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Keywords:

  • Fas ligand;
  • transforming growth factor β;
  • apoptosis;
  • tumor microenvironment;
  • immune privilege

Abstract

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

Despite the fact that expression of Fas ligand (FasL) in cytotoxic T lymphocytes (CTLs) and in natural killer (NK) cells plays an important role in Fas-mediated tumor killing, During tumor progression FasL-expressing tumor cells are involved in counterattacking to kill tumor-infiltrating lymphocytes (TILs). Soluble FasL levels also increase with tumor progression in solid tumors, and this increase inhibits Fas-mediated tumor killing by CTLs and NK cells. The increased expression of FasL in tumor cells is associated with decreased expression of Fas; and the promoter region of the FASL gene is regulated by transcription factors, such as neuronal factor κB (NF-κB) and AP-1, in the tumor microenvironment. Although the ratio of FasL expression to Fas expression in tumor cells is not strongly related to the induction of apoptosis in TILs, increased expression of FasL is associated with decreased Fas levels in tumor cells that can escape immune surveillance and facilitate tumor progression and metastasis. Transforming growth factor β (TGF-β) is a potent growth inhibitor and has tumor-suppressing activity in the early phases of carcinogenesis. During subsequent tumor progression, the increased secretion of TGF-β by both tumor cells and, in a paracrine fashion, stromal cells, is involved in the enhancement of tumor invasion and metastasis accompanied by immunosuppression. Herein, the authors review the clinical significance of FasL and TGF-β expression patterns as features of immune privilege accompanying tumor progression in the tumor microenvironment. Potential strategies for identifying which molecules can serve as targets for effective antitumor therapy also are discussed. Cancer 2004. © 2004 American Cancer Society.

Apoptosis is a genetically controlled mechanism that plays an important role in the regulation of tumor progression and metastasis.1 Tumor growth depends not only on uncontrolled proliferation but also on the suppression of apoptosis. Many triggers, such as irradiation,2 antitumor drugs,3 and features of the immune response to tumor cells,4 can lead to the induction of apoptosis, including the stimulation of cell membrane–bound death receptors for the death ligands Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). In addition, there is significant evidence showing that tumor cell apoptosis is triggered by antitumor surveillance of the immune system and by the antitumor drugs and irradiation that are used to treat patients. Induction of apoptotic signals by chemotherapeutic agents is an important step in apoptosis, and some antitumor agents induce both FasL expression and Fas expression. These agenst also may cause activation-induced cell death (AICD) in tumor cells.5 Despite the fact that regulation of FasL expression, rather than Fas expression, in tumor cells is critical to the induction of apoptosis by any trigger, the increased expression of FasL in tumor cells also can mediate a counterattack against cytotoxic T lymphocytes (CTLs).6 Soluble FasL (sFasL), which is a proteolytically processed form of FasL produced by tumor cell metalloproteinases during disease progression, can inhibit the death receptor–dependent pathway associated with the Fas/FasL system. Thus, the expression of FasL in tumor cells plays a critical role in the induction of apoptosis by several triggers, such as DNA-damaging agents and immune surveillance.

Transforming growth factor β (TGF-β) is involved in the proliferation and differentiation of cells, embryonic development, and angiogenesis.7 TGF-β has a growth-factor-like ability to stimulate fibroblast growth, although it actually is a potent inhibitor of epithelial cell proliferation, which is regulated by the cell microenvironment that signals cell cycle arrest.8 The potential involvement of TGF-β in carcinogenesis has been characterized ever since its growth-inhibitory properties were discovered. However, this essential signaling pathway becomes inactivated during the development of malignant disease. Furthermore, in some cells, TGF-β stimulates cell proliferation rather than inhibiting cell growth.9 Thus, TGF-β may play a critical role in the escape or progression of some malignancies from host immunity. Although TGF-β is a biphasic molecule, its increased secretion by tumor cells plays an important role in disease progression by conferring immune privilege to CTLs, and TGF-β also stimulates tumor proliferation by promoting paracrine growth in the tumor microenvironment.

