TNFα cooperates with IFN-γ to repress Bcl-xL expression to sensitize metastatic colon carcinoma cells to TRAIL-mediated apoptosis

Background

TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

Methodology/Principal Findings

The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL) ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo.

Conclusions/Significance

TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression. Combined CTL immunotherapy and TRAIL therapy hold great promise for further development for the treatment of metastatic colorectal cancer.     

Regulation of FasL by NF-kappaB and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.     

Positive and negative consequences of soluble Fas ligand produced by an antigen-specific CD4(+) T cell response in human carcinoma immune interactions.

The influence of a human CD4(+) T cell response in anti-carcinoma immune reactions remains largely uncharacterized. Here, we made use of a major histocompatibility complex (MHC) class-II-restricted, anti-ras oncogene-specific CD4(+) T cell line produced previously in vivo from a patient with metastatic carcinoma in a peptide-based phase I trial. Using this patient-derived T cell line as a potentially relevant cell type, we examined the consequences of the anti-carcinoma CD4(+) T cell response, with emphasis on specific lymphokines potentially important for the regulation of Fas/Fas ligand (FasL) interactions. Antigen (Ag)-specific CD4(+) T cells produced substantial amounts of IFN-gamma following recognition of MHC class-II-matched Ag-presenting cells expressing the cognate peptide. The IFN-gamma promoted significant upregulation of Fas on the surface of colon carcinoma cells and sensitized these targets to Fas-mediated apoptosis and Ag-specific CD8(+) cytotoxic T lymphocyte (CTL)-mediated lysis involving a Fas-based effector mechanism. Moreover, Ag-stimulated CD4(+) T cells secreted soluble FasL (sFasL), which induced the death of TNF-resistant/refractory colon, breast, and ovarian carcinoma cells. Interestingly, although CD4(+)-derived sFasL expressed cytotoxic activity, the recovery of carcinoma cells which resisted Fas-mediated lysis displayed enhanced metastatic ability in vivo, compared with the unselected parental population, in an athymic mouse model. Thus, a tumor-specific CD4(+) T cell response may have both positive and negative consequences in human carcinoma via the production of proinflammatory cytokines such as IFN-gamma and/or sFasL that may (1) improve or facilitate CTL-target engagement, contact-independent effector mechanisms, and the overall lytic outcome and (2) potentially select for Fas-resistant tumor cells that escape immune destruction, which may thus impact the metastatic process.     

Perforin and Fas act together in the induction of apoptosis, and both are critical in the clearance of lymphocytic choriomeningitis virus infection

In this report we questioned the current view that the two principal cytotoxic pathways, the exocytosis and the Fas ligand (FasL)/Fas-mediated pathway, have largely nonoverlapping biological roles. For this purpose we have analyzed the response of mice that lack Fas as well as granzyme A (gzmA) and gzmB (FasxgzmAxB(-/-)) to infection with lymphocytic choriomeningitis virus (LCMV). We show that FasxgzmAxB(-/-) mice, in contrast to B6, Fas(-/-), and gzmAxB(-/-) mice, do not recover from a primary infection with LCMV, in spite of the expression of comparable numbers of LCMV-immune and gamma interferon-producing cytotoxic T lymphocytes (CTL) in all mouse strains tested. Ex vivo-derived FasxgzmAxB(-/-) CTL lacked nucleolytic activity and expressed reduced cytolytic activity compared to B6 and Fas(-/-) CTL. Furthermore, virus-immune CTL with functional FasL and perforin (gzmAxB(-/-)) are more potent in causing target cell apoptosis in vitro than those expressing FasL alone (perfxgzmAxB(-/-)). This synergistic effect of perforin on Fas-mediated nucleolysis of target cells is indicated by the fact that, compared to perfxgzmAxB(-/-) CTL, gzmAxB(-/-) CTL induced (i) an accelerated decrease in mitochondrial transmembrane potential, (ii) increased generation of reactive oxygen species, and (iii) accelerated phosphatidylserine exposure on plasma membranes. We conclude that perforin does not mediate recovery from LCMV by itself but plays a vital role in both gzmA/B and FasL/Fas-mediated CTL activities, including apoptosis and control of viral infections.     

