The "Fas counterattack" is not an active mode of tumor immune evasion in colorectal cancer with high-level microsatellite instability.

Microsatellite instability (MSI) is an alternative pathway of colorectal carcinogenesis. It is found in 10% to 15% of sporadic colorectal neoplasms and is characterized by failure of the DNA mismatch-repair system. High-level MSI (MSI-H) is associated with tumor-infiltrating lymphocytes (TILs) and a favorable prognosis. Expression of Fas ligand (FasL/CD95L) by cancer cells may mediate tumor immune privilege by inducing apoptosis of antitumor immune cells. The aim of this study was to investigate the relationship between FasL expression and MSI status in primary colon tumors. Using immunohistochemistry, we detected FasL expression in 91 colorectal carcinoma specimens, previously classified according to the level of MSI as MSI-H (n = 26), MSI-low (MSI-L) (n = 29), and microsatellite stable (n = 36). Tumor-infiltrating lymphocyte density was quantified by immunohistochemical staining for CD3. MSI-H tumors were significantly associated with reduced frequency (P = .04) and intensity (P = .066) of FasL expression relative to non-MSI-H (ie, microsatellite stable and MSI-L) tumors. Higher FasL staining intensity correlated with reduced TIL density (P = .059). Together, these findings suggest that the abundance of TILs found in MSI-H tumors may be due to the failure of these tumor cells to up-regulate FasL and may explain, in part, the improved prognosis associated with these tumors.

Addressing the "Fas counterattack" controversy: blocking fas ligand expression suppresses tumor immune evasion of colon cancer in vivo.

Fas ligand (FasL/CD95L) is a transmembrane protein belonging to the tumor necrosis factor superfamily that can trigger apoptotic cell death following ligation to its receptor, Fas (CD95/APO-1). Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of antitumor immune effector cells-the "Fas counterattack." However, the ability of FasL to mediate tumor immune privilege is controversial due to studies that indicate FasL has both pro- and anti-inflammatory activities. To resolve this controversy and functionally define the role of FasL in tumor immune evasion, we investigated if suppression of endogenously expressed FasL in colon tumor cells resulted in reduced tumor development and improved antitumor immune challenge in vivo. Specifically, FasL expression in CMT93 colon carcinoma cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immunocompetent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells led to increased lymphocyte infiltration. The overall level of neutrophils present in all of the tumors examined was low, with no difference between the tumors, irrespective of FasL expression. Thus, down-regulation of FasL expression by colon tumor cells results in an improved antitumor immune challenge in vivo, providing functional evidence in favor of the "Fas counterattack" as a mechanism of tumor immune evasion.

Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Crystal structure of the priming beta-ketosynthase from the R1128 polyketide biosynthetic pathway.

ZhuH is a priming ketosynthase that initiates the elongation of the polyketide chain in the biosynthetic pathway of a type II polyketide, R1128. The crystal structure of ZhuH in complex with the priming substrate acetyl-CoA reveals an extensive loop region at the dimer interface that appears to affect the selectivity for the primer unit. Acetyl-CoA is bound in a 20 A-long channel, which placed the acetyl group against the catalytic triad. Analysis of the primer unit binding site in ZhuH suggests that it can accommodate acyl chains that are two to four carbons long. Selectivity and primer unit size appear to involve the side chains of three residues on the loops close to the dimer interface that constitute the bottom of the substrate binding pocket.

The Fas signalling pathway and its role in the pathogenesis of cancer.

Tumor cells frequently exhibit de novo expression of Fas ligand (FasL/CD95L). Coupled with resistance to Fas-mediated apoptosis, FasL expression enables many cancers to deliver a pre-emptive strike or 'counterattack' against the immune system. New studies also indicate that FasL expression on tumor cells could confer a double advantage to these cells by stimulating their own proliferation. However, pro-inflammatory effects of FasL have also been observed. New findings are beginning to reconcile the paradoxical effects of FasL, with the clinical significance of the Fas counterattack only beginning to emerge.

Activation of the p38 MAPK and ERK1/2 pathways is required for Fas-induced IL-8 production in colonic epithelial cells.

This paper aims to examine the contribution of all three of the MAP kinase signaling pathways to Fas-induced IL-8 up-regulation in HT29 colon epithelial cells.

Type I insulin-like growth factor receptor expression on colorectal adenocarcinoma cell lines is decreased in response to the chemopreventive agent N-acetyl-l-cysteine.

Increased expression of the type I insulin-like growth factor receptor (IGF-1R) is associated with colon cancer, while the antioxidant N-acetyl-l-cysteine (NAC) is known to suppress colonic proliferation. We demonstrate that NAC down-regulates the expression of IGF-1R on three colorectal adenocarcinoma cell lines (HT29, SW480, and LoVo). NAC also abrogates the proliferative effect of IGF-I on HT29 cells. This indicates a novel mechanism for the therapeutic effects of NAC.

