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. 2015 Apr 13;27(4):561-73.
doi: 10.1016/j.ccell.2015.02.014. Epub 2015 Apr 2.

Cancer Cell-Autonomous TRAIL-R Signaling Promotes KRAS-driven Cancer Progression, Invasion, and Metastasis

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Free PMC article

Cancer Cell-Autonomous TRAIL-R Signaling Promotes KRAS-driven Cancer Progression, Invasion, and Metastasis

Silvia von Karstedt et al. Cancer Cell. .
Free PMC article

Abstract

Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.

Figures

Figure 1
Figure 1. KRAS-driven lung and pancreatic cancers are promoted via endogenous mTRAIL-R
(A) Images of representative lungs of the indicated 6 months-old KP-mice. (B) Lungs from A were weighed and their weight was plotted in comparison to age-matched C57BL/6 mice. (C) Kaplan-Meier survival curve of mice with indicated genotypes, statistics were determined by log-rank test; *** p=0.0008. (D) Images of representative pancreata of the indicated 4.5 months-old KC-mice. (E) Fixed pancreata were stained with H&E and the number of PanINs per pancreatic section was determined by histopathological examination. The bottom and top of the box are the 25th and 75th percentile of the data, respectively. The whiskers represent maximum and minimum values; * P < 0.05 (Student’s t-test); scale bar, 100 μm. (F) Representative H&E, αCK19 and DAPI stainings of pancreas of mice of indicated genotypes; scale bar, 25 μm.
Figure 2
Figure 2. Human TRAIL-R2 drives proliferation, migration and invasion
(A) A549 shControl, shTRAIL-R1 and shTRAIL-R2 were subjected to soft agar colony formation assays. Relative colony formation was determined normalized to A549-luc pLKO.1 after 4 weeks. (B) KRAS-mutated Panc Tu I, A549-luc and DLD-1 cells were subjected to control or TRAIL-R2 knockdown for 72 hr and then BrdU proliferation assays. (C) Six KRAS-mutated and five KRAS-WT cell lines were transfected with siRNAs silencing TRAIL-R2 or mTRAIL-R and subsequently subjected to migration assays. Migration was normalized to control transfected cells in each of the cell lines. (D) KRAS-mutated cells as in C apart from H460 and KRAS-WT cells as in C were transfected with siRNAs silencing human or murine KRAS and subjected to migration assays. (E) DLD-1 and DKO4 cells were transfected as indicated and subjected to migration assays. Migration was normalized to control transfected DKO4 cells. (F) Panc Tu I cells were transfected as indicated and subjected to migration assays. (G) A549-luc, DLD-1 and Colo357 cells were subjected to invasion assays. (H) 2 x 106 A549-luc pLKO.1 or shTRAIL-R2 were injected i.v. into SCID beige mice (n=9 mice/group). Tumor burden was quantified in paraffin sections of H&E-stained lungs by pathological inspection. (I) 1 x 106 PancTu-I shCtr (n=9) or shTRAIL-R2 (n=8) were orthotopically implanted into pancreata of SCID beige mice. After 10 days, primary tumors were resected and the number of liver macrometastases was determined. (J) A schematic representation of TRAIL-R2 mutants. (K) A549 shTRAIL-R2 cells were transiently transfected with either vector (pcDNA3.1) or with the indicated TRAIL-R2 version and subsequently subjected to migration assays. Migration was normalized to A549-luc pLKO.1 cells. Representative Western blots are shown. Figures represent means of three independent experiments +/- SEM; individual dots represent the results of measurements from a single cell line shown as a mean of three independent experiments or sample from a single mouse when applicable. Ctr, control; TR2, TRAIL-R2; ICD, intracellular domain; DD, death domain; CTD, C-terminal domain. ns, not significant, * p<0.05; ** p < 0.01 and *** p<0.001 [ANOVA (A, B,E,F,G,K); Student’s t-test (C,D,H), Mann-Whitney test (I)]. See also Figure S1 and Table S1.
Figure 3
Figure 3. mTRAIL-R promotes KRAS-driven pancreatic cancer progression and metastasis
(A) Kaplan-Meier survival curve, Log-rank test ** p=0.004. (B) Animals with metastasis in the liver and lungs as determined by H&E staining at time of death were quantified. Three of KPC-TRAIL-RWT/WT could not be determined due to tissue decay. Fisher’s exact test *** p >0.001 and * p > 0.05. (C) Representative H&E stainings of pancreatic adenocarcinomas and lung tissues. Met=Metastasis; scale bar, 50 μm. (D) Representative images of isotype, αPDX-1, αSp-C and αCC10 stainings of KPC lung metastases; scale bar, 25 μm. (E) Representative images of αCC10 stainings of KP lung tumors; scale bar, 25 μm. See also Figure S2.
