The Mechanism Of Secretion Of BMP-2 By Tumor Associated Macrophages(TAMs) In Promoting Microcalcifications And Metastasis In Invasive Breast Cancer

Objective:Breast microcalcifications are small deposits of calcium(diameter < 0.5mm)in breast tissues,and are well known as an important diagnostic feature of breast cancer,especially in the early detection of non-palpable breast cancer.It was shown that approximately 55% of non-palpable breast cancer were found with microcalcifications and mammography is the main detection method for microcalcifications.Although many studies have indicated that microcalcifications in breast cancer is correlated with HER-2 overexpression and predicts poor prognosis,the mechanism underlying microcalcifications in breast cancer is still unclear.Recent studies show it may be correlated with the osteoimmunological disorder and the abnormal of bone morphogenetic protein 2(BMP-2).As a member of the transforming growth factor(TGFβ)superfamily,BMP-2 plays an important role in breast tissue development in embryos and osteoblast differentiation.It can promote osteoblast differentiation and chondrogenesis through up-regulation of runt-related transcription factor 2(RUNX2).Hence,we hypothesized that in breast cancer,BMP-2 can also up-regulate RUNX2 expression and make breast cancer cells achieve osteoblastic characteristics to promote microcalcifications.In tumor tissues,BMPs also play an important role in cell division,survival,migration and differentiation.Meanwhile,the disorder of BMP signaling pathway may cause a variety of diseases,including tumors.Although,BMPs and its downstream Smad1/5/8signaling have been confirmed to be related to EMT in many studies,there are few studies on the mechanism of BMPs induction of EMT and whether it is involved in the regulation of EMT-TFs.We found that BMP-2 was positively correlated with the expression of classical EMT transcription factor TWIST1,through TGCA database analysis.It was demonstrated that BMP-2 is mainly produced by cells in the tumor microenvironment,but not by the tumor cells themselves.As one of the main cells of tumor microenvironment,tumor-associated macrophages(TAMs)have long been proved to secrete BMP-2.Hence,we speculated that TAMs can secrete BMP-2 to upregulate RUNX2 and cause microcalcification of breast cancer cells.At the same time,it could up-regulate TWIST1 to induce EMT,leading to poor prognosis of patients with microcalcifications.This study intends to verify the secretion of BMP-2 by TAMs on the formation of breast cancer microcalcifications and biological behavior by up-regulating RUNX2 and TWIST1 respectively.Meanwhile,BMP receptor inhibitor LDN193189(LDN)was used to inhibit the effect of TAMs on microcalcification formation,invasion and metastasis.Methods:1.Immunohistochemistry of paraffin sections of invasive breast cancer patients.272 invasive breast cancer patients from the first affiliated hospital of China medical university from January 2010 to January 2012 was included.Immunohistochemical analysis of BMP-2,TAMs markers(CD163 and CD68),RUNX2 and TWIST1 was performed,and the presence of microcalcification by detection of mammography and prognosis were analyzed in combination with other clinicopathological features.2.Induction of M2-type macrophages and co-culture with breast cancer cells.Thp-1 cells were added into the lower chamber of Transwell plate(6-well Transwell plate,0.4μm,Corning),PMA induction for 24 hours,LPS+ IFN-Gamma induction for 48 hours,IL-4 induction for 48 hours and flow cytometry was used to verify the induction effect.Breast cancer cells were added into the upper chamber of transwell plate and marked as co-culture group.Additionally,breast cancer cells were added into 6-well plates and marked as negative control group(NC group).In addition,cells in the coculture group were added with BMP receptor inhibitor LDN and marked as co-culture+ inhibitor group.All the cells were cultured for 48 hours for the next experiment.3.Alizarin red S staining: Alizarin red S staining was used to detect the deposition of calcium in each group,and Image J software was used to analyze the average optical density value in order to compare the difference of calcium deposition in each group.4.Detection of cell protein changes in each group: The expression of BMP-2,Smad1/5/8,p-Smad1/5,AKT,p-AKT,RUNX2,TWIST1 and EMT markers were detected by Western-blot.Meanwhile,RT-PCR was used to detect the m RNA expression levels of BMP-2,Smad5,AKT,RUNX2,TWIST1 and EMT markers.5.To verify the changes of cell function in each group: CCK-8 assay,scratch healing assay and Transwell invasion assay were used to detect the ability of proliferation,migration and invasion in each group.6.In vivo study to verify the secretion of BMP-2 by TAMs on microcalcification and pro-tumor effect of breast cancer with/without inhibiton of BMP-2 by LDN,fifteen5-week-old BALB/ C mice were randomly divided into three groups with five mice in each group:(1)The co-culture group of 4T1 cells and M2 macrophages were digested and prepared into cell suspension,which was inoculated under the fourth pair of fat pads on the right breast of 5 mice,marked as co-culture group;(2)The co-culture group of 4T1 cells and M2 macrophages were digested and prepared into cell suspension,which was inoculated to the other 5 mice under the fourth pair of fat pads on the right breast.At the same time,LDN(2.5mg/kg)was intraperitoneally injected once a day and labeled as co-culture + inhibitor group.