ADAMTS-1 expression decreased in human breast tumors in vivo, mainly in triple negative cases (ER-, PR-, and Her-2), and ADAMTS-1 knockdown was shown to stimulate migration, invasion and invadopodia formation in breast cancer cells in vitro. Our series of experiments further suggest that VEGF is involved in these effects of ADAMTS-1 in breast cancer cells. To our knowledge, this is the first report establishing this relationship in human breast cancer cells.
ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motifs) was the first described isoform in this family , and this identification was based on its elevated expression in cachexia-inducing adenocarcinomas in mice. Its multidomain structure links this secreted protein to various cellular functions; for example, it is a potent inhibitor of angiogenesis and plays an important role in follicular rupture and ovulation  and urogenital development, as demonstrated by the characteristics of knockout animal models .
Altered ADAMTS-1 expression has been reported in different types of tumors, including breast cancer [11, 12]. However, the role of ADAMTS-1 in human breast cancer is not fully understood and requires further investigation. Lu et al. (2009)  reported that ADAMTS-1 was overexpressed in 39.7% of breast tumors, and these authors further demonstrated that ADAMTS-1 overexpression was associated with an increased risk of bone metastasis. Similarly, we observed variable levels of ADAMTS-1 mRNA expression in a series of primary breast tumors. However, the immunolocalization of ADAMTS-1 showed that the expression of this molecule was lower in triple negative tumors as compared to normal tissues. In this study, we compared the distribution of ADAMTS-1 in tumors of different clinical stages, which may explain the apparent discrepancies between our findings and previous data. The previous study by Lu et al. analyzed an array of breast cancer tissues without any consideration for the stage of the tumor. Therefore, we considered tumor stage in our analysis and also evaluated the expression of ADAMTS-1 in the tumor stroma, which was reduced in higher-staged tumors.
Immunoblots comparing normal tissue and cancer tissue revealed that ADAMTS-1 could be detected in four different bands: the 110 kDa band, which likely represents total protein; the 80 kDa, which may characterize the protease without the pro-domain or the activated form; and two smaller bands, which may correspond to activated ADAMTS-1 with additional proteolytic processing [23, 24]. We also observed that the 80 kDa ADAMTS-1 band was decreased in breast tumors as compared to adjacent normal tissue samples. Thus, a reduction in ADAMTS-1, as determined by immunohistochemistry, may represent a decrease in the 80 kDa ADAMTS-1 band. With regard to the in vitro expression of ADAMTS-1, both the MCF7 and MDA-MB-231 cell lines exhibited a prominent ADAMTS-1 80 kDa band in conditioned medium.
Our results showed that MDA-MB-231 cells with reduced ADAMTS-1 expression demonstrated increased migration, velocity and invasion. In cancer progression, cell-to-cell detachment from the primary tumor and the acquisition of a motile phenotype are required for cells to become invasive and colonize distant organs, thereby producing a metastasis . The spread of cancer cells to distant sites in the body is the major cause of death for cancer patients [26, 27], and one major challenge in cancer therapy is to inhibit the spread of tumor cells from the primary tumor site to distant organs .
Previous reports have acknowledged the role of ADAMTS-1 in cell migration. Krampert et al. (2005)  studied the role of ADAMTS-1 in the healing of skin wounds. In this model, they observed that ADAMTS-1 played different roles in fibroblast migration depending on the concentration; a decrease in the level of this protein stimulated cell migration via the proteolytic activity of ADAMTS-1.
The effects of ADAMTS-1 knockdown on cell migration and invasion seem to be related to VEGF, as MDA-MB-231 cells with reduced ADAMTS-1 expression showed increased levels of VEGF in conditioned medium. The relationship between VEGF and ADAMTS-1 was recently reported, and the carboxyl-terminal domain of ADAMTS-1 was shown to be responsible for binding and sequestering VEGF . This sequestration of VEGF by ADAMTS likely inhibits various functions of VEGF, such as its role in cell migration and invasion.
