GRP78参与肿瘤细胞和肿瘤相关巨噬细胞之间正反馈调节的机制研究
发布时间：2019-06-01 14:38
【摘要】：肿瘤相关巨噬细胞(TAM)是肿瘤微环境中重要的间质细胞,主要表现M2型巨噬细胞的特征,能够通过分泌多种细胞因子和蛋白酶,为肿瘤细胞创造一个利于其发展的微环境。葡萄糖调节蛋白78(GRP78)是内质网中重要的应激蛋白,由于肿瘤细胞处于缺氧、缺糖以及酸性的微环境,所以常常导致GRP78在肿瘤细胞中的高表达,过量的GRP78能够突破内质网的限制,进入细胞核、线粒体、细胞质基质、细胞膜以及细胞的分泌物中,参与调控胞内信号并影响肿瘤微环境,促进肿瘤的发生发展。TAM是肿瘤微环境中促进肿瘤发展的重要影响因素,高表达的GRP78也从多个方面发挥促肿瘤作用,两者在功能上具有相似之处;TAM受肿瘤微环境的招募和驯化常常聚集于肿瘤的缺氧部位,GRP78的高表达也常发现于肿瘤中心的缺氧区域,两者在定位上有相似之处;TAM出现于肿瘤发展的中后期,而GRP78的高表达也是由肿瘤中后期的恶劣环境所致,两者在出现的时间上具有相似之处;肿瘤相关巨噬细胞与GRP78在功能、空间和时间上的“巧合”,预示着GRP78可能参与了肿瘤细胞与肿瘤相关巨噬细胞之间的相互调控。探明GRP78在其中发挥的作用以及调控机制,对肿瘤靶向药物的开发具有重要的指导意义。本研究通过人巨噬细胞分化模型和GRP78分泌模型深入探究了TAM与GRP78的相互调控作用,并且针对GRP78在肿瘤细胞中的特性设计了靶向药物。具体研究内容如下:第一部分:M2型巨噬细胞能够诱导肿瘤细胞中GRP78高表达,而高表达的GRP78在M2型巨噬细胞的促肿瘤迁移过程中发挥了重要作用。研究发现M2型巨噬细胞在促进肿瘤细胞迁移的同时能够诱导肿瘤细胞中GRP78高表达。进一步的机制研究发现,M2型巨噬细胞分泌的CCL17和CCL22能够与肿瘤细胞表面的CCR4受体结合,进而激活下游的PI3K/Akt信号。p-Akt能够抑制内质网Ca2+通道蛋白IP3R的活性,使Ca2+在内质网中发生聚集,进而促进ATF-6的剪切以及入核,最终导致GRP78表达的升高。如果利用shRNA干扰技术敲低GRP78的表达会明显减弱M2型巨噬细胞的促迁移效应。这部分研究结果,不仅表明高表达的GRP78在M2型巨噬细胞的促肿瘤迁移过程中发挥了重要作用,同时也突破了对“肿瘤的恶劣微环境是诱导GRP78高表达的主要原因”的一贯认知,揭示了GRP78在肿瘤细胞中高表达的新机制。第二部分:肿瘤细胞中高表达的GRP78激活了自身的炎症反应,进而促进了肿瘤细胞的迁移。研究发现,肿瘤细胞中高表达的GRP78能够与STAT3和JAK2形成蛋白复合物,从而拉近STAT3与JAK2的距离,促进STAT3的磷酸化。p-STAT3能够进入细胞核并引起以IL-1β和TNF-α高表达为代表的肿瘤细胞自身炎症反应,而敲低IL-1β和TNF-α的表达也能够明显减弱M2型巨噬细胞的促迁移效应。这一发现不仅揭示了GRP78促进肿瘤细胞转移及恶化的新机制,还表明巨噬细胞可以通过诱发肿瘤细胞自身的炎症反应来维持肿瘤的炎症微环境,为“肿瘤相关巨噬细胞的免疫抑制特性”和“肿瘤的炎症微环境”之间的动态平衡提供了新的解释。第三部分:肿瘤细胞中高表达的GRP78能够增强肿瘤细胞与基质之间的粘附,促进肿瘤细胞的上皮间质转化(EMT)。高表达的GRP78能够促进TGF-β1的表达和分泌,进而激活Smad2/3信号,p-Smad2/3又能够进一步促进Snail-2的表达,而Snail-2作为EMT过程的重要转录因子,调控了N-cadherin、Vimentin和E-cadherin等EMT关键标志物的表达,最终诱发肿瘤细胞EMT。另外,高表达的GRP78还激活了Fibronectin-integrin-β1-FAK信号,增强了肿瘤细胞与基质之间的粘附。这一发现阐明了GRP78通过改变细胞结构进而增强肿瘤细胞迁移能力的新机制。第四部分:肿瘤分泌型GRP78能够诱导巨噬细胞向M2型极化。利用浓度梯度的DLD1细胞培养基或者纯化的GRP78蛋白处理RAW264.7细胞,能够使RAW264.7细胞呈现明显的分化形态。进一步的qRT-PCR、western blot和代谢组学检测发现,经GRP78处理后,RAW264.7细胞中M2型巨噬细胞标志物(CD206、Arg-1和IL-10)的表达明显升高,而M1型巨噬细胞标志物(CD80、iNOS和IL-12)的表达没有明显的变化,同时细胞中的糖酵解代谢减弱,脂肪酸代谢明显增强,表明经GRP78处理后,RAW264.7细胞呈现M2型巨噬细胞特征。这一研究结果首次证明肿瘤细胞分泌型GRP78能够诱导巨噬细胞向M2型极化,通过改造微环境发挥促肿瘤作用。第五部分:针对GRP78构建的GBP-SubA融合蛋白能够靶向结合肿瘤细胞表面的GRP78,进而破坏细胞内的GRP78,最终导致肿瘤细胞凋亡。针对GRP78能够特异存在于肿瘤细胞表面并且能够促进肿瘤发展的双重特性,构建并表达纯化了GBP-SubA融合蛋白。GBP-SubA能够特异靶向肿瘤细胞表面GRP78,并破坏细胞内GRP78,最终导致细胞凋亡。GBP-SubA不但针对GRP78单一分子实现了靶向和杀伤肿瘤细胞的双重作用,而且还能通过影响肿瘤微环境发挥抗肿瘤效应,极具开发潜力。本研究表明,TAM能够促进肿瘤细胞中GRP78的高表达,高表达的GRP78一方面能够通过刺激炎症、促进EMT、增强细胞与基质的粘附,提高肿瘤细胞的迁移能力;另一方面能够分泌到细胞外,促进微环境中巨噬细胞的M2型分化。在肿瘤细胞与TAM形成的正反馈调节中,GRP78发挥了重要作用。这一发现为以GRP78和肿瘤相关巨噬细胞为对象的研究提供新的思路。本研究中针对GRP78双重特性的重组靶向药物的构建和制备,也为后续抗肿瘤靶向药物的开发提供了可能。
[Abstract]:The tumor-related macrophage (TAM) is an important mesenchymal cell in a tumor microenvironment, which is mainly characterized by the characteristics of the M2-type macrophage, and can be used for creating a microenvironment for the development of the tumor cell by secreting a plurality of cytokines and a protease. The glucose regulation protein 78 (GRP78) is an important stress protein in the endoplasmic reticulum, and because the tumor cells are in the microenvironment of hypoxia, lack of sugar and acid, the GRP78 frequently leads to the high expression of the GRP78 in the tumor cells, and the excessive GRP78 can break through the restriction of the endoplasmic reticulum, enter the nucleus and the mitochondria, The cytoplasmic matrix, the cell membrane and the secretion of the cells are involved in regulating the intracellular signal and influencing the microenvironment of the tumor and promoting the development of the tumor. TAM is an important factor to promote the development of the tumor in the micro-environment of the tumor, and the high-expression GRP78 also plays a role in promoting the tumor from a plurality of aspects, The high expression of GRP78 is often found in the hypoxic region of the center of the tumor, and the two are similar in position; TAM