TLR4 Signaling Promotes Immune Escape And Apoptosis Resistance Of Tumor Cells And The Underlying Mechanisms

Toll-like receptors(TLRs)are expressed in many kinds of immune cells,including antigen-presenting cells dendritic cells(DC),macrophages and B cells.Also,recent studies show that TLRs can be expressed by tumor cells,and TLR signaling contributes to tumor immune esacpe and promotes tumor metastasis.However,the mechanisms by which TLR-triggered tumor immune esacpe and tumor progression remain to be fully understood.Inspired by the data that the chronic inflammation may induce tumorigenesis of colon cancer,in this study,we investigated the TLR expression in colon cancer cells and then what's the function of TLR signaling in the immune esacape and apoptosis resistance of colon cancer cells.Our findings demonstrate that both human HT29 and mouse CT26 colon cancer cells express high level of TLR4,suggesting TLR4 signaling might contribute to immune escape.Firstly, we screened production of the immune suppressive cytokines by TLR4-ligated colon cancer cells through RT-PCR and ELISA.The data showed that the expression of IL-6, COX-2 and production of IL-6,PGE2 were significantly upregulated by CT26 colon cancer cells stimulated with LPS.It's well known that IL-6 and PGE2 can suppress the function of immune cells and promote tumor cell migration,invasion and survival. Then,we investigated whether Rapamycin,anti-inflammatory regent,affected TLR4-mediated IL-6 and PGE2 production by colon cancer cells.The data of RT-PCR, real-time PCR and ELSIA demonstrated that Rapamycin inhibited IL-6 and COX-2 expression,and IL-6 and PGE2 production by CT26 cells stimulated with LPS.These data indicated that Rapamycin could effectively suppress TLR4-mediated production of immunosuppressive mediators by colon cancer cells.As IL-6 and PGE2 are both the important mediators that regulate cell proliferation, apoptosis,migration and invasion of several cell types,so we wondered whether LPS could promote migration and invasion of CT26 cells,and whether inhibition of LPS-induced IL-6 and PGE2 production by Rapamycin was responsible for the deceased mobility of CT26 cells.We added recombinant mIL-6 or PGE2 in the lower chamber and observed both exogenous IL-6 and PGE2 could reverse the inhibitory effect of Rapamycin on CT26 cell migration and invasion,and simultaneous addition of IL-6 and PGE2 almost totally reversed the inhibitory effect of Rapamycin.These results indicated that LPS-induced IL-6 and PGE2 can promote invasion of colon cancer cells,and Rapamycin may inhibit TLR4-promoted invasion of colon cancer cells by suppressing cancer cell autocrine production of IL-6 and PGE2.Our previous study demonstrated that TLR4 could induce apoptosis resistance of lung cancer cells.We wondered whether Rapamycin could reduce TLR-induced apoptosis resistance of cancer cells.Annixin V/PI analysis demonstrated LPS stimulation could significantly reduce the apoptosis of both human HT29 and murine CT26 colon cancer cells induced by OXL or DXR.In addition,Rapamycin could increase apoptosis of LPS-induced CT26 cancer cells to OXL or DXR treatment,indicating that Rapamycin could reduce TLR4-evoked apoptotic resistance in colon cancer cells, and enhance the sensitivity of cancer cells to anti-tumor drugs,and thus exhibiting anti-tumor effect.Then we explored the mechanism for Rapamycin to inhibite TLR4-triggered IL-6 and PGE2 production and apoptosis resistance of CT26 cells.Firstly,Western blot and FACS analysis demonstrated that Rapamycin could inhibit significantly surface expression of TLR4 in CT26 cells stimulated by LPS without alternation TLR4 mRNA expression,suggesting that TLR4 distribution might be modulated via post-transcriptional regulation such as TLR4 membrane transport or its glycosylation modification.These data suggested that Rapamycin-mediated downregulation of TLR4 expression may contribute to the suppressive effect of Rapamycin on tumor immune escape.Then we investigated which TLR4 signaling pathway involved in the inhibitory effect of Rapamycin on immune escape.Western blot analysis showed that LPS-mediated phosphorylation of p38,JNK,ERK1/2 MAP kinase remained unchanged in the presence of Rapamycin,but the activation of Akt and NF-κB were significantly disrupted by Rapamycin,indicating that Akt and NF-κB inactivation were involved in the process.To confirm the inhibitory effect of Rapamycin,CT26 cells were incubated with the PI3K/Akt(LY294002)and NF-κB specific inhibitor (PDTC)in presence LPS.Similar to Rapamycin,both LY294002 and PDTC significantly inhibited LPS-induced IL-6 and PGE2 secretion,and reverse the apoptosis resistance to OXL and DXR treatment for colon cancer cells.These results demonstrated that inactivation of Akt and NF-κB pathways is involved in the suppressive effect of Rapamycin on the LPS-increased IL-6,PGE2 secretion and apoptosis resistance.As above demonstrated,Rapamycin inhibited Akt and NF-κB activation triggered by LPS.Rapamycin is the potent inhibitor of PI3K/Akt pathway.Up to now,how Rapamycin inhibits LPS-induced NF-κB activity remains to be fully understood. NF-κB activation is regulated by I-κBαphosphorylation and subsequent I-κBαdegradation,allowing NF-κB to translocate into the nucleus.I-κBαphosphorylation is regulated by I-κB kinase(IKKα/β).It is generally accepted that activation of IKKα/βis the regulatory target for Akt,which involved in cellular signaling in response to pro-inflammatory stimuli.We found that Rapamycin suppresses the LPS-induced Akt/IKKα/β/NF-κB cascade pathway,thus blocking TLR-triggered the immune escape and apoptosis resistance of tumor cells.In summary,Rapamycin could suppress the TLR4-induced IL-6,PGE2 secretion and inhibit autocrine IL-6,PGE2-mediated migration and invasion,and reverse TLR4-induced apoptosis resistance of colon cancer cells through downregulation of surface TLR4 expreesion of colon cancer cells and inhibition of Akt/IKKα/β/NF-κB pathways.Our results have provided the new insight into the mechansim for the antitumor effect of Rapamycin,and indicate the potential application of Rapamycin in colon cancer treatment. Surgery,radiotherapy and chemotherapy are all conventional approaches for cancer treatments.However,clinical observations suggest that sometimes radiotherapy and chemotherapy may promote progression of hepatocellular carcinoma(HCC).However, up to now,the underlying mechanisms remain unknown.Therefore,to improve treatment outcomes of HCC,a better understanding of mechanisms that underlie the behavior of HCC and contribute to HCC progression induced by radiotherapy and chemotherapy is necessary.The primary aim of this study is to explore the mechanisms for HCC to escape immune surveillance after radiation and biotherapy,leading to HCC pathogenesis and development.Many studies demonstrate that the presence of proinflammatory cytokines and persistent chronic inflammation in the tumor microenvironment lead to or promote cancer pathogenesis and development.Recently,studies have showed that Toll-like receptor 4 (TLR4)signaling in tumor cells can mediate tumor cell immune escape and tumor progression,companied by immune suppressive cytokine production and apoptosis resistance.High-mobility group box-1(HMGB1)was originally identified as a DNA-binding protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells.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 an proinflammatory 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.So,HMGB1 has also been implicated in disease states,including Alzheimer's,sepsis,ischemia-reperfusion, autoimmune diseases.Recent studies indicate that HMGB1 is associated with the tumor proliferation and metastasis,and HMGB1 has been defined as an endogenous ligand of TLR4.So,we investigated whether HMGB1 released by dying HCC cells is a paracrine factor for HCC development after radiotherapy,chemotherapy or biotherapy.In our study,we found that supernatants from dying HCC cell line SMMC-7721 induced by radiation(30 Gy-co-ray)or TRAIL treatment could significantly induce production of immunpsuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 by SMMC-7721 cells.To determine whether HMGB1 released from dying SMMC-7721 cells could contribute to the effects,silencing of HMGB1 in SMMC-7721 cells was performed.HMGB1 knock-down dying cells have a greatly reduced ability to promote MIP-3αprodcution,which proves that the release of HMGB1 can signal the demise of a cell to its neighbors.To further confirm the role of HMGB1 in dying cell supernatant for the induction of MIP-3α,stimulation of SMMC-7721 cells with recombinant HMGB1 also could dramatically induce the MIP-3αexpression,indicating HMGB1 in the supematant responsible for the induction of immunosuppressive cytokines and chamokines.To clarify whether HMGB1 was released by apoptotic or dying SMMC-7721 cells,Annexin V/PI apoptosis assay of SMMC-7721 ceils with radiation and TRAIL treatment showed that the dying cells rather than apoptotic cells could release HMGB1 responsible for the tumor immune escape,because the supernatant from dying cells could promote MIP-3αexpression in SMMC-7721 but apoptotic cells could not. These data show that HMGB1 in the supernatant from dying HCC cells after radiotherapy or TRAIL treatment is responsible for the induction of the immunosuppressive factors by HCC cells in paracrine manner.Since HMGB1 could mediate the growth and spread of gliomas,so we are wondering whether HMGB1 in the supernatant from dying HCC cells could promote HCC proliferation.We found HMGB1 promoted the growth of SMMC-7721 cells as detected by MTT assay.The evaluation of cell proliferation by BrdU incorporation also confirmed the promotion of the growth of SMMC-7721 cells by HMGB1 stimulation.Then we investigated whether HMGB1 could promote the cell cycles and thus increasing the cell proliferation.Cell cycle analysis by PI staining revealed that recombinant HMGB1 could promote transition of G1 to S phase entry,which might be responsible for the HCC growth.These evidences suggested that the dying cell with radiation or biotherapy contribute to the tumor cell growth by releasing high levels of HMGB1 which can promote cell cycle.Since cyclin A,D E are required for progression through the G1 and S entry,we then examined by Western blot whether cyclin A,D and E were affected by HMGB1 stimulation.We found that cyclin D was dramatically increased in SMMC-7721 cells after HMGB1 stimulation,without alternation of cyclin A and cyclin E.In addition,as p53,p21 and p27 all negatively regulate cell cycle progression by inhibition of the cyclin E/cyclin-dependent kinase activity,we speculated that these negative regulators might play a role in the process of cell cycle entry promoted by HMGB1.Western blot showed that HMGB1 led to significant down-regulation of p27,but not p21 and p53. Because only phosphorylated p27 on T187 can be recognized by ubiquitin protein ligase for consequent ubiquition and degradation,we therefore tested whether p27 phosphorylation was affected by HMGB1 stimulation.Western blot showed that phosphorylation on p27(T187)increased in SMMC-7721 cells in response to HMGB1. Together,the data suggest that decrease of p27 and increase of cyclinD might be responsible for the promotion of cells cycle and consequent HCC cell proliferation and growth by HGMB1 released from dying HCC cells.For the effectiveness of chemotherapy or radiotherapy,one of major obstacles is the resistance of cancer cells to anti-tumor drugs.We speculated that HMGB1 might be involved in the increased anti-apoptosis effect of HCC cells after chemotherapyor radiotherapy.SMMC-7721 cells were pretreated by HMGB1,and then induced by TRAIL.Annexin V/PI analysis revealed that HMGB1 reduced apoptosis induction of HCC cells by TRAIL.To further elucidate the mechanism for the induction of apoptosis resistance by HMGB1,pro-or anti-apoptotic proteins were analyzed by Western blot.We found that upon stimulation of HMGB1,anti-apoptotic protein expression of Bcl-xL,but not Bcl-2,Bax or Bak,was significantly upregulated in SMMC-7721 cells.These data suggest that upregulation of Bcl-xL expression by HMGB1 might be the mechanism to avoid the TRAIL-induced apoptosis and contribute to HCC cell survival.As observed above,recombinant HMGB1 and the HMGB1-containing supernatant from dying HCC cells promote the production of immunosuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 by SMMC-7721 HCC cells.In addition,HMGB1 is identified as one of the endogenous ligands of TLR4.To further confirm that TLR4/MyD88 signaling pathway mediates HMGB1-induced immune escape,we established siRNA TLR4 and siRNA MyD88 of SMMC-7721 cells.RT-PCR showed that, in response to HMGB1 or supernatant from dying HCC cells,TLR4 or MyD88-silenced SMMC-7721 cells have a greatly reduced ability to express MIP-3αmRNA.Together, these results suggest that the increase of MIP-3αexpression induced by recombinant HMGB1 and supernatant from dying cells is mediated through TLR4/MyD88 pathway. In addition,similarly to LPS stimulation,HMGB1 could activate p38 MAPK,ERK1/2, JNK,and NF-κB pathways in SMMC-7721 HCC cells.Therefore,we investigated whether MAPK or NF-κB signal pathways were involved in the cell cycle progression and apoptotic resistance in human HCC cells in response to HMGB1.Western blot assay showed that HMGB1-induced phosphorylation of p27 and subsequent degradation,and cyclin D expression were significantly abrogated by the specific inhibitor of MEK1/2 (PD98059)and ERK1/2(U0126).However,the NF-κB inhibitor PDTC decreased the HMGB1-upregulated Bcl-xL,So,MEK1/2 and ERK1/2 kinase activation is essential for HMGB1-promoted cell cycle progress,and NF-κB activation is required for apoptotic resistance of HCC cells.These data demonstrate that HMGB1/TLR4-mediated MEK1/2/ ERK1/2 and NF-κB activation is responsible for the promotion of HCC cell production of immunosuppressive factors and induction of apoptosis resistance.In conclusion,our findings provide the direct evidence that,during chemotherapy or radiotherapy,HMGB1 released by dying HCC cells could induce production of the immunosuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 expression in HCC cells.HMGB1 promotes the cell cycle transition of G1-S phase by enhancing cyclinD expression,and phosphorylation of p27-T187 and subsequent proteasomal degradation in HCC cells.Furthermore,up-regulation of anti-apoptotic protein Bcl-xL contributes to the recombinant HMGB1-triggered HCC cell resistance of TRAIL-induced apoptosis,indicating that HMGB1 is closely related to tumor immune escape and development.HMGB1 fails to induce MIP-3αexpression in HCC cells with either silence of HMGB1 or TLR4/MyD88,further confirming the HMGB1/TLR4/MyD88-dependent pathway mediates induction of immune escape and progression of HCC after chemotherapy or radiotherapy.Therefore,our findings provide the direct evidence that,during radiotherapy or chemotherapy,the necrotic cells-induced immune escape and apoptosis resistance promotes HCC progression by HMGBI/TLR4/MyD88 dependant pathways,which imply a potential application of HMGB1 neutralization or interfering in combination with chemotherapy in the treatment of hepatocellular carcinoma.