The Role And Molecular Mechanism Of G-CSF In Adipocyte-mediated Migration And Invasion Of Breast Cancer

Cancer metastasis is the leading cause of death in breast cancer patients.It is currently believed that cancer development is the result of information exchange between cancer cells and their microenvironmnment.Adipocytes around breast cancer can be activated by cancer cells and transformed into "cancer-associated adipocytes(CAAs)",which have been demonstrated to be closely involved in cancer microenvironmnment remodeling,therefore promoting breast cancer progression.Yet,the molecular mechanism is still unclear.By establishing a co-culture model of adipocytes and breast cancer cells in vitro,we found that the conditioned medium of adipocytes co-cultured with breast cancer cells could significantly promote the migration and invasion of breast cancer cells.To explore the underlying molecular mechanisms,we profiled the differentially expressed genes(DEGs)between adipocytes and breast cancer-induced CAAs by transcriptome sequencing(RNA-Seq).G-CSF was identified one of the most highly enhanced genes in CAAs.Q-PCR experiments confirmed that the expression of G-CSF in CAAs was significantly up-regulated when compared with that in adipocytes,and ELISA results showed that the secreting protein level of G-CSF in CAA-CM was significantly increased.Subsequent studies have found that adipose-derived G-CSF was able to induce breast cancer cell EMT,migration and invasion,by activating Stat3 signaling.In line with it,anti-G-CSF neutralizing antibody or the Stat3 inhibitor Stattic were capable of inhibiting CAA-or rh G-CSF-induced EMT,migration and invasion of breast cancer cells.In addition,compared with normal adipose tissues,the expression of G-CSF in clinical adipose tissues adjacent to breast cancer was dramatically increased.Therefore,we came to the conclusion that the interaction between adipocytes and breast cancer cells increased the secretion of adipose-derived G-CSF,which induces breast cancer cell EMT,migration and invasion of breast cancer cells,by activating Stat3.Method: 1.Primary human mammary preadipocytes were characterized by immunofluorescence and flow cytometry.The adipocyte-induction medium was used to induce pre-adipocytes differentiation into mature adipocytes.This was verified by differentiation marker detection using Q-PCR,and lipid droplet examination by Oil red O staining.2.We established a co-culture system of adipocytes and breast cancer cells in vitro with transwells.After co-culture for 36 hours,the upper chambers in transwells were removed,and the adipocytes were cultured with serum-free DMEM.After 24 hours,the control supernatants(Adi-CM)without co-culture and the supernatants from co-cultured adipocytes(CAA-CM)were collected,respectively.Then,the effects of Adi-CM or CAA-CM on breast cancer cell migration,invasion and proliferation were detected by wound healing assay,transwell migration assay,matrigel invasion assay or MTT.3.Three experimental groups were set up in the experiment.(1)adipocytes cultured alone(Control group).(2)adipocytes treated with the conditioned medium of MDA-MB-231 breast cancer cells(CM group).(3)adipocytes co-cultured with MDA-MB-231 breast cancer cells(Co-culture group).After 36 hours,the total RNAs of adipocytes in each group were extracted,and subjected to RNA-Seq.The differentially expressed genes(DEGs)between adipocytes and CAAs were verified by Q-PCR experiments.The secreting levels of G-CSF in adipocytes/CAAs conditioned media were detected by ELISA.4.Breast cancer cell lines were treated with Adi-CM,CAA-CM in the presence of absence of anti-G-CSF neutralizing antibody(α-G-CSF),and 0.2% serum-containing DMEM was used as a control.The effects of α-G-CSF on CAA-CM-induced breast cancer cell migration and invasion were detected by cell scratch,transwell migration and invasion assay,respectively.Western blot was used to analyze the effect of α-G-CSF on CAA-CM-induced Stat3 phosphorylation in breast cancer cells.5.Breast cancer cells were treated with human recombinant G-CSF(rh G-CSF)protein,or G-CSF was stably expressed in MDA-MD-231 cells.The effects of G-CSF on migration and invasion of breast cancer cells were examined by cell scratch,transwell migration and Matrigel invasion assays.The effect of G-CSF on Stat3 signaling in breast cancer cells was detected by Western blot.Further,breast cancer cells were treated with α-G-CSF or Stattic,and migration and invasion of breast cancer cells were detected by