is present in the middle and late stage of the development of the tumor, while the high expression of the GRP78 is caused by the severe environment in the middle and later stages of the tumor, and the two are similar in the time of occurrence; The "coincidence" of tumor-related macrophages and GRP78 in function, space and time indicates that GRP78 may be involved in the mutual regulation of tumor cells and tumor-associated macrophages. It is of great significance to explore the role of GRP78 in the development of tumor targeting drugs. In this study, the interaction between TAM and GRP78 was investigated by human macrophage differentiation model and GRP78 secretion model, and targeted drug was designed for the characteristics of GRP78 in tumor cells. The results are as follows: The first part: M2-type macrophage can induce the high expression of GRP78 in tumor cells, while the high-expression GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages. It was found that M2-type macrophages were able to induce high expression of GRP78 in tumor cells while promoting the migration of tumor cells. The further mechanism study found that the CCL17 and the CCL22 secreted by the M2-type macrophages can be combined with the CCR4 receptor on the surface of the tumor cell, thereby activating the downstream PI3K/ Akt signal. P-Akt can inhibit the activity of the endoplasmic reticulum Ca2 + channel protein IP3R, so that the Ca2 + can aggregate in the endoplasmic reticulum, thereby promoting the shearing of the ATF-6 and the entry of the core, and finally, the expression of the GRP78 is increased. The knockdown of the expression of the low GRP78 by using the shRNA interference technique will significantly reduce the pro-migration effect of the M2-type macrophages. The results of this study not only show that the highly expressed GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages, but also breaks through the consistent cognition of the "The harsh microenvironment of the tumor is the main cause of high expression of GRP78.", and reveals the new mechanism of the high expression of the GRP78 in the tumor cells. The second part: The high-expression GRP78 in the tumor cells has activated its own inflammatory response, thus promoting the migration of the tumor cells. It was found that the high expression of GRP78 in tumor cells could form a protein complex with STAT3 and JAK2, so as to close the distance between STAT3 and JAK2 and to promote the phosphorylation of STAT3. P-STAT3 is able to enter the nucleus and cause the autoinflammatory response of the tumor cells represented by the high expression of IL-1 and TNF-1, while the expression of the knockdown of IL-1 and TNF-1 can also significantly reduce the migration-promoting effect of M2-type macrophages. This finding not only reveals the new mechanism of GRP78 to promote the metastasis and deterioration of the tumor cells, but also shows that the macrophage can maintain the inflammatory microenvironment of the tumor by inducing the inflammatory reaction of the tumor cells to provide a new explanation for the dynamic equilibrium between the "Immunosuppression characteristics of tumor-associated macrophages" and the "The inflammatory microenvironment of the tumor". The third part: The high-expression GRP78 in the tumor cells can enhance the adhesion between the tumor cells and the matrix, and promote the epithelial-mesenchymal transition (EMT) of the tumor cells. The high-expression GRP78 can promote the expression and secretion of TGF-CD1, and then activate the Smad2/3 signal, and p-Smad2/3 can further promote the expression of Snail-2, while Snail-2, as an important transcription factor in the EMT process, regulates the expression of EMT key