Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells.
Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In addition, chromatin immunoprecipitation assay further confirmed the results of reporter gene studies by showing the specific binding of NFAT2 to the -201 to +71 region.
In the present study, we demonstrated that NFAT2 directly stimulates transcription of FasL in high level CORT-treated mLTC-1. In conclusion, the present study provides further evidence for our finding that CORT-induced FasL expression in Leydig cells is mediated by NFAT.
The FAS and FASL system plays a substantial role in apoptosis and immune escape of cells. Three polymorphisms located in the promoter regions of FAS (-1377G/A and -670A/G) and FASL (-844T/C) have been shown to alter the transcriptional activity of the genes, respectively. This study was conducted to evaluate the effects of these polymorphisms on the susceptibility of neuroblastoma in the Chinese population. A total of 203 patients with neuroblastoma and 411 controls were recruited in this case-control study. Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) was applied for genotyping. Unconditional logistic regression was used to estimate cancer risk by calculating odds ratios (ORs) and their 95% confidence intervals (95% CIs). It was observed that significantly increased risks of neuroblastoma associated with FAS -1377G/A and FASL -844T/C polymorphisms, with ORs equal to 1.55 (95% CI, 1.10–2.20) for FAS -1377 A allele and 2.90 (95% CI, 2.04–4.12) for FASL -844CC genotype carriers compared with non-carriers, respectively. However, no association was found between the polymorphisms of FAS -670A/G and risk of neuroblastoma. In addition, the cumulative effect of FAS and FASL polymorphisms on risk of neuroblastoma was observed (P for trend = 2.502×10−10), with OR for the carriers of both FAS -1377A allele and FASL -844CC genotypes equaled to 3.95 (95% CI, 2.40–6.51). This work reveals that polymorphisms of FAS -1377G/A and FASL -844T/C but not FAS -670A/G are associated with risk of neuroblastoma in Chinese. These findings support the hypothesis that genetic polymorphism in FAS/FASL death system may influence individual susceptibility to neuroblastoma.
AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect.
METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay.
RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P<0.01). SGC-7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC-7901 cells (z = 2.06-44.30, P<0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs 30.5±2.3%, P<0.05).
CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.
Gastric cancer; FasL gene; B7-1 gene; Gene therapy; Synergistic effect
Background: The FAS and FASL system plays a key role in regulating apoptotic cell death and corruption of this signalling pathway has been shown to participate in immune escape and tumorigenesis. There is reduced expression of FAS but elevated expression of FASL in many types of human cancers including lung cancer. We recently reported an association between functional polymorphisms in FAS (–1377G→A) and FASL (–844T→C) and risk of oesophageal cancer.
Objective: To examine the contribution of these polymorphisms to risk of developing lung cancer.
Methods: Genotypes of 1000 lung cancer patients and 1270 controls were analysed by PCR based restriction fragment length polymorphism. Associations with risk of lung cancer were estimated by logistic regression.
Results: Compared with non-carriers, there was a 1.6 fold excess risk of developing lung cancer for carriers of the FAS –1377AA genotype (odds ratio (OR) 1.59, 95% confidence interval (CI) 1.21 to 2.10; p = 0.001), and 1.8 fold excess risk (OR 1.79, 95% CI 1.26 to 2.52; p = 0.001) for carriers of FASL –844CC. Gene–gene interaction of FAS and FASL polymorphisms increased risk of lung cancer in a multiplicative manner (OR for the carriers of both FAS –1377AA and FASL –844CC genotypes 4.18, 95% CI 2.83 to 6.18). Gene–environment interaction of FAS or FASL polymorphism and smoking associated with increased risk of lung cancer was also found.
Conclusion: These results are consistent with our initial findings in oesophageal cancer and further support the hypothesis that the FAS and FASL triggered apoptosis pathway plays an important role in human carcinogenesis.
