TLR4 Signaling Induces Tumor Cell MIP-3alpha Production Promoting Tumor Immune Escape

Chronic infection and inflammation have been regarded as one of main courses of cancer pathogenesis and development. Many studies demonstrate that the presence of proinflammatory cytokines and persistent chronic inflammation in the tumor microenvironment may lead to or promote cancer pathogenesis and development. For example, it has been shown that chronic bacterial infection may drive inflammation to cancer pathogenesis. However, the underlining mechanisms by which chronic inflammation leads to cancer pathogenesis remain to be fully understood. So, the mechanistic study about the relation between chronic inflammation and cancer pathogenesis is one of hot topics in the field of tumor biology and tumor immunology. Recently, the studies about the roles of Toll-like receptors (TLRs) expressed by tumor cells in tumor development and immune escape attract much attention.TLRs play critical roles in the innate recognition of pathogen-associated molecule patterns (PAMPs) and in triggering acquired immunity. Innate immune cells recognize PAMPs conserved in microbes by TLRs to initiate host immune response against infectious pathogens. The family of TLRs senses conserved structure found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. Up to now, more than 10 human TLRs have been discovered, TLRs are conserved between human and mouse. TLRs are thought to be restricted to immune cells, such as dendritic cells (DC), macrophages, T and B lymphocytes etc, which initiate innate and adaptive immune responses through producing cytokines, increasing expression of cell adhesion molecules, and secreting proinflammatory mediators. While optimal production of the proinflammatory cytokines is necessary for eliminating invading pathogens, uncontrolled TLR activation might result in the immunopathological injury to host. One of the most severe conditions is LPS-induced endotoxin shock, in which lethal amount of proinflammatory cytokines are produced and damages of tissues inducedupon LPS challenge. Another issue is the pathogenesis of autoimmune diseases. So, the control of TLR signaling is very important for the immune system.In the past years, the studies of TLRs focused on TLR expression on immune cells and the effects of TLR signaling on the immune response and inflammation. As we know, MAPKs and NF-κB pathways are all involved in tumor survival and cell apoptosis, and such pathways are triggered by TLRs. So, in recent years, some researchers investigated the expression of TLRs on tumor cells and proposed the concept that TLR signaling in tumor cells may promote immune escape of tumor cells. Therefore, the primary aim of this study is to investigate the expression of TLRs on cancer cells and to demonstrate what's the role of TLR4 signaling in the tumor cell immune escape.Almost every cancer cell that has been examined secretes chemokines, strongly suggesting that they may contribute to tumor growth or progression. In part, this may be a consequence of its ability to attract mononuclear cells to cancer sites, where they provide growth or angiogenic factors that promote cancer development. Accumulating evidence also points to a direct effect of chemokines on the migration and outgrowth of cancer cells which express chemokine receptors. In particular, some chemokines can activate anti-apoptotic pathways in these cells. By either mechanism, tumor cells that secrete and/or respond to chemokines would have a selective advantage to grow or metastasis. Macrophage inflammatory protein (MIP)-3a/CCL20 is a CC chemokine which is constitutively expressed by epithelial cells from many different tissues. CC-chemokine receptor (CCR) 6 is the only known receptor for MIP-3a, a CC chemokine chemotactic for lymphocytes and DC. Many solid tumors infiltrated by immature DC have been observed to express chemokines, such as MIP-3a, which can chemoattract immature DC, and as a consequence, immature DC can promote tumor immune escape.Our findings demonstrate that many kinds of tumor cells, including mouse colon cancer cell line CT26, mouse lung cancer cell line 3LL, melanoma cell lines B16 and B16F10, fibrosarcoma cell line L929 and mouse breast cancer cell line 4T1, express high level of TLR4 mRNA. Consistent with the RT-PCR result, Western