Regulating the suppressors: apoptosis and inflammation govern the survival of tumor-induced myeloid-derived suppressor cells (MDSC)

Immune suppressive myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit innate anti-tumor immunity and are a significant obstacle to cancer immunotherapy. Inflammation is a known inducer of Gr1(+)CD11b(+) MDSC; however, the factors/conditions that regulate MDSC survival and half-life have not been identified. We have used mass spectrometry (MS) and proteomic analysis to identify proteins and pathways that regulate MDSC survival. This analysis revealed high expression of caspase family proteins and the Fas-FasL, p38 MAPK, and TGFβ pathways, suggesting that Fas-FasL apoptosis regulates MDSC survival. Flow cytometry, confocal microscopy, and western blot analyses confirmed the MS findings and demonstrated that Fas(+) MDSC are susceptible to Fas-mediated killing in vitro. In vivo studies with FasL-deficient and Fas-deficient mice demonstrated that Fas-FasL interactions are essential for MDSC apoptosis and for rejection of established metastatic disease and survival and that FasL(+) T cells are the effector population mediating MDSC apoptosis. MS findings validated by biological experiments demonstrated that inflammation increases MDSC levels by protecting MDSC from Fas-mediated apoptosis, possibly by activating p38 MAPK. These results demonstrate that MDSC half-life in vivo is regulated by FasL(+) T cells and that inflammation increases MDSC levels by conferring resistance to Fas-mediated apoptosis and identifies T cells as the relevant effector cells causing MDSC apoptosis in vivo. This newly recognized mechanism for regulating MDSC levels identifies potential new targets for decreasing MDSC in cancer patients.     

Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL

We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-gamma treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-gamma pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-gamma pretreatment rendered SW480 cells sensitive to both Fas-dependent and -independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas- and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-gamma and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential "escape" of Fas-resistant carcinoma cells.     

CTL adoptive immunotherapy concurrently mediates tumor regression and tumor escape

Tumor escape and recurrence are major impediments for successful immunotherapy. It is well-documented that the emergence of Ag-loss variants, as well as regulatory mechanisms suppressing T cell function, have been linked to inadequate antitumor activity. However, little is known regarding the role of Fas-mediated cytotoxicity by tumor-specific CD8(+) CTL in causing immune evasion of Fas resistant variants during adoptive immunotherapy. In this study, we made use of an adoptive transfer model of experimental lung metastasis using tumor-specific CTL as a relevant immune-based selective pressure, and wherein the Fas ligand pathway was involved in the antitumor response. Surviving tumor cells were recovered and examined for alterations in antigenic, functional, and biologic properties. We showed that diminished susceptibility to Fas-mediated cytotoxicity in vivo was an important determinant of tumor escape following CTL-based immunotherapy. Tumor escape variants (TEV) recovered from the lungs of CTL-treated mice exhibited more aggressive behavior in vivo. However, these TEV retained relevant MHC class I and tumor Ag expression and sensitivity to CTL via the perforin pathway but reduced susceptibility to Fas-mediated lysis. Moreover, TEV were significantly less responsive to eradication by CTL adoptive immunotherapy paradigms as a consequence of increased Fas resistance. Overall, we identified that Fas(low)-TEV emerged as a direct consequence of CTL-tumor interactions in vivo, and that such an altered neoplastic Fas phenotype compromised immunotherapy efficacy. Together, these findings may have important implications for both tumor progression and the design of immunotherapeutic interventions to confront these selective pressures or escape mechanisms.     

Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit T cell-mediated cytotoxicity by inhibiting Fas ligand expression

We reported recently that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect CD4+ T cells against Ag-induced apoptosis by down-regulating the expression of Fas ligand (FasL). Because the cytotoxic activity of CD8+ CTLs is mediated through two mechanisms, which involve the perforin/granzyme and the FasL/Fas pathways, in this study we investigated the effects of VIP/PACAP on the generation and activity of allogeneic CTLs, of CD8+ T1 and T2 effector cells and of alloreactive peritoneal exudate cytotoxic T cells (PEL) generated in vivo. VIP/PACAP did not affect perforin/granzyme-mediated cytotoxicity, perforin gene expression, or granzyme B enzymatic activity, but drastically inhibited FasL/Fas-mediated cytotoxicity against allogeneic or syngeneic Fas-bearing targets. VIP/PACAP inhibit CTL generation, but not the activity of competent CTLs. The inhibition is associated with a profound down-regulation of FasL expression, and these effects are mediated through both VPAC1 and VPAC2 receptors. VIP/PACAP inhibit the FasL/Fas-mediated cytotoxicity of T1 effectors and do not affect T2 cytotoxicity, which is entirely perforin/granzyme mediated. Similar effects were observed in vivo. Both the FasL/Fas-mediated cytotoxicity and FasL expression of cytotoxic allogeneic PELs generated in vivo in the presence of VIP or PACAP were significantly reduced. We conclude that, similar to their effect on CD4+ T cells, the two structurally related neuropeptides inhibit FasL expression in CD8+ cytotoxic T cells and the subsequent lysis of Fas-bearing target cells.     