Apoptosis through Fas/Fas ligand (FasL) is an important regulator of immune system homeostasis but its role in bone homeostasis is elusive. We systematically analyzed: a) the expression of Fas/FasL during osteoblastogenesis and osteoclastogenesis in vitro, b) the effect of FasL on apoptosis and osteoblastic/osteoclastic differentiation, and c) osteoblastogenesis and osteoclastogenesis in mice deficient in Fas or FasL. The expression of Fas increased with osteoblastic differentiation. Addition of FasL weakly increased the proportion of apoptotic cells in both osteoclastogenic and osteoblastogenic cultures. In a colony forming unit assay, FasL decreased the proportion of osteoblast colonies but did not affect the total number of colonies, indicating specific inhibitory effect of Fas/FasL on osteoblastic differentiation. The effect depended on the activation of caspase 8 and was specific, as addition of FasL to osteoblastogenic cultures significantly decreased gene expression for runt-related transcription factor 2 (Runx2) required for osteoblastic differentiation. Bone marrow from mice without functional Fas or FasL had similar osteoclastogenic potential as bone marrow from wild-type mice, but generated more osteoblast colonies ex vivo. These colonies had increased expression of the osteoblast genes Runx2, osteopontin, alkaline phosphatase, bone sialoprotein, osteocalcin, and osteoprotegerin. Our results indicate that Fas/FasL system primarily controls osteoblastic differentiation by inhibiting progenitor differentiation and not by inducing apoptosis. During osteoclastogenesis, Fas/FasL system may have a limited effect on osteoclast progenitor apoptosis. The study suggests that Fas/FasL system plays a key role in osteoblastic differentiation and provides novel insight into the interactions between the immune system and bone.
Apoptosis; Cell Differentiation; Monocytes/Macrophages; Stromal Cells; Transgenic/Knockout Mice
FAS and its ligand FASL are crucial in apoptotic cell death. Loss of FAS and gain of aberrant FASL expression are common features of malignant transformation. This study was designed to investigate whether the functional polymorphisms of FAS -1377G/A (rs2234767) and FASL -844T/C (rs763110) affect the risk of developing breast cancer. Genotypes were analyzed by a polymerase chain reaction-restriction fragment length polymorphism assay in 436 breast cancer patients and 496 healthy controls. In this study, as compared to the wild-type homozygote and heterozygote, the distribution of the FAS -1377GG, GA and AA genotypes among breast cancer patients were significantly different from those among healthy controls (P=0.011), with the AA genotype being more prevalent among patients than the controls (P=0.003). Similarly, the frequencies of the FASL -844TT, TC and CC genotypes also significantly differed among breast cancer patients and healthy controls (P<0.001), with the CC genotype being significantly over-represented in breast cancer patients compared with the controls (P<0.001). In the unconditional logistic regression model following adjustment for age, the subjects carrying the FAS -1377AA genotype had a 1.75-fold increased risk [95% confidence interval (CI), 1.13–2.69] for development of breast cancer compared with patients carrying the GG genotype. Similarly, in the recessive model, the FASL -844CC genotype significantly increased the risk of breast cancer with an odds ratio (OR) of 1.92 (95% CI 1.46–2.54) compared with the TT or TT + TC genotypes. Our results suggest that functional polymorphisms in the death pathway genes FAS and FASL significantly contribute to the occurrence of breast cancer.
breast cancer; FAS; FASL; risk
Fas signaling promotes metastasis of gastrointestinal (GI) cancer cells by inducing epithelial-mesenchymal transition (EMT), and EMT acquisition has been found to cause cancer chemoresistance. Here, we demonstrated that the response to chemotherapy of GI cancer patients with higher expression of FasL was significantly worse than patients with lower expression. Fas-induced activation of the ERK1/2-MAPK pathway decreased the sensitivity of GI cancer cells to chemotherapeutic agents and promoted the expression of P-glycoprotein (P-gp). FasL promoted chemoresistance of GI cancer cell via upregulation of P-gp by increasing β-catenin and decreasing miR-145. β-catenin promoted P-gp gene transcription by binding with P-gp promoter while miR-145 suppressed P-gp expression by interacting with the mRNA 3′UTR of P-gp. Immunostaining and qRT-PCR analysis of human GI cancer samples revealed a positive association among FasL, β-catenin, and P-gp, but a negative correlation between miR-145 and FasL or P-gp. Altogether, our results showed Fas signaling could promote chemoresistance in GI cancer through modulation of P-gp expression by β-catenin and miR-145. Our findings suggest that Fas signaling-based cancer therapies should be administered cautiously, as activation of this pathway may not only lead to apoptosis but also induce chemoresistance.
Fas signaling; epithelial-mesenchymal transition; chemoresistance; gastrointestinal cancer
AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line.
METHODS: An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice.
RESULTS: SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91±8 vs 60±5, P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60±5 vs 50±2, P<0.05). In addition, G1-phase arrest (67.75±0.39 vs 58.03±2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%, P<0.0001).