cell scratch,transwell migration and Matrigel invasion assays,respectively.Stat3 activity in breast cancer cells was detected by Western blot.6.The changes of EMT markers in breast cancer cells upon rh G-CSF and Stattic treatments were detected by Q-PCR and Western blot.F-actin reorganization in breast cancer cells was detected by staining with phalloidin.7.The expression levels of the two pro-invasive genes,MMP2 and MMP9,were detected by Q-PCR.8.Hematoxylin-eosin(HE)staining was used to detect the morphology of mammary adipose tissues from clinical human breast cancer samples.The expression of G-CSF in paracancerous adipose tissues was detected by Q-PCR.Results: 1.The primary preadipocytes were characterized by immunofluorescence.They were shown to be CD45-negative and S100-positive.The expressions of CD44 and CD90 on the surface of preadipocytes were both positive.After induction with adipocyte-induction medium for 15-16 days,adipocyte differentiation rate reached 50-60% and lipid droplets were detected by oil red O staining.By Q-PCR,it was found that the adipocyte markers PPAR-γ,C/EBP-α,and FABP4 were significantly increased,while HSL and PREF1 were significantly decreased in mature adipocytes.2.After co-culture with breast cancers cells,adipocytes exhibited decreased levels of PPAR-γand C/EBP-α,whereas HSL and the expressions of pro-inflammatory factors IL-6 and IL-1β were up-regulated.Using cell scratch and transwell migration assyas,CAA-CM was shown to significantly promote the migration of MDA-MB-231 and BT549 cells.Transwell matrigel invasion experiments showed that CAA-CM was able to significantly promote the invasive ability of MDA-MB-231 breast cancer cells.MTT assay showed that CAA-CM had no significant effect on breast cancer cell proliferation.3.By RNA-Seq,195 differentially expressed genes were identified between adipocytes and CAAs.Among them,30 genes encode secretory proteins.Q-PCR experiment was carried out to verify the differentially expressed genes.Both the G-CSF m RNA expression and its secreted protein level in conditioned medium were up-regulated upon co-culture with breast cancer cells,as assessed by Q-PCR and ELISA respectively.4.Cell scratch and transwell assays showed that CAA-CM could promote breast cancer cell migration and invasion significantly.Western blot analysis showed that CAA-CM significantly promoted the phosphorylation of Stat3-Y705 in breast cancer cells.G-CSF-neutralizing antibody was able to specifically inhibit the effects of adipose-derived G-CSF,in terms of breast cancer cell migration and invasion,and also Stat3-Y705 phosphorylation.5.Cell scratch and transwell migration experiments showed that rh G-CSF significantly promoted the migratory ability of breast cancer cells.Matrigel invasion assay showed that rh G-CSF significantly promoted the invasion ability of breast cancer cells.Western blot analysis showed that rh G-CSF significantly promoted the phosphorylation of Stat3-Y705 in breast cancer cells.Treatment of breast cancer cells with α-G-CSF antibody or Stattic inhibited the migration and invasion of breast cancer cells induced by CAA-CM or G-CSF.6.By Western blot and Q-PCR,G-CSF was found to promote the expression of EMT marker genes(N-cadherin,vimentin,fibronectin,α-SMA),while Stattic inhibited the expression of these EMT marker genes significantly.Phalloidin staining experiments demonstrated that rh G-CSF were capable of inducing F-actin reorganization in MDA-MD-231 cells.7.By Q-PCR,it was found that the expressions of MMP2 and MMP9 in breast cancer cells increased significantly upon treatments with CAA-CM or rh G-CSF.However,these effects were inhibited by α-G-CSF antibody or Stattic treatment.8.By hematoxylin-eosin(HE)staining,adipocytes adjacent to breast cancer exhibited altered morphology when compared with normal adipocytes.The expressions of G-CSF,IL-6 and IL-1β in paracancerous adipocytes were significantly increased by Q-PCR.Conclusion: 1.Co-culture with breast cancer cells led to conversion of adipocytes into CAAs,which were able to promote the migration and invasion of MDA-MB-231 and BT549 breast cancer cells.2.We profiled the differentially expressed genes between adipocytes and CAAs by RNA-Seq,and G-CSF was found highly expressed in CAAs.3.Adipose-derived G-CSF induces breast cancer cell EMT,migration and invasion by activating Stat3.4.The expression of G-CSF increased in paracancerous adipose tissues of clinical human breast cancer tissue samples,when compared with normal adipose tissues.