Decitabine and vorinostat cooperate to sensitize colon carcinoma cells to Fas ligand-induced apoptosis in vitro and tumor suppression in vivo

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.     

Activated T cell exosomes promote tumor invasion via Fas signaling pathway

Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.     

Cutting edge: a novel role for Fas ligand in facilitating antigen acquisition by dendritic cells

Fas ligand (FasL)-expressing tumor cells are found to effectively mediate rejection of the coinoculated FasL negative parental cells while having no effect on the growth of histologically distinct tumor cells. These observations indicate that FasL induces a specific immune response against Ag derived from FasL-bearing tumors and suggest a possible role for FasL in tumor Ag presentation. Indeed, tumor cells expressing FasL can efficiently interact with dendritic cells (DCs) and this interaction requires the expression of membrane-bound FasL on tumors and Fas on DCs. Moreover, DCs cocultured with FasL-expressing tumors are able to elicit a tumor-specific immune response in vivo, suggesting that DCs acquire tumor Ag during the Fas/FasL-mediated DC-tumor contact. These results identify a novel role for FasL in augmenting tumor-DC interactions and subsequent tumor Ag acquisition by DCs, and suggest that FasL-expressing tumor cells could be used to generate tumor-specific DC vaccines.     

Fas-mediated suicide of tumor-reactive T cells following activation by specific tumor: selective rescue by caspase inhibition

CD8+ T lymphocytes that specifically recognize tumor cells can be isolated and expanded ex vivo. While the lytic properties of these cells have been well described, their fate upon encounter with cognate tumor is not known. We performed reverse 51Cr release assays in which the lymphocyte effectors rather than the tumor cell targets were radioactively labeled. We found that melanoma tumor cells caused the apoptotic death of tumor-specific T cells only upon specific MHC class I-restricted recognition. This death was entirely blockable by the addition of an Ab directed against the Fas death receptor (APO-1, CD95). Contrary to the prevailing view that tumor cells cause the death of anti-tumor T cells by expressing Fas ligand (FasL), our data suggested that FasL was instead expressed by T lymphocytes upon activation. While the tumor cells did not express FasL by any measure (including RT-PCR), functional FasL (as well as FasL mRNA) was consistently found on activated anti-tumor T cells. We could successfully block the activation-induced cell death with z-VAD-fmk, a tripeptide inhibitor of IL-1 beta-converting enzyme homologues, or with anti-Fas mAbs. Most importantly, these interventions did not inhibit T cell recognition as measured by IFN-gamma release, nor did they adversely affect the specific lysis of tumor cell targets. These results imply that Fas-mediated activation-induced cell death could be a limiting factor in the in vivo efficacy of adoptive transfer of class I-restricted CD8+ T cells and provide a means of potentially enhancing their growth in vitro as well as their function in vivo.     

T cell–tumor cell: a fatal interaction?

Fas (Apo-1/CD95) is a cell-surface protein that is responsible for initiating a cascade of proteases (caspases) culminating in apoptotic cell death in a variety of cell types. The function of the Fas/FasL system in the dampening of immune responses to infectious agents through the autocrine deletion of activated T cells has been well documented. More recently, it has been proposed that tumor cells express FasL, presumably to avoid immune detection. In this review, we focus on the role of the interaction of Fas and FasL in the modulation of antitumor responses. We critically examine the evidence that FasL is expressed by tumor cells and explore alternative explanations for the observed phenomena in vitro and in vivo. By reviewing data that we have generated in our laboratory as well as reports from the literature, we will argue that the Fas/FasL system is a generalized mechanism used in an autocrine fashion to regulate cell survival and expansion in response to environmental and cellular cues. We propose that FasL expression by tumor cells, when present, is indicative of a perturbed balance in the control of proliferation while “immune privilege” is established by “suicide” of activated antitumor T cells, a form of activation-induced cell death. Received: 5 May 1998 / Accepted: 20 May 1998     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.     

High levels of Fas ligand and MHC class II in the absence of CD80 or CD86 expression and a decreased CD4<Superscript>+</Superscript> T cell Infiltration,enables murine skin tumours to progress

It is still not clear why some tumours will be recognized and destroyed by the immune system, and others will persist, grow, and eventually kill the host. It has been hypothesized that tumour cells might evade immunological destruction by expressing Fas ligand (FasL), a molecule which induces apoptosis in Fas(+) target cells. However, the role of FasL in creating an immune privileged status within a tumour remains controversial. To determine whether FasL is associated with skin tumour progression, we developed a tumour model enabling us to compare two squamous cell carcinomas (SCC). One is a regressor SCC which spontaneously regresses after injection into syngeneic mice. The other is a progressor SCC which evades immunological destruction. Detailed flow cytometric analysis was used to study tumour cell expression of FasL, Fas, CD80, CD86 and MHC class II. We also analysed the percentage of apoptotic tumour cells in vivo using annexin V and correlated skin tumour progression with CD4 and CD8 T cell infiltration. Progressor tumours expressed high levels of FasL in vivo, which was virtually absent from regressor tumours. The percentage of progressor tumours expressing MHC II was significantly greater than regressor tumours, while neither tumour expressed CD80 or CD86 costimulatory molecules. Consistent with a regressor phenotype, the percentage of viable tumour cells was significantly lower for regressor compared to progressor tumours which coincided with a significantly larger CD4(+) T cell infiltrate into the tumour mass. The results suggest that progression of skin tumours occurs if tumour cells express high levels of MHC II but not costimulatory molecules such as CD80 or CD86. This implies that tumours may induce anergy in CD4(+) T cells via MHC II antigen presentation in the absence of costimulation. To ensure escape from the immune system, tumours may then kill these T cells via a FasL-dependent mechanism.     

