Study Of The Effect And Mechanism Of Chaperone-mediated Autophagy On Breast Cancer Angiogenesis

Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide,many factors contribute to initiation and progression of breast cancer.The survival rate has been improved due to the wide application of combination of surgery with radiotherapy and chemotherapy in the clinical treatment,however,the long-term survival rate of breast cancer has not been significantly improved.There are still about 40% patients died due to the recurrence of breast cancer who has received postoperative radiotherapy and chemotherapy.Therefore,it is very important to elucidate the molecular mechanism in the development and progression of breast cancer,in order to find a new target on diagnosis and treatment of breast cancer,improve the prevention and treatment and extend the life of patients.Angiogenesis,the process of new blood vessel sprouting from the existing vasculature,was recognized as a necessary part of wound healing,development of placenta,tissue growth and regeneration as well as cancer progression.This process is induced by low tissue oxygenation and it is a crucial prerequisite for rapid tissue growth,providing proper oxygen supply and removal of toxic metabolites.Suppression of angiogenesis as a way of slowing down tumor progression continues to be one of the most important areas of c ancer research.The angiogenic process is relatively complex and it is regulated by numerous pro-and anti-angiogenic factors.Hypoxia-inducible factor 1-alpha(HIF1A)and abundant lactic acid production by glycolysis can regulate the expression of vascular endothelial growth factor A(VEGFA),promote angiogenesis,and maintain the supplyment of oxygen and nutrient consumption.Cells undergo degradation of intracellular components by lysosomes,also known as autophagy,which is crucial for cellular homeostasis and adaptation to various forms of stress.Of the three different kinds of autophagic pathways,chaperone-mediated autophagy(CMA)is unique for the selective degradation of damaged soluble cytosolic proteins by recognizing peptide sequence motif(KFERQ)via molecular haperone heat shock cognate protein of 70 kDa(HSC70),then the targeted substrate binds to lysosomes through the lysosome-associated membrane protein type 2A(LAMP2A),key protein in the CMA,and undergoes degradation.Recently,there has been growing interest in how CMA impacts cancer cell pathogenesis and progression.Blockage of CMA reduces tumor growth by reducing glycolytic flux and/or the accumulation of glycolytic intermediates in lung cancer cells and melanoma.CMA can also promote lung cancer cell survival through selective stabilization of the pro-survival protein,MCL.CMA sustain rapid proliferation of gastric cancer by constant degradation of RND3.However,the role of CMA activity in tumor angiogenesis of breast cancer remains largely unknown.In this study,lentivirus-mediated shRNA and overexpression,tissue microarray,immunofluorescence,quantitative PCR,protein prokaryotic expression and purification,lysosomal extraction,lysosome phagocytosis,Western Blot and siRNA transfection,tube formation,clony formation,MTT,would healing,Transwell migration,subcutaneous tumorigenesis of nude mice,ELISA,ECAR,drug resistan ce and other in vitro and in vivo models were used,respectively,to study of the effect and mechanism of chaperone-mediated autophagy on breast cancer angiogenesis from the cell and animal levels.Main contents and results:First,CMA promotes breast cancer cell proliferation,metastasis and drug resistance1.On the basis of our formal work,the relationship between CMA and the degree of malignancy,metastasis and prognosis of breast cancer was analyzed by immunohistochemistry with 21 cases of adjacent normal tissues and 166 cases of breast cancer tissue.The results showed that the expression of LAMP2 A,key protein in the CMA pathway,was significantly related with the malignancy and the lymph node tissue of breast cancer.Higher expression of LAMP2 A was corelated with higher pathological grading.Besides,the expression of LAMP2 A is related with the overall survival rate of LAMP2 A,The overall survival rate of patients with low LAMP2 A expression was much longer than that with higher LAMP2 A expression.2.MDA-MB-231 cells of LAMP2 A low expression was successfully constructed by infection of another lentivirus-mediated LAMP2 A shRNA.The proliferation and migration of breast cancer cells were observed by 7-day MTT proliferation curve and Transwell migration assay.The results showed that CMA promoted the proliferation and metastasis of MDA-MB-231 breast cancer cells.3.The mammary epithelial cell line MCF10 A with low LAMP2 A expression was successfully constructed by infection of lentivirus-mediated LAMP2 A sh RNA,and verified by Western Blot.The effect of CMA on the proliferation and migration of MCF10 A was observed by 7-day MTT proliferation curve and Transwell migration assay.We found that CMA had no effect on