The current review focuses on the role of FasL and TGF-β as potential antitumor therapeutic targets. We discuss the ways in which tumor cells can escape from immune surveillance during tumor progression and how this conferred immune privilege affects the difficulty associated with the treatment of malignant disease. Changes in the tumor microenvironment are discussed in terms of exploring new strategies for eradicating tumor cells.

Expression of FasL and Fas in Tumor Cells

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

FasL is a 40 kilodalton (kD) transmembrane protein that is classified as a type II protein of the TNF family. FasL induces apoptosis of susceptible cells by cross-linking its receptor, Fas.10 In general, FasL is expressed in T lymphocytes, such as cytotoxic T cells (CTLs) in tumor-infiltrating T cells (TILs), and in natural killer (NK) cells and macrophages. Despite the fact that FasL is expressed in specific organs, including the testis and eye,11 overexpression of FasL also is found in various tumors, including melanoma,12 lymphoma,13 esophageal carcinoma,14 gastric carcinoma,15 colon carcinoma,16 and breast carcinoma.17 FasL can be cleaved proteolytically to form a bioactive, soluble trimer,18 and it can associate with lipid microvesicles that stabilize its bioactivity and prolong its biologic half-life.19 High levels of FasL expression in the membrane (mFasL), soluble, and microvesicle-associated fractions (vFasL) have been reported in cultures of activated B cells, T cells, and NK cells.20

Fas is a 45 kD type I transmembrane glycoprotein that was first identified using antibodies that induced the rapid death of tumor cells by binding to the natural ligand of Fas, FasL. Fas is expressed ubiquitously in many tissues, with particularly high expression levels found in the thymus, liver, heart, and kidney.21 Fas is characterized by three extracellular-region cysteine-repeat domains, and the intercellular region of the death domain is critical for apoptosis signaling.22 The expression of Fas can be induced by cytokines, such as interferon-gamma (IFN-γ) and TNF-α, and also by the activation of lymphocytes.23 Fas-mediated apoptosis is triggered by FasL. The mechanism of the Fas-receptor/ligand death system has been clarified as follows: After binding of FasL to the Fas receptor, Fas associates with two specific proteins, Fas-associated death domain (FADD) and caspase-8, to form the death-inducing signal complex (DISC).24 FADD recruits the cysteine protease caspase-8, which directly or indirectly activates other members of the caspase family, resulting in cleavage of cellular proteins and subsequent apoptosis. Genetic defects that impair DISC formation can severely impair the induction of apoptosis by Fas. Thus, because the overexpression of caspase-8 is sufficient to induce apoptosis, caspase activation plays a central role in Fas signaling–mediated apoptosis.

The TGF-β Signaling Pathway and Alterations in Tumor Cells

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

Three mammalian TGF-β isoforms, TGF-β1, TGF-β2, and TGF-β3, have been identified, and these isoforms possess similar functions in vitro with respect to cell growth regulation and immune modulation.25 However, each isoform appears to have distinct activities in vivo, as indicated by the distinct phenotypes of mice that lack specific TGF-β ligands.26 TGF-β ligands bind to and signal via a heteromeric complex of type I and II receptors that have serine/threonine kinases.27 Because the TGF-β type II receptor (T-βRII) is necessary for specific ligand binding, this receptor forms an oligomeric complex that is accompanied by type I receptor phosphorylation. Through the identification and characterization of signal transduction pathways downstream of the serine/threonine kinase TGF-β receptors, nine members of the human TGF-β intracellular signaling pathways have been identified. These nine molecules are referred to as ‘Smad1’ through ‘Smad9’.28 Smad1, Smad5, Smad8, and Smad9 transduce signals from the bone morphogenetic proteins (BMPs). Smad2 and Smad3 are important substrates of the TGF-β type I receptor (T-βRI). Upon phosphorylation by T-βRI, Smad2 and Smad3, which are associated with Smad 4, translocate to the nucleus, associate with a DNA-binding partner, and activate the transcription of specific target genes. Smad6 and Smad7 inhibit the signaling function of the receptor-activated Smads, which are involved in both TGF-β and BMP signaling, by preventing receptor-mediated phosphorylation. The TGF-β serine/threonine kinase receptors also may activate certain members of the mitogen-activated protein kinase (MAPK) family in tumor cells.