markers such as N-cadherin, Vimentin and E-cadherin, and finally induces the tumor cell EMT. In addition, the highly expressed GRP78 also activates the Fibronectin-integrin-1-FAK signal, enhancing the adhesion between the tumor cells and the matrix. This finding illustrates a new mechanism for GRP78 to enhance the ability to migrate tumor cells by changing the cell structure. The fourth part: The tumor-secreted GRP78 can induce the macrophage to polarize the M2-type. RAW264.7 cells were treated with a DLD1 cell culture medium or purified GRP78 protein with a concentration gradient, and the RAW264.7 cells were able to present a distinct differentiation form. The expression of M2-type macrophage marker (CD206, Arg-1 and IL-10) in RAW264.7 cells was significantly increased after treatment with GRP78 and the expression of M1-type macrophage marker (CD80, iNOS and IL-12) was not significantly changed. In the same time, the metabolism of glycolysis in the cells is weakened, and the metabolism of the fatty acid is obviously enhanced. After the treatment with the GRP78, the cells of the RAW264.7 have the characteristics of the M2-type macrophage. The results of this study first proved that the tumor cell secretory type GRP78 can induce the macrophage to be polarized to the M2 type, and can play the role of promoting the tumor through the transformation of the microenvironment. The fifth part: The GBP-SubA fusion protein, which is constructed for GRP78, can target the GRP78 on the surface of the tumor cell, and then destroy the GRP78 in the cell, and finally lead to the apoptosis of the tumor cells. GBP-SubA fusion protein was constructed and expressed for GRP78 to be specific to the surface of tumor cells and to promote the development of tumor. The GBP-SubA can specifically target the tumor cell surface GRP78 and destroy the intracellular GRP78, and eventually lead to the apoptosis of the cells. The GBP-SubA not only realizes the double action of targeting and killing the tumor cells for the GRP78 single molecule, but also can play an anti-tumor effect by influencing the tumor microenvironment, and has great development potential. The research shows that TAM can promote the high expression of GRP78 in tumor cells, and the high-expression GRP78 can improve the migration ability of the tumor cells by stimulating the inflammation, promoting the adhesion of the cells to the matrix, and improving the migration ability of the tumor cells; and on the other hand, can be secreted outside the cells, And the M2-type differentiation of the macrophages in the microenvironment is promoted. GRP78 plays an important role in the positive feedback regulation of tumor cells and TAM formation. This finding provides a new way of thinking for the study of GRP78 and tumor-related macrophages. The preparation and preparation of the recombinant targeting drug for GRP78 dual-character in the study also provides a possibility for the development of subsequent anti-tumor targeting drugs.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R73-3
，
本文编号：2490345
[Abstract]:The tumor-related macrophage (TAM) is an important mesenchymal cell in a tumor microenvironment, which is mainly characterized by the characteristics of the M2-type macrophage, and can be used for creating a microenvironment for the development of the tumor cell by secreting a plurality of cytokines and a protease. The glucose regulation protein 78 (GRP78) is an important stress protein in the endoplasmic reticulum, and because the tumor cells are in the microenvironment of hypoxia, lack of sugar and acid, the GRP78 frequently leads to the high expression of the GRP78 in the tumor cells, and the excessive GRP78 can break through the restriction of the