CONCLUSION: Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.
FasL gene; Gene transfer; Apoptosis; Carcinoma; Gastrocellular; Gene therapy
Cervical cancer is one of the most important cancers in African women. Polymorphisms in the Fas (FasR) and Fas ligand (FasL) genes have been reported to be associated with cervical cancer in certain populations. This study investigated whether these polymorphisms are associated with cervical cancer or human papillomavirus (HPV) infection in South African women.
Participants were 447 women with invasive cervical cancer (106 black African and 341 women of mixed-ancestry) and 424 healthy women controls, matched by age, (101 black African and 323 women of mixed-ancestry) and domicile (rural or urban). Two polymorphisms in Fas gene (FasR-1377G/A, FasR-670A/G) and one in FasL gene (FasL844T/C) were genotyped by TaqMan. None of the polymorphisms, or the Fas haplotypes, showed a significant association with cervical cancer. There was also no association with HPV infection in the control group. However, on analysis of the control group, highly significant allele, genotype and haplotype differences were found between the two ethnic groups. There were generally low frequencies of FasR-1377A alleles, FasR-670A alleles and FasL-844C alleles in black women compared to the women of mixed-ancestry.
This is the first study on the role of Fas and FasL polymorphisms in cervical cancer in African populations. Our results suggest that these SNPs are not associated with cervical cancer in these populations. The allele frequencies of the three SNPs differed markedly between the indigenous African black and mixed-ancestry populations.
During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L) expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period.
RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat) and three mouse (CMT93, CT26, B16F10) cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody.
Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent.
These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.
Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E2 (PGE2), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE2 increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E2-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE2 positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE2.
Fas ligand; prostaglandin E2; regulation; tumour immune evasion
The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.
The Wnt signaling pathway plays a crucial role during embryonic development and in the maintenance of stem cell populations in various organs and tissues. Aberrant activation of this pathway through different mechanisms participates in the onset and progression of several types of human cancers. In the presence of Wnt ligands, stabilized β-catenin acts as a transcriptional activator to induce the expression of target genes through binding to the TCF/LEF family of transcription factors. Using in vitro and in vivo models, we show that TCF/LEF proteins can be activated independently of β-catenin through cooperation with members of the ATF2 subfamily of transcription factors. This novel alternative mechanism of TCF/LEF activation is constitutively up-regulated in certain hematopoietic tumor cells, where it regulates the expression of TCF/LEF target genes and promotes cell growth.
being immunogenic, gastric cancers overcome antitumour immune responses
by mechanisms that have yet to be fully elucidated. Fas ligand (FasL)
is a molecule that induces Fas receptor mediated apoptosis of activated
immunocytes, thereby mediating normal immune downregulatory roles
including immune response termination, tolerance acquisition, and
immune privilege. Colon cancer cell lines have previously been shown to
express FasL and kill lymphoid cells by Fas mediated apoptosis in
vitro. Many diverse tumours have since been found to express FasL
suggesting that a "Fas counterattack" against antitumour immune
effector cells may contribute to tumour immune escape.
Aim—To ascertain if
human gastric tumours express FasL in vivo, as a potential mediator of
immune escape in stomach cancer.
paraffin wax embedded human gastric adenocarcinomas.
was detected in gastric tumours using immunohistochemistry; FasL mRNA
was detected in the tumours using in situ hybridisation. Cell death was
detected in situ in tumour infiltrating lymphocytes using terminal
deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL).
expression of FasL was detected in all 30 resected gastric
adenocarcinomas examined. In the tumours, FasL protein and mRNA were
co-localised to neoplastic gastric epithelial cells, confirming
expression by the tumour cells. FasL expression was independent of
tumour stage, suggesting that it may be expressed throughout gastric
cancer progression. TUNEL staining disclosed a high level of cell death
among lymphocytes infiltrating FasL positive areas of tumour.
gastric adenocarcinomas express the immune downregulatory molecule,
FasL. The results suggest that FasL is a prevalent mediator of immune
privilege in stomach cancer.
Fas ligand; gastric cancer; immune escape; apoptosis; tumour; mRNA
Fas ligand (FasL) induces apoptosis in Fas-bearing target cells, such as leukocytes, and up-regulation of FasL expression on the endothelium may contribute to anti-inflammatory reactions that attenuate leukocyte extravasation during inflammation. Since oxidants generated during inflammation and cigarette smoking may modulate endothelial function, we examined the effect of H2O2 and cigarette smoke on endothelial FasL expression.