the proliferation and metastasis of MCF10 A.Furthermore,quantitative PCR assay showed that CMA could not affect the mRNA level of ATG5.4.MTT proliferation assay was used to detect the effect of CMA on chemotherapeutic drug resistance of breast cancer cells.It was found that reducing the expression of LAMP2 A could enhance the killing effect of doxorubicin and paclitaxel on breast cancer cell lines MDA-MB-231 and MCF7.Similarly,LAMP2 A overexpression can enhance the resistance of breast cancer cell lines to paclitaxel and doxorubicin.In addition,the killing effect of doxorubicin and paclitaxel on MDA-MB-231 and MCF7 cells was dose-dependent.Second,the role of CMA in the angiogenesis of breast cancer1.Construction of breast cancer cell lines with different expression levels of LAMP2 A and detection of their CMA activity(1)Breast cancer cell lines MDA-MB-231,MDA-MB-436,MCF-7 and T47 D with LAMP2 A knockdown and overexpressing were successfully constructed by infection of lentivirus-mediated LAMP2 A shRNA and overexpression vector and verified by Western Blot.Quantitative PCR assay were performed to verify the specificity of the selected sh RNA against LAMP2 A.(2)Immunofluorescence method was used to observe the CMA activity of breast cancer cell lines with different expression of LAMP2 A.The results showed less amount of LAMP2A-active lysosomes and an increase in the distance of between LAMP2A-positive lysosomes and the nucleus after downregulation of LAMP2 A in MDA-MB-231,MDA-MB-436,T47 D and MCF7 breast cancer cells,indicating that the CMA activity was downregulated.While LAMP2 A overexpression showed the opposite manner.(3)Ultrasonic crushing and ultra-high speed centrifugation was used to collect the lysosomes,and β-hexosaminidase release assay was used to detect the membrane integrity of lysosomes.The results showed that the membrane integrity of lysosomes was more than 95%.The expression of HSP60,β-Actin and LAMP2 A lysosomal was tested by Western Blot,and the results showed that the extracted lysosomal components are very specific.High purified proteins of GAPDH and HSC70 were obtained by prokaryotic expression of human GAPDH and HSC70 cDNAs and purification.The CMA activity was detected by GAPDH phagocytosis in vitro in breast cancer cells with different LAMP2 A expression.The results showed that MDA-MB-231 cells with low expression of LAMP2 A had significantly reduced the ability of phagocytosis of GAPDH in vitro,while,the ability of phagocytosis of GAPDH in vitro was significantly enhanced in LAMP2 A overexpressing MDA-MB-231 cells.2.The effect of CMA on breast cancer angiogenesis was detected by in vitro assay(1)The effect of breast cancer cell supernatants with different CMA activities on angiogenesis was observed by the tube formation of vascular endothelial cells.The results showed that high expression of LAMP2 A in MDA-MB-231,T47 D,MCF7 and MDA-MB-436 breast cancer cells can significantly enhance the ability of tube formation of vascular endothelial cells,while downregulation of LAMP2 A showed the opposite manner.(2)The effect of breast cancer cell supernatants on the proliferation of vascular endothelial cells was observed by clony formation assay.The results showed that downregulation of LAMP2 A in MDA-MB-231,T47 D,MCF7 and MDA-MB-436 breast cancer cells could significantly inhibit the clonal formation ability of endothelial cells,while upregulation of LAMP2 A in MDA-MB-231,T47 D,MCF7 and MDA-MB-436 cells showed the opposite manner.(3)The effect of breast cancer supernatants on the proliferation of vascular endothelial cells was observed by 7-day MTT growth curve.The results showed that downregulation of LAMP2 A in MDA-MB-231,T47 D,MCF7 and MDA-MB-436 breast cancer cells could significantly inhibit the clonal formation ability of endothelial cells,while upregulati on of LAMP2 A in MDA-MB-231,T47 D,MCF7 and MDA-MB-436 cells showed the opposite manner.(4)The effect of breast cancer supernatants on the migration ability of vascular endothelial cells was detected by Transwell migration assay.The results showed that downregulation of LAMP2 A in MDA-MB-231 and MCF7 cells could significantly inhibit the migration ability of endothelial cells,while upregulation of LAMP2 A in MDA-MB-231 and MCF7 cells showed the opposite manner.