Control of ocular tumor growth and metastatic spread by soluble and membrane Fas ligand

Fas ligand (FasL) can be either membrane bound, or cleaved by metalloproteinases (MMP) to produce a soluble protein. The two different forms of FasL are reported to have opposite functions-membrane-bound FasL (mFasL) is proinflammatory and soluble FasL (sFasL) is antiinflammatory. We previously showed that, within the immune-privileged eye, tumors expressing high levels of mFasL overcame the suppressive ocular environment, triggered an inflammatory response, and were subsequently rejected. By contrast, eye tumors expressing low levels of mFasL grew progressively. To evaluate the effect of sFasL on the tumor growth and metastatic potential of ocular FasL-expressing tumors, we compared tumor cell clones that expressed equal amounts of (low) mFasL in the presence or absence of sFasL. Tumor cells transfected with a modified FasL gene expressed only mFasL (noncleavable), grew progressively within the eye, and induced systemic protective immunity that prevented metastatic spread of tumor cells to the liver. Unexpectedly, tumors transfected with wild-type FasL (wtFasL; cleavable), which could produce both sFasL and mFasL, elicited considerably more inflammation and grew more slowly within the eye. However, the cleavable wtFasL eye tumors failed to trigger protective immunity and gave rise to liver metastases. Interestingly, exposure to the ocular environment was required for the wtFasL tumors to gain metastatic potential. We conclude that the fate of FasL-expressing tumors is determined by a combination of the following: (a) the relative proportion of membrane and sFasL, and (b) the local environment that determines the extent of FasL cleavage.     

肿瘤坏死因子相关的凋亡诱导配体对胰腺癌细胞抗肿瘤作用的研究

Objective To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL).Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry.Procaspase-8 and procaspase-3 were determined by Western blot. Results The stable overexpression of TRAIL was detected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore,co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P ＜ 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and themechanisms involved in this effect may be proapoptosis and bystander effect.     

Fas ligand expression in metastatic renal cell carcinoma during interleukin-2 based immunotherapy: no in vivo effect of Fas ligand tumor counterattack.

PURPOSE: It has been hypothesized that tumor cells expressing Fas ligand (FasL) might be able to counterattack and neutralize tumor-infiltrating lymphocytes. We assessed the effect of FasL tumor counterattack on the clinical outcome of interleukin-2 (IL-2)-based immunotherapy in metastatic renal cell carcinoma. EXPERIMENTAL DESIGN: Tumor core needle biopsies were obtained before IL-2-based immunotherapy in 86 patients and repeated within the first cycle in 57 patients. Tumor cells expressing FasL and intratumoral lymphocyte subsets expressing CD4, CD8, CD56, and CD57 were analyzed by immunohistochemistry. RESULTS: At baseline, negative FasL staining in tumor cells was seen in 10 of 86 (12%) biopsies, whereas intense FasL staining was seen (a) in fewer than 10% of tumor cells in 26 (30%) biopsies; (b) in 11 to 50% of tumor cells in 25 (29%) biopsies; (c) in 51 to 90% of tumor cells in 18 (21%) biopsies; and (d) in >90% of tumor cells in 7 (8%) biopsies. On treatment, tumor FasL expression did not change from baseline levels. Moreover, tumor FasL expression was not correlated with objective response or survival whereas the absolute number of CD4(+), CD8(+), CD56(+), and CD57(+) cells per mm(2) tumor tissue at baseline was significantly higher in responding patients compared with nonresponding patients (P = 0.01, P = 0.008, P = 0.015, and P < 0.001, respectively). During the first course of immunotherapy, the absolute number of CD4(+), CD8(+), and CD57(+) cells per mm(2) tumor tissue was significantly higher in responding patients compared with nonresponding patients (P = 0.034, P < 0.001, and P < 0.001, respectively). However, no correlation was observed between the number of intratumoral lymphocytes and tumor FasL expression level. CONCLUSION: These observations do not support the hypothesis that FasL tumor "counterattack" has an effect on the clinical outcome in metastatic renal cell carcinoma during IL-2-based immunotherapy.     

