The Effects Of Semaphorin 3A On Bone Rebuilding In The Process Of Cancer Osteoblastic Metastases

BackgroundBreast cancer and prostate cancer are the most common diagnosed female cancer and male cancer in the United States, which are all responsible for the second leading cause of cancer deaths in women and men respectively. Cancer cells have a strong propensity to from a secondary tumor in the bone microenvironment. It metastasizes to bone in more than 80% of breast cancer patients and 90% of prostate cancer patients with advanced disease. Bone metastases are associated with skeletal related events(SREs), such as severe bone pain, pathological fractures, hypercalcemia, and nerve compression. The patients with bone metastases have a high risk of SER: 60% will suffer pathological fractures, 20% will develop hypercalcemia and 10% will experience spinal cord compression. They are incurable in most cases and the majority of current therapies are palliative.Bone metastases are characterized by the interaction between tumor cells and bone microenvironment. Many cytokines and other factors derived by cancer cells, such as parathyroid hormone related protein(PTHrP), could regulate the differentiation and activity of the osteoblasts and osteoclasts directly or indirectly. On the other hand, transforming growth factor beta(TGF-beta) and so on from bone dissolution or bone cells could further promote the proliferation, migration, invasion and angiogenesis of cancer cells. This is known as the ‘‘vicious cycle’’ of bone metastases. The drug for resistance bone resorption, such as diphosphonate and RANKL antibody have been developed to the prevention and treatment of cancer bone metastases. Although such most treatments could directly relieve pain, they could not slow the progress of the disease.According to the pathologic and radiological appearance, bone metastases are traditionally divided into three types, including osteolytic metastases that cause excess bone resorption, osteoblastic metastases that cause excess bone formation and “mixed” subtypes. Breast cancers commonly lead to osteolytic metastases, while prostate cancers typically cause osteoblastic metastases. However, it must be noted that 15%~25% of breast cancer patients would have osteoblastic bone lesions. The interaction between the tumor cells and osteoblasts, osteoclasts and stromal cells in the bone microenviroment, disturb the balance between bone formation induced by osteoblasts and osteolysis induced by osteoclasts. It is widely recognized that cancer cells produce factors like bone morphogenic proteins, endothelin-1 to contribute new bone formation. However, the precise mechanisms of bone osteoblastic metastases are not fully understood. More researches are absolutely necessary to further study the factors associated to the cancer bone metastases.Sema 3A, a secreted axon guidance molecule, has been proved to have an important influence on bone rebuilding. A recent study by Hayashi et al demonstrated that Sema 3A produce by osteoblasts has the bidirectional regulating ability for osteoblasts and osteoclasts. Sema 3A could combine to its specific receptor and stimulates the osteoblastic differentiation through the canonical Wnt/β-catenin signaling pathway. Furthermore, Sema 3A could inhibit osteoclastic differentiation by suppressing RANKL-induced immunoreceptor tyrosine-based activation motif(ITAM) signaling. Meanwhile, it has showed that Sema 3A is correlated with cancer cell migration, invasion and angiogenesis. So, it is therefore conceivable that Sema 3A produced by cancer cells could significantly influence bone rebuilding in which cancer cells, osteoblasts, and osteoclasts interact and stimulate bone metastases development.The role of Sema 3A in the process of cancer osteoblastic metastases and the related potential mechanisms remain unknown up to now. People still don’t have enough knowledge about whether Sema 3A/NRP1 signaling is involved in the regulation of differentiation of osteoblasts and osteoclasts by cancer cells. In this study, a steady cell culture model in vitro for research the interaction between cancer cells and osteoblasts, osteoclasts was established, for further explore the effects of Sema 3A in bone metastases process.Methods1 The Sema 3A expression levels in various kinds of bone metastases cancer cellsThe expression of Sema 3A in breast cancer cell line(MDA-MB-231 and MCF-7) and prostate cancer cell line(LNCa P、PC-3、C4-2) were detected by real-time fluorescent quantitative PCR, Western blot and cell immunofluorescence method.2 The