In intestinal epithelial cells, TGF-β appears to play a major growth-inhibitory role that is associated with G1 cell-cycle arrest, down-regulation of cyclin D1, and inhibition of Cdk4-associated Rb kinase activity.29 However, the regulation of TGF-β in epithelial cell proliferation is altered by transformation. Studies of colon carcinoma cell lines have documented a correlation between the degree of differentiation of the tumor and sensitivity to TGF-β with respect to the inhibition of proliferation and differentiation.30 Furthermore, loss of responsiveness to the growth-inhibitory effects of TGF-β has been observed in many tumor cell types, including colorectal carcinoma,31 breast carcinoma,32 and pancreatic carcinoma.33 Thus, it is likely that loss of the growth-inhibitory effect of TGF-β is common and is a critical event in malignant transformation and tumor progression. The loss of the growth-inhibitory effect of TGF-β may occur via down-regulation or mutation of T-βRII, because the loss of TGF-β function abolishes its negative growth regulation. Previous reports have indicated that the decreased expression of T-βRII is an important step in the malignant transformation of epithelial cells resulting from the activation of Ras protein and from antigen-presenting cell (APC) mutations.34, 35 In addition, down-regulation of T-βRII has been observed among the 13% of human colon carcinomas with microsatellite instability.36 The mutational loss of TGF-β signal transduction has been noted in colorectal carcinoma as indicated by the observed levels of Smad237 and Smad4.38 TGF-β receptor activation, in turn, can promote tumor progression and invasion, although the mutations of T-βRII appear to be rare in the majority of colorectal carcinomas that do not possess the replication error phenotype.

Because TGF-β actually may promote tumor progression in several cell types under certain circumstances, TGF-β expression is increased in a variety of tumor types. Studies of colorectal carcinomas have documented that high-level expression of TGF-β1 in the primary tumor is associated with advanced tumor stage and is an independent prognostic factor.39 It has been reported that TGF-β is a potent modulator of epithelial cell phenotype for keratinocyte and mammary epithelial cells,40 and there is important evidence that autocrine TGF-β expression by tumor cells plays a critical role in the epithelial-to-fibroblastoid conversion in mammary cells41 and in keratinocytes.42 Although TGF-β1 overexpression in transgenic mouse keratinocytes inhibits the growth of carcinogen-induced benign skin tumors, this overexpression promotes the progression of advanced lesions to the malignant phenotype.43 The TGF-β-induced mesenchymal transition in Ha-Ras-transformed mammary epithelial cells induces the disruption of cell-cell adhesion and the loss of epithelial polarity.44 In an experimental rat liver model, chronic exposure to a high concentration of TGF-β1 resulted in loss of the growth-inhibitory effect of TGF-β1 and loss of contact inhibition. These losses were associated with a fibroblastlike cell morphology and anchorage-independent growth.45

