
In summary, in this study we have identified Bsp as a novel mediator of the prometastatic effects of TGF-β on the tumor cell. Since Bsp expression in metastases is down-regulated by systemic treatment with anti-TGF-β antibodies, local suppression of Bsp in the tumor cell may contribute to the efficacy of this therapeutic approach. In other model systems, systemic antagonism of TGF-β has been shown to enhance immune surveillance and suppress angiogenesis (7
). Treatment with anti-TGF-β antibodies enhanced the ability of dendritic cell-based vaccines to inhibit the growth of 4T1 primary tumors (48
), so immune mechanisms are likely also to contribute to the anti-metastatic efficacy of TGF-β antagonism in the 4T1 model. Indeed, we have preliminary data suggesting that efficacy is at least partially dependent on the presence of CD8+ T-cells (J-SN and LW, unpublished observations). The relative contributions of the tumor- and stroma-targeted mechanisms are likely to vary among tumor types, depending on which steps in the metastatic cascade pose the biggest barrier to efficient metastasis, and the extent to which they are TGF-β-dependent. A more complete understanding of the molecular mediators of the various pro-metastatic effects of TGF-β should help guide the clinical application of TGF-β antagonists.