The Mechanisms Of Tumor-secreted GRP78 Recruits Macrophages And The Intervention Effect Of Berberine

Glucose regulated protein 78(GRP78),a conserved molecular chaperone protein in the endoplasmic reticulum(ER),usually facilitates proper folding and assembly of proteins in normal cells.Moreover,it serves as a calmodulin to bind calcium and regulates calcium homeostasis.It has shown that the tumor microenvironment characterized by glucose deprivation,acidosis and hypoxia drives expression of GRP78.Increased intracellular GRP78 may escape from ER to the mitochondria,nucleus,cytoplasm and cell membrane surface to promote tumor development.More importantly,GRP78 can be secreted extracellularly,which accelerates tumor progression by transforming microenvironment.Tumor-associated macrophages(TAMs)are the most abundant types of immune cells in the tumor microenvironment,which provide favorable conditions for tumor growth and survival by secreting cytokines and chemokines;Secreted GRP78 transforms microenvironment by regulating stromal cells and further to promote tumor progression,indicating they have similar function of promoting tumor development.TAMs are often gathered in the hypoxic areas of tumor tissues by the recruitment of various chemokines and cytokines;GRP78 also over-expressed under the stimulation of microenvironment such as hypoxia,indicating they have similar position.The aggregation of TAMs is more tend to occur in the advanced stage of tumor;GRP78 secretion is closely correlated with malignant degree of tumors,indicating they have similar time of emergence.Based on the similarity of TAMs and GRP78 in the function,stage of tumor development and location in the tumor,we speculate that GRP78 plays an important role in the recruitment of TAMs into tumor tissues.This study aims to explore the molecular mechanism of secreted GRP78 recruiting macrophages,and discovers the inhibitor that block the secretion of GRP78.These findings notonly uncover the new function of GRP78 as a chemokine,and also provide new ideas for targeted therapy of tumor microenvironment.The detailed research contents are as follows:(1)Tumor-secreted GRP78 had the function of recruiting macrophages.Co-culture models of secreted GRP78 and macrophages were established in vitro by using tumor-conditioned medium with different malignant degrees and prokaryotic expression and purified His-GRP78,and the results showed that secreted GRP78 promoted the migration of macrophages.Furthermore,the results of “recruitment model in vitro" showed that macrophages were more likely to migrate to tumor cells with higher GRP78 secretion,indicating macrophages have the tendency for secreted GRP78.Importantly,more infiltrating macrophages were detected in tumor tissues using DLD1 cells with over-expressed GRP78.The results of GRP78-containing matrigel injection into the skin of mice also revealed that macrophage was easier to infiltrate into GRP78-abundant regions.Mechanistically,secreted GRP78 activated Fibronectin-integrin-?1-FAK and MMP2/9 signaling,which promoted the adhesion role of macrophages and matrix and degradation role of extracellular matrix,respectively.(2)Secreted GRP78 was able to enter into macrophages relied on different endocytosis pathways and localized to ER and mitochondria.The results of FITC-/Bition-labeled His-GRP78 and His antibody staining showed that secreted GRP78 transported into macrophages and lasting 6 h.When the synthesis of ATP was inhibited or membrane traffic events were shut down,the blockage of secreted GRP78 entrance was observed,indicating secreted GRP78 internalization was endocytosis-dependent.Further studies showed that phagocytosis and clathrin,caveolin-1 and micropinocytosis-mediated endocytosis pathways contributed to internalization of secreted GRP78.The internalized GRP78 entered into mitochondrion and ER.(3)The internalized GRP78 bound to intracellular Ca2+,which caused cytoskeletal remodeling and promoted directional migration of macrophages.The concentration gradient of GRP78 formed a concentration difference at the front and the rear of macrophage migration,resulting in more GRP78 entering in front protrusion.The internalized GRP78 bound to intracellular Ca2+,causing uneven distribution of Ca2+ and subsequent polarization of macrophages.The polarization of macrophages up-regulated mi R-200b-3p,which activated the effector proteins Rho A,Rac1 and Cdc42 and ultimately led to cytoskeleton remodeling,further guiding macrophages migration into GRP78-expressing tumor cells.(4)Berberine improved the tumor microenvironment by inhibiting GRP78 secretion.Lower concentrationa of berberine(10 ?M)inhibited the secretion of GRP78 in tumor cells.Molecular docking,fluorescence titration and competitive binding experiments showed that berberine interacted with GRP78 directly at the ATP-binding site of protein.Berberine regulated its function by competing with ATP binding,resulting in the inhibition of ATPase activity.Berberine binding caused the conversion of GRP78 from its active monomer to the inactive dimer forms,which led to GRP78 cannot be sorted into exosomes and secreted extracellularly.(5)Berberine induced autophagic death by elevating GRP78 levels in cancer cells.Higher concentrations of berberine promoted autophagic death in tumor cells.Mechanistically,berberine enhanced GRP78 level by up-regulation of ATF6 and suppression of ubiquitination/proteasomal degration of GRP78.Overexpressed GRP78 activated excessive autophagy by interacting with more intracellular VPS34,which ultimately led to tumor cells death.In summary,secreted GRP78 entered into macrophages by different endocytosis,and combined with intracellular Ca2+ to promote the polarity of macrophages and remodeling of cytoskeleton,and finally the macrophages migrated into tumor.Berberine improved the tumor microenvironment by inhibiting the secretion of GRP78 in low doses.The high concentrations of berberine caused excessive accumulation of GRP78,which induced tumor cell death by activating autophagy program.This study revealed the new function of secreted GRP78 as a chemokine.Meanwhile,based on the therapeutic target in the microenvironment,the specific inhibitor of secreted GRP78-berberine was discovered.These findings not only establish the foundation for the tumor clinical application of berberine,but also provide a new idea for the development of tumor-targeted drug based on microenvironment.