The Roles of Fas and FasL in Tumor Cells and in Immune Privilege

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

FasL appears to play roles in immune privilege (i.e., the so-called tumor counterattack phenomenon) and in inflammation. FasL expression on the cell surface may trigger an inflammatory response, leading to tumor rejection. In contrast, FasL confers immune privilege to tumors by inducing apoptosis in infiltrating lymphocytes. In the case of tumor grafts, FasL expression in graft tumors results in a neutrophilic inflammatory response that leads to graft rejection,46 but this is not sufficient to confer immune privilege. Rather, sFasL levels increase during inflammation, and sFasL may play a regulatory role in inducing Fas-mediated apoptosis, although the capacity of sFasL to induce apoptosis is lower than that of mFasL.47 In tumor progression, it has been reported that tumor cells in various organs can express FasL and induce apoptosis in T cells that express Fas.48 Thus, tumor cells can counterattack Fas-bearing immune cells not only to avoid rejection by the immune system but also to participate in their destruction. Tumor cells are more effective in counterattacking activated T cells, because these tumor cells are resistant to the cytotoxic effects of FasL, whereas TILs are sensitive to FasL-mediated apoptotic cell death. In fact, various human colon adenocarcinoma cell lines express FasL mRNA and protein, whereas normal colon epithelial cells do not.49 In Jurkat T cells, FasL has the functional role of inducing apoptosis,50 which is inhibited by the addition of neutralizing anti-FasL monoclonal antibodies. In vivo, human carcinoma tissues have an immune privilege, and a significant reduction of TILs has been observed in esophageal carcinoma51 coincident with a significant increase in TIL apoptosis in FasL-expressing tumor cells. This finding suggests that the apoptotic cell death of TILs is mediated by FasL in response to the increase in FasL expression that occurs in esophageal carcinoma progression. Similarly, FasL expression in gastric carcinoma has been reported in several studies,52–54 and tumor FasL expression was associated with increased apoptosis of Fas-positive TILs in metastatic gastric carcinoma.54 Furthermore, investigations of colorectal carcinoma have shown that FasL expression is observed not only on the cell surface but also in the cytoplasm of tumor cells,55, 56 and apoptosis of TILs was observed more frequently in FasL-positive tumor cells than in FasL-negative tumor cells.56, 57 Similar findings of a close correlation between apoptosis of TILs and FasL-positive tumors have been reported in patients with head and neck malignancies58 and ovarian carcinoma.59 Moreover, among patients with colorectal carcinoma, FasL expression levels are higher in metastatic lesions than in primary lesions,16 and high FasL expression levels are associated with lymph node metastasis grade in breast carcinoma.60 In addition, a high ratio of FasL to Fas can be indicative of poor prognosis in patients with ovarian carcinoma61 and hepatic carcinoma.62 These findings suggest that high FasL expression levels in the tumor microenvironment may facilitate metastasis.

The loss of Fas expression may cause decreased sensitivity of the tumor cells to T-cell cytotoxicity in association with increased FasL expression by tumor cells at their borders with neighboring lymphocytes. In addition, Fas expression in esophageal carcinoma has been proposed as an independent prognostic factor, and decreased Fas expression in tumors may represent a good therapeutic target.63 It appears that decreased Fas expression coupled with increased FasL expression in tumor cells may be involved in immune privilege during tumor progression. Nevertheless, the molecular mechanism by which FasL expression in tumor cells induces inflammation, resulting in tumor rejection or, in the case of tumor progression, immune privilege, remains unknown. One noteworthy report indicated that FasL-transfected colon carcinoma cells introduced subcutaneously into mice were rejected due to the induction of neutrophil infiltration. However, the same cells can survive within the intraocular space because of the presence of TGF-β, which inhibits neutrophil activation.64 This finding indicates that sFasL may play an inhibitory role by inducing neutrophil infiltration in response to the inflammatory response. Taken together, these results suggest that TGF-β and FasL cooperatively regulate immunologic tolerance according to the tumor microenvironment. The regulation depends on the type of tumor tissue and the sensitivity of the cell to Fas/FasL-mediated cell death.

A mutated Fas molecule can help tumor cells escape immune surveillance, resulting in resistance to FasL, which inhibits the attacking T-cells and NK cells. Fas mutations have been identified in many human malignancies, including non-Hodgkin lymphoma65 and bladder carcinoma,66 which exhibited a high incidence of Fas mutations. In colon and gastric carcinomas, which have a microsatellite mutator phenotype, Fas mutations are observed with a frequency of approximately 10%.67 In contrast, in other malignancies, such as hepatocellular carcinoma and melanoma,68 Fas mutations are rare. Resistance to Fas-mediated apoptosis allows tumor cells to increase the expression of FasL and to mount a counterattack against Fas-sensitive immune cells, resulting in tumor progression and metastasis.