Figure 4
Figure 4. TRAIL-R2 promotes activation of a pro-migratory Rac1/PI3K signaling axis
(A) A549-luc pLKO.1 and shTRAIL-R2 cells were fixed and stained using Texas red-coupled phalloidin and DAPI. Representative confocal images; scale bar, 25 μm. (B) A549-luc pLKO.1 and shTRAIL-R2 were subjected to pulldown of GTP-bound Rac1 using PAK1-beads and Western blotting. (C) A549-luc cells were transfected as shown and subjected to migration assays. (D) A549-luc cells were transfected with GFP, Rac1, or dnRac1 (Rac1T17N) and subjected to immunoprecipitation via addition of Flag-tagged TRAIL after lysis and Western blot analysis. Asterisks indicate the IP antibody heavy chain. (E) Endogenous Rac1 in A549-luc cell lysates was loaded with GDP/GTPγS in vitro, and GTP-bound Rac1 was isolated using PAK1-RBD beads and analyzed by Western blot. (F) The indicated cell lines were subjected to KDs or NSC23766 treatment [100 μM] for 72 hr and then BrdU proliferation assays. (G) A549-luc cells were subjected to the indicated KDs and migration assays or Western blot analysis in the presence or absence of GDC-0941 [1 μM]. (H) FADD-deficient and proficient A549-luc cells were transfected with TRAIL-R2-targeting siRNAs and A549 shTRAIL-R2 cells were reconstituted with either ΔICD or ΔDD/CTD. All cells were subjected to Western blotting. (I) A549-luc, PancTu-I and DLD-1 cells were transfected as indicated and subjected to migration assays or Western blot analysis. (J) Proposed model of migration signaling mediated by endogenous TRAIL/TRAIL-R2 in KRAS-mutated cells. Representative Western Blots are shown. Values are means +/- SEM of three independent experiments. ICD, intracellular domain; DD, death domain; CTD, C-terminal domain; Ctr, Control; TR2, TRAIL-R2; * p < 0.05, ** p<0.01 and *** p<0.0001 (ANOVA). See also Figure S3.
Figure 5
Figure 5. mTRAIL-R and mTRAIL promote Rac1 activity in KPC tumors in vivo
(A) Intravital imaging was performed on mice bearing either KPC-pLKO (n=3) or –shmTRAIL-R (23, 25 n=3 each) tumors and average lifetime fluorescence was quantified. Representative Western blots are shown. (B) Lifetime maps of representative single KPC-pLKO.1 or -shmTRAIL-R (sh25) cells in vivo. SHG=second harmonic generation; scale bar, 50 μm. (C) Intravital imaging was performed on mice bearing KPC-tumors before and 1 hr after treatment with either vehicle, or mTRAIL-R-Fc. Average lifetime fluorescence was quantified. (D) Parental KPC cells were transiently transfected with a Rac-FRET reporter and treated with mTRAIL-R-Fc for the indicated times in vitro. Single cells were analyzed as in A. Values are means +/- SEM of three independent experiments. ns, not significant, * p < 0.05, ** p <0.01 and *** p<0.0001 (Mann-Whitney test); [ns], nano seconds. See also Figure S4.
Figure 6
Figure 6. High TRAIL-R2 expression correlates with markers of malignancy in patients with KRAS-mutated cancers
(A) TRAIL-R1 and TRAIL-R2 expression in human PDAC samples of 95 patients was quantified. Percentage >20% of positive [intensity of a minimum of 1 on a scale of 1-3 (Ganten et al., 2009)] cancer cells within one patient sample was considered high expression and included into the figure which depicts percentage of high TRAIL-R expression within the patient population. Representative stainings from two patients are shown; scale bar, 25 μm; Fisher’s exact test *** p<0.0001. (B) TRAIL-R2 expression was determined in a human PDAC cohort (n=106). All tumors were positive ranging from score 3 to 7 (median=5). Data were dichotomized by the median 5 and the percentage of cases above (black) and below (white) median was calculated separately for cases with lymph vessel invasion (L1, n=71) and cases without lymph vessel invasion (L0, n=30). In 5 patients, lymph vessel invasion had not been determined, although tumors also stained positive. Fisher’s exact test ** p=0.001. (C) TRAIL-R1 and TRAIL-R2 expression levels (H-score) in lung metastases of KRAS-mutated and KRAS-WT CRC. The central line represents the mean, error bars are +/- SEM. (D) Metastasis-free (Kaplan-Meier)-survival according to high or low TRAIL-R1/2 expression (i. e. above or below respective H-score median) in patients with KRAS-mutated CRC. Log-rank test ns= not significant; * p < 0.02. (E) As in D, in patients with KRAS-WT CRC. Log-rank test ns= not significant; * p < 0.02.

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