TRAIL-R deficiency in mice promotes susceptibility to chronic inflammation and tumorigenesis (original) (raw)
Monoallelic loss of TRAIL-R promotes lymphomagenesis and metastasis. Loss of p53 or p19ARF is a common event in _c-myc_–driven lymphomas, leading to a more aggressive disease and to resistance to treatment with cyclophosphamide in vivo (17, 18). We hypothesized that aberrations in the p53-dependent regulation of the immune-mediated extrinsic cell death pathway, through the deletion of TRAIL-R, could affect _myc_-driven lymphomagenesis by loss of a putative tumor suppressor activity. To test this hypothesis we crossed mice bearing the Eμ-myc transgene with mice lacking TRAIL-R (the unique proapoptotic TRAIL receptor in the mouse genome). Hemizygous deletion of TRAIL-R (TRAIL-R+/–; n = 30) in the Eμ-myc background led to an increased rate of lymphoma formation as compared with WT (n = 11) animals, whereas no obvious difference with regard to disease-free survival between TRAIL-R+/– and TRAIL-R–/– (n = 14) animals could be found (Figure 1A). Furthermore, the median disease-free survival was significantly different between the TRAIL-R+/– mice and WT littermates as well as between the TRAIL-R–/– mice and WT littermates (87 days for TRAIL-R+/–, 82 days for TRAIL-R–/–, and 119 days for WT mice) in the Eμ-myc background (log-rank test, P = 0.0023 and P = 0.0003, respectively), with an associated increased lymphoma risk of 2.6-fold (95% CI, 1.5–5.9) for TRAIL-R+/– and 3.4-fold (95% CI, 2.4–17.4) for TRAIL-R–/– mice for lymphoma development (Figure 1A).
Monoallelic loss of TRAIL-R promotes _c-myc_–driven lymphomagenesis. (A) Kaplan-Meier survival curves for the different genotypes (TRAIL-R–/–, n = 14; TRAIL-R+/–, n = 30; and WT, n = 11) on the Eμ-myc genetic background. Survival was markedly decreased in TRAIL-R+/– and TRAIL-R–/– mice (log rank test, P = 0.023 and P = 0.003, respectively) relative to WT littermates. Immunohistochemistry for c-myc+ (α-myc) (B and C) and B220+ showed increased metastasis of lymphoma cells (B and data not shown; n = 6/genotype) to liver (n = 5/genotype; average tumor emboli area percentage ± SEM; Student’s t test, P < 0.05) and lung (box-and-whisker plot shows relative median number of B220+ cells/field; Mann-Whitney U test, P < 0.05) in both TRAIL-R+/– and TRAIL-R–/– animals relative to WT animals on the Eμ-myc genetic background.
More aggressive disease was evident as well with increased metastasis, as detected by immunohistochemical analysis for c-Myc (Figure 1, B and C) and B220+ (B cell marker; data not shown) cells in nonlymphoid organs such as the livers and lungs of _TRAIL-R_–deficient animals. Quantitative analysis of immunohistochemically stained sections from the liver and lungs revealed an increased frequency of B220+ cells in the lungs of TRAIL-R+/– and TRAIL-R–/– mice (median B220+, 39 cells/field and 43 cells/field, respectively) relative to that of WT mice (median B220+, 7 cells/field) (Figure 1B). The average percent of liver surface occupied by metastatic lymphoma cells was assessed (Figure 1B) in animals that developed lymphoma. Analysis of H&E-stained sections revealed that TRAIL-R+/– and TRAIL-R–/– had a significantly (P < 0.05, Student’s t test) larger liver area occupied by invading lymphoma cells (13.0% and 12.8% of total analyzed liver area) relative to WT animals (1.4% of total analyzed liver area) on the Eμ-myc genetic background. Thus loss of one allele of TRAIL-R was sufficient to contribute to an increased metastatic potential of _c-myc_–driven lymphomas to lung and liver.
Next we investigated changes in lymphoma proliferation and cell death by performing immunohistochemistry for Ki-67 and cleaved caspase-3 (Figure 2A). Approximately 90%–95% of all cells present in the lymphomas stained positive for Ki-67, in concordance with the previous reports on the aggressiveness of the disease (21). However, we were unable to document any consistent changes in Ki-67 labeling that correlated with any particular genotype. In contrast, monoallelic loss of TRAIL-R led to a marked loss of cells labeling for cleaved (active) caspase-3 (Figure 2, A and B) in lymphomas, suggesting decreased levels of apoptosis may have contributed to the increased aggressiveness of the lymphomas arising in _TRAIL-R_–/– Eμ-myc animals.