The Effect of the Tumor Microenvironment on Immune Privilege

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

In the setting of tumor progression, the microenvironment of solid tumors is characterized by hypoxia and subsequent acidosis.69 Although tumor cells under these conditions require several angiogenic factors to be induced via the activation of hypoxia-inducible factor 1 in tumor angiogenesis, tumor cells also can gain immune privilege for tumor progression in the microenvironment. Tumor cells can down-regulate Fas, which is associated with p53 mutation,70 making them less susceptible to FasL-mediated apoptosis. They also can up-regulate FasL expression via proinflammatory cytokines, such as TGF-β, interleukin-10 (IL-10), prostaglandins, and reactive oxygen metabolites. In turn, the production of cytokines from stromal cells, which are distinct from the general antiinflammatory microenvironment, activates Akt phosphorylation, which promotes tumor survival through down-regulation of Fas expression and up-regulation of FasL in tumor cells.71 In addition, increased production of TGF-β can result in down-regulation of FasL in CTLs, inhibiting AICD in tumor cells.72 Furthermore, it has been reported that in gastric carcinoma, the production of H2O2 by tumor-associated macrophages leads to T-cell dysfunction, which is coupled with the production of intracellular cytokines, such as IL-10, in the tumor microenvironment, depending on disease progression.73 It appears that the induction of TGF-β and IL-10 and the activation of Akt following changes in the tumor microenvironment, including hypoxia and glucose starvation, play an important role in tumor progression and metastasis by conferring immune privilege (Fig. 1).

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Figure 1. Molecular mechanisms of immune privilege in tumor cells via the Fas/Fas ligand (FasL) system in the tumor microenvironment. Changes in the tumor microenvironment with hypoxia and glucose starvation induce several Th2 cytokines, such interleukin-4 (IL-4) and IL-10, in stromal cells in a paracrine fashion, leading to enhancement of tumor growth and angiogenesis. The tumor microenvironment increases expression of FasL via transcription factors, including AP-1 and nuclear factor κB (NF-κB), in tumor cells that counterattack cytotoxic T lymphocytes (CTLs) as a part of tumor immune privilege. In tumor cells, FasL, which is expressed at increased levels, is cleaved by matrix metalloproteinases to form soluble FasL (sFasL), which is involved in the down-regulation of Fas expression and the inhibition of Fas-mediated apoptosis by CTLs. Increased secretion of transforming growth factor β (TGF-β) from tumor cells and macrophages accompanies tumor progression and inhibits the proinflammatory response mediated by cytokines such as IL-1α and IL-6. These cytokines are involved in tumor rejection in concert with sFasL. TGF-β not only inhibits secretion of interferon-gamma (IFN-γ) from effector cells but also down-regulates FasL expression in tumor cells. Despite the fact that the activated T lymphocytes mediated by T-cell receptor (TCR)/CD3 and IL-2 increase expression of Fas and FasL during the immune response, CTLs can be counterattacked by FasL in tumor cells. This counterattack leads to apoptosis, which is associated with decreased levels of TCR-ζ. DC: dendritic cell; MHC: major histocompatibility complex; PGE2: prostaglandin E2; MMP: matrix metalloprotease; Mϕ: macrophage.