It has been described that ADAMTS-1 sequesters VEGF . VEGF is known to enhance migration and invasion [13, 15, 16, 18]. We then carried out combined multi-tiered experiments to relate the role of ADAMTS and VEGF during cell migration and invasion. ADAMTS-1 knockdown in MDA-MB-231 cells resulted in a decrease in ADAMTS-1 protease activity in conditioned medium. Furthermore, cells with reduced ADAMTS-1 expression demonstrated increased levels of VEGF in conditioned medium. Taken together, these results suggest that ADAMTS-1 knockdown decreased the presence of this protease in the conditioned medium of MDA-MB-231 cells, thus preventing the sequestration of VEGF and rendering this growth factor available to exert its cellular effects, including migration and angiogenesis [13, 15, 16, 18].
To analyze the putative role of VEGF in the cell migration of MDA-MB-231 cells, we carried out migration and invasion assays in MDA-MB-231 cells with reduced ADAMTS-1 expression. To assess the effect of VEGF, we used a VEGF blocking antibody and found that ADAMTS-1 knockdown increased migration and invasion, as expected. However, treatment with blocking antibodies partially rescued both cell migration and invasion, and these results suggest a close relationship between ADAMTS-1 and VEGF in regulating cell migration and invasion.
The evidence presented here establishes a relationship between ADAMTS-1 and VEGF, and our results also indicated that VEGF in conditioned medium from MDA-MB-231 cells with ADAMTS-1 silenced initiated tubulogenesis in HUVEC cells. ADAMTS-1 has been described as a protease with angioinhibitory properties  that significantly blocks VEGFR2 phosphorylation and suppresses endothelial cell proliferation. In addition, the inhibition of ADAMTS-1-related angiogenesis is related to the sequestering of VEGF.
VEGF also induces invadopodia formation by increasing the activity of MMP-2, MMP-9 and MT1-MMP . MDA-MB-231 cells with ADAMTS-1 knockdown demonstrated increased invasion in Boyden chambers, and cells with reduced ADAMTS-1 expression also demonstrated increased invadopodia formation. Therefore, MDA-MB-231 cells with depleted levels of ADAMTS-1 may increase the availability of VEGF, which could enhance invadopodia formation and/or activity.
Various authors have demonstrated invadopodia formation and/or activity in MDA-MB-231 cells using a variety of approaches. For example, invadopodia activity in MDA-MB-231 cells has been reported in src-transformed cells [30, 31], in cells cultured on fibronectin  and in cells treated with growth factors . Most of these studies were carried out in cells grown for at least 16 hours; in contrast, our results revealed invadopodia activity over a short timeframe as well as increased matrix digestion in MDA-MB-231 cells when ADAMTS-1 is knocked down.
Cancer cells rely on invadopodia to initiate invasive activity [30, 34], as these formations are enriched with actin filaments (F-actin) and components needed for actin assembly, including neural-Wiskott Aldrich Syndrome protein (N-WASP) and cortactin [30, 35–37]. Therefore, it was not surprising that the functional knockdown of ADAMTS-1 stimulated the migratory and invasive activity of MDA-MB-231 cells. On the other hand, ADAMTS-1 knockdown had the opposite effect on MCF7 cells and reduced their migratory activity. This discrepancy could be the result of different biological behaviors between these cell lines. It is well known that MCF7 cells express estrogen and progesterone receptors, while MDA-MB-231 cells do not. Thus, MDA-MB-231 cells may be more aggressive and invasive as compared to MCF7 cells.
Another possible explanation for these differences in migration and invasion could be related to VEGF and VEGF receptor levels. VEGFR2 expression levels in MDA-MB-231 cells were 1.5-fold higher in comparison to MCF7 cells, whereas MDA-MB-231 cells with reduced ADAMTS-1 expression showed augmented VEGF levels in the conditioned medium as compared to the controls. Taken together, these results suggest that MDA-MB-231 cells possess suitable machinery to interact with VEGF, a growth factor important for migration and invasion [13, 15–18].