Human umbilical vein endothelial cells (HUVECs) were exposed to nontoxic concentrations of H2O2 and cigarette smoke extracts (CSE). Membrane FasL expression was assessed by immunostaining with anti-FasL antibody followed by either monolayer-cell-based spectrofluorimetry or flow cytometry. Soluble FasL in culture supernatants was measured by enzyme-linked immunosorbent assay. For the cytotoxic assay, HUVECs were exposed to H2O2 and co-cultured with neutrophils. Neutrophils were stained by a peroxidase/diaminobenzidine-based reaction, and apoptosis was evaluated on the basis of nuclear morphology after Giemsa staining. To analyze in vitro FasL expression in arteries, rat thoracic aortas were incubated with H2O2, and paraffin-embedded sections were prepared for immunohistochemistry with anti-FasL antibody.
Exposure of HUVECs to H2O2 dose-dependently increased their levels of both membrane and soluble forms of FasL expression. CSE exposure also caused increased levels of FasL expression, but the increase was partially inhibited by the addition of catalase. When co-cultured with neutrophils, HUVECs exposed to H2O2 significantly promoted neutrophil apoptosis. Rat thoracic aortas incubated with H2O2 exhibited increased FasL expression on their endothelium.
Low levels of oxidative stress increase FasL expression on endothelial cells, thereby potentially reducing leukocyte extravasation and tissue damage.
Fas/Fas ligand (FasL) system is one of the key apoptotic signaling entities in the extrinsic apoptotic pathway. De-regulation of this pathway, i.e. by mutations may prevent the immune system from the removal of newly-formed tumor cells, and thus lead to tumor formation. The present study investigated the association between −1377 G/A (rs2234767) and −670 A/G (rs1800682) polymorphisms in Fas as well as single nucleotide polymorphisms INV2nt −124 A/G (rs5030772) and −844 C/T (rs763110) in FasL in a sample of Iranian patients with breast cancer. This case-control study was done on 134 breast cancer patients and 152 normal women. Genomic DNA was extracted from whole blood samples. The polymorphisms were determined by using tetra-ARMS-PCR method. There was no significant difference in the genotype distribution of FAS rs2234767 polymorphism between cases and controls. FAS rs1800682, FASL rs5030772, and FASL rs763110 genotypes showed significant associations with an increasing risk of breast cancer (odds ratio OR = 3.18, P = 0.019; OR = 5.08, P = 0.012; OR = 2.40, P = 0.024, respectively). In conclusion, FAS rs2234767 was not associated with breast cancer risk. Though, FAS rs1800682, FASL rs5030772, and FASL rs763110 polymorphisms were associated with the risk of breast cancer in the examined population.
Background and Aim
Defective apoptosis is a hallmark of cancer development and progression. Death receptors (DR4, FAS) and their ligands (TRAIL, FASL) are thought to mediate the major extrinsic apoptotic pathway in the cell. SNPs in these genes may lead to defective apoptosis. Hence, the present study aimed to investigate the association of functional SNPs of DR4 (rs20575, rs20576 and rs6557634), FAS (rs2234767) and FASL (rs763110) with gallbladder cancer (GBC) risk.
This case-control study included 400 GBC and 246 healthy controls (HC). Genotyping was carried out by Taqman genotyping assays. Statistical analysis was performed by using SPSS ver16. Meta-analysis was performed using Comprehensive Meta-analysis software (Version 2.0, BIOSTAT, Englewood, NJ) to systematically summarize the possible association of SNP with cancer risk. Functional prediction of these variants was carried out using Bioinformatics tools (FAST-SNP, F-SNP). False discovery rate (FDR test) was used in multiple comparisons.
The DR4 Crs20575Ars20576Ars6557634, Grs20575Ars20576Grs6557634 and Grs20575Crs20576Grs6557634 haplotypes conferred two-fold increased risk for GBC. Among these, the DR4 Crs20575Ars20576Ars6557634 haplotype emerged as main factor influencing GBC susceptibility as the risk was not modulated by gender or gallstone stratification. Our meta-analysis results showed significant association of DR4 rs6557634 with overall cancer risk, GI cancers as well as in Caucasians. We didn't find any association of FAS and FASL SNPs with GBC susceptibility.
