The role for LPA in ovarian, endometrial, cervical, melanoma, and prostate cancers is currently emerging (7
). Despite studies reporting the effects of LPA on breast cancer cells in vitro (12
), the role of LPA in breast cancer in vivo was not known. In the present study, we demonstrated that LPA promoted breast cancer progression in vivo. This contention was first supported by the fact that the mitogenic activity of LPA on a series of human breast cancer cell lines correlated with the expression of LPA receptors. We also observed that overexpression of LPA1
specifically sensitized MDA-BO2 breast cancer cells in culture to the mitogenic action of LPA. Secondly, the growth of subcutaneous breast tumor xenografts or skeletal breast tumor metastases was markedly increased when LPA1
overexpression in MDA-BO2 breast cancer cells was turned on by doxycycline withdrawal. Moreover, the mitotic index of MDA-BO2 cells in situ was also increased when LPA1
overexpression was turned on. These results do not preclude the possibility that LPA2
receptors may also play a role in breast cancer progression in vivo. However, because the overexpression of LPA1
did not induce any modification in the expression levels of LPA2
in MDA-BO2 cells, our results strongly suggested that the increased in vivo growth of MDA-BO2 cells upon doxycycline withdrawal was directly related to the overexpression of LPA1
. Local production of bioactive LPA in the tumor microenvironment in vivo should therefore support LPA1
-dependent breast tumor cell proliferation.
It has been previously reported that MDA-MB-231 breast cancer cells do not directly produce LPA (13
). However, ATX in MDA-MB-231 and MDA-MB-435S breast cancer cells can induce the production of bioactive LPA, which in turn stimulates cell migration, invasion, and proliferation (13
). In the present study, MDA-BO2 cells and transfectants did not directly produce LPA or express ATX.
We therefore focused our attention on human blood platelets. Several factors suggested that platelets could locally produce bioactive LPA in vivo in the tumor bed. First, thrombin-activated platelets (19
) and lyso-PLDs (20
) are major sources of LPA in the serum. Second, platelets play a major role in the metastatic dissemination of tumor cells in vivo (27
), and, more recently, platelet aggregation was shown to be essential for successful formation of B16 melanoma bone metastases in animals (21
). Third, because of the leaky vasculature of angiogenic tumors (36
), platelets are in contact with tumor cells and are therefore able to secrete multiple factors upon activation (37
). Fourth, MDA-MB-231 breast cancer cells (as well as other tumor cell lines) interact with platelets and stimulate platelet aggregation in vitro (38
), suggesting that the platelet-aggregating activity of breast cancer cells might induce the release of LPA from activated platelets. In agreement with the latter findings (38
), we observed here that our MDA-BO2 parental and transfected breast cancer cell lines strongly stimulated platelet aggregation and the subsequent release of LPA from activated platelets. In addition, platelet-derived LPA stimulated in vitro the proliferation of parental MDA-BO2 cells, and this activity was further increased upon LPA1
overexpression. In vivo, a highly specific platelet aggregation inhibitor (Integrilin) inhibited by 50% the extent of bone metastases caused by MDA-BO2 cells and markedly blocked the progression of osteolytic lesions and skeletal tumor burden in animals bearing LPA1
-overexpressing cells. Moreover, this observation was not restricted to our MDA-BO2 breast cancer bone metastasis model since Integrilin also inhibited the progression of bone metastases caused by CHO-β3wt ovarian cancer cells. Thus, it is most likely that platelet-derived LPA promotes breast cancer growth in vivo. The experiments presented here provide evidence of LPA-dependent tumor promotion in MDA-BO2 and CHO-β3wt models. It is therefore important to test additional tumors to determine whether paracrine signaling by LPA is a general mechanism of metastatic tumor growth.
Because subcutaneous and skeletal MDA-BO2 breast tumors are highly vascularized (39
), these data strongly support the idea that platelets from the blood stream come into contact with the tumor bed in vivo, and then aggregate and secrete LPA, which stimulates the proliferation of breast cancer cells. Interestingly, compared with human serum, mouse serum contains relatively little LPA (8
); more importantly, in contrast to human platelets, mouse platelets do not aggregate in response to LPA in vitro (40
). Therefore, we anticipate that the contribution of LPA to the progression of the bone metastatic disease in patients could be even more important than that observed here in animals.
Besides the fact that LPA and its receptor LPA1
promoted the growth of primary or metastatic breast tumors, we also showed evidence indicating that LPA could indirectly contribute to the bone destruction associated with MDA-BO2 skeletal metastases. LPA was recently shown to stimulate the production of IL-6 and IL-8 by ovarian and breast cancer cells (23
). Tumor cells do not directly destroy bone (21
). Instead, IL-6 and IL-8 produced by breast cancer cells stimulate osteoclast-mediated bone resorption (41
). We showed here that platelet-derived LPA, as well as purified LPA, stimulated the secretion of these cytokines by MDA-BO2 breast cancer cells. In addition, production of IL-6 and IL-8 was markedly increased when tumor cells overexpressed LPA1
. It is, therefore, conceivable that beyond its tumor growth–promoting effect, LPA indirectly stimulates osteoclast-mediated bone resorption. Thus, in addition to the vicious cycle related to the direct reciprocal interaction between breast cancer cells and osteoclast-mediated bone resorption (15
), we propose that there exists an LPA-dependent cycle wherein bone-residing tumor cells stimulate LPA production by platelets, which in turn enhances tumor growth and cytokine-mediated bone resorption (Figure ).
Figure 11 Schematic representation of the LPA effects on progression of osteolytic bone metastases. Breast cancer cells produce factors (PTHrP, cytokines) that stimulate osteoclast-mediated bone resorption. In turn, bone resorption releases growth factors (IGFs, (more ...)
Although a role for LPA in cancer was emerging, there was a paucity of experimental evidence to support it. This study is, to the best of our knowledge, the first to demonstrate a role for LPA and its receptor LPA1 in the growth of breast cancer bone metastasis.