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Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor β (PDGFRβ) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRβ signaling using soluble PDGFRβ (sPDGFRβ) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRβ. A stable transfectant (TNBCT/Bo-sPDGFRβ) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRβ cells. Intratibial injection of TNBCT/Bo-sPDGFRβ cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRβ is useful for the treatment of breast cancer bone metastasis. (Cancer Sci 2011; 102: 1904–1910)
Breast cancer is the leading cause of cancer death in women,(1) and the skeleton is the preferred site for breast cancer metastasis.(2,3) Breast cancer bone metastases are usually incurable, and only 20% of patients with breast cancer are still alive 5 years after the discovery of bone metastases.(4) Of the several subtypes of breast cancer, triple-negative breast cancers do not express estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptor 2 (HER2),(5) and are associated with a poor prognosis as they do not respond to endocrine therapy or HER2-targeted therapy.(6) Thirteen percent of triple-negative breast cancer patients have bone metastases and the median survival is only 0.8 years.(6)
Metastatic cancer cells in the bone microenvironment release factors and cytokines that accelerate bone destruction. In turn, other factors liberated from the bone matrix promote cancer cell growth and proliferation.(7) Of these cytokines and growth factors, the platelet-derived growth factor (PDGF) family is thought to contribute to the regulation of bone turnover.(8) Members of the PDGF family are well-known inducers of cell migration, proliferation, and transformation through activation of their cognate receptors.(9–11) Interactions between PDGF and PDGF receptor (PDGFR) are multiple and complex, however, PDGF-AA and PDGF-CC interact with PDGFRα,(12–14) and PDGF-BB and PDGF-DD interact with PDGFRβ.(15,16) Both PDGF-BB and PDGF-DD are abundantly expressed in many human cancer cells and tissues, including prostate,(17–19) kidney,(20–22) lung,(23) and breast.(24) In addition, a large number of breast tumors display a positive PDGFR phenotype. It is reported that invasive mammary carcinomas express PDGFRα in 65% and PDGFRβ in 75% of cases,(25) and PDGFRβ expression is significantly higher in tumor-associated stromal cells.(26–28) Both PDGF-BB and -DD enhance tumor angiogenesis by stimulating vascular endothelial growth factor expression.(29,30) In addition to its angiogenic effects, PDGF also recruits cancer-associated stromal cells (CAS). Increasing evidence suggests that cancer cells recruit stromal cells and endothelial cells into the tumor, and tumor–stromal cell interactions in the microenvironment are essential for directing tumor progression.(31,32) Human bone matrix is a reservoir of growth factors such as transforming growth factor β (TGF-β), PDGF,(33) and basic fibroblast growth factor.(34) During the process of bone remodeling, TGF-β and PDGF are released from the bone matrix and TGF-β signaling through Smad2/3/4 promotes PDGF-BB secretion by tumor cells.(35) Furthermore, several studies show that PDGFRβ is activated by both autocrine and paracrine mechanisms.(9,26) These data support a critical role of PDGFRβ signaling in cancer bone metastasis.
Several strategies to block PDGF/PDGFR-mediated tumor growth are currently being tested in basic research and clinical trials. These strategies fall into two categories: (i) neutralizing antibodies directed against PDGF ligands or receptors;(9) and (ii) inhibitors of PDGFR kinase activity (e.g. imatinib), which inhibit phosphorylation of PDGFR and increase apoptosis of tumor cells.(36) However, neutralizing antibodies are not completely specific,(9) and imatinib has potential disadvantages in that it is active against multiple receptor tyrosine kinases(9) and no benefit was reported in metastatic breast cancer patients.(37) Therefore, more specific and effective strategies must be developed.
Here, we propose a novel approach for neutralizing PDGFRβ signaling by using soluble PDGFRβ (sPDGFRβ), and show evidence of inhibited growth, angiogenesis, and bone lysis of triple-negative breast cancer cells after blockade of PDGFRβ signaling.
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- Materials and Methods
- Disclosure Statement
- Supporting Information
In this study, the bone-seeking triple-negative breast cancer cell line, TNBCT/Bo, was transfected with a mammalian expression vector encoding the extracellular domain of PDGFRβ, and a stable transfectant, TNBCT/Bo-sPDGFRβ, was established. The CM from TNBCT/Bo-sPDGFRβ cells neutralized the growth of human fibroblasts stimulated with PDGF-BB, and the tumor incidence in TNBCT/Bo-sPDGFRβ-injected athymic nude mice was significantly lower than in control mice (P < 0.01), as was cancer cell proliferation, angiogenesis, and the recruitment of stromal cells. However, the number of apoptotic cells increased.
Many characteristics of bone tissue make it an ideal environment for breast cancer cell migration and colonization.(42) When tumor cells reach the bone, stromal cells within the bone microenvironment (including fibroblasts, myofibroblasts, pericytes, and endothelial cells) contribute to the development of micrometastatic foci.(43,44) Because the reciprocal interaction between breast cancer cells and the bone microenvironment results in a vicious cycle that increases the tumor burden,(45) blocking this interaction is one potential therapeutic strategy for treating bone metastases. The present study focused on PDGFRβ signaling for the following reasons: (i) PDGFRβ is expressed in human mammary carcinomas and correlates with a malignant, invasive phenotype,(25) and PDGF-BB expression is also increased in advanced breast cancer patients;(16) (ii) paracrine PDGFR signaling is commonly observed in breast cancer, which triggers stromal cell recruitment, thereby affecting tumor growth, angiogenesis, and metastasis;(9) and (iii) the PDGFR tyrosine kinase inhibitor, imatinib, has been used to treat breast cancer bone metastases; however, the side-effects included myelosuppression.(46) Therefore, we attempted to specifically block PDGFRβ-signaling to treat breast cancer bone metastasis.
The triple-negative breast cancer cell line TNBCT was chosen because neither endocrine therapy nor HER2-targeted therapy is effective against triple-negative breast cancer;(6) therefore, other treatment strategies must be developed. The TNBCT cells showed epithelial-like growth in vitro, and formed bone tumors in nude mice after direct intratibial injection of cancer cells. Expression of ER, PR, and HER2 was not detected on the tumor cells by immunohistochemistry (Fig. S1). Because the incidence of TNBCT bone tumors was not very high, we tried to select bone-seeking cancer cells in vivo. The selected subclone was designated TNBCT/Bo, and showed a bone tumor incidence greater than that of TNBCT (data not shown).
Using TNBCT/Bo, we established a stable transfectant, TNBCT/Bo-sPDGFRβ. This cell line expressed mRNA encoding the sPDGFRβIg1–3 sequence (Fig. 1a). Secretion of IgG-fusion proteins by TNBCT/Bo-mock and TNBCT/Bo-sPDGFRβ cells was confirmed by ELISA, although the concentration of the protein from TNBCT/Bo-mock cells was approximately 8.5-fold higher than that from TNBCT/Bo-sPDGFRβ cells.
Next, a proliferation assay was carried out to examine the biological activity of sPDGFRβIg1–3. Primary human fibroblasts were used for this assay, in which growth was accelerated by PGDF-BB but not by PGDF-AA. Treatment with PDGF-BB significantly induced cell growth in the presence of CM from TNBCT/Bo-mock cells. In contrast, no growth induction was observed in the presence of CM from TNBCT/Bo-sPDGFRβ cells (Fig. 2). These results indicated that the PDGF-BB–PDGFRβ interaction was blocked by sPDGFRβIg1–3. The growth rates of TNBCT/Bo-mock and -sPDGFRβ cells were also examined. Despite constitutive expression of endogenous PDGFRα and PDGFRβ mRNA (Fig. 1a), both cells lines grew at similar rates under normal culture conditions (Fig. 1b). A PDGFRβ-mediated autocrine loop might not exist in these cells, as sPDGFRβIg1–3 did not show any inhibitory effect on the growth of TNBCT/Bo-sPDGFRβ cells.
The TNBCT/Bo-mock or TNBCT/Bo-sPDGFRβ cells were injected into the tibias of nude mice to assess the therapeutic benefit of sPDGFRβIg1–3. The histologically-determined tumor incidence at week 12 was 10/13 (77%) in TNBCT/Bo-mock-injected mice and 1/15 (7%) in TNBCT/Bo-sPDGFRβ-injected mice (P < 0.01; Table 2). Radiographic and histological analyses revealed that TNBCT/Bo-sPDGFRβ cells showed limited growth with minimal osteolysis. In contrast, TNBCT/Bo-mock cells showed invasive growth with severe osteolysis (Fig. 3a,b). Positive immunostaining of TNBCT/Bo-mock and -sPDGFRβ cells for human IgG (Fig. 3c) confirmed continued expression of the IgG-fusion proteins. Taken together, these results suggest that sPDGFRβ is useful for the treatment of triple-negative breast cancer bone metastases.
Immunohistochemical analysis also showed that sPDGFRβ inhibited the proliferation of tumor cells and induced apoptosis in vivo; however, in vitro data suggested that the proliferation of TNBCT/Bo cells was independent of autocrine PDGFRβ signaling. Taken together, these results suggest that PDGFRβ-mediated paracrine stimulation in the bone microenvironment facilitates the progression of TNBCT/Bo tumors. In agreement with a direct role for PDGFRβ signaling as a switch toward endothelial cell development and induction of endothelial cell differentiation,(47,48) MVD was significantly decreased in TNBCT/Bo-sPDGFRβ tumors compared with that in the controls. As PDGFRβ expression in tumor-associated endothelial cells was upregulated in the bone microenvironment in our previous study,(36) sPDGFRβ could be an effective inhibitor of tumor-associated angiogenesis, especially in bone metastases. In addition to its pro-angiogenic effects, PDGFRβ signaling is associated with the recruitment of stromal cells.(31,32) The tumor stroma has been viewed as a passive component of tumors in bone metastasis.(29) It is widely accepted that tumor–stromal cell cross-talk promotes a tumor-promoting phenotype in the recruited stromal cells. Paracrine stimulation of tumor pericytes is associated with a decrease in tumor cell apoptosis.(40) Emerging data suggest, however, the CAS may affect the malignant behavior of neoplastic cells and contribute to tumor progression.(49) Expression of PDGFRβ is significantly higher in tumor-associated stromal cells.(26,27) Based on these data, we analyzed stromal cell recruitment by TNBCT/Bo-mock and -sPDGFRβ tumors. The number of CAS per field was significantly less in the TNBCT/Bo-sPDGFRβ tumor, suggesting another mechanism underlying the antitumor effects of sPDGFRβ: inhibition of tumor-associated stromal cell recruitment.
In conclusion, we have shown that sPDGFRβ inhibits the growth of triple-negative breast cancer cells in the bone microenvironment through the inhibition of tumor cell proliferation, induction of tumor cell apoptosis, and reduced CAS recruitment and neovascularization. These findings suggest that sPDGFRβ could be a useful agent for blocking the vicious PDGFRβ-mediated cycle of bone metastasis.