TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development

Abstract

TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R-deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R-expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.

Figure 1. Generation of Trail-r^–/– mice.

(A) Strategy to target exon 2 of the Trail-r gene. (i) Functional protein domains of TRAIL-R encoded by the Trail-r WT allele (ii). SP, signal peptide; CRD, cysteine-rich domain; TM, transmembrane domain; DD, death domain. Locations of the external (ext) 3′ and 5′ and the internal (int) probes and of genotyping primers mTR-a (a), mTR-b (b), and mTR-d (d) are indicated. (iii) Targeting construct. Open triangle, FRT sites; gray triangles, loxP sites; tkneo, selection cassette; DTA, diphtheria-toxin A-cassette. (iv) Null allele. (B) Southern blot analysis of targeted G418-selected ES cell clone after correct homologous recombination. (C) Constitutive deletion of exon 2 of Trail-r in different tissues. PCR analysis of genomic DNA of tail, lymph node, kidney, lung, heart, and brain of Trail-r^–/–, Trail-r+^/–, and Trail-r^+/+ mice using primers a, b, and d is shown. The primers a and b amplified a 300-bp fragment of the Trail-r WT allele, and primers a and d amplified a 235-bp fragment of the Trail-r–null allele.

Figure 2. TRAIL-R does not influence primary skin tumorigenesis.

(A) Papilloma incidence rates. (B) Tumor initiation rates, as reflected in the average number of papillomas over time. (C) Tumor growth rates, represented by combined average papilloma area over time. (D) Carcinoma incidence rates. Vertical bars indicate deaths of mice with carcinomas. (E) Papilloma-to-carcinoma conversion rate per week of papilloma exposure. The 95% confidence intervals are displayed. (F) Cell surface–expressed TRAIL-R was examined using a TRAIL-R–specific antibody (MD5-1) on WT immortalized keratinocytes (cell line C-50), papilloma cells (cell line 308), and carcinoma cells (cell lines DT02 and DT12). Histological analysis (G–J) of tumor sections did not reveal any differences between the different Trail-r genotypes. Representative tissue sections (H&E staining and brown keratin staining) from Trail-r^–/– mice are shown. (G) Keratin-positive papilloma. Among all genotypes, primary malignant tumors were (H) SCC, (I) spindle cell variant of SCC (SVSCC), or (J) fibrosarcomas, which is shown with keratin-positive hair follicles (HF). (K) Tissue sections of malignant carcinomas (fibrosarcoma or SCC) were stained with antibodies against active caspase-3 or Ki-67. Representative fields are shown (original magnification, ×400).

Figure 3. TRAIL-R suppresses lymph node metastases of skin carcinomas.

(A) Time course of detection of metastatic lymph nodes per total mice in study analyzed (left panel). Significantly higher incidence of lymph node metastases in TRAIL-R–deficient mice compared with TRAIL-R–expressing mice was detected upon necropsy (right panel), as determined by Pearson χ² test. (B–E) Tissue sections were stained with H&E and a keratin-specific antibody. Lymph nodes of Trail-r^–/– mice with keratin-positive metastatic lesions with SCC morphology (B, D, and E) or keratin-negative metastatic lesions (met) with fibrosarcoma (FSA) morphology (C). All stainings shown were from specimens obtained from tumors of Trail-r^–/– mice.

Figure 4. Detachment sensitizes skin carcinoma cells specifically to TRAIL and CD95L.

(A) Subconfluent cells were incubated in the absence (control) or presence of TRAIL (iz-muTRAIL, 1 μg/ml) and/or bortezomib (20 nM) for 24 hours. Cell death was determined by PI staining and flow cytometric analysis. (B and C) Cells were trypsinized and cultured either on an uncoated (attached) or a poly-HEMA–coated surface (detached) in the absence or presence of TRAIL (B; 1 μg/ml) or at the indicated concentrations (C). (D) Subconfluent cells (DT02) were treated with or without 1 μM thapsigargin (Tgg), 30 μg/ml oxaliplatin (Oxp), 5 μg/ml cycloheximide (Chx), or 43°C heat shock (HS) or irradiated with 15 Gy (IR) and were incubated in the absence or presence of TRAIL (1 μg/ml) for 24 hours. (E) Attached (att) or detached (det) cells were incubated in the absence or presence of TRAIL (1 μg/ml) or CD95L (100 ng/ml) for 24 hours. Each data point represents one of the cell lines DT01–DT06 or DT11. The mean percentage of dead WT cells is indicated by a horizontal line. (F) Attached or detached cells were incubated in the absence or presence of TRAIL (1 μg/ml), CD95L (100 ng/ml), 5-FU (200 μg/ml), or etoposide (10 μM) for 24 hours. Standard deviation for duplicates or triplicates is shown. Results are representative of at least 2 independent experiments.

Figure 5. Detachment-induced inactivation of ERK sensitizes to TRAIL- and CD95L-induced apoptosis.

(A) Cells were incubated attached or detached for 12 hours. Cell surface–expressed TRAIL-R and CD95 were detected on the indicated skin carcinoma cell lines. Representative flow cytometry profiles of at least 2 independent experiments are shown. (B) DT02 cells and (C) DT01 cells were trypsinized (0 h) and incubated either detached or attached for the indicated times. Western blots were incubated with the indicated antibodies. In B, note that c-FLIP_L, but not c-FLIP_S, was detected. (D) DT02 cells were trypsinized and seeded into noncoated 24-well plates either with or without U0126 (40 μM) and TRAIL or CD95L at the indicated concentrations for 24 hours. Cell death was determined as described in Figure 4. Standard deviation for duplicates is shown. Results are representative of 3 independent experiments.