(5)The effect of breast cancer supernatants on the migration ability of vascular endothelial cells was observed by would healing test.The results showed that the wound healing rate of endothelial cells was significantly decreased after LAMP2 A was silenced in T47 D and MDA-MB-436 cells,and the wound healing rate of endothelial cells was significantly accelerated after overexpression of LAMP2 A in T47 D and MDA-MB-436 cells.3.The effect of CMA on breast cancer angiogenesis was detected by in vivo assayThe expression level of LAMP2 A in the subcutaneous tumor tissue was detected by Western Blot,and the results showed that LAMP2 A was still low in tumor tissue.The expression of CD31 and CD34 was detected by immunohistochemistry in MDA-MB-231 subcutaneous tumor tissue.The results showed that CD31 and CD34 were significantly decreased after knockdown of LAMP2 A,indicating that CMA could promote angiogenesis.Third,effect of CMA on pro-angiogenesis factors1.The expression of VEGFA in breast cancer cells was detected by quantitative PCR.The results showed that the expression of VEGFA was significantly decreased after LAMPA knockdown in MCF7 and MDA-MB-231 cells,while VEGFA level showed a significant increase after LAMP2 A was overexpressed in MCF7 and MDA-MB-231 cells.2.The expression of VEGFA levels in MDA-MB-436 and MCF7 cells were determined by Western blot.The results showed that the expression of VEGFA was significantly decreased after LAMP2 A knockdown in MDA-MB-436 and MCF7 cells,while the expression of VEGFA was significantly enhanced after LAMP2 A overexpression in MDA-MB-436 and MCF7 cells.3.The expression of VEGFA in the supernatant of MDA-MB-231 cells was detected by ELISA,and the results showed that the expression of VEGFA was significantly decreased after LAMP2 A knockdown in MDA-MB-231 cells,while the expression of VEGFA was significantly enhanced after LAMP2 A overexpression in MDA-MB-231 cells.4.The expression of VEGFA in subcutaneous tumor tissue of nude mice was detected by Western Blot.The results showed that the expression of VEGFA was significantly decreased after LAMP2 A knockdown in subcutaneous tumor tissue of MDA-MB-436 cells,while the expression of VEGFA was significantly enhanced after LAMP2 A overexpression in subcutaneous tumor tissue of MDA-MB-436 cells.Four,CMA could promote the HK2 expression level and enhance glycolysis1.Effects of CMA on glycolysis and its key enzymes(1)The expression of lactic acid in the supernatant of MDA-MB-231 was detected by lactate kit.The results showed that the expression level of lactic acid in MDA-MB-231 cells was significantly decreased after LAMP2 A expression The level of lactic acid in the lactate detecting kit in MDA-MB-231 cells.The results showed that the lactate production was significantly decreased after LAMP2 A knockdown in MDA-MB-231 cells,while the lactate production was significantly enhanced after LAMP2 A overexpression in MDA-MB-231 cells.(2)Ecidification rate of MDA-MB-231 cells was detected by extracellular acidification rate assay.The results showed that baseline glucose metabolism was significantly decreased after LAMP2 A knockdown in MDA-MB-231 cells,while baseline glucose metabolism was significantly enhanced after LAMP2 A overexpression in MDA-MB-231 cells.(3)The expression levels of HK2 m RNA and protein in MDA-MB-231 cells was detected by quantitative PCR and Western Blot.The expression level of HK2 in MDA-MB-231 cells was significantly decreased after LAMP2 A expression,while the expression level of HK2 in MDA-MB-231 cells was significantly increased after LAMP2 A overexpression.2.HK2 knockdown can inhibit angiogenesis(1)HK2 siRNA transfection and quantitative PCR assay were used to verify the effect of HK2 knockdown,and the results showed that HK2 si RNA can successfully knock down HK2 m RNA level in MDA-MB-231 cells.(2)The lactate kit was used to detect the extracellular lactate level after HK2 knockdown,and it was found that downregulation of HK2 could successfully reduce the expression level of extracellular lactic acid.(3)The effect of HK2 knockdown on angiogenesis was detected by tube formation assay.It was found that knocking down HK2 could significantly reduce the ehancing effec t of CMA to angiogenesis.(4)The effect of HK2 knockdown on the proliferation and migration of vascular endothelial cells was detected by MTT proliferation assay and Transwell migration assay.It was found that HK2 knockdown could significantly reduce the ehancing effect of CMA to the proliferation and migration of vascular endothelial cells.3.Effects of reduced glycolysis on proliferation and migration of breast cancer cells with different expression levels of LAMP2A(1)The effect of damaged glycolysis on the proliferation of breast cancer cells was detected by MTT proliferation assay.The results showed that the promoting effect of CMA on the proliferation was significantly reduced after glycolysis was inhibited in MDA-MB-231 breast cancer cells.(2)The effect of damaged glycolysis on the migration ability of breast cancer cells was detected by Transwell migration assay.The results showed that the promoting effect of CMA on the migration of MDA-MB-231 cells was decreased after the glycolysis was reduced.Conclusion: CMA could promote breast cancer angiogenesis.The underlying mechanism is related to VEGFA signaling pathway regulated by HK2-mediated glycolysis.And we also found that inhibiting glycolysis can reduce the promoting effect of CMA in the proliferation and metastasis of breast cancer cells.This study provides a new idea for the study of breast cancer angiogenesis and may help us find new methods to the diagnosis and treatment of breast cancer.