The Roles Of HMGB1 In The Colon Cancer Cell Escape Of Immunological Surveillance And The Underlying Mechanisms

Malignant neoplasms have become the main cause of human death, so the effective approaches for the prevention and treatment of cancer need to be identified. More and more recent evindences show that the interaction of tumor and host plays a key role in the tumorigenesis. Tumors invade host but also need the blood supply from host. Altough there are many effector mechsnisms of immune system for the control fo tumor occurrence, tumor cells have developed several methods to escape immunological attack by host. The mechanisms of tumor immune escape to tumor-induced immunosuppression are very complicated, including immunoselection, antigen modulation mask of the tumor surface antigen, the leak out of tumor cells and the immunostimulation of tumor. Fas/FasL counterattack will induce the apoptosis of tumor antigen-specific CTL. Besides above, immunosuppressive cytokines such as IL-10, TGF-β, PGE2, NO and regulatory T cell are also involved in the tumor immune escape.The clinical epidemiological investigations reveal that there is a positive correlation between HMGB1 and the development, invasion and metastasis of the tumor, even the prognosis of cancer patients. Due to the mobility in the SDS-PAGE, HMGB1 protein was designated as its name. There are two domains namely A-box and B-box and negative charged carboxyl terminal. HMGB1 enhances the interaction of other proteins with DNA. Through its L-shaped A-box and B-box, which are each-70 amino acids, it enhances transcriptional activation and in this way, regulates several families of DNA-binding protein.During the immunologic challenge, inflammatory cells and immune cells are activated, resulting in HMGB1 being packed into lysosomes and liberation into the extracellular environment; HMGB1 also can be''passively released''into the extracellular milieu by necrotic and damaged somatic cells Once in the extracellular milieu, HMGB1 can act as a inflammatory cytokine to activate endothelial cells, promoting angiogenesis and extravascular emigration of inflammatory cells and stem cells, thereby initiating inflammation by activation key signaling pathways involved in the regulation of cell differentiation, growth, motility and death. HMGB1 has a closed relationship with the tumorigenesis and tumor growth, metastasis. In colon cancer, HMGB1 protein is excessively expressed in 90 percent of patients. In gastric carcinoma, HMGB1 and RAGE is positive in 85 percent and 65 percent of samples respectively. ,the expression of RAGE is positive. And more importantly ,the expression of RAGE is always gather in invasive tumor. In hepatocarcinoma, there is statistical correlation between HMGB1 with hepatitis, cirrhosis and hepatocarcinoma. HMGB1 has been identify as the endogenous ligand of TLR4.Our previous studies has demonstrated the activation of TLR4 signal will promote immune escape of human lung cancer cells and mouse colon cancer cells. There are different mechanisms in different cells. In lung cancer cells, TGF-β,VEGF,IL-8 is induced and involved in the immune escape; In colon cancer cells, the activation the TLR4 signal will activate the EEK and NF-κB Signaling pathways to promote the secretion of MIP-3α.which can chemoattract the immature dendritic cells.The evidences from clinic remind us that the high level of HMGB1 in cancer patients may have signification in the tumorigenesis. And the HMGB1 is the endogenous ligand of TLR4.Is it possible that the HMGB1 autocrined by tumor promotes the immune escapse via TLR4?To test our hypothesis, we screened lots of cell strain from many kinds of tissues. We need a model that autocrine HMGB1, so the colon cancer cells CT26 was selected because of high production of HMGB1. Considering that HMGB1 will release from death cells, So Trypan blue was used to exclude the possibility that the high production of HMGB1 from CT-26 colon cancer clels was not due to the cell death.To evaluate the effect of HMGB1, we interfered the HMGB1 expression in CT-26 colon cacner cells by siRNA. The silence of HMGB1 was assessed 48h after transfection by immunoblot analysis and ELISA, and we proved that the secretion of HMGB1 decreased 30-40% 48h after transfection.IL-6 and PGE2 are involved in cell proliferation, apoptosis resistance, invasion of cancer cells. So, we tested the effect of HMGB-1 siRNA on the secretion of IL-6 and PGE2. We found the HMGB1siRNA could suppress production of IL-6, but not PGE2..Then, we stimulated CT26 cells by rmHMGB1. Similar to LPS, rmHMGB1 could promote the secretion of IL-6 and PGE2.Our previous study demonstrated that TLR4 could induce apoptosis resistance of lung cancer cells and colon cancer cells. There are several receptors described to bind HMGB1, such as TLR4,TLR2 and RAGE. So, we wonder whether HMGB1 could induce apoptosis colon cancer cells. Annexin V/PI analysis demonstrated that HMGB1siRNA will increase the apoptosis of CT26 colon cancer cells induced by OXL. To confirm the effect of HMGB1, CT26 cells were incubated with rmHMGB1 for 24h, then Annexin V/PI analysis demonstrated that rmHMGB1 could reduce the apoptosis of CT26 cells induced by OXL.It have been proved that HMGB1 released by death cells could promote the proliferation of SMMC-7721 HCC cells. So we wonder whether HMGB1 secreted by CT26 cells could promote CT26 cells proliferation. We found HMGB1siRNA reduced the growth of CT26 cells . The evaluation of cell proliferation by BrdU incorporation also confirmed that HMGB1siRNA could inhibit the growth of CT26 cells. Cell cycle analysis by PI staining revealed that HMGB1siRNA could reduce the proportion of S phase. To confirm the effect of HMGB1 we stimulated CT26 cells by rmHMGB1, then the growth of CT26 cells was detected. We proved that rmHMGB1 could promote the proliferation of CT26 cells by increase the proportion of S phase cells.As described above, we proved that the HMGB1 secreted by CT26 cells could promote the cell proliferaton, the secretion of IL-6 and PGE2, apoptosis resistance of CT-26 cells. We went further investigate the underlying molecular mechanism. Our previous study demonstrated that the promotion of cell growth and apoptosis resistance induced by LPS could been abrogated by PDTC (NF-κB specific inhibitor) or LY294002(Akt/PI3K specific inhibitor) So, we observed the ffects of several kinds of cell signal specific inhibitors on CT26 cells stimulated with rmHMGB1. We found that the secretion of IL-6 and PGE2 induced by rmHMGB1 was partially abrogated by U0126(ERK specific inhibitor) or PDTC (NF-κB specific inhibitor). The mechanisms of apoptosis resistance and cell growth related with HMGB1 remain to be studied. In addition, we once showed that rapamycin could suppress the TLR4-induced IL-6, PGE2 secretion through inhibition of Akt/IKKα/β/NF-κB pathway. So, we observed the effect of rapamycin on the biological effect of HMGB1.We found that the secretion of IL-6 and PGE2 by CT-26 cells induced by rmHMGB1 could be inhibited by rapamycin.In summary,we demonstrated that autocrine HMGB1 could promote growth, apoptosis resistance, immunosuppressive cytokine secretion of colon cancer cells CT26. Our results provide possible mechsnistic explanation for the mechanisms of the immune escape of colon cancer cells, and also outline a new approach for the prevention and treatment of cancers by blocking HMGB1.