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Volume 146, Issue 1, Pages 278-290 (January 2014)
Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells Verena Haselmann, Alexandra Kurz, Uwe Bertsch, Sebastian Hübner, Monika Olempska–Müller, Jürgen Fritsch, Robert Häsler, Andreas Pickl, Hendrik Fritsche, Franka Annewanter, Christine Engler, Barbara Fleig, Alexander Bernt, Christian Röder, Hendrik Schmidt, Christoph Gelhaus, Charlotte Hauser, Jan–Hendrik Egberts, Carola Heneweer, Anna Maria Rohde, Christine Böger, Uwe Knippschild, Christoph Röcken, Dieter Adam, Henning Walczak, Stefan Schütze, Ottmar Janssen, F. Gregory Wulczyn, Harald Wajant, Holger Kalthoff, Anna Trauzold Gastroenterology Volume 146, Issue 1, Pages (January 2014) DOI: /j.gastro Copyright © 2014 AGA Institute Terms and Conditions
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Figure 1 TRAIL-R2 interacts with hnRNPA1, NF45, p68, and NF90/NF110. (A) TRAIL-R2 was immunoprecipitated from whole-cell lysates of Panc89 cells. Immunoprecipitates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis or by 2-dimensional electrophoresis. Protein bands/spots excised from the gel were analyzed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. (B) TRAIL-R1 and TRAIL-R2 were precipitated from whole-cell lysates of pancreatic ductal adenocarcinoma (Panc89, Panc1, BxPC3), lung carcinoma (KNS62), renal carcinoma (A498), breast carcinoma (MDA-MB-231), and colon carcinoma cells (HCT116) using either mapatumumab (Mapa) or lexatumumab (Lexa). Protein complexes were analyzed by Western blotting. As controls, cell lysates without antibodies (ctrl beads) and antibodies alone (ctrl Mapa, ctrl Lexa) were analyzed in parallel. n.s., nonspecific. (C) Protein lysates (20 μg) used for immunoprecipitation analyzed in Western blotting. (D) Intracellular distribution of TRAIL receptors analyzed by indirect immunofluorescence and confocal LSM using antibodies against TRAIL-R1 (R1; mapatumumab) and TRAIL-R2 (R2; lexatumumab). (E) TRAIL-receptor presence in soluble nuclear fractions (NF) and cytoplasmic fractions (CF) of various tumor cells analyzed by Western blotting. Expression of lamin A/C (NF marker) and α-tubulin (CF marker) were analyzed in parallel. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
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Figure 2 nTRAIL-R2 interacts with the microprocessor complex and with accessory proteins NF90, p68, NF45, and hnRNPA1. (A) TRAIL receptors were precipitated from nuclear extracts of Panc1 cells. Protein complexes were analyzed by Western blotting. As controls, nuclear extracts incubated without antibodies (ctrl beads) and antibodies alone (ctrl Mapa, ctrl Lexa) were analyzed in parallel. Immunoprecipitations also were performed with nuclear extracts pretreated with RNase. n.s., nonspecific. (B) Protein lysates (20 μg) used for immunoprecipitation analyzed in Western blotting with lamin A/C detection as gel loading control. (C–E) Co-localization of TRAIL-R1 (R1) and TRAIL-R2 (R2) with the indicated proteins studied in Panc1 cells by immunofluorescence and confocal LSM. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
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Figure 3 TRAIL-R2 regulates the levels of let-7. (A and B) Panc1 cells were transfected with control siRNA (ctrl-si) or TRAIL-R2 siRNA (R2-si) for 40 hours. (A) Left: TRAIL-R2 expression studied by Western blot. (A) Right: Levels of mature let-7 species determined by reverse-transcription PCR and normalized to RNU6B levels. Effects of R2-siRNA were normalized to the expression levels (set to 1) in cells treated with the ctrl-si. Bars (± SEM) represent the average of biological triplicates each with 2 technical replicates. *Significant changes. (B) Levels of pri–let-7 determined by reverse-transcription PCR. Three biological replicates in technical triplicates were analyzed. Relative expression was calculated by the delta-delta CT method and normalized to glyceraldehyde-3-phosphate dehydrogenase with ctrl-si set to 1. Shown are means ± SEM. (C) Flow cytometric analysis of EGFP expression in Panc1 cells simultaneously transfected for 40 hours with ctrl-si or R2-si and expression plasmids carrying EGFP–pri-miR fusion sequences. The green fluorescence intensity of ctrl-si–treated cells was set as 100%. MFI, mean fluorescence intensity. Shown are means ± SEM (n = 3). (D) TRAIL-R2 was precipitated from lysates of untreated Panc1 cells. RNA was extracted and analyzed for the presence of pri-miRNAs by reverse-transcription (RT) PCR with genomic DNA (gDNA) as positive control. (E) Expression of HMGA2 and Lin28B analyzed by Western blot in Panc1 cells transfected with ctrl-si or R2-si. (F) Western blot analyses of HMGA2 and Lin28B expression in Panc 1 cells transfected with ctrl-si or R2-si together with either control LNA oligonucleotides (ctrl-LNA) or anti–let-7–LNA (let-7-LNA). Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
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Figure 4 TRAIL-R2 enhances cell proliferation. Panc1 cells were transfected with siRNA specific for TRAIL-R2 (R2-si) or with control siRNA (ctrl-si). (A) Proliferation was determined by cell counting 72 hours later. Shown are means ± SD (n = 3). (B) In parallel, 48 hours after transfection the cells were harvested, counted, seeded at a density of 104/well in 96-well plates, and proliferation was determined by 3H-thymidine assay 24 hours later. Shown are means ± SD (n = 6). (C) Panc1 cells were stably transfected with an expression vector encoding TRAIL-R2 or with the empty vector (pCR3.1). Levels of TRAIL-R2, HMGA2, and Lin28B were determined by Western blotting in whole-cell lysates, in cytoplasmic fractions (CF) and nuclear fractions (NF). Proliferation was determined by cell counting 72 hours after seeding of 1 × 105 cells per 6 wells. Shown are means ± SD (n = 3). (D) Panc1 cells were cultured for 48 hours with or without neutralizing anti-TRAIL antibodies (α-TRAIL, anti-TRAIL) or soluble TRAIL-R2-Fc (R2-Fc). Cell numbers were determined by cell counting. Shown are means ± SD (n = 3). The levels of TRAIL-R2 and HMGA2 were determined by Western blotting. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
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Figure 5 TRAIL-R2 is down-regulated during differentiation. (A) A818-6 cells were grown as a monolayer or hollow spheres. Scale bars: 100 μm. (B) Protein expression was analyzed by Western blot. (C) Intracellular distribution of TRAIL-R2 (R2) and hnRNPA1 was analyzed by confocal LSM. Scale bars: 50 μm. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
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Figure 6 Knockdown of TRAIL-R2 inhibits orthotopic tumor growth in severe combined immunodeficiency disease (SCID) beige mice. (A) PancTuI cells with stably down-regulated expression of TRAIL-R2 (Western blot) were injected orthotopically into SCID beige mice (n = 12/group). Mice were killed 26 days later. (B) Tumor growth was monitored on postoperative days 12, 18, and 25 by ultrasound imaging with mice in the supine position and sagittal transducer orientation. The tumors (T) are outlined. L, liver; GI, gastrointestinal tract. Scale bar: 1 mm. (C) Tumor weights are shown as box plots with medians. Top of box, 75th percentile; bottom of box, 25th percentile. Circles and asterisk represent extreme values. