NE/?₂-AR/NGF Positive Feedforward Loop Promotes The Malignant Progression Of Triple-negative Breast Cancer

Sympathetic nerve system is a primary part of the tumor micro-environment,which plays an important role in tumorigenesis.The sympathetic nerve fibers in the tumor tissue are regulated by the sympathetic nerve system(SNS)to promote tumorigenesis;moreover,the sympathetic nerve fibers stimulated by cytokines which are secreed by the tumor cells,induce neurogenesis in tumor tissue for adaption to survival demand.However,in the microenvironment of the triple negative breast cancer(TNBC),either the roles of the sympathetic nerves in the malignant progression of TNBC or the interaction between the sympathetic nerves and the breast cancer cell is still unclear.According to reports,there were 90%of the pathological types of TNBC belong to invasive ductal carcinoma,and normal breast tissue is innervated by the 4th to 6th thoracic sympathetic nerves.According to epidemiological studies,breast cancer patients have bad moods such as severe depression and anxiety,resulting in excitation of SNS,which promotes the sympathetic nerve fiber releasing a large amount of norepinephrine(NE)in the breast tissue.NE primarily binds to the ?₂-adrenergic receptor(?₂-AR)on the surface of tumor cells,thereby exerts its tumor biological function.In clinical treatment,the application of?₂-AR blockers can improve the prognosis of breast cancer patients.Another study showed that tumor cells secrete nerve growth factor(NGF)to induce neuro proliferation in tumor tissues.As an important neurotrophic factor,NGF plays a key role in the differentiation,advance and regeneration of neurons.Breast cancer tissues secrete more NGF than that in normal breast tissue.Therefore,we hypothesize that the interaction between sympathetic nerves and breast cancer cells leads to sympathetic hyperplasia in tumor tissues,which in turn promotes the malignant progression of TNBC.Objective:TNBC patients'serum,breast cancer tissue samples,triple-negative breast cancer cells and DRG neuronal cells were used to explore the role of NE/?₂-AR/NGF positive feedforward loop in the malignant progress of TNBC.immunosorbent assay(ELISA),IHC,Western Blot were performed to provide experimental data and the new perspectives for the clinical diagnosis and treatment of TNBC.Methods:(1)Collect 120 serum samples of patients with breast hyperplasia and TNBC,and compare the expression levels of NE and NGF in the serum of the two groups by enzyme-linked ELISA,and explore the role of NE and NGF in TNBC.(2)At the organizational level,collect the normal breast tissues of 45 cases of breast hyperplasia patients and 120 cases of breast cancer tissues of TNBC patients,and compare the distribution of sympathetic nerves,the expression levels of?₂-AR,NGF,tropomyosin receptor kinase A(TrkA)by IHC,to explore the correlation between sympathetic nerves and the characteristics of TNBC clinical cases and the interaction mechanism between sympathetic nerves and breast cancer cells.(3)At the cell and molecular level,we selected normal breast epithelial cell lines,triple-negative breast cancer cells(simultaneous expression of?₂-AR and NGF)and non-triple-negative breast cancer cell lines for follow experiments.Cells and NE/isoproterenol(ISO)/inhibitor of?₂-AR/inhibitor of extracellular signal-regulated protein kinase(U0126)/PKA blocker(KT-5720)were cocultured.The proliferation ability was tested by cell proliferation test(CCK-8 method).The expression of?₂-AR/NGF/p-ERK/p-CERB were tested by Western blot(Western Blot).The level of NGF secreted by triple-negative breast cancer cells was analysed by ELISA.(4)Triple-negative breast cancer cells and neuronal cells of the dorsal root ganglion(DRG)in SD rats born 2 days old were cocultured.Using NE and?₂-AR,NGF/TrkA blocker pretreatment,the axon growth of each group of DRG neuron cells was detected by immunofluorescence double staining,to explore the mechanism of NE/?₂-AR signal on the axon growth of DRG neurons.Results:(1)The levels of NE and NGF in the serum of TNBC patients were significantly higher than those of the control group,and the levels of NE and NGF showed a positive correlation.(2)IHC results showed that in normal breast tissue,sympathetic nerves,arteries,veins,and lymphatic vessels were located in the fibrous interstitium surrounding the lobules of the breast,while in triple-negative breast cancer tissues,the sympathetic nerve fibers significantly proliferated,located in the tumor lesions,as an important part of the tumor microenvironment,it was tightly surrounded by breast cancer cells.Both normal breast epithelial cells and TNBC could express?₂-AR and NGF.However,the expression level of?₂-AR and NGF in TNBC was significantly higher than that of normal breast epithelial cells.In addition,nerve fibers in both groups express TrkA.But compared with the nerve fibers of normal breast tissue,the expression level of TrkA in the nerve fibers of triple-negative breast cancer tissue was significantly stronger.(3)The sympathetic adrenergic neurotransmitter-NE binding with?₂-AR which was on the surface of TNBC,activated the ERK signaling pathway,promoted the proliferation and stimulated the secretion of NGF.?₂-AR blockers significantly reverse the above effects.(4)The sympathetic adrenergic NE/?₂-AR signal stimulated TNBC to secrete NGF.NGF binding with TrkA in DRG neuron cells,induced the axon growth.?₂-AR and NGF/TrkA blockers reversed the above effects.Conclusion:In the tumor microenvironment of TNBC,SNS regulated the proliferation of sympathetic nerve fibers in tumor tissue and incresed release of NE.NE bond to?₂-AR on the surface of breast cancer,activating the ERK signal pathway and promoting the proliferation and release of NGF,thereby induced sympathetic nerve hyperplasia in tumor tissues,leading to NE/?₂-AR/NGF positive feedback loop,which promoted the malignant progression of TNBC.