Coordinate regulation of IFN consensus sequence-binding protein and caspase-1 in the sensitization of human colon carcinoma cells to Fas-mediated apoptosis by IFN-gamma

Interferon-gamma is thought to be essential for the regulation of antitumor reactions. However, the degree of responsiveness of malignant cells to IFN-gamma may have a profound influence on the overall efficacy of an antitumor response. In this study, we examined the molecular basis by which IFN-gamma differentially sensitized human primary and metastatic colon carcinoma cells to Fas-mediated apoptosis. To that end, we analyzed IFN-gamma-induced gene expression at the genome scale, followed by an analysis of the expression and function of specific genes associated with IFN-gamma- and Fas-mediated signaling. We found that although both cell populations exhibited a similar gene expression profile at the genome scale in response to IFN-gamma, the expression intensities of the IFN-gamma-regulated genes were much greater in the primary tumor. Noteworthily, two genes, one involved in IFN-gamma-mediated signaling, IFN consensus sequence-binding protein (ICSBP), and one involved in Fas-mediated signaling, caspase-1, were clearly shown to be differentially induced between the two cell lines. In the primary tumor cells, the expression of ICSBP and caspase-1 was strongly induced in response to IFN-gamma, whereas they were weakly to nondetectable in the metastatic tumor cells. Functional studies demonstrated that both caspase-1 and ICSBP were involved in Fas-mediated apoptosis following IFN-gamma sensitization, but proceeded via two distinct pathways. This study also reports for the first time the expression of ICSBP in a nonhemopoietic tumor exhibiting proapoptotic properties. Overall, in a human colon carcinoma cell model, we identified important functional contributions of two IFN-gamma-regulated genes, ICSBP and caspase-1, in the mechanism of Fas-mediated death.     

Influence of interferon γ on modulation of Fas expression by human colon carcinoma cells and their subsequent sensitivity to antigen-specific CD8+ cytotoxic T lymphocyte attack

The inability of certain neoplastic populations to undergo Fas-mediated death by immune effector mechanisms may confer a selective survival advantage, which may contribute to tumor escape. In this study, we examined the role of Fas-mediated lysis in a human-antigen (Ag)-specific cytotoxic T lymphocyte (CTL)/colon carcinoma cell model, and the regulation of the lytic phenotype by interferon γ (IFNγ). Previously, we have identified mutated ras peptides reflecting the valine-for-glycine substitution at position 12 as unique HLA-A2-restricted, CD8⁺ CTL neo-epitopes. Peptide-specific CTL, established from both normal and carcinoma-bearing individuals, lysed in vitro a HLA-A2⁺ primary colon adenocarcinoma cell line, SW480, harboring the naturally occurring ras mutation. Pretreatment of SW480 cells with IFNγ was necessary to promote efficient Ag-specific CTL killing, although the mechanisms by which IFNγ influenced the lytic outcome remains to be elucidated. Here, we show, by phenotypic analysis of SW480 cells, a significant up-regulation of HLA-A2, ICAM-1 and Fas molecules after IFNγ pretreatment, which paralleled their sensitivity to lysis with anti-Fas stimuli. Moreover, nearly half of the lytic response to IFNγ-treated SW480 cells was inhibited by neutralizing anti-Fas or anti-Fasligand (FasL) mAb, revealing for the first time an important functional role for Fas/FasL interactions in carcinoma cell killing by human Ag-specific CTL. mAb against HLA-A2, ICAM-1, the αβ T cell receptor (TCR) and Fas molecules inhibited lysis; however, if these CTL were preactivated to express functional FasL and then used as effectors, only anti-Fas mAb efficiently blocked lysis. IFNγ also increased pro-caspase-3 protein expression and its subsequent activation in SW480 cells following Ag-specific CTL attack. Peptide-based caspase inhibitors blocked both caspase-3 activation and CTL-mediated lysis. Overall, these data suggested that IFNγ (a) facilitated both Ag-dependent and Ag-independent events as a prerequisite for efficient CTL/target interactions, FasL up-regulation and triggering of Fas-dependent, as well as Fas-independent lysis (perforin); and (b) enhanced or restored a Fas-sensitive phenotype in SW480 cells, reflecting modulation of cell-surface and intracellular elements of the Fas pathway. Thus, IFNγ may play an important role in the regulation of a human neoplastic cell death phenotype, which may have implications for our understanding of the processes of both tumor evasion and tumor regression following Ag-specific CTL attack. Received: 20 December 1999 / Accepted: 1 February 2000     

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.