effects of Sema 3A and related mechanisms in cancer-induced osteoblastic differentiationSema 3A sh RNA lentivirus was transfected into high expression breast cancer cells and prostate cancer cells, then screened by puromycin. Neutralizing antibody for Sema 3A was used in order to counteract the effects of Sema 3A on the osteoblasts differentiation induced by cancer cell conditioned medium(CM). MC3T3-E1 cells were treated with osteogenic medium in the absence of presence of cancer cells CM.2.1 Alikaline phosphatase(ALP) activity and ALP staining of were determined by alkaline phosphatase assay kit and BCIP/NBT alkaline phosphatase colour development kit respectively.2.2 The bone nodule formation of osteoblasts was detected by Alizarin Red S staining.2.3 The relative m RNA expression levels of Col1α1, OCN, RUNX2, etc were measured by qPCR.2.4 The protein expressional levels of NRP1 and activation of β-catenin of osteoprogenitor cells were determined by Western-blot analysis.3 The effects of Sema 3A and related mechanisms in cancer-induced osteoclastic differentiationRAW264.7 cells and bone marrow-derived macrophages(BMMs) were treated with RANKL and M-CSF in the absence of presence of cancer cells CM.1. The number of TRAP-positive multinucleated cells(MNCs) was detected by TRAP staining.2. The bone resorption activity was evaluated by scanning electron microscope3. The relative mRNA expression levels of CK, TRAP and CTR were measured by q PCR.4. The protein expressional levels of NRP1 and PLC of RAW264.7 cells were determined by Western-blot analysis.Results1 The Sema 3A expression levels in various kinds of bone metastases cancer cells1.1 The mRNA and protein expression of Sema 3A were higher in osteoblastic bone metastases breast cancer cell line MCF-7 than in osteolytic bone metastases breast cancer cell line MDA-MB-231;1.2 The mRNA and protein expression of Sema 3A were higher in osteoblastic bone metastases prostate cancer cell line C4-2 than in osteolytic bone metastases prostate cancer cell line PC-3.2 The effects of Sema 3A and related mechanisms in cancer-induced osteoblastic differentiation2.1 Breast cancer MCF-7 cell stimulated alkaline phosphatase activity and mineralization of MC3T3-E1 cells obviously, but treatment with MDA-MB-231 CM blocked these processes.2.2 Sema 3A shRNA could effectively down-regulated Sema 3A m RNA and protein expression level in breast cancer MCF-7 cells and prostate cancer C4-2 cells, and Sema 3A down-expression cancer cell lines were successfully established;2.3 Down-expression of Sema 3A by shRNA can significantly inhibit the osteoblastic differentiation induced by MCF-7 cells and C4-2 cells respectively. Neutralizing antibody for Sema 3A has the similar effects.2.4 Treating MC3T3-E1 cells with MCF-7 cells CM greatly induced NRP1 expression, which was partially but significantly inhibited by Sema 3A shRNA down-regulation of Sema 3A expression. MCF-7 cells CM significantly increased nuclear β-catenin accumulation in MC3T3-E1 cells compared with OS alone, whereas MCF-7Sema 3A- CM had a weak effect on stimulating β-catenin nuclear accumulation.3 The effects of Sema 3A and related mechanisms in cancer-induced osteoclastic differentiation3.1 MCF-7 cells CM significantly increased osteoclastic formation compared to samples treated with RANKL alone.3.2 Down-regulating Sema 3A expression in MCF-7 cells by sh RNA further enhanced osteoclastic differentiation stimulation induced by MCF-7 cells CM.3.3 MCF-7 cell-derived Sema 3A stimulates NRP1 expression but inhibits PLCγ phosphorylation in RAW 264.7 cellsConclusionIn this study, the expression levels of Sema 3A, an important factor for bone rebuilding, were detected in various kinds of bone metastases cancer cells. Then the potential role of Sema 3A in regulation of osteoblastic and osteoclastic differentiation was investigated.1 Sema 3A is higher expressed in osteoblastic metastases cancer cells including breast cancer MCF-7 cells and prostate cancer C4-2 cells.2 Osteoblastic metastases cancer cell-derived Sema 3A stimulates NRP1 expression and β-catenin nuclear accumulation in MC3T3-E1 cells, and then promotes the osteoblastic differentiation.3 MCF-7 cell-derived Sema 3A stimulates NRP1 expression but inhibits PLCγ phosphorylation in RAW 264.7 cells, and attenuates MCF-7 cells CM-induced osteoclastic differentiation stimulation.In conclusion, our data suggest that MCF-7 cell-derived Sema 3A plays a causative role in osteoblastic bone metastases progression by stimulating osteoblastic differentiation and inhibiting osteoclastogenesis.