blottingrevealed that TLR4 protein was also expressed on the surface of all of mouse cancer cells we analyzed. Then, we analyzed whether activation of TLR4 signaling in tumor cells by lipopolysaccharide can induce the synthesis and secretion of chemokines. RT-PCR results showed that four tumor cell lines expressed constructively MCP-1. RNATES is expressed constructively by B16 and CT26 cells. Stimulation of LPS enhanced expression of RNATES as well as IP-10, VEGF in 4T1 cells. The expressing of MIP-3a was induced by LPS in 3LL, B16 and CT26 cells. Then, we selected CT26 colon cancer cells which express TLR4 as tumor cellular model to investigate the role of TLR4 signaling and the function of TLR4 signaling-induced MIP-3a in the tumor cell immune escape. We analyzed the expression of MIP-3a in CT26 cells stimulated with or without LPS. As clearly demonstrated in RT-PCR and ELISA results, high level of MIP-3a expression could be induced by LPS in CT26 cells, although MIP-3a expression was very low in CT26 cells without LPS stimulation.Many cellular stress stimuli such as LPS can activate both NF-κB and MAPK pathways. So, we examined the activation of the TLR4 signal pathway by analyzing the phosphorylation of downstream signaling molecules such as MAPK or NF-κB in CT26 colon cancer cells after LPS stimulation. We found that LPS stimulation significantly induced activation of MAPK and NF-κB pathways in CT26 colon cancer cells. However, MAPKs, including ERK, JNK, and p38, play different roles in MIP-3a production. We pretreated CT26 cells with P38 inhibitor SB203580, ERK inhibitor U0126, JNK inhibitor SP600125, NF-κB inhibitor PDTC respectively, and then stimulated CT26 with LPS. We found that pretreatment with ERK inhibitor U0126 or NF-κB inhibitor PDTC significantly blocked MIP-3a production in CT26 after LPS stimulation, suggesting that TLR4-activated ERK and NF-kB pathways in CT26 cells might contribute to the increased production of MIP-3a. Interestingly, JNK inhibitor SP600125 increased LPS-induced MIP-3a production in CT26. To further study the role of JNK activation in the regulation of MIP-3a production, CT26 cells were stimulated with LPS in the presence of JNK inhibitor SP600125. We found that SP600125 pretreatment also enhanced LPS-induced ERK activation in CT26 cells,suggesting that SP600125 might affect MIP-3a production through reversing the activation of the ERK pathway in CT26 cells, thus providing additional evidence that the ERK pathway is involved in the MIP-3a production in CT26 colon cancer cells.It is well established that chemokines are involved in the promotion of cancer migration and metastasis. Several specific chemokines have been shown to be able to promote tumor cell growth. Thus we tested the proliferation of CT26 cells after LPS stimulation for 24h, 48h or 72h, however, we found LPS could not affect CT26 cell proliferation. It has been shown that MIP-3a not only recruits inflammatory cell migration, but also directs cancer cell migration and invasion. DC have been demonstrated to express CCR6, the receptor of MIP-3a, and DC could be chemoattracted by MIP-3a. So, we wonder whether TLR4-activated tumor cells would chemoattract more immature DC through the increased production of MIP-3a. By using a Boyden chamber invasion assay, we found that TLR4-activated tumor cells could chemoattract more immature DC through the increased production of MIP-3a induced by LPS, because neutralizing antibody against MIP-3a could block the chemoattraction of immature DC by the tumor cells.In conclusion, our findings indicate that TLR4 is expressed by mouse colon cancer cells. TLR4 signaling induces CT26 colon cancer cells to secret higher level of MIP-3a. LPS-induced activation of ERK and NF-kB pathways are involved in the increased production of MIP-3a by CT26 cells. Then tumor cells-derived MIP-3a could chemoattract more immature DC surrrounding the tumor cells, which may contribute the tumor immune escape. Therefore, our results demonstrate one important mechanism for the role of tumor TLR4 signaling in the tumor immune escape by showing that tumor cells can express functional TLR4, and TLR4 signaling can induce tumor cells to secrete chemokines, such as MIP-3a, which can chemoattract immature DC into tumor tissue promoting tumor cell escape of immunological surveillance.