TRAIL-R+/– lymphomas show apoptotic defects and reduced TRAIL-R mRNA expression. (A) Immunohistochemistry shows abundant labeling of the proliferation marker Ki-67 (DAB, brown staining) in both WT (+/+) and _TRAIL-R_–deficient (+/– and –/–) lymphomas. Immunohistochemical staining (A) for cleaved caspase-3 and quantification thereof (B) shows fewer median number of cells expressing active caspase-3 (DAB, brown staining) in TRAIL-R+/– and TRAIL-R–/– lymphomas compared with that of WT lymphomas (Mann-Whitney U test, P < 0.05). (C) LOH was detected by PCR analysis of DNA isolated from Eμ-myc TRAIL-R+/– lymphomas in 20% (2 of 10) of the lymphomas (data not shown). (D) RT-PCR analysis on RNA isolated from Eμ-myc lymphomas of different genotypes shows decreased TRAIL-R mRNA expression in Eμ-myc _TRAIL-R+/–_lymphomas compared with RNA isolated from WT Eμ-myc lymphomas. (E) Relative quantitative RT-PCR analysis and densitometry shows that loss of 1 TRAIL-R allele reduced the mean (± SEM) TRAIL-R expression to approximately 60% that in WT lymphomas (n = 4 each genotype; Student’s t test, P < 0.05). (F) Immunofluorescence (FITC, green) on living Eμ-myc lymphoma cells of different TRAIL-R genotypes using the MD-5 antibody suggest expression on WT Eμ-myc lymphoma cells (α–TRAIL-R) with a heterogenous (speckled) membrane distribution as evident from the 2 different focal images. However, expression was barely detectable in TRAIL-R+/– Eμ-myc lymphoma cells and not present in TRAIL-R–/– Eμ-myc lymphoma cells. Representative images are shown from 2 independent lymphomas per genotype. Original magnification, ×100. (G) Flow cytometry analysis on Eμ-myc lymphoma cells suggests expression of TRAIL-R in WT cells but not TRAIL-R+/– and TRAIL-R–/– Eμ-myc lymphoma cells. Eμ-myc lymphoma cells from at least 2 different animals/genotype were analyzed.
Loss of heterozygosity (LOH) is a common event for many well-established tumor suppressor genes. Both INK4A/ARF and Trp53 are subject to LOH in the Eμ-myc model (18). A similar analysis on DNA isolated from enlarged lymph nodes from TRAIL-R+/– animals suggested that loss of the remaining TRAIL-R allele could only be detected in 20% (2/10) of the tumors analyzed (Figure 2C). Although this suggests that signaling through TRAIL-R could be modulated by additional mechanisms and that a TRAIL-resistant phenotype was selected for, LOH constitutes a rare mechanism that is unlikely to explain the lack of effect of TRAIL-R gene dosage. Using RT-PCR analysis to monitor changes in expression levels of TRAIL-R (Figure 2, D and E), we found that expression of TRAIL-R mRNA was intact in lymphomas lacking one allele of TRAIL-R. However, loss of one allele of TRAIL-R reduced expression of TRAIL-R mRNA by 60% of WT lymphomas (Figure 2E) and was associated with a near complete loss of surface expression of TRAIL-R as detected by flow cytometry and immunofluorescence (Figure 2, F and G). Potentially, reduced TRAIL-R expression (together with downstream mechanisms) may be sufficient to quench signaling through the TRAIL-R to the point at which it mimics complete loss of TRAIL-R on the Eμ-myc genetic background.