Fas and Fas ligand interactions in malignant disease

Apoptosis has been implicated in tumor development and progression. Fas (CD95) and Fas ligand (FasL) are an interacting receptor ligand pair that elicits apoptosis in many cell types. Although originally described as proteins regulating peripheral immune tolerance, accumulating evidence suggests that Fas/FasL may play an important role in carcinogenesis, tumor outgrowth, and metastasis. This review summarizes our current knowledge about the regulation of Fas and FasL expression, Fas signaling, soluble Fas production, the role(s) of Fas and FasL in hematopoietic and non-hematopoietic tumorigenesis and progression, and the potential application of Fas-induced apoptosis in cancer therapy.     

Human pancreatic cancer cells express non-functional Fas receptors and counterattack lymphocytes by expressing Fas ligand; a potential mechanism for immune escape

The aim of this study was to investigate the expression and functional status of Fas ligand (FasL) and its receptor (Fas) in human pancreatic cancers. Using RT-PCR and Western blotting, Fas and FasL were expressed in seven surgically resected pancreatic cancer specimens and five cell lines; Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2. In the resected specimens, pancreatic cancer cells induced apoptosis in the surrounding lymphoid cells. In coculture experiments of pancreatic cancer and Jurkat T cells, 50% of Jurkat T cells underwent apoptosis after 2 days, however, almost all pancreatic cancer cells remained viable. In addition, by testing Fas function using anti-Fas antibody (CH11), all cell lines were resistant to Fas-mediated apoptosis except Capan-1 cells which showed sensitivity similar to that of Jurkat T cells. These results suggest that pancreatic cancer cells evade immune surveillance by expression of FasL and non-functioning Fas that allow them to activated T-cells. These tumor escape mechanisms may contribute to the rapid fatal course of pancreatic cancer.     

Fas and Fas ligand expression in tumor cells and in vascular smooth-muscle cells of colonic and renal carcinomas.

CD95/APO-1 ligand (FasL) is implicated in the maintenance of immune privileged sites by inducing apoptosis of activated infiltrating T lymphocytes. Therefore, progressive tumors might express high levels of FasL and develop as immune privileged sites. In this study, we investigated the expression of FasL and CD95/APO-1 (Fas, the FasL-receptor) in vitro in rat adenocarcinoma cell lines and the localization in situ in normal human kidney and colon and in their adenocarcinomas. The rat cell line PROb (a progressive tumor in vivo) expressed a higher level of FasL than the sister cell line REGb (a regressive tumor in vivo), as detected by flow cytometry. The 2 cell lines expressed the same level of Fas, but were resistant to FasL-induced apoptosis. In human tissue, both kidney and colon extracts expressed FasL by Western blot. Further investigations, using immunohistochemical staining of paraffin sections, showed that normal colon mucosa expressed Fas and FasL in crypt epithelial cells in the subnuclear compartment. Normal kidney showed Fas and FasL labeling mostly restricted to epithelial cells of proximal tubules and Henlé's loop, showing that this expression is not uniform throughout the organ. Smooth-muscle cells of muscularis propria and blood vessels in and around the tumors were also intensely but more uniformly labeled. In colon-cancer cells, FasL expression remained strong, whereas Fas expression was significantly reduced. A similar reduction in Fas expression was noted in renal-cancer cells. Tumor-infiltrating immune cells of the macrophage lineage do not express FasL. Our results show that smooth-muscle cells of muscularis propria and blood vessels are able to express FasL and to a slight extent Fas. In normal epithelial cells of colon and kidney, Fas and FasL are often co-expressed. The reduced expression of Fas in corresponding cancer cells in combination with the ability to express FasL might facilitate immune escape.     

Reciprocal co-expression of Fas and Fas ligand in human cholangiocarcinoma

We have previously characterized the role of Fas in tumorigenesis using two cholangiocarcinoma cell lines expressing high (Fas(H)) and low (Fas(L)) levels of Fas. Here we further characterize Fas ligand (FasL) expression and function in these two cell lines. The Fas(L) cells expressed a high level of FasL, whereas the Fas(H) cells expressed a low level of FasL showing reciprocal expression of Fas and FasL in tumor cells. FasL released from the Fas(L) cells is capable of inducing apoptosis of lymphocytes, which is blocked by neutralizing Fas antibody. To study the underlying mechanism for the reciprocal expression of Fas and FasL, we examined the activities of both the Fas and FasL promoters. The activity of the Fas promoter is suppressed and the activity of the FasL promoter is stimulated in the Fas(L) cells compared to the Fas(H) cells. The inverse activities of Fas and FasL promoter in tumor cells are regulated by NF-kappaB, which inhibits Fas expression and increases FasL expression through binding to their respective promoters. The inverse expression of Fas and FasL in tumor cells is partially reversed by an NF-kappaB inhibitor. In conclusion, human cholangiocarcinoma cells reciprocally co-express functional Fas and FasL, which are the result of the activities of the Fas and FasL promoters being regulated by NF-kappaB. These findings provide a potential unifying molecular mechanism for modulating tumorigenesis via Fas/FasL expression.     

Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium

Summary Introduction: T cell presence in TIL, and the ratio of CD8⁺ and CD4⁺ T cell subsets in particular, can correlate with tumor prognosis in some tumors, although the significance of such infiltration into glioma is controversial. However, gliomas represent a lower extreme in their extent of T cell infiltration, and are thus useful in assessing factors that can decrease T cell presence within tumor tissue. Fas ligand, a pro-apoptotic cell surface protein, may play a key role in reduction of T cells in tumor tissue. Objective: To assess the level of FasL expression on brain tumor endothelium and to correlate this with relative levels of CD4⁺ and CD8⁺ T cell subsets in TIL from brain tumors. Methods: CD3⁺, CD4⁺, and CD8⁺ cells were quantified in fresh TIL by flow cytometry. Paraffin embedded sections of tumors, including meningiomas and gliomas as well as extracranial malignancies, underwent immunohistochemical staining for FasL and Von-Willebrand’s factor (Factor VIII) to determine expression levels of endothelial FasL. Results: FasL expression was high in aggressive intracranial malignancies compared to more indolent neoplasms, and correlated inversely with CD8⁺/CD4⁺ TIL ratios in all tumor classes combined (ANOVA,p<0.05). Conclusion: Low levels of T cells within TIL, as well as low CD8⁺/CD4⁺ TIL ratios appear to be a property of parenchymal tumor presence. Together with the inverse correlation seen between FasL expression and CD8⁺/CD4⁺ TIL ratios, the high levels of endothelial FasL expression in gliomas suggests that FasL decreases T cell presence in brain tumors in a subset-selective manner, thus contributing to glioma immune privilege.     

Fas ligand breaks tolerance to self-antigens and induces tumor immunity mediated by antibodies

The role of Fas ligand (FasL) in programmed cell death via interaction with its receptor Fas is well characterized. It has been proposed that expression of FasL can confer immune privilege to some organs, allowing them to kill infiltrating lymphocytes and inflammatory cells. However, a number of studies have shown that when tumors or transplants express FasL, rejection often occurs as a consequence of proinflammatory functions of FasL. Here we demonstrate that FasL elicits tumor immunity in a murine melanoma model with weak immunogenicity and low expression of major histocompatibility complex (MHC) class I. We show that protected mice recognize melanocyte differentiation self-antigens. Importantly, tumor immunity is mediated by antibodies, as it can be transferred by serum from protected mice.     

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells

We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2-7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.     

Expression of Fas and Fas ligand in Hodgkin's disease.

Fas and Fas ligand expression were investigated in twenty two cases of classical Hodgkin's disease (HD) by immunohistochemistry. While Reed-Sternberg (RS) cells in 7/22 (32%) cases expressed Fas ligand, reactive lymphoid cells expressed Fas ligand in only 2 (9%) cases. In 20/22 (91%) cases, the RS cells expressed Fas. A higher proportion of RS cells in the nodular sclerosis subtype expressed Fas as compared to the mixed cellularity subtype. In 18/22 (82%) cases, Fas expression was also noted in the reactive lymphoid cells. In eight cases, the reactive lymphoid cells were also analyzed by flow cytometry and a majority of them were CD4+CD45RO+. Most of these activated T-cells expressed Fas but were negative for Fas Ligand. To investigate the co-expression of Fas and Fas Ligand in the RS cells, six cases were subjected to Fas and Fas ligand immunostaining on consecutive sections. The co-expression was documented in the RS cells in four of six cases. These six cases with expression of both Fas and Fas ligand were investigated for the incidence of apoptosis. There was no statistically significant relationship between expression of Fas on reactive cells, expression of FasL on RS cells and the proportion of apoptotic reactive cells. In all these cases apoptosis was not observed in the RS cells. Thus Fas - FasL interactions may not lead to apoptosis of the RS cells.     

Autocrine secretion of Fas ligand shields tumor cells from Fas-mediated killing by cytotoxic lymphocytes

Mechanisms responsible for resistance of tumors to death receptor-mediated damage by cytotoxic lymphocytes are not well understood. Uveal melanoma cells expressed Fas but were insensitive to Fas triggering induced by bystander cytotoxic T lymphocytes or a Fas-specific agonistic antibody; this could not be ascribed to tumor counterattack against T cells or general resistance of the tumors to apoptosis. Treatment with inhibitors of metalloproteases rendered uveal melanomas sensitive to Fas-mediated cytotoxicity. Metalloprotease inhibitors did not affect the expression of Fas but increased the surface expression of Fas ligand (FasL), which correlated with the disappearance of soluble FasL from culture supernatants of tumor cells. FasL eluted from the surface of uveal melanomas specifically inhibited cytotoxic T lymphocyte lysis of tumor cells pretreated with an inhibitor of metalloproteases. In addition to uveal melanomas, a number of other tumor cell lines of various cellular origins were sensitized to Fas-mediated cytotoxicity by metalloprotease inhibitors. Our results show that autocrine secretion of FasL shields tumor cells from Fas-mediated killing by cytotoxic lymphocytes. This defines a novel mechanism of tumor escape from immune surveillance.     

Expression of Fas ligand in retinoblastoma

BACKGROUND: The importance of the Fas-Fas ligand (FasL) mechanism for the immune evasion by tumors provided a strong rationale for the examination of FasL expression in retinoblastoma. In an earlier publication, the authors reported that invasive retinoblastomas decreased Fas expression. Because to the authors' knowledge there is not much information regarding the effect of FasL expression on retinoblastoma, the authors studied the expression of FasL in retinoblastoma and correlated it with invasiveness. METHODS: Thirty-six archival retinoblastoma specimens were divided into 2 groups. Group A (n = 17) was comprised of specimens from tumors with no invasion and Group B (n = 19) was comprised of specimens from tumors with invasion of the choroid (focal, diffuse), optic nerve (laminar, postlaminar, surgical end), and orbit. Sections were immunostained with a monoclonal antibody to FasL and the immunoreactivity was assessed. RESULTS: In Group A, FasL was negative in 100% (17 of 17) of the tumor specimens. In Group B, FasL was expressed in 79% (15 of 19) of the tumor specimens (positive in 9 tumors and heterogeneous in 6 tumors). The difference in FasL expression between the two groups was significant (P < 0.001) CONCLUSIONS: Increased expression of FasL was observed in specimens taken from patients with aggressive tumors. Thus, Loss of Fas and gain of aberrant FasL expression were common features of malignant transformation. The data suggested that the Fas/FasL pathway is potentially immunosuppressive and may be involved in the escape of retinoblastoma cells from immune destruction.     

Clinicopathological significance of Fas and Fas ligand expressions in esophageal cancer

Esophageal carcinomas have recently been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. However, the prognostic importance of Fas/FasL and their correlation with clinicopathological characteristics are yet to be delineated in this highly malignant carcinoma. Specimens from 106 esophageal squamous cell carcinoma patients were used for immuno-histochemical evaluation of Fas, FasL, and CD8 expressions. Fifty-two (49%) and 34 (32%) patients were positive for FasL and Fas, respectively. There were no associations between FasL expression and clinicopathological characteristics except lymph vessel invasion. Strong FasL expression correlated with significant (P < 0.001) decrease in tumor nest CD81 cells. However, neither FasL nor CD81 had any impact on patient survival. Strong Fas expression was correlated with depth of invasion (40.3% in pT1, T2 versus 20.5% in pT3, T4; P5 0.0308), histological differentiation (45.7% in well versus 25.4% in nonwell; P < 0.05), and lymph node metastasis (22.6% in positive versus 45.5% in negative; P < 0.01). Fas expression was one of the independent favorable prognosticators for patients’ survival (risk ratio, 3.26; P < 0.01) in esophageal SCC. Fas expression was an independent prognosticator for recurrencefree survival, whereas FasL expression did not influence the survival in esophageal squamous cell carcinoma. Down-regulation of tumor Fas may be the hallmark of immune privilege for the tumor, thus causing the patients’ poorer outcome. Tumor FasL may counterattack the host immune cells to such an extent that the prognosis is not affected.     

Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity

Recent evidence suggests that one mechanism whereby cytotoxic drugs, such as doxorubicin, kill tumors is the induction or up-regulation of Fas ligand (FasL) expression on the tumor cell surface. The ensuing engagement of Fas by FasL on adjacent cells leads to apoptosis. However, despite cytotoxic drug-induced FasL expression, Fas-sensitive tumors frequently resist chemotherapy, suggesting that they may possess a mechanism that prevents or inactivates Fas-FasL interactions. In the present work, we addressed the involvement of the FasL/Fas signaling pathway in doxorubicin-induced apoptosis and the ability of matrix metalloproteinases (MMPs) to proteolytically cleave FasL in tumor cells. Doxorubicin-induced apoptosis was inhibited by expression of soluble Fas or incubation of the tumor cells with MMP-7 but not with MMP-2 or MMP-9. Resistance to doxorubicin was also induced by expression in the tumor cells of constitutively active MMP-7 but not of a catalytically inactive mutant. Conversely, inhibition of MMP-7 expression in tumor cells by transfection of MMP-7 cDNA in antisense orientation resulted in sensitization to doxorubicin. MMP-7 efficiently cleaved recombinant FasL in vitro and reduced cell surface FasL expression. Our observations provide evidence that one mechanism whereby MMP-7 may promote tumor survival and resistance to doxorubicin is by cleaving FasL and reducing its effectiveness in triggering Fas-mediated apoptosis.     

Up-regulation of Fas ligand and down-regulation of Fas expression in oral carcinogenesis

An important molecule involved in delivering the death signal that initiates apoptosis is called Fas, or Apo-1, which sits on the cell surface. When another molecule called the Fas ligand (FasL) binds to it, Fas triggers a series of events inside the cell that leads to apoptosis. In order to investigate the mechanism of immune escape and the expression of Fas and FasL in oral premalignant lesions (OPLs) and oral squamous cell carcinomas (OSCCs), a total of 64 samples were evaluated by an immunohistochemical method using a labelled streptavidin–biotin assay. These samples comprised nine hyperkeratotic and 24 oral premalignant lesions (nine of mild, moderate, and six of severe dysplastic lesions), and 24 OSCCs, together with seven healthy controls. The results demonstrated that the majority of invasive OSCCs showed down-regulation of Fas expression but up-regulation of FasL expression. These phenomena were also detected in OPLs. The results indicate that the expression of Fas and FasL is involved in oral carcinogenesis and this may be a mechanism by which the cancer cells evade the host immune assault. Perhaps, in future, Fas/FasL system may be used as a prognostic biomarker in predicting the behavior of oral premalignant lesions.     

Fas/Fas ligand system and apoptosis induction in testicular carcinoma

BACKGROUND: Tumor-infiltrating, Fas ligand (FasL)-expressing lymphocytes are able to eliminate Fas-bearing tumor cells by apoptosis induction. Activated cytotoxic T-cells that express Fas may enter apoptosis in the presence of FasL tumor cells. To date, no studies of patients with testicular carcinoma have correlated the differential expression of Fas and FasL in both cell types with the corresponding apoptotic index (AI). METHODS: Fas and FasL were investigated immunohistochemically in paraffin embedded tissue sections from 25 patients with nonseminomatous testicular tumors. The percentages of positive cells and the ratios of Fas cells to FasL cells were correlated with the AI of tumor cells and lymphocytes, respectively, using Spearman correlations. RESULTS: No association was found between the rate of FasL positive cells and AI of the other cell type or between the rate of Fas positive cells and the AI of the same cell type. Ratios between Fas positive cells and FasL positive cells were not correlated with the AI; however, a significant positive correlation was found between the AI of tumor cells and the AI of lymphocytes. CONCLUSIONS: It seems unlikely that the Fas/FasL system is responsible for immune escape of the tumor in testicular carcinoma. Rather, the significant positive correlation between the AIs of tumor cells and lymphocytes implicate a previously unknown mechanism of apoptosis induction in both cell types.