1 Introduction
Breast cancer is one of the major causes of death in women. However, most mortality and morbidity does not arise from the primary tumor, but from distant metastasis. High morbidity is especially caused by bone metastasis. In order to metastasize a cancer cell must shed many of its epithelial characteristics, invade the surrounding tissue to enter the circulation, subsequently survive in the circulation, extravasate and proliferate in the metastatic niche [
1]. Invasion is therefore a key step in the metastatic cascade. Several classes of proteins have been shown to play a role in this process, such as the integrin family of adhesion receptors. Integrins, which consist of an α and β subunit, are important for adhesion of cells to the extracellular matrix [
2]. Cancer cells express specific integrin combinations, whose signaling favors migration and invasion [
3]. Integrin α
vβ
3 is strongly expressed by breast cancer cells, especially by cells residing in bone metastasis [
4]. Its expression promotes bone metastasis of breast cancer cells [
5,
6], whereas inhibition of α
vβ
3 diminishes their osteotropism [
7].
Transforming Growth Factor (TGF)-β has a dual role in carcinogenesis. In the early stages it has a growth inhibitory and pro-apoptotic effect, whereas at the later stages of cancer, TGF-β promotes invasion and metastasis [
8,
9]. In line with its stimulatory role in cancer progression, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis [
10‐
12]. Moreover, studies in mouse models have demonstrated that inhibition of TGF-β-signaling in breast cancer cells reduces metastasis [
13‐
16].
TGF-β and Bone Morphogenetic Proteins (BMPs) signal via comparable mechanisms. Upon binding of the ligand, the type II receptor phosphorylates the type I receptor. The type I receptor, in turn, phosphorylates the receptor-regulated (R)- Sma and Mad related gene product (Smad). Phosphorylated R-Smads form heteromeric complexes with the common mediator Smad (Co-Smad) Smad4. These complexes then translocate to the nucleus, where they regulate transcription of target genes in collaboration with other transcription factors [
17]. In most cell types, TGF-β signals through the TGF-β receptor type II (TβRII) and the TGF-β type I receptor, also termed activin receptor-like kinase 5 (ALK5). ALK5 mediates the phosphorylation of Smad2 and Smad3 [
18]. BMPs signal through their specific BMP receptor type II (BMPRII) and BMP type I receptors (BMPRI) which induce the activation of Smad1, Smad5 and Smad8 [
19]. In addition to the previously described Smad pathways, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways [
20].
TGF-β is thought to be pro-invasive by inducing epithelial-to-mesenchymal transition (EMT). During this process, carcinoma cells acquire a more motile, mesenchymal phenotype [
21]. In normal breast and kidney epithelial cells BMP-7 can inhibit TGF-β-induced EMT [
22]; BMP-7 has also been shown to inhibit EMT and bone metastasis in breast and prostate tumors [
23,
24]. However, the effect of BMP-7 on tumor invasion is unknown.
Previously, we have set up a spheroid invasion model to study TGF-β-induced invasion [
25,
26]. In this assay, spheroids made from MCF10A series of cell lines invade in response to TGF-β. The MCF10A cell lines originate from MCF10A1, a spontaneously immortalized breast cell line [
27]. This cell line was transformed with oncogenic RAS, resulting in MCF10AT (hereafter referred to as M-II), which forms premalignant lesions in mice that upon grafting progressed into carcinoma [
28]. From these carcinomas MCF10CA1a was isolated (hereafter referred to as M-IV), which metastatasizes to the lung [
29]. Using these cell lines in the invasion assay, we have shown the importance of Smad signaling for TGF-β-induced invasion [
25]. In the current paper, we have analyzed the role of BMP-7. We show that BMP-7 can inhibit TGF-β-induced invasion through inhibition of TGF-β-induced integrin β
3 expression.
4 Discussion
In order to understand how BMP-7 exerts its anti-metastatic effects reported before [
23,
24], we investigated whether BMP-7 interferes with TGF-β-induced invasion of human breast cancer cells. We showed that BMP-7 inhibits TGF-β-induced invasion in the metastatic breast cancer cell line M-IV through inhibition of integrin β
3 expression. In addition, we showed that BMP-7 specifically inhibits the invasion of this metastatic cell line, and not the invasion of its premalignant precursor, M-II. Moreover, the inhibitory effect was specific for BMP-7, as its closest homolog, BMP-6 did not have any effect on TGF-β-induced invasion. To our knowledge, this is the first report that shows that BMP-7 inhibits TGF-β-induced invasion.