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A substantial amount of evidence has been reported showing that immune effector cells, including lymphocytes, monocytes, and dendritic cells, undergo apoptosis in the tumor microenvironment.73 Apoptotic cell death associated with the activation of caspases is observed in immune cells in the vicinity of tumor cells, rather than in inflammatory lesions or normal tissue. In addition, spontaneous apoptotic cell death was observed in peripheral blood mononuclear cells (PBMCs) in a group of patients with malignant disease but not in a healthy control group.74 In patients with metastatic melanoma compared with normal blood donors, significantly greater numbers of apoptotic T cells, which were preferentially positive for CD3, Fas, and annexin V, were observed in circulating PBMCs.75 These Fas-positive T cells are sensitive to apoptosis. Because T-cell receptor ζ (TCR-ζ) is a substrate for caspase-3,76 the apoptotic Fas-positive T cells exhibited decreased expression of the TCR-ζ chain, which also was associated with the loss of Fas expression from the surface of CD3-positive/Fas-positive T cells in patients with melanoma.75 Similarly, in patients with head and neck malignancies compared with individuals with no evidence of disease, a significant increase in CD3-positive/Fas-positive T cells among PBMCs was observed in association with decreased serum levels of sFasL.77 Furthermore, CD8-positive T cells were apoptotic to a greater extent than were CD4-positiveT cells, suggesting that CD8-positive T cells are more sensitive to apoptosis in patients with head and neck malignancies.77 It also has been reported that increased apoptotic cell death of peripheral T lymphocytes occurs in patients with gastric carcinoma, and this finding was associated with decreased TCR-ζ chain levels.78 The TCR-ζ chain is an important step in the activation of effector T cells in signal transduction of the tumor immune response mediated by TCR and APCs.79 These observations of increased apoptosis of CD3-positive/Fas-positive T cells in circulating peripheral blood and in the vicinity of tumor cells are not restricted to a specific disease type. Thus, tumor progression via immune privilege appears to be a universal phenomenon among patients with malignant disease. Despite the fact that sFasL is less effective than mFasL in cross-linking its receptor, low levels of sFasL in the serum may affect the contribution of tumor counterattack attributable to Fas-mediated apoptosis of CTLs in active disease. The other explanation for the counterattack carried out by FasL-overexpressing tumor cells is that microvesicle-associated FasL can induce apoptosis in T cells more efficiently, because microvesicle-associated FasL, rather than sFasL, reportedly is bioactive in ovarian carcinoma cells.80 It appears that the functional clipping of CTLs via tumor FasL–mediated apoptosis of TILs, as well as circulating lymphocytes, induces the overall immune competence of patients with malignant disease.

Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

Fas-dependent and Fas-independent apoptosis

Treatment with IFN-γ increases the expression of Fas in many cell types. Sensitization of colon carcinoma cells to 5-fluorouracil/leucovorin (5-FU/LV) has been reported following the induction of increased expression of Fas by IFN-γ.81 Sensitization to 5-FU/LV via susceptibility to IFN-γ-induced thymineless death on Fas signaling represents a typical situation in which such agents and IFN-γ combine to have cytotoxic effects. Clinical evaluation of 5-FU/LV has been conducted for patients with colorectal carcinoma in a Phase I study, and subsequent studies are underway to evaluate other therapeutic modalities.82 Furthermore, combination treatment with 5-FU and IFN-α enhanced tumor responses in patients with advanced hepatocellular carcinoma,83 in which the mechanism of enhanced antitumor effect appears to involve the p53-mediated signal transduction pathway induced by IFN-α.84 It appears that the role of death receptors and ligands in the antitumor activity of chemotherapeutic agents is complex, and death ligands may kill drug-resistant cells by sensitizing them to the immune response through increased expression of Fas. The cytotoxic ligands involved in TRAIL-induced apoptosis can be enhanced by several antitumor drugs, including etoposide, doxorubicin, cisplatin, and 5-FU,85, 86 which mediate increases in the expression of the TRAIL receptor DR5. The combination of cytokines or ligands with anticancer drugs may enhance death receptor signaling pathways.

Inhibition of MMPs to inhibit sFasL production

sFasL trimerizes and efficiently binds to its membrane-bound receptor, resulting in rapid internalization and down-regulation of the surface Fas receptor.87 This suggests that sFasL has an antagonistic rather than agonistic function, despite the finding that sFasL has less apoptosis-inducing activity than does mFasL. The role of sFasL in tumor cells is to inhibit Fas-mediated apoptosis caused by CTLs; in this setting, apoptosis occurs via internalization of FasL after the down-regulation of Fas. Inhibition of matrix metalloproteases (MMPs) to increase the production of sFasL in Fas-sensitive cells may increase the Fas-mediated apoptosis of CTLs by leading to the accumulation of FasL at the tumor surface. In this regard, a previous study showed that treatment with an MMP inhibitor (MMPI) sensitized neuroblastoma cells to Fas-activating antibody and doxorubicin-induced apoptosis in Fas-sensitive cell lines by increasing expression levels of FasL and Fas on tumor cell surfaces.88 In addition, doxorubicin-induced apoptosis can be augmented specifically by transfection with antisense MMP7, because doxorubicin-induced apoptosis is inhibited by the expression of sFas and by coincubation of tumor cells with MMP-7, but not with MMP-2 or MMP-9, in colon carcinoma and sarcoma cells.89 MMP-7 is expressed widely in human solid tumors, including esophageal, gastric, and colorectal carcinomas and metastatic tumor specimens. The combination of MMPIs with antitumor agents may sensitize tumor cells, because synthetic MMPIs also inhibit FasL shedding. The synthetic MMPIs increased apoptosis in Fas-sensitive cells, but Fas-resistant cells had increased levels of Fas and FasL, suggesting a potential role for MMPIs in treatments that combine them with apoptosis-inducing chemotherapeutic agents.

