Fas Signal Promotes Lung Cancer Growth By Recruiting Myeloid-derived Suppressor Cells

Host immune system can surveillance and kill the mutated cells,however,cancer patients,especially the advanced cancer patients,usually exhibit the impaired immunity against cancer.In order to initiate the anti-tumor immune response,several approches for the cancer immunotherapy such as injecting tumor vaccine and adoptive transfer of CTL have been used in clinic trials,however the therapeutic effect of current immunotherapy of cancer is far from that we expected.The poor curative effect may result from immunosuppressive status of cancer patients. Acummulated evidences demonstrate that tumor can consistently release tumor-derived factors(TDFs),which promote the generation of immunosuppressive cells,constituting immunosuppressive environment,thus inhibiting the host immunity against cancer.During this process,the roles of myeloid-derived suppressor cells (MDSC)and regulatory T cells(Treg)in the tumor immune esacpe attach more attraction,because of their essential role for establishing the immunosuppressive tumor microenvironment,inducing tumor escape from host immune defence. MDSC represent a heterogeneous population comprising different myeloid cells at the early-stage of differentiation,including granulocytes,monocytes and a pool of immature cell of myelomonocytic lineage that retain the ability to form colonies on agar.Majority of MDSC inhabit in the bone marrow,and markedly increase during the vaccination,inflammation,transplantation,radiation and tumor progression.They play important roles in the immune tolerance and also in many physiological and pathological process as described above.Once immune response is trigged,MDSC mobilized form bone marrow and accumulate in the lymph organs and tissues where they exert as immune suppressor cells.The tumor associated MDSC were described for the first time in the bone marrow of Lewis lung cancer-bearing mice,and the counterpart of MDSC in human are mostly CD34+,which reported by Pak et al in patients with head and neck cancer.Subsequently,human MDSC have been characterized in the peripheral blood of patients with squamous cell carcinoma, non-small cell lung cancer and breast cancer.After the removal of tumor,MDSC in cancer patient decreased.In tumor-bearing mice,conventional MDSC(cMSC)could further differentiate into a novel subset of MDSC under the tumor microenvironment, which was named by us as the differentiated MDSC(dMSC)with the phenotype of CD11b^lowGr1^low.dMSC highly express MMP-9 and produce IL-6,showing strong potential to promotie tumor metastasis(our unpublished data).Other than promoting tumor metastasis and protecting the tumor from immune attack,tumor MDSC also participate in the tumor angiogenesis by directly differentiating into vascular endothelial cells.Thus,MDSC accumulation in tumor tissue promotes tumor progression,however,which factor(s)is responsible for the MDSC migration and accumulation in tumor tissue remain unclear.In 1980,Berendt et al reported that they found a group of T cells in tumor tissue exerting inhibitory effect on tumor immunity.Due to the limitation of bio-techniques, the features of these cells have not been well identified.Until to 1995,these T suppressor cells were characterized by Sakguchi et al as CD4⁺CD25^highcells,the so-called regulatory T cells(Treg)now.And in search of specific Treg cell markers, transcription factor Foxp3 was identified to be uniquely expressed in Treg cells.In patients with cancer including pancreatic cancer,gastric enterologic cancer,non-small cell lung cancer and ovary cancer,increased Tregs were found in tumor tissue and peripheral blood.Consistent with the change of MDSC,Treg recover to normal level after removing tumor,but rebound when tumor recumce.In a murine fibrosarcoma model,selective accumulation of Treg cells in tumor tissue was studied,where the majority of tumor-infiltrating lymphocytes(TIL)in late-stage progressive tumor were Tregs.Furthermore,local depletion of CD4⁺T cells in tumor led to eradication of well-established tumors and development of long term anti-tumor memory.This study suggested Tregs-mediated immune suppression occurs predominantly at tumor site, local reversaling Treg cells in tumor tissue,even the late stage tumor,can effectly inhibit tumor growth.Increased Treg cells in tumor was a characteristic feature in most tumors,but little is know about the mechanism which is responsible for the elevated Tregs in tumor?Fas belongs to the TNFR super-family,which can recognize and transmit death signal.However,other than transmiting death singal,Fas also mediates inflammatory signal.We and other research group have proved Fas-ligated DC can release amounts of pro-inflammatory cytokines and chemokines,promoting inflammation.In tumors, Fas is widely expressed,and various tumor show diverse response to Fas stimulation. Some cancer cells are sensitive to Fas-mediated apoposis,but majority of them show apoptosis resistance and even grow fast upon Fas ligation.Upon to now,previous reports on Fas-induced tumor growth focused on the direct promoting effect of Fas such as tumor proliferation,motility and invasion.While the influence of Fas signal in modulating host immunity was ignored.The aim of current work is to study the role of Fas signal in lung cancer growth and its influence on the tumor immunity.Firstly,we demonstrated that 3LL Lewis lung cancer cells constitutively expressed certain level of Fas.Upon stimulation of agonistic anti-Fas antibody Jo2, even at high concentration up to 5μg/ml,3LL cells show resistant to Fas-induced apoptosis.In vitro stimulating 3LL cells with Jo2 did not affect the 3LL growth,as detected by MTT methods.In order to investigate the effect of Fas signal on lung cancer growth in vivo,we transfected the wild type Fas(Fas-WT)vector or dominant negative Fas(Fas-DN)which lacking endocellular domain of Fas into 3LL cells,and selected stable 3LL transfectant clones highly expressing Fas-WT(3LL/Fas-WT)or Fas-DN(3LL/Fas-DN)with the pressure of G418.Prior to the in vivo experiments, we tested whether overexpression of Fas on 3LL cells affected the growth rates of these cells in vitro.In vitro growth rates of the Fas-transfectants and parental cells were compared by MTT method.No difference in in vitro growth rate was observed between parental 3LL cells and 3LL Fas-transfectants,demonstrating that the basal proliferation rate of the 3LL cells was not altered by Fas-overexpression.In contrast, when 3LL/Fas-WT cells,3LL/Fas-DN cells or parental 3LL cells,were subcutaneously inoculated into C57BL6/J mice,we observed the accelerated growth of 3LL/Fas-WT and reduced growth of 3LL/Fas-DN compared with parental 3LL cells respectively.Accordingly,the survival percentage of 3LL/Fas-WT-bearing mice cells reduced more significantly than that of mice bearing parental 3LL cells or 3LL/Fas-DN cells.In order to confirm the specific role of Fas signal on promoting in vivo 3LL lung cancer cells growth,we subcutaneously inoculated 3LL/Fas-WT cells, 3LL/Fas-DN cells or parental 3LL cells,into FasL-deficient gld mice,and found there was no significant difference in the tumor growth and mice survive among these groups.Collectively,we can conclude that Fas over-expression in lung cancer cells promotes lung cancer growth in vivo,and which is dependent on the Fas/FasL interaction.Next,we wanted to know what's the mechanisms for the promotion of lung cancer growth in vivo by Fas signal in lung cancer cells.Because accumulation of MDSC and Treg in tumor is involved in the tumor immune escape and tumor progression,we analyzed the accumulation of MDSC and Treg in the tumor tissues 7 days and 14 days after inoculation with 3LL/Fas-WT,3LL/Fas-DN,or parental 3LL into C57BL/6J mice.In C57BL/6J mice model,MDSC appeared to infiltrate tumor 7 days later,and increased 14 days after cancer cell inoculation.Among three groups,more markedly infiltration of MDSC was observed in tumors formed by 3LL/Fas-WT than that tumors formed by parental 3LL cells and 3LL/Fas-DN cells at either 7^thday or 14^th day.No obvious infiltration of Foxp3⁺Treg cells was observed in three groups of tumors 7 days later,but appeared at 14^thday.Among threee groups of tumors the most significant infiltration of Foxp3⁺Treg cells was observed in 3LL/Fas-WT transplanted tumor.Further,we analyzed the percentages of dMSC(a MDSC subset identified in our previous work)and Treg cells in the tumor-infiltrating cells isolated by Percoll gradient centrifugation.We found the more infiltration of dMSC in tumor with Fas-overexpression lung cancer cells.Together with above data,these results demonstrate that Fas signal in lung cancer cells can recruit more MDSC and Foxp3⁺ Treg cells into tumor tissues.Considering that MDSC can induce generation of Treg cells and the Treg accumulation in the Fas-overexpressing tumor was later than MDSC accumulation as observed above,we wondered whether the rapid accumulation of MDSC could induce subsequent accumulation of Treg cells in the Fas-overexpressing tumor.Through in vivo depleting Gr-1+ cells in mice beating 3LL/Fas-WT,we found the ratio of tumor-infiltrating Treg cells decreased significantly,thus confirming our hypothesis.As more accumulation of MDSC and Treg cells was observed in Fas-overexpressing lung cancer,we examined whether Fas-ligated 3LL lung cancer cells can chemoattract more MDSC or Treg cells in vitro.We found that supematant of Fas-ligated 3LL cells was more effective in chemoattracting Gr1⁺CD11b⁺MDSC than control supernatant derived from isotype-treated 3LL lung cancer cells.However, no significant chemoattraction of CD4⁺Foxp3⁺Treg cells was observed.So,Fas ligation can promote 3LL lung cancer cells to chemoattract more MDSC but not Treg cells.Then,we went further to look for which factor(s)derived from Fas-ligated lung cancer cells was responsible for the enhanced chemoattraction of MDSC.As reported, Fas signaling is associated with inflammation by increasing chemokines secretion and chemoattracting inflammatory cells,and VEGF,MCP-1 have been shown to be crucial for MDSC migration.Therefore,we detected the expression of VEGF,MCP-1 in the supernatant of 3LL lung cancer cells stimulated by Jo2,however,no significant secretion of MCP-1 and VEGF was observed.Also,pro-inflammatory mediators produced by cancer cells can recruit and activate inflammatory cells,which further stimulate tumor progression.Thus,we also detected the pro-inflammatory cytokines in the supernatant of Jo2-stimulated 3LL cells,and found that Fas ligation could significantly induce 3LL cells to secrete PGE2.Accordingly,Fas ligation also significantly increased COX2 expression in 3LL lung cancer cells.It was known that COX-2-derived PGE2 can promote migration of cells including tumor cells,DC, endothelial cells.We wondered whether the increased production of PGE2 by Fas-ligated cancer cells was responsible for the increased chemoattraction of MDSC? When COX2 selective inhibitor SC-58125 was used to pre-treat the Fas-ligated 3LL cancer cells,the chemoattracting effect of Fas-ligated 3LL cancer cells on MDSC was abrogated.These data suggest that Fas signal can induce lung cancer cells to secrete more PGE2 which contributes to the enhanced chemoattraction of MDSC.RT-PCR results showed the expression of PGE2 receptors EP2,EP4 in MDSC,supporting the conclusion that PGE2-mediated chemoattraction for MDSC.Finally,we wanted to know which pathway(s)responsible for the increased PGE2 production in Fas-ligated lung cancer cells.Activation of MAPK and NF-κB pathways has been shown to contribute to the Fas-mediated pro-inflammatory factor production and to be associated with tumor growth and invasion.Jo2 stimulation can activate the ERK,p38,NF-κB pathways in 3LL lung cancer cells,stat3 signaling activation in tumor is essential for downregulation of the host immunity.Then we also detected the Star3 signaling pathway upon Fas ligation,and found Jo2 stimulation promoted the nuclear tranlocation of Stat3,indicating Stat3 activation may also be involved in the Fas-mediated lung cancer immune escape.To elucidate which pathway(s)was essential for the increased production of PGE2 by Fas ligation, specific inhibitors for signaling pathways were used to pretreat 3LL lung cancer cells before Jo2 stimulation.We found p38 MAPK specific inhibitor SB203580 could markedly suppress the Jo2-induced PGE2 production,indicating Fas-activated p38 MAPK signaling pathway is responsible for the increased PGE2 production by Fas-ligated lung cancer cells.In summary,we demonstrate that Fas signal can activate the MEK,p38 MAPK and NF-ΚB signaling pathway in lung cancer cells,and p38 MAPK mediates the Fas ligation-induced PGE2 production,which contribute to Fas-induced MDSC and Tregs accumulation in tumor and subsequently promote lung cancer growth in vivo.Our study elucidates the novel immune regulatory role of Fas signal in tumor immune escape,providing new mechanistic explanation for blockade of COX2 in the treatment of tumor growth.