Since the antagonism between TGF-β and BMP-7 has been described before [
22‐
24,
35‐
37], the question arises whether the process that we describe occurs in other cell types as well. BMP-7 inhibits bone metastasis of the MDA-MB231 breast cancer cell line [
23] and PC3 prostate cancer cell line [
24]. However, in these cell lines BMP-7 inhibited TGF-β/Smad signaling [
23,
24], whereas under our experimental conditions we did not observe an inhibitory effect of BMP-7 on the TGF-β-Smad pathway. On the other hand, BMP-7 inhibited tumor progression of the uveal melanoma cell line OCM-1 without inhibiting the TGF-β-Smad pathway [
37]. Thus, BMP-7 is able to inhibit the tumor-promoting effects of TGF-β through either Smad- and non-Smad- mechanisms, depending on the cellular context.
Our data indicate that the inhibitory effect of BMP-7 on breast cancer invasion in part can be explained by inhibition of TGF-β-induced expression of integrin β
3. Integrin β
3 is induced by TGF-β and enhances EMT in a feed-forward loop by inducing Src [
38]. It is therefore tempting to speculate that the inhibition of TGF-β-induced integrin β
3 expression by BMP-7 might also play a role in the observed antagonistic effects between TGF-β and BMP-7 in EMT.
As mentioned before we did not detect a significant effect of BMP-7 on TGF-β Smad signaling, although we have shown that TGF-β-induced invasion is dependent on Smad3 and Smad4 [
25]. Since TGF-β-induced integrin β
3 expression has been reported to be dependent on p38, but not on Smads [
39], the inhibitory effect of BMP-7 on TGF-β-induced invasion might be mediated via non-Smad pathways. However, we could not find a significant effect of BMP-7 on (TGF-β-induced) p38 phosphorylation that could explain the inhibition of BMP-7 on TGF-β-induced integrin β
3 expression (data not shown). It is therefore likely that the effect is mainly due to BMP-7 transcriptional targets, such as transcription factors or microRNAs, that interfere with TGF-β-induced integrin β
3 expression.
One striking finding is that BMP-7 is able to inhibit TGF-β-induced invasion, whereas BMP-6 is not, despite the fact that they share 73% amino acid homology and are able to bind to the same receptors [
40‐
43]. Generally, BMP-6 is regarded as more potent than BMP-7 in inducing bone differentiation [
40,
44,
45]. However, BMP-7 is inhibited by Noggin, whereas BMP-6 is resistant to inhibition by Noggin due to an additional lysine at residue 60 [
45]. On the other hand, we found that BMP-6 and BMP-7 induce Smad1 phosphorylation and BMP-Smad transcriptional activity to the same extent in MIV cells. Possibly, BMP-6 or BMP-7 have different effects on non-Smad signaling pathways. However, we did not observe any significant differences between BMP-7 and BMP-6 on (TGF-β-induced) p38 and ERK phosphorylation in MIV cells (data not shown).
BMP-7 did not inhibit TGF-β-induced invasion of the premalignant precursor cell line M-II. Interestingly, overexpression of a dominant-negative TGF-β-receptor also had differential effects on these two cell lines in vivo [
46]. It is likely that upon tumor progression the response to TGF-β is altered in such a manner that BMP-7 is able to inhibit certain TGF-β responses. On the genetic level M-IV has an extra copy of the long arm of chromosome 1, a type of alteration often observed in breast cancer [
29]. A detailed survey of the genetic changes in the MCF10A series of cell lines revealed several genetic changes in M-IV compared to M-II, as well as differences in global gene expression [
47]. It is likely that the ability of BMP-7 to inhibit TGF-β-induced invasion is highly dependent on these additional genetic changes.
In conclusion, we have shown that BMP-7 inhibits TGF-β-induced invasion through inhibition of TGF-β-mediated integrin β3 expression. These data reinforce the rationale to either use BMP-7 or target integrin β3 in breast cancer metastasis.
Acknowledgements
We thank our colleagues for valuable discussion. We thank M. van Dinther for excellent technical assistance, Ken Iwata (OSI Pharmaceuticals, New York, USA) and Kuber Sampath (Creative Biomolecules, Inc, Hopkinton, USA) for reagents and Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, USA) for the cell lines. This work was supported by the Tumor Host Genomics (518198), Centre for Biomedical Genetics and Swedish Cancerfonden (09 0773).