endoplasmic reticulum, enter the nucleus and the mitochondria, The cytoplasmic matrix, the cell membrane and the secretion of the cells are involved in regulating the intracellular signal and influencing the microenvironment of the tumor and promoting the development of the tumor. TAM is an important factor to promote the development of the tumor in the micro-environment of the tumor, and the high-expression GRP78 also plays a role in promoting the tumor from a plurality of aspects, The high expression of GRP78 is often found in the hypoxic region of the center of the tumor, and the two are similar in position; TAM is present in the middle and late stage of the development of the tumor, while the high expression of the GRP78 is caused by the severe environment in the middle and later stages of the tumor, and the two are similar in the time of occurrence; The "coincidence" of tumor-related macrophages and GRP78 in function, space and time indicates that GRP78 may be involved in the mutual regulation of tumor cells and tumor-associated macrophages. It is of great significance to explore the role of GRP78 in the development of tumor targeting drugs. In this study, the interaction between TAM and GRP78 was investigated by human macrophage differentiation model and GRP78 secretion model, and targeted drug was designed for the characteristics of GRP78 in tumor cells. The results are as follows: The first part: M2-type macrophage can induce the high expression of GRP78 in tumor cells, while the high-expression GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages. It was found that M2-type macrophages were able to induce high expression of GRP78 in tumor cells while promoting the migration of tumor cells. The further mechanism study found that the CCL17 and the CCL22 secreted by the M2-type macrophages can be combined with the CCR4 receptor on the surface of the tumor cell, thereby activating the downstream PI3K/ Akt signal. P-Akt can inhibit the activity of the endoplasmic reticulum Ca2 + channel protein IP3R, so that the Ca2 + can aggregate in the endoplasmic reticulum, thereby promoting the shearing of the ATF-6 and the entry of the core, and finally, the expression of the GRP78 is increased. The knockdown of the expression of the low GRP78 by using the shRNA interference technique will significantly reduce the pro-migration effect of the M2-type macrophages. The results of this study not only show that the highly expressed GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages, but also breaks through the consistent cognition of the "The harsh microenvironment of the tumor is the main cause of high expression of GRP78.", and reveals the new mechanism of the high expression of the GRP78 in the tumor cells. The second part: The high-expression GRP78 in the tumor cells has activated its own inflammatory response, thus promoting the migration of the tumor cells. It was found that the high expression of GRP78 in tumor cells could form a protein complex with STAT3 and JAK2, so as to close the distance between STAT3 and JAK2 and to promote the phosphorylation of STAT3. P-STAT3 is able to enter the nucleus and cause the autoinflammatory response of the tumor cells represented by the high expression of IL-1 and TNF-1, while the expression of the knockdown of IL-1 and TNF-1 can also significantly reduce the migration-promoting effect of M2-type macrophages. This finding not only reveals the new mechanism of GRP78 to promote the metastasis and deterioration of the tumor cells, but also shows that the macrophage can maintain the inflammatory microenvironment of the tumor by inducing the inflammatory reaction of the tumor cells to provide a new explanation for the dynamic equilibrium between