Inhibition of proteasome degradation

Because activation of the proteasome is an inevitable phenomenon in the microenvironment of solid tumors that is involved in the degradation of several targets of antitumor agents as well as attenuation of the presentation of specific tumor antigens by dendritic cells, it appears that the inhibition of proteasome degradation is critical for the restoration of antitumor effect and immune response. The proteasome inhibitor PS-341 inhibits neuronal factor κB (NF-κB) activation in tumor cells, including multiple myeloma cells90 and breast,91 colon,92 and pancreatic carcinoma cells.93 NF-κB is a prominent instigator of chemoresistance, and protease inhibition has proven to be an effective means of preventing NF-κB activation in multiple myeloma and in solid tumors in a number of studies. Overexpression of Bcl-2 cannot prevent PS-341-induced apoptosis.94 The proteasome inhibitor also affects the presentation of tumor-associated antigen via the antigen transporter machinery.95 Down-regulation of these proteasome subunits has been suggested in lymphoma, breast carcinoma, and renal cell carcinoma.96 The peptides produced by the proteasome are bound in the binding pocket groove of major histocompatability complex Class I molecules.97 Thus, proteasome activity plays an important role in the presentation of antigen peptides and in peptide transport into the endoplasmic reticular lumen. The use of proteasome inhibitors may be effective not only in enhancing cellular sensitivity to antitumor agents but also in restoring the immune response of CTLs in tumor cells.

Inhibition of TGF-β function to restore immune response

TGF-β plays a dual role in tumor progression. In the late phase of tumor growth, the alteration of Smad-mediated signal transduction pathways via T-βRII is critical for tumor progression and metastasis through autocrine and paracrine tumor regulation. The blockade of autocrine and paracrine TGF-β signaling, which is involved in tumor angiogenesis, stroma formation and remodeling, and immunosuppression, may enhance the therapeutic efficacy of treatments for patients with malignant disease. Experimental results obtained using the mammary tumor virus–polyomavirus middle T antigen transgenic mouse model indicated that the blockade of TGF-β with soluble Fc:T-βRII enhanced apoptosis and that tumor cell mobility and lung metastases were blocked by the inhibition of Akt activity in metastatic mammary cell lines.98 Another report indicated that transgenic mice that stably expressed soluble Fc:T-βRII, the circulating form of Fc:T-βRII, not only exhibited reduced formation of melanoma metastases but also experienced a decrease in metastases to the lung from endogenous mammary tumors.99 Nonetheless, Fc:T-βRII had no effect on the proliferative rate of primary tumor cells in either of those two reports. These results indicate that Fc:T-βRII decreases tumor cell intravasation and/or decreases the survival of tumor cells in the circulation, because the number of circulating tumor cells was lower in Fc:T-βRII-treated mice than in control mice. In addition, these findings suggest that soluble TGF-β antagonists can inhibit metastases significantly in models of breast carcinoma and melanoma.