切割Fas mRNA核酶的构建及其体内外切割活性的鉴定

白血病细胞表面可高表达FasL ,诱导表达Fas的T细胞凋亡 ,从而降低其抗白血病的作用 .以高表达Fas的小鼠淋巴瘤细胞系Yac 1作为模型 ,设计并合成了针对FasmRNA 5 96位点的锤头状核酶基因 ,用T7/SP6体外转录系统检测了该核酶对FasmRNA的体外切割效率 ,通过电穿孔转染法将其导入Yac 1细胞 ,通过RT PCR、Western印迹法检测细胞上Fas的表达 .细胞经抗Fas的抗体作用后 ,通过MTT法测细胞的增殖 ,annexin Ⅴ凋亡检测试剂盒测细胞凋亡 .该核酶体外切割活性达6 0 % ,且能有效降低细胞表面Fas的表达 ;细胞经抗Fas的抗体作用后 ,转染核酶的细胞增殖活性较对照增高 ,而凋亡率明显降低 .结果表明 ,构建的切割FasmRNA核酶在体内外均具备良好的切割FasmRNA的活性 ,使细胞免于Fas途径的凋亡 ,为研究抑制T细胞的凋亡从而增强其抗白血病效应提供实验基础 .     

Topotecan enhances immune clearance of gliomas

Despite aggressive surgery, radiation therapy, and chemotherapy, glioblastoma multiforme (GBM) is refractory to therapy, recurs quickly, and results in a median survival time of only 14 months. The modulation of the apoptotic receptor Fas with cytotoxic agents could potentiate the response to therapy. However, Fas ligand (FasL) is not expressed in the brain and therefore this Fas-inducing cell death mechanism cannot be utilized. Vaccination of patients with gliomas has shown promising responses. In animal studies, brain tumors of vaccinated mice were infiltrated with activated T cells. Since activated immune cells express FasL, we hypothesized that combination of immunotherapy with chemotherapy can activate Fas signaling, which could be responsible for a synergistic or additive effect of the combination. When we treated the human glioma cell line U-87 and GBM tumor cells isolated from patients with TPT, Fas was up regulated. Subsequent administration of soluble Fas ligand (sFasL) to treated cells significantly increased their cell death indicating that these Fas receptors were functional. Similar effect was observed when CD3⁺ T cells were used as a source of the FasL, indicating that the up regulated Fas expression on glioma cells increases their susceptibility to cytotoxic T cell killing. This additive effect was not observed when glioma cells were pre-treated with temozolomide, which was unable to increase Fas expression in tumor. Inhibition of FasL activity with the antagonistic antibody Nok-1 mitigated these effects confirming that these responses were specifically mediated by the Fas-FasL interaction. Furthermore, the CD3⁺ T cells co-cultured with topotecan treated U-87 and autologous GBM tumor cells showed a significant increase in expression in IFN-γ, a key cytokine produced by activated T cells, and accordingly enhanced tumor cytotoxicity. Based on our data we conclude that drugs, such as topotecan, which cause up regulation of Fas on glioma cells can be potentially exploited with immunotherapy to enhance immune clearance of tumors via Fas signaling. Jun Wei and Guillermo DeAngulo are Co-lead authors.     

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.     

Cutting edge: the tumor counterattack hypothesis revisited: colon cancer cells do not induce T cell apoptosis via the Fas (CD95, APO-1) pathway

The counterattack hypothesis, suggesting that cancer cells express Fas ligand (FasL) and are able to kill Fas-expressing tumor-infiltrating activated T cells, was supported by reports of the killing of Jurkat cells by FasL-expressing human colon cancer cell lines. Through the use of an improved cytotoxic assay in which soluble FasL and FasL-transfected KFL9 cells were used as positive controls, we show that none of seven human colon cancer cell lines induce apoptosis of two Fas-expressing target cell lines, Jurkat and L1210-Fas cells. Moreover, in coculture experiments, cancer cell monolayers do not inhibit the growth of Fas-expressing lymphoid cells. Although FasL mRNA and protein were detected in the extracts of the colon cancer cell lines, flow cytometry and confocal microscopy failed to detect the protein on the surface of tumor cells. These results suggest that the counterattack of tumor-infiltrating T lymphocytes by cancer cells may not account for immune tolerance toward tumor cells.     

增殖诱导配体(APRIL)研究进展

增殖诱导配体 (aproliferation inducingligand ,APRIL)是肿瘤坏死因子 (TNF)家族的新成员 ,在多种肿瘤组织中有高表达 ,能促进肿瘤细胞增殖 ,防止肿瘤细胞受CD95L、FasL等诱导的凋亡 ;调节体液免疫 ;并在T、B淋巴细胞的成熟和活化中起一定作用。因此 ,APRIL与肿瘤的发生、发展以及免疫系统的调节有密切关系。     

Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.     

The Fas/Fas ligand system and cancer: immune privilege and apoptosis

The Fas/FasL system has been suggested to play an important role in the establishment of immune privilege status for tumors by inducing Fas-mediated apoptosis in tumor-specific lymphocytes. However, the role of cell-surface expressed FasL in tumor cell protection has recently become controversial. Our laboratory has focused on the study of the role of the Fas/FasL system in the normal tissue remodeling of the female reproductive tract and in immune-privileged organs. Our studies have demonstrated a connection between sex hormones and the regulation of the Fas/FasL pathway in immune and reproductive cells. More recently, we have investigated the resistance of tumor cells to Fas-mediated apoptosis. We have also characterized a new form of FasL, different from the classical membranal form, which is secreted by ovarian cancer cells. In this review we describe the main techniques used in these studies.