Fas ligand promotes tumor immune evasion of colon cancer in vivo.

The study of the role of Fas ligand (FasL/CD95L) in tumor immune evasion has been complicated by the discovery that FasL may trigger cytokine secretion and induce inflammation. Antisense suppression of FasL expression by colon tumor cells was used to investigate if a reduction in endogenously expressed FasL in tumors resulted in reduced tumor development and improved anti-tumor immune challenge in vivo. Downregulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immune-competent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells was associated with increased lymphocyte infiltration. Moreover, constitutively expressed FasL was not pro-inflammatory. This study indicates that upregulation of FasL expression by colon tumor cells results in an improved anti-tumor immune challenge in vivo, providing functional evidence in favor of the 'Fas counterattack' as a mechanism of tumor immune evasion.

Targeting the Fas/Fas ligand pathway in cancer.

Fas is a transmembrane receptor that can induce apoptosis after cross-linking with either agonistic antibodies or with Fas ligand (FasL). Although originally described as an important regulator of peripheral immune homeostasis, accumulating evidence suggests that the Fas/FasL system plays an important role in tumour development. In addition to its proapoptotic functions, accumulating evidence demonstrates that Fas can activate numerous nonapoptotic signalling pathways, and that activation of these pathways can result in increased tumourigenicity and metastasis. This review summarises the current understanding of the Fas/FasL system in tumorigenesis and discusses attempts to utilise the Fas/FasL system in the treatment of cancer.

Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR.

OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease. METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene. RESULTS: E. coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections. CONCLUSIONS: E. coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease.

Fas ligand expressed in colon cancer is not associated with increased apoptosis of tumor cells in vivo.

Expression of Fas ligand (FasL/CD95L) may help to maintain colon cancers in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Colon tumor-derived cell lines appear to be relatively insensitive to apoptosis mediated by their own or exogenous FasL in vitro, despite expression of cell surface Fas. In our present study, we sought to investigate if FasL upregulated in human colon cancers leads to any increase in apoptosis of the tumor cells in vivo. FasL and Fas receptor (APO-1/CD95) expression by tumor cells were detected immunohistochemically. Apoptotic tumor cell death was detected by immunohistochemistry for caspase-cleaved cytokeratin-18. FasL expression did not correlate with the extent of apoptosis of tumor cells. There was no significant local difference in the frequency of apoptosis of tumor cells between tumor nests that expressed FasL (mean = 2.4%) relative to those that did not (mean = 2.6%) (p = 0.625, n = 10; Wilcoxon signed rank). FasL expressed by the tumor cells appeared to be functional, since FasL expression in tumor nests correlated with diminished infiltration of tumor-infiltrating lymphocytes (TILs). TILs were detected using immunohistochemistry for CD45. Expression of FasL by tumor nests was associated with a mean 4-fold fewer TILs relative to FasL-negative nests (range 2.4-33-fold, n = 10, p < 0.003). Together, our results indicate that colon tumors are insensitive to FasL-mediated apoptosis in vivo.

Neurokinin-1 receptor (NK-1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P.

We have previously shown that the receptor for substance P (SP), neurokinin-1 receptor (NK-1R), is a marker of human mucosal but not peripheral mononuclear cells. In the present study, we investigate NK-1R expression in the human colonic mucosa in vivo, particularly in the epithelial cells. We investigate the influence of proinflammatory Th1 cytokines and SP on expression and function of NK-1R in colonic epithelial cells in vitro. Using in situ hybridization to detect NK-1R mRNA, and immunohistochemistry to detect NK-1R protein, colonic epithelial cells were found to express NK-1R in vivo. In contrast, colon epithelial cell lines (Caco-2, HT29, SW620, T84) were negative for NK-1R mRNA and protein. However, stimulation with a proinflammatory cytokine cocktail containing IFN-gamma, TNF-alpha, and IL-1beta, caused induction of NK-1R expression. Expression of NK-1R in human colonic epithelial cells in vivo may therefore reflect cytokine conditioning by the mucosal microenvironment. SP did not alter ion transport in monolayers of cytokine-treated T84 cells. While SP stimulated epithelial ion transport in colonic mucosae ex vivo, this was not a direct effect of SP on the epithelial cells, and appeared to be neurally mediated. However, SP (10(-10)-10(-8) M) elicited a dose-dependent proliferative effect on cytokine-stimulated, but not unstimulated, SW620 cells. Proliferation of the epithelial cells in response to SP was mediated specifically via cytokine-induced NK-1R, since an NK-1R-specific antagonist (Spantide 1) completely blocked SP-mediated proliferation in the cytokine-treated cells. Our results therefore demonstrate that proinflammatory cytokines induce expression of NK-1R in human colonic epithelial cell lines, and that SP induces proliferation of the epithelial cells via cytokine-induced NK-1R.