The DR4 haplotype Crs20575Ars20576Ars6557634 represents an important factor accounting the patients susceptibility to GBC probably due to decreased apoptosis. However, additional well-designed studies with larger sample size focusing on different ethnicities are required to further validate the results.
Malignant glioma is a lethal form of brain cancer that is very difficult to treat. The aggressive behavior of these neoplasms and their limited responsiveness to therapy has been attributed in part to the ability of these tumors to evade the immune system. Gliomas, like many other solid tumors, express components of numerous immune escape mechanisms, including immunosuppressive proteins such as TGF-β, IL-10, and FasL. Here, we show that FasL expression can support the growth of experimental intracranial glioma. We show that FasL is readily detected in human glioblastoma multiforme clinical specimens. FasL was found to be expressed by three well-characterized rat glioma cell lines (9L, F98, and C6) and glioma cell-derived FasL mediated the death of phytohemagglutinin-stimulated Jurkat T-lymphocytes when cocultured with glioma cells in vitro. We asked if inhibiting 9L-derived FasL altered the growth of experimental glioma. FasL expression knockdown using shRNA reduced the growth of subcutaneous and intracranial 9L gliomas by ∼50% in immune competent Fisher 344 rats. In contrast, FasL expression knockdown had no affect on the growth of intracranial 9L glioma in T-cell deficient athymic rats. Intracranial tumors derived from FasL knockdown 9L glioma cells contained up to 3-fold more tumor infiltrating T-cells than tumors derived from control 9L cells. These results demonstrate that down-regulating FasL expression and/or function in glial malignancies can enhance T-cell tumor infiltration and inhibit tumor growth. The findings suggest that targeting endogenous FasL in glial malignancies could enhance the efficacy of emerging immune-based treatment strategies.
CD95L; Fas ligand; glioma; immunotherapy; immunity
Recent epidemiological studies demonstrated that the incidence of esophageal squamous cell carcinoma (ESCC) is on the increase. Although neoadjuvant chemoradiotherapy (CRT) followed by surgery may improve long-term survival and reduce local recurrence in patients with esophageal cancer, the overall cure rate of esophageal cancer is low. Fas/Fas ligand (FasL) signaling initiates the cell death pathway. The roles of FasL in tumor growth, progression and resistance to treatment have been demonstrated in several malignancies. The aim of this preliminary study was to evaluate Fas/FasL expression in ESCC with neoadjuvant CRT. A total of 20 patients who received neoadjuvant CRT (30–40 Gy; 5-fluorouracil plus cisplatin followed by surgery) were enrolled. We evaluated the expression of Fas, FasL and Ki67 (a proliferative marker) using immunohistochemistry and analyzed the correlations between their expression and clinical outcomes. Additionally, we investigated the association of Fas/FasL expression with peritumoral immune CD8-positive and Foxp3-positive cells. High FasL expression was significantly correlated with disease recurrence (P=0.0134). Patients with high FasL expression exhibited poorer recurrence-free and overall survival (P=0.0102 and 0.0385, respectively). Patients with low Fas and high FasL exhibited significantly poorer recurrence-free survival (P=0.0035). Although statistical significance was not reached, Fas expression appeared to be inversely correlated with Foxp3-positive cells and FasL expression appeared to be inversely correlated with CD8-positive cells. In conclusion, FasL expression was associated with tumor relapse and poor prognosis in patients with ESCC following CRT. Pharmacological control of Fas/FasL signaling may improve therapeutic efficacy and outcome in ESCC patients receiving preoperative CRT.
Fas; Fas ligand; esophageal squamous cell carcinoma; neoadjuvant chemoradiotherapy; prognosis
Background and Objectives
Disruption of apoptosis has been implicated in carcinogenesis. Specifically, various single-nucleotide polymorphisms (SNPs) in apoptotic genes, such as FAS-1377 G/A SNP, have been associated with cancer risk. FAS-1377 G/A SNP has been shown to alter FAS gene promoter transcriptional activity. Down-regulation of FAS and cell death resistance is key to many cancers, but an association between FAS-1377 G/A SNP and cancer risk is uncertain. Therefore, we conducted a meta-analysis of the current literature to clarify this relationship.