TGF-β antibodies also inhibit Akt activity in mammary tumor cells, suggesting that the inhibition of Akt may a requirement for the antitumor activity of TGF-β. A recent study also indicated that the reconstitution of T-βRI in breast carcinoma cells that expressed a kinase-inactive T-βRII enhanced survival if there was increased Akt and ERK activity but if there was Smad2 phosphorylation.100 Thus, the tumor progression associated with TGF-β can be activated by a Smad-independent pathway and is characterized by a low level of receptor activation. It nonetheless is possible to grow tumor cells that carry SMAD mutations. Treatment with the monoclonal TGF-β antibody and the transfection of soluble forms of T-βRII or T-βRIII produce antitumor and antiangiogenic effects, whereas treatment with antisense TGF-β1 and TGR-β2 induces partial rejection of tumors transplanted into immunocompetent mice by restoring the immunogenicity of the tumors.101 These results indicate that the down-regulation of TGF-β activity in tumor cells can restore the tumor-specific cellular immunity that mediates tumor rejection.

Several TGF-β inhibitors are being developed for clinical applications; among these inhibitors are the humanized monoclonal antibodies CAT-192102 and CAT-152,103 which are specific for TGF-β1 and TGF-β2, respectively. A Phase II study of the monoclonal antibody CAT-192 as a therapeutic agent against diffuse systemic sclerosis is underway, as is a Phase III trial of CAT-152 for treatment of glaucoma. The pan-TGF-β antibodies 1D11104 and 2G7105 currently are being evaluated in preclinical studies for the treatment of diffuse scleroderma and malignant disease, respectively. Another class of TGF-β antagonist blocks the catalytic activity of the receptor. Because small-molecule inhibitors, which compete with adenosine triphosphate (ATP) for the ATP-binding site of the T-βRI kinase,106 cannot inhibit TGF-β function completely, including T-βRII kinase activity, the development of bifunctional receptor inhibitors will be required to completely inhibit TGF-β function. Antisense oligonucleotides that targeted TGF-β2 were found to inhibit malignant mesothelioma growth.107 Several possible strategies targeting the Fas/FasL system and TGF-β to enhance apoptosis in tumor cells are summarized in Figure 2.

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Figure 2. Possible strategies for the enhancement of apoptosis in tumor cells mediated by the Fas/Fas ligand (FasL) system are described in terms of the molecular mechanisms of immune privilege in tumor progression. Treatment with anticancer agents and interferon (IFN)-γ or IFN-α increases Fas expression in a p53-dependent manner in tumor cells, and this increase in expression plays a role in Fas-mediated apoptosis. Such treatment also increases FasL expression in a p53-independent manner, leading to activation-induced cell death. Cotreatment with a transforming growth factor β (TGF-β) antagonist or with a matrix metalloprotease (MMP) inhibitor that suppresses the function of TGF-β and soluble FasL (sFasL) may enhance the antitumor effect of chemotherapeutic agents used in cancer therapy. In addition, cotreatment with a proteasome inhibitor enhances antitumor effects via modulation of the tumor microenvironment and also via an increase in immune response mediated by activated T lymphocytes. CTLs: cytotoxic T lymphocytes; NF-κB: nuclear factor κB; sFasL: microvesicle-associated FasL.

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Concluding Remarks

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES

Altered expression of Fas and FasL in tumor cells is critical to the acquisition of immune privilege in tumor progression and metastasis. Such alterations in expression occur in cooperation with increased secretion of TGF-β in both an autocrine fashion and a paracrine fashion in the tumor microenvironment. The increased production of TGF-β inhibits the secretion of IFN-γ from CTLs and induces expression of FasL in tumor cells. The multiple signaling networks, which are supported by the escape of the tumor from the host immune system, represent the most important aspect of the malignant cycle in tumor progression. Several molecularly targeted treatments designed to modulate the malignant cycle may be somewhat effective in promoting tumor regression. In particular, the specific inhibition of tumor growth through paracrine signaling networks, as well as the targeting of tumor cells, may be useful in overcoming drug resistance and curing solid tumors.

REFERENCES

  1. Top of page
  2. Abstract
  3. Expression of FasL and Fas in Tumor Cells
  4. The TGF-β Signaling Pathway and Alterations in Tumor Cells
  5. The Roles of Fas and FasL in Tumor Cells and in Immune Privilege
  6. The Effect of the Tumor Microenvironment on Immune Privilege
  7. Future Prospects for Restoring Fas-Mediated Apoptosis in Tumor Cells
  8. Concluding Remarks
  9. REFERENCES