(3)The NC group 4T1 cells were digested and was inoculated under the fourth pair of fat pads on the right breast of the remaining5 mice,which were labeled as NC group.Five weeks later,the mice were examined by PET-CT for visceral metastasis and pathological verification was performed.Alizarin red S staining and immunohistochemical staining were used to verify the calcification and protein expression.Results:1.The expression of BMP-2 in invasive breast cancer with microcalcifications was significantly higher than that without microcalcifications(P=0.000),and both microcalcifications and high expression of BMP-2 indicate a poor prognosis(P=0.010 and 0.000).2.Patients with high expression of CD68 and CD163 were more likely to be with microcalcifications,with a P value of 0.002 and 0.000,respectively.The expression of BMP-2 was positively correlated with the expression of CD68 and CD163(P= 0.000 and 0.000,respectively),and the high expression of CD163 indicates a poor prognosis(P=0.003).3.The expression of BMP-2 was significantly correlated with the expression of RUNX2 and TWIST1(P = 0.004 and 0.005,respectively),and the co-high expression of RUNX2 and TWIST1 indicates a poor prognosis(P =0.005).4.The secretion of BMP-2 by TAMs upregulates RUNX2 expression in MCF-7cells and MDA-MB-231 cells,which promotes microcalcifications in breast cancer through p-Smad1/5 and p-AKT pathway(P=0.0002 and 0.0173)and LDN could inhibit its promotion effect in both cells.(P=0.0005 and 0.0247).5.The secretion of BMP-2 by TAMs upregulates the expression of TWIST1 in breast cancer cells through p-Smad1/5 and p-AKT pathway.Meanwhile,the expression of vimentin was upregulated,which promotes the occurrence of EMT in breast cancer and the LDN could inhibit the occurrence of EMT.Western-blot and RT-PCT analysis showed that analysis showed that after co-cultured with TAMs,the expressions of pSmad1/5,p-AKT,RUNX2,TWIST1 and vimentin were significantly increased in MCF-7 and MDA-MB-231 cells,while the expressions of Smad1/5/8,AKT and Ecadherin were not significantly changed.Adding LDN could inhibit the expression of P-Smad1/5,P-AKT,TWIST1 and vimentin in co-cultured group.6.The secretion of BMP-2 by TAMs could enhance the proliferation,migration and invasion of breast cancer cells,which can be inhibited by LDN.The proliferation,migration and invasion abilities of cells in each group were detected by CCK-8,scratch healing test and Transwell invasion test,respectively.The results showed that the proliferation,migration and invasion abilities of cells in co-culture group were significantly enhanced compared with NC group,and LDN could inhibit the proliferation,migration and invasion abilities.7.The mouse in situ metastasis model showed similar results.After co-cultured with TAMs,the metastasis ability of breast cancer in mice was significantly enhanced,while LDN could inhibit its metastasis ability.Meanwhile,the immunohistochemical results also showed similar results to those of cell experiments.Conclusion: 1.The expression of BMP-2 is significantly increased in invasive breast cancer patients with microcalcifications,and high expression of BMP-2 and microcalcifications indicates a poor prognosis.2.The expression of BMP-2 was significantly associated with TAMs,RUNX2,and TWIST1.The high expression of CD163 and the co-high expression of RUNX2+TWIST1 indicates a poor prognosis.。3.The secretion of BMP-2 by TAMs could up-regulate RUNX2 and promote the formation of breast cancer microcalcification through p-Smad1/5 and p-AKT signaling,which can be inhibited by the BMP receptor inhibitor,LDN.4.The secretion of BMP-2 by TAMs could up-regulate TWIST1 to promote the expression of vimentin which induce EMT of breast cancer cells through p-Smad1/5and p-AKT signaling.The proliferation,migration and invasion of breast cancer cells were also upregulated,but the effect could be inhibited by LDN.5.LDN therapy is not suitable for all breast cancer patients,and it may only be suitable for patients with TAMs aggregation or high BMP-2 expression.In clinical practice,LDN therapy may be more suitable for breast cancer patients with microcalcifications.