the "Immunosuppression characteristics of tumor-associated macrophages" and the "The inflammatory microenvironment of the tumor". The third part: The high-expression GRP78 in the tumor cells can enhance the adhesion between the tumor cells and the matrix, and promote the epithelial-mesenchymal transition (EMT) of the tumor cells. The high-expression GRP78 can promote the expression and secretion of TGF-CD1, and then activate the Smad2/3 signal, and p-Smad2/3 can further promote the expression of Snail-2, while Snail-2, as an important transcription factor in the EMT process, regulates the expression of EMT key markers such as N-cadherin, Vimentin and E-cadherin, and finally induces the tumor cell EMT. In addition, the highly expressed GRP78 also activates the Fibronectin-integrin-1-FAK signal, enhancing the adhesion between the tumor cells and the matrix. This finding illustrates a new mechanism for GRP78 to enhance the ability to migrate tumor cells by changing the cell structure. The fourth part: The tumor-secreted GRP78 can induce the macrophage to polarize the M2-type. RAW264.7 cells were treated with a DLD1 cell culture medium or purified GRP78 protein with a concentration gradient, and the RAW264.7 cells were able to present a distinct differentiation form. The expression of M2-type macrophage marker (CD206, Arg-1 and IL-10) in RAW264.7 cells was significantly increased after treatment with GRP78 and the expression of M1-type macrophage marker (CD80, iNOS and IL-12) was not significantly changed. In the same time, the metabolism of glycolysis in the cells is weakened, and the metabolism of the fatty acid is obviously enhanced. After the treatment with the GRP78, the cells of the RAW264.7 have the characteristics of the M2-type macrophage. The results of this study first proved that the tumor cell secretory type GRP78 can induce the macrophage to be polarized to the M2 type, and can play the role of promoting the tumor through the transformation of the microenvironment. The fifth part: The GBP-SubA fusion protein, which is constructed for GRP78, can target the GRP78 on the surface of the tumor cell, and then destroy the GRP78 in the cell, and finally lead to the apoptosis of the tumor cells. GBP-SubA fusion protein was constructed and expressed for GRP78 to be specific to the surface of tumor cells and to promote the development of tumor. The GBP-SubA can specifically target the tumor cell surface GRP78 and destroy the intracellular GRP78, and eventually lead to the apoptosis of the cells. The GBP-SubA not only realizes the double action of targeting and killing the tumor cells for the GRP78 single molecule, but also can play an anti-tumor effect by influencing the tumor microenvironment, and has great development potential. The research shows that TAM can promote the high expression of GRP78 in tumor cells, and the high-expression GRP78 can improve the migration ability of the tumor cells by stimulating the inflammation, promoting the adhesion of the cells to the matrix, and improving the migration ability of the tumor cells; and on the other hand, can be secreted outside the cells, And the M2-type differentiation of the macrophages in the microenvironment is promoted. GRP78 plays an important role in the positive feedback regulation of tumor cells and TAM formation. This finding provides a new way of thinking for the study of GRP78 and tumor-related macrophages. The preparation and preparation of the recombinant targeting drug for GRP78 dual-character in the study also provides a possibility for the development of subsequent anti-tumor targeting drugs.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R73-3
，
本文编号：2490345
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