From PubMed and Chinese language (CNKI and WanFang) databases, we located articles published up to March 5, 2013, obtaining 44 case-control studies from 41 different articles containing 17,858 cases and 24,311 controls based on search criteria for cancer susceptibility related to the FAS gene -1377 G/A SNP. Odds ratios (ORs) and 95% confidence intervals (CI) revealed association strengths. Data show that the -1377 G allele was protective against cancer risk. Similar associations were detected in “source of control,” ethnicity and cancer type subgroups. Lower cancer risk was found in both smokers with a GG+GA genotype and in non-smokers with the GG+GA genotype, when compared to smokers and nonsmokers with the AA genotype. Males carrying the -1377G allele (GG+GA) had lower cancer incidence than those with the AA genotype. Individuals who carried both FAS-1377(GG+GA)/FASL-844(TT+TC) genotypes appeared to have lower risk of cancer than those who carried both FAS-1377 AA/FASL-844 CC genotypes.
The FAS-1377 G/A SNP may decrease cancer risk. Studies with larger samples to study gene-environment interactions are warranted to understand the role of FAS gene polymorphisms, especially -1377 G/A SNP, in cancer risk.
Although corticotropin-releasing hormone (CRH) and Fas ligand (FasL) have been documented in ovarian carcinoma, a clear association with tumour progression and immuno-escape has not been established. FasL plays an important role in promoting tumour cells' ability to counterattack immune cells. Here, we examined immunohistochemically the expression of CRH, CRHR1, CRHR2 and FasL in 47 human ovarian cancer cases. The ovarian cancer cell lines OvCa3 and A2780 were further used to test the hypothesis that CRH might contribute to the immune privilege of ovarian tumours, by modulating FasL expression on the cancer cells. We found that CRH, CRHR1, CRHR2 and FasL were expressed in 68.1, 70.2, 63.8 and 63.8% of the cases respectively. Positivity for CRH or FasL expression was associated with higher tumour stage. Finally, CRH increased the expression of FasL in OvCa3 and A2780 cells through CRHR1 thereby potentiated their ability to induce apoptosis of activated peripheral blood lymphocytes. Corticotropin-releasing hormone produced by human ovarian cancer might favour survival and progression of the tumour by promoting its immune privilege. These findings support the hypothesis that CRHR1 antagonists could potentially be used against ovarian cancer.
CRH; Fas ligand; ovarian cancer; immune privilege; apoptosis
The FAS receptor–ligand system plays a key role in regulating apoptotic cell death, and corruption of this signaling pathway has been shown to participate in tumor-immune escape and carcinogenesis. We have recently demonstrated (Sun, T., X. Miao, X. Zhang, W. Tan, P. Xiong, and D. Lin. 2004. J. Natl. Cancer Inst. 96:1030–1036; Zhang, X., X. Miao, T. Sun, W. Tan, S. Qu, P. Xiong, Y. Zhou, and D. Lin. 2005. J. Med. Genet. 42:479–484) that functional polymorphisms in FAS and FAS ligand (FASL) are associated with susceptibility to lung cancer and esophageal cancer; however, the mechanisms underlying this association have not been elucidated. We show that the FAS –1377G, FAS –670A, and FASL –844T variants are expressed more highly on ex vivo–stimulated T cells than the FAS –1377A, FAS –670G, and FASL –844C variants. Moreover, activation-induced cell death (AICD) of T cells carrying the FASL –844C allele was increased. We also found a threefold increased risk of cervical cancer among subjects with the FASL –844CC genotype compared with those with the –844TT genotype in a case-control study in Chinese women. Together, these observations suggest that genetic polymorphisms in the FAS–FASL pathway confer host susceptibility to cervical cancers, which might be caused by immune escape of tumor cells because of enhanced AICD of tumor-specific T cells.
Activation-induced transcriptional upregulation of the ligand for Fas (FasL) and the resulting apoptosis of Fas-bearing cells constitute essential steps in a host of normal and pathological processes. Here we describe an activation-inducible cis-acting regulatory element in the fasL promoter that is required for gene expression. Oligonucleotide competition and antibody supershift analyses identified two activation-induced DNA-binding species: Egr-1 (NGFI-A, krox-24, zif268, TIS-8), a transcription factor that has been implicated in growth, differentiation, and apoptosis; and Egr-3 (PILOT), a transcription factor of no previously known function. Activation-induced expression of Egr-3, like that of FasL, was inhibited by cyclosporin A, whereas expression of Egr-1 was unaffected. Transient expression of Egr-3 alone increased fasL promoter activity in a cyclosporin A-insensitive manner, whereas expression of Egr-1 had little effect. Moreover, endogenous fasL mRNA was induced in nonlymphoid cells by forced expression of Egr-3 in the absence of any other stimulus. These studies identify a critical Egr family-binding site in the fasL promoter and demonstrate that activation-induced Egr-3, but not Egr-1, directly upregulates fasL transcription in response to activating stimuli.