Sensitivity to TRAIL may be circumvented by several mechanisms, and our data on TRAIL-R+/– lymphomas on the Eμ-myc genetic background suggest that such mechanisms might occur in this model and that this may hamper the response to TRAIL. Therefore, we investigated TRAIL sensitivity in lymphomas in vitro. Indeed, challenging a WT Eμ-myc lymphoma cell line (p54) with recombinant murine TRAIL did not trigger death and only weakly triggered caspase activation as determined by FLICA analysis (Figure 3, A and B). No synergistic killing or caspase activation was observed in the presence of etoposide and TRAIL. We investigated biochemical changes in enlarged thymuses and spleens from WT and TRAIL-R+/– animals suffering from lymphoma. Cellular FLICE-inhibitory protein (c-FLIP) has been proposed to protect cells from TRAIL-induced cell death and has been shown to be directly repressed by c-Myc (22). Thus we reasoned that in this model c-FLIP expression might be selected for during lymphoma development. Changes in c-FLIP protein levels correlated with changes in the TRAIL-R genotype, where decreased expression of c-FLIPs was evident in enlarged thymuses and spleens from TRAIL-R+/– animals as compared with WT animals (Figure 3, C and D). This suggests that high levels of c-FLIPS could be selected for in order to circumvent TRAIL-mediated killing in lymphomas arising in WT animals.
WT Eμ-myc lymphomas are resistant to TRAIL in vitro and express high levels of FLIPs. (A) Challenging isolated WT Eμ-myc lymphoma cells in vitro with TRAIL resulted in lack of cell death (as determined by tryphan blue staining) in comparison with treatment with etoposide. (B) In vitro cell death assays. Propidium iodide uptake (red staining) and fluorescent-labeled inhibitor of caspase activity (FLICA or FITC-VAD-FMK; green staining) suggest that TRAIL (1 μg/ml) only weakly triggers caspase activity and cell death relative to control in comparison with treatment with etoposide (1 μM) in living Eμ-myc lymphoma cells. (C) Total RNA isolated from WT western blot of splenic and thymic lymphomas shows increased expression of c-FLIPS in WT relative to TRAIL-R+/– lymphomas. (D) Densitometric analysis using NIH ImageJ of western blots from lymphomas (splenic and thymic) from WT (n = 10) and TRAIL-R+/– (n = 10) mice. The ratios of the FLIPs band in relation to the actin band (loading control) is shown. The red horizontal band represents median, and the red cross represents the mean. WT lymphomas have a higher FLIPs/actin ratio (P < 0.05, Student’s t test), suggesting a relatively higher FLIPs expression compared with TRAIL-R+/– lymphomas.
We hypothesized that changes in gene expression in Eμ-myc WT lymphomas relative to _TRAIL-R_–deficient lymphomas could reflect _TRAIL-R_–dependent downstream changes in gene expression or changes that may provide protective adaptations to intact TRAIL-R signaling. In order to address these questions, we performed expression profiling on a subset of lymphomas from WT, TRAIL-R+/– and TRAIL-R–/– animals. Fifty-nine genes were found to be differentially expressed and common among WT and _TRAIL-R_–/– animals (Supplemental Figure 1 and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI29900DS1). Using the gene lists of differentially expressed genes for supervised expression-based clustering discriminated between WT and _TRAIL-R_–deficient lymphomas but did not cluster the different _TRAIL-R_–deficient genotypes (i.e., TRAIL-R+/– and TRAIL-R–/–) into distinct groups (Supplemental Figure 1). In addition, 4 genes were TRAIL-R dependently regulated with a false discovery rate of less than 26.7% and were annotated as signal transducers, oncogenes, or involved in cell death (according to the classification of the Gene Ontology Consortium) (Figure 4A). Interestingly, a number of genes were apoptosis and proliferation regulators (i.e., Hspa1b, Socs3, and p21), whereas others implicated a role in transcription (Zfpm1, also known as Fog-1). Hsp70 and Socs3 are downstream targets for the Jak/Stat pathway and have been implicated in the regulation of apoptosis (23, 24), whereas upregulation of the cyclin-dependent kinase inhibitor p21 is a common feature of activated Stat signaling and protection from apoptosis (25, 26). Thus we further hypothesized that TRAIL-R may either regulate Stat-signaling or that increased Stat-signaling may protect some WT lymphomas from immune-mediated cell death by TRAIL. Indeed, western blotting revealed increased levels of phosphorylated (Tyr705) Stat3 present in WT lymphomas as compared with those deficient in TRAIL-R (Figure 4B), and immunohistochemistry suggested that the lymphoma cells expressed phosphorylated Stat3 (Figure 4B). To the best of our knowledge, no report has previously described Stat3 phosphorylation as a result of TRAIL-R signaling, and treatment of the TRAIL-sensitive murine L929 cell line with recombinant TRAIL did not produce any increased levels of phosphorylated Stat3 (Supplemental Figure 2A). However, treating murine L929 cells and human colon cancer HCT116 cells with a combination of the pharmacologic Stat3 inhibitor JSI-124 (cucurbitacin I) at doses below or near the EC50 in combination with recombinant TRAIL triggered synergistic caspase-3 cleavage and cell death that was associated with a reduced expression of the Stat3-responsive gene product survivin in HCT116 cells (Supplemental Figure 2, B–E). This suggests that phosphorylated Stat3 may provide cell-intrinsic protection of cells from TRAIL-induced cell death.
Expression profiling of Eμ-myc lymphomas suggests that differentially expressed genes are regulated independent of TRAIL-R gene dosage as well as a role for Stat3 in WT lymphomas. RNA samples from TRAIL-R+/– and TRAIL-R–/– Eμ-myc lymphomas (n = 5/genotype) were hybridized to MOE430A 2.0 Affymetrix arrays. Differentially expressed genes (upregulated >2-fold) were subjected to statistical analysis. A shows the top 4 genes that showed consistent changes over the different probe sets. Hspa1b, heat-shock protein 70; Socs3, suppressor of cytokine signaling 3; Zfpm1, friend of GATA1 (also known as Fog-1); Cdkn1a, cyclin-dependent kinase inhibitor 1a (p21). *P < 0.05, Student’s t test. (B) Immunoblotting suggests that Tyr705-phosphorylated Stat3 is elevated in WT Eμ-myc lymphomas compared with _TRAIL-R_–deficient lymphomas. Tyr705-phosphorylated Stat3 is expressed in a subset of cells within the lymphomas, as detected by immunohistochemistry. (C) CD244 is expressed at higher levels in Eμ-myc lymphomas of TRAIL-R+/– animals (Mann-Whitney U test, P < 0.05; n = 5/genotype). Red horizontal lines indicate medians; red crosses indicate means. (D and E) _TRAIL-R_–deficient lymphomas (n = 3/genotype; Mann-Whitney U test) show frequent infiltration of CD244+/c-myc cells (Cy3, green; Cy2, red) compared with WT lymphomas, suggesting expression of CD244 on nonlymphoma cells. Representative pictures are shown. Original magnification, ×60 (B); ×100 (E).
Because low levels of phosphorylated Stat3 have been associated with antitumor immunity (27), we hypothesized that blockage of Stat3 phosphorylation might correlate with increased infiltration of immune cells in _TRAIL-R_–deficient lymphomas. Indeed, analysis of the microarray data identified a subset of the TRAIL-R–deficient lymphomas expressing high levels of CD244/2B4 (Figure 4C), a surface molecule on NK cells, CD8+ T cells, eosinophils, and basophils (28). Indeed, _c-myc_–negative cells expressing CD244 were frequently identified in _TRAIL-R_–deficient lymphomas but not in WT lymphomas (Figure 4, D and E), suggesting increased presence of immune infiltrating cells in _TRAIL-R_–deficient Eμ-myc lymphomas that correlated with low levels of Tyr705-phosphorylated Stat3.
TRAIL-R–deficient animals are susceptible to sublethal γ-irradiation–induced pneumonitis. Loss of TRAIL-R has been associated with an attenuated apoptotic response to irradiation, particularly in the spleen and thymus (9). The long-term relevance for the lack of apoptosis in these organs in the context of radiation therapy is unclear. However, ionizing radiation is an established carcinogen in a number of animal models as well as in humans (reviewed in ref. 29). In order to investigate a putative protective effect of TRAIL-R in radiation-induced carcinogenesis, mice were subjected to a sublethal dose of 4 Gy of whole-body irradiation at the age of 4–5 weeks (14 WT, 13 TRAIL-R+/–, and 23 TRAIL-R–/– mice) and monitored for up to 18 months.
