The Mechanisms Of GRP78 Involving In Positive Feedback Between Tumor Cells And Tumor-associated Macrophages

Tumor-associated macrophage(TAM)is an important stroma cell in tumor microenvironment.Due to its characteristics of M2 macrophages,TAM can secrete many cytokines and proteases to create a favorable microenvironment for tumor development.Glucose-regulated protein 78(GRP78)is an important chaperone in endoplasmic reticulum(ER).However,because tumor cells are often in hypoxia,glucose deficiency and acidosis environmental,the expression of GRP78 in tumor cells is often higher than the adjacent normal tissue.The excessive GRP78 can break the binding of ER and translocate into the nucleus,mitochondria,cytoplasmic matrix,cell membrane,and cell secretions.The ubiquitous GRP78 not only regulate the intracellular signal to promote the development of the tumor,but also affect the microenvironment to facilitate tumor progression.TAM is an important factor in tumor microenvironment during tumor development,and GRP78 also promotes tumor development in different ways,thus they have similarities in function;TAM is recruited and re-educated by tumor microenvironment,and often gathers in the hypoxic areas of tumor.Similarly,the high expression of GRP78 locates in the hypoxic site of tumor center;TAM arises in the late stages of tumor development,and the high expression of GRP78 is also induced by the poorly-perfused tumor microenvironment in advanced tumor,thus they have similarities in the time of their occurrence.The series of "coincidences" in the function,position and time,indicate the possible association between TAM and GRP78,and the exploration of relationship and regulatory mechanisms has significance for development of tumor targeting drugs.In this thesis,the relationship between TAM and GRP78 was investigated with human macrophage differentiation and GRP78 secretion models.Furthermore,an effective tumor targeting drug was designed according to the characteristics of GRP78 in tumor cells.The main research contents are described as follows:Part one: M2 macrophages could induce high expression of GRP78 in tumor cells,while the excessive GRP78 played an important role in M2 macrophages-induced tumor migration.The results showed that M2 macrophages could induce the high expression of GRP78 in tumor cells which further promoted the migration of tumor cells.The mechanism studies revealed that CCL17 and CCL22 secreted by M2 macrophages could bind to CCR4 receptor on the surface of tumor cells and further activated PI3K/Akt signal.The p-Akt could inhibit the activity of Ca2+ channel protein IP3 R,leading to Ca2+ accumulation in ER,which resulted in the ATF-6 cleavage and its nucleus entrance,and increased GRP78 expression eventually.Using shRNA knocked down GRP78,the effect on the migration promotion of M2 macrophage was weakened significantly.These results not only suggested that the excessive GRP78 played an important role in M2 macrophages-induced tumor migration,but also changed the consistent cognition that “the harsh microenvironment is the main inducer of GRP78 high level expression in tumor”,revealing a new mechanism for GRP78 high expression in tumor cells.Part two: The high expression of GRP78 in tumor cells activated its own inflammatory response,which in turn promoted the migration of tumor cells.The excessive GRP78 could form protein complexes with STAT3 and JAK2 in tumor cells,causing the interaction between STAT3 and JAK2,and inducing phosphorylation of STAT3.p-STAT3 entered into the nucleus and ultimately led to inflammatory response that represented by IL-1? and TNF-? high level expression.Furthermore,knocking down IL-1? and TNF-? reduced the pro-migration effect of M2 macrophages significantly.This finding not only revealed a new mechanism of how the high level GRP78 expression promotes tumor cells metastasis and deterioration,it also showed that macrophages could induce the tumor cell inflammatory response to maintain the tumor inflammatory microenvironment,and provided a new insight of the dynamic balance between the “immunosuppressive properties of TAM” and “the inflammatory microenvironment of the tumor”.Part three: The high expression of GRP78 enhanced the adhesion between tumor cells and matrix,promoted the epithelial-mesenchymal transition(EMT).The excessive GRP78 could up-regulate the expression and secretion of TGF-?1,which activated the Smad2/3 signal and further promoted the expression of Snail-2.As an important transcription factor,Snail-2 triggered EMT through the regulation of N-cadherin,Vimentin and E-cadherin expression.Moreover,the excessive GRP78 could activate Fibronectin-integrin-?1-FAK signal,and enhance adhesion between tumor cells and the matrix.This finding shed light on the new mechanism of increase of tumor cell migration by GRP78 through the cell structural alteration.Part four: Tumor cells secretory GRP78 could polarize macrophages to M2 type.Treatment with DLD1 cell culture medium or recombinant GRP78 protein in a concentration gradient resulted in a distinct differentiation of RAW264.7 cells.The results of qRT-PCR,western blot and metabolomic tests showed that the expression of M2 macrophage markers(CD206,Arg-1 and IL-10)increased significantly after GRP78 treatment,but the expression of M1 macrophage markers(CD80,iNOS and IL-12)had no obvious changes.In addition,the glycolysis was reduced and the fatty acid metabolism was significantly enhanced after GRP78 treatment.All above-mentioned changes proved that GRP78 could drive RAW264.7 polarization into M2 macrophages.These results indicated that the GRP78 secreted by tumor cell was a vital inducer of M2 macrophages polarization in tumor microenvironment,and also proved that tumor cells could transform the microenvironment through their high level of GRP78 secretion.Part five: GBP-SubA fusion protein could efficiently target GRP78 on the tumor cell surface,resulting destroy the intracellular GRP78 and ultimately inducing tumor cell apoptosis.GBP-SubA fusion protein was designed according to GRP78 dual characteristics of which its specifical existence on the tumor cell surface and its promotion of the tumor development.The fusion protein of GBP-SubA could specifically target tumor cells and destroy the intracellular GRP78,and eventually lead to apoptosis.The results suggested that GBP-SubA not only achieved a dual function of targeting and then killing tumor cells as our desired,but also affected tumor microenvironment to exert anti-tumor effect,which implies its potential to develop drugs for tumor targeting.This study revealed that TAM could promote GRP78 high expression in tumor cells.On the one hand,the excessive GRP78 could improve the migration ability of tumor cells by stimulating inflammatory response,promoting EMT and enhancing the adhesion between tumor cells and matrix.On the other hand,the excessive GRP78 could be secreted into microenvironment to polarize macrophage to M2 type.All of these results implicated that GRP78 played an important role in the positive feedback regulation between tumor cells and TAM.These findings provided new insights for further study of pathophysiology of GRP78 and TAM.Furthermore,results given from this study also lay a scientific foundation for the designation of effective drugs with precision targets of tumor cells.