Tumors escape immunological rejection by a diversity of mechanisms. In this report, we demonstrate that the colon cancer cell SW620 expresses functional Fas ligand (FasL), the triggering agent of Fas receptor (FasR)-mediated apoptosis within the immune system. FasL mRNA and cell surface FasL were detected in SW620 cells using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining, respectively. We show that SW620 kills Jurkat T cells in a Fas-mediated manner. FasR-specific antisense oligonucleotide treatment, which transiently inhibited FasR expression, completely protected Jurkat cells from killing by SW620. FasL-specific antisense oligonucleotide treatment of SW620 inhibited its Jurkat-killing activity. FasL has recently been established as a mediator of immune privilege in mouse retina and testis. Our finding that colon cancer cells express functional FasL suggests it may play an analogous role in bestowing immune privilege on human tumors. HT29 and SW620 colon cancer cells were found to express FasR mRNA and cell surface FasR using RT-PCR and immunofluorescence flow cytometry, respectively. However, neither of these cells underwent apoptosis after treatment by the anti-FasR agonistic monoclonal antibody CH11. Our results therefore suggest a Fas counterattack model for immune escape in colon cancer, whereby the cancer cells resist Fas-mediated T cell cytotoxicity but express functional FasL, an apoptotic death signal to which activated T cells are inherently sensitive.
Fas/Fas Ligand (FasL) interactions play a significant role in peripheral T lymphocyte homeostasis and in certain pathological states characterized by T cell depletion. In this study, we demonstrate that antigen-presenting cells such as monocyte-derived human macrophages (MDM) but not monocyte-derived dendritic cells express basal levels of FasL. HIV infection of MDM increases FasL protein expression independent of posttranslational mechanisms, thus highlighting the virus-induced transcriptional upregulation of FasL. The in vitro relevance of these observations is confirmed in human lymphoid tissue. FasL protein expression is constitutive and restricted to tissue macrophages and not dendritic cells. Moreover, a significant increase in macrophage-associated FasL is observed in lymphoid tissue from HIV (+) individuals (P < 0.001), which is further supported by increased levels of FasL mRNA using in situ hybridization. The degree of FasL protein expression in vivo correlates with the degree of tissue apoptosis (r = 0.761, P < 0. 001), which is significantly increased in tissue from HIV-infected patients (P < 0.001). These results identify human tissue macrophages as a relevant source for FasL expression in vitro and in vivo and highlight the potential role of FasL expression in the immunopathogenesis of HIV infection.
Despite impressive results obtained in animal models, the clinical use of Fas ligand (FasL) as an anticancer drug is limited by severe toxicity. Systemic toxicity of death ligands may be prevented by using genes encoding membrane-bound death ligands and by targeted transgene expression through either targeted transduction or targeted transcription. Selective induction of tumor cell death is a promising anticancer strategy. A fusion protein is created by fusing the extracellular domain of Fas ligand (FasL) to the peptide arginine-glycine-aspartic acid (RGD) that selectively targets avβ3-integrins on tumor endothelial cells. The purpose of this study is to evaluate the effects of RGD-FasL on tumor growth and survival in a murine hepatocellular carcinoma (HCC) tumor model. Treatment with RGD-FasL displaying an obvious suppressive effect on the HCC tumor model as compared to that with FasL (p < 0.05) and resulted in a more additive effect on tumor growth delay in this model. RGD-FasL treatment significantly enhanced mouse survival and caused no toxic effect, such as weight loss, organ failure, or other treatment-related toxicities. Apoptosis was detected by flow cytometric analysis and TUNEL assays; those results also showed that RGD-FasL is a more potent inducer of cell apoptosis for H22 and H9101 cell lines than FasL (p < 0.05). In conclusion, RGD-FasL appears to be a low-toxicity selective inducer of tumor cell death, which merits further investigation in preclinical and clinical studies. Furthermore, this approach offers a versatile technology for complexing target ligands with therapeutic recombinant proteins. To distinguish the anti-tumor effects of FasL in vivo, tumor and liver tissues were harvested to examine for evidence of necrotic cells, tumor cells, or apoptotic cells by Hematoxylin and eosin (H&E) staining.
FasL; RGD-FasL; apoptosis; tumor targeting