Lethality was only observed in the TRAIL-R–/– group of animals at 28–52 weeks following irradiation (Kaplan-Meier test, P = 0.010; Figure 5A), with lower body weight, labored breathing, and hunched body posture recorded on sex- and age-matched _TRAIL-R_–deficient animals at 28 weeks following ionizing irradiation as compared with WT controls (Figure 5B). This revealed systemic disease in irradiated TRAIL-R+/– and TRAIL-R–/– animals relative to WT littermates manifesting as lethality in the more severe cases. Histological examination of the lungs from control (nonirradiated WT), TRAIL-R–/–, and irradiated WT mice (39 weeks) (Figure 5C) showed no abnormalities, whereas TRAIL-R–/– mice that succumbed following sublethal irradiation showed emboli in respiratory bronchioles (Figure 5D) and loss of interstitial alveolar space accompanied by the deposition of fibronectin and collagen, as shown by immunohistochemistry and Masson’s trichrome staining (Figure 5, D and E). Lethality correlated with pulmonary CD3+ and B220– infiltrates, suggesting a T cell origin of the infiltrates (Figure 6A). The infiltrates were accompanied by a subset of cells expressing membrane/cytoplasmic TRAIL (Figure 6A). Substantial proliferation in alveolar/nonlymphoid cells was found as judged by increased labeling by the Ki-67 proliferation marker (Figure 6A), suggesting that the inflammation triggered hyperplasia. Evaluation classified the overall pulmonary condition as moderate to severe chronic bronchopneumonia.
TRAIL-R–/– animals show decreased survival following exposure to a single sublethal dose (4 Gy) of ionizing radiation. (A) Survival following 4 Gy of whole-body irradiation was decreased in the TRAIL-R–/– group (n = 23) at 28–52 weeks following irradiation in comparison with the group of WT (n = 14) animals and TRAIL-R+/– (n = 13) animals (Kaplan-Meier log-rank analysis, P = 0.010). (B) However, decreased body weight was observed in both TRAIL-R+/– (n = 3) and TRAIL-R–/– (n = 4) animals relative to WT (n = 4) animals at 28 weeks following 4 Gy of ionizing irradiation (P < 0.05, Mann-Whitney U test). No weight difference was detected between genotypes in nonirradiated animals (data not shown). (C) A representative H&E staining of the lungs from lethargic TRAIL-R–/– animals irradiated with 4 Gy at 39 weeks prior to sacrifice. Extensive inflammatory emboli in the respiratory bronchioles and increased cellularity in the interstitial space was observed (C; ×20, lower left and right panels). Lungs were histochemically stained with Masson’s trichrome (D) in order to detect the presence of collagen (bright blue) and were analyzed by immunofluorescence for fibronectin (E; Cy3, red). Lungs from irradiated and lethargic TRAIL-R–/– animals showed severe pneumonitis (D) and extensive deposition of collagen and fibronectin (E).
Loss of TRAIL-R leads to increased infiltration of CD3+ cells and tumorigenesis following sublethal irradiation. (A) Immunohistochemistry on lungs from TRAIL-R–/– animals with infiltrates show increased numbers of CD3+ cells but little to no positive staining for B220. Infiltrates contained a number of TRAIL-positive cells expressing cytoplasmic and membrane-bound TRAIL and focal areas that stained positive for Ki-67. (B) Mice lacking 1 or 2 alleles of TRAIL-R show an increased incidence of pulmonary adenomas (top row). Microphotographs show a normal spleen and a splenic lymphoma with metastasis to the liver (bottom row). Lungs from 12 WT, 9 TRAIL-R+/–, and 18 TRAIL-R–/– mice were examined and stained immunohistochemically_._ (C) The frequency per mouse of pulmonary adenomas in relation to bronchopneumonia is shown. WT, n = 12; TRAIL-R+/–, n = 9; TRAIL-R–/–, n = 18. (D) Irradiated TRAIL-R–/– animals show correlation between bronchopneumonia and hyperplasia in their lungs. Hyperplastic adenomatous focal lesions (left panel) and pulmonary adenomas (middle and right panel) in the lungs of irradiated TRAIL-R–/– animals showed positive immunohistochemistry for NF-κB p65. Staining was observed in the cytoplasm and nucleus (right panel) of adenomatous cells. Representative photographs of investigated animals are shown. (E) Lungs of irradiated animals of the different TRAIL-R genotypes (WT, n = 7; TRAIL-R+/–, n = 5; TRAIL-R–/–, n = 10) were blindly classified according to inflammatory grade (bronchopneumonia grade) by the use of histological examination and immunohistochemistry for CD3 and fibronectin. Preneoplasia/neoplasia (hyperplastic) grading was based on combined macroscopic observations, histological findings (H&E staining), and immunohistochemistry for Ki-67. Grade 0, no or only scattered stained cells constituting less than 2% of the section; grade 1, heterogeneous staining with at least 20% of the section showing 2%–10% positive cells; grade 2, at least 20% of the section showing 11%–50% positive cells; grade 3, and at least 20% of the section showing more than 50% positive cells. For hyperplasia/neoplasia, presence of adenoma was classified as grade 4. The _n_-value represented by each data point is shown.
Overall, most organ sites affected (including any manifestation of inflammation, fibrosis, or neoplastic growth not present in nonirradiated animals) by sublethal irradiation were markedly different between TRAIL-R–/– and WT animals (Table 1). A tumor mass found unique to irradiated TRAIL-R–/– animals was a splenic lymphoma with extensive involvement of the liver and lungs observed in an irradiated TRAIL-R–/– animal. This lymphoma did not express surface markers of either B cells (B220) or T cells (CD3) (Figure 6B and data not shown). However, only lesions in the lung showed a statistically significant difference in rate per animal between irradiated TRAIL-R–/–(0.555 tumors per mouse) and WT (0) animals (Fisher’s exact test, P < 0.05). Interestingly, _TRAIL-R–/–_mice showed 0.136 tumors per mouse in the lungs, whereas the corresponding number for TRAIL-R+/– mice was 0.111 (see Table 1 and Figure 6, B and C). However, inflammation and/or fibrosis in the lung (i.e., bronchopneumonia) was more frequently observed in TRAIL-R–/– animals as compared with TRAIL-R+/– (0.500 compared with 0.111 tumors per mouse) mice. Hyperplastic adenomatous focal lesions and pulmonary adenomas (Figure 6D) in the lungs of irradiated TRAIL-R–/– animals frequently overexpressed NF-κB p65, a proinflammatory protein that is associated with tumor progression and metastasis (reviewed in ref. 30). Lungs of irradiated animals of the different TRAIL-R genotypes (WT, n = 7; TRAIL-R+/–, n = 5; TRAIL-R–/–, n = 10) were blindly classified according to inflammatory grade (bronchopneumonia grade) through the use of histological examination and immunohistochemistry for CD3 and fibronectin. The determination of preneoplasia/neoplasia (hyperplastic grade) was based on combined macroscopic observations, histological findings (H&E staining) and immunohistochemistry for Ki-67 (for more detail on classification, see Methods). TRAIL-R–/– animals showed correlation between the severity (grade) of bronchopneumonia and grade of hyperplasia in their lungs. (Figure 6E), suggesting that the presence of hyperplasia was intimately linked to chronic inflammation in the _TRAIL-R_–deficient genetic background. Thus the murine TRAIL-R plays an important role in regulating the long-term tissue response in the lung following a single sublethal dose of ionizing radiation.
Most commonly affected organ sites following sublethal irradiation (4 Gy)
TRAIL-R–/– animals also showed chronic enterocolitis with some areas of ulceration, increased epithelial atrophy, erosion, and inflammatory infiltrates (Figure 7, A and B). These were typical radiation-induced lesions that, at sublethal doses, mostly heal 2–6 weeks after irradiation but may persist for some time. Indeed, we found some lesions in irradiated WT animals at similar time points following irradiation, but with significantly less severity (lower average percentage of total area) in the GI tract compared with irradiated TRAIL-R–/– animals (Figure 7B). Ulceration is, however, a highly abnormal feature after several months following irradiation, and its presence suggests that the TRAIL-R–/– animals suffer from chronic inflammation and impaired healing in the GI tract as well as in the lungs following tissue damage induced by ionizing irradiation. Infiltrates in the GI tract consisted of predominantly CD3+ cells and only a small number of B220+ cells (Figure 7C), suggesting increased infiltration of T cells.
TRAIL-R suppresses chronic colitis following sublethal irradiation. (A) The small intestine (ileum) of WT and TRAIL-R–/– animals sublethally irradiated 32 weeks prior to sacrifice show increased lymphoid infiltration and luminal protrusions in the TRAIL-R–/– animals compared with WT mice. The proximal part of the small bowel (duodenum) showed epithelial atrophy and erosion in an irradiated TRAIL-R–/– animal at 32 weeks following irradiation. Normal colon of a WT animal at 32 weeks following 4 Gy of irradiation is compared with TRAIL-R–/– animals, which showed chronic enterocolitis following the same treatment. Original magnification, ×4 (first and second row, first column); ×10 (first and second row, second column); ×20 (third row); ×40 (fourth row) (fifth row, first column); ×60 (fifth row, second column). (B) The surface area covered with either lymphoid infiltrates or atrophic lesions was assessed from tissue sections of the small intestine and colon from WT (n = 4), TRAIL-R+/– (n = 3), and TRAIL-R–/– (n = 4) mice subjected to 4 Gy of whole-body irradiation. *P < 0.05, Student’s t test. (C) Representative immunohistochemistry for B220 and CD3 on a TRAIL-R–/– small intestine with atrophy shows abundant infiltration of CD3+ cells.
Loss of TRAIL-R leads to susceptibility to chemically induced hepatocarcinogenesis. Ten male 7-day-old littermate WT and TRAIL-R–/– pups were challenged with the DNA-damaging hepatocarcinogen DEN. DEN has been shown to elicit a prominent activation of p53 in the mouse liver when administered i.p. (31). Given that TRAIL-R is a p53 target gene and is induced in the liver following DNA damage (ref. 10 and our unpublished observations), we reasoned that loss of TRAIL-R may contribute to increased initiation of hepatocarcinogenesis, as apoptosis and potentially removal of subsequent mutated cells in the organ may be perturbed. However, we observed only a slight increase in the incidence of DEN-induced HCC in TRAIL-R–/– mice compared with WT mice, where 80% (8/10) of the _TRAIL-R_–deficient animals developed HCC compared with 60% (6/10) of the WT animals at 10 months of age. TRAIL-R–/– mice developed a larger number of macroscopically visible liver nodules by gross observation (depicted in Figure 8A) than WT animals (11 and 8 per animal, respectively), indicating increased presence of neoplastic changes in the liver of TRAIL-R animals following DEN treatment. Detailed histological analysis of the livers from DEN-treated _TRAIL-R_–deficient and WT animals suggested that an increased number of lesions with a radius in excess of 1.0 mm was present in TRAIL-R–/– livers (Figure 8, B and C). However, no significant increase in the number of the DEN treatment–related preneoplastic lesions with a radius of less than 0.5 mm was observed in _TRAIL-R_–null animals (Figure 8C). Immunohistochemical analysis using the proliferation marker Ki-67 and staining for apoptotic cells using TUNEL suggested that the HCCs in the livers of TRAIL-R–/– mice had reduced numbers of TUNEL+ cells/mm2 compared with HCCs from WT animals (Figure 8D), whereas the number of Ki-67+ cells in the HCCs did not differ significantly (Figure 8E). This suggests that apoptosis is reduced in HCCs of TRAIL-R–/– animals compared with WT HCCs, whereas proliferation remains unaltered by the loss of the TRAIL-R.
Loss of TRAIL-R renders mice susceptible to DEN-induced hepatocarcinogenesis. (A and B) TRAIL-R–/– mice (n = 10) injected with DEN (0.30 mmol/kg body weight) at 7 days of age showed an increased liver tumor load compared with WT littermates (n = 10) at 10 months after treatment. (A) Gross observations of small (top row, demarcated with arrows and arrowheads) and larger liver tumors in DEN-treated animals (bottom row, demarcated with arrowheads). (B) Microscopy of H&E-stained histological sections shows the location of a liver tumor (T) with surrounding normal (N) liver tissue. (C) Detailed histological analysis of livers (>0.5 cm2 liver area analyzed, excluding macroscopic lesions) from randomly selected littermates (n = 3/genotype) shows an increased number of lesions exceeding a radius of 1.0 mm in TRAIL-R–/– animals. *P < 0.05, Student’s t test. Means ± SD are shown. (D and E) Immunohistochemical analysis of proliferation (Ki-67 labeling; E) and cell death (TUNEL staining; D) in HCCs from WT (n = 17) and TRAIL-R–/– (n = 17) animals treated with DEN show reduced TUNEL labeling in TRAIL-R–/– HCCs (Mann-Whitney U test, P < 0.05) compared with WT HCCs, whereas similar levels of Ki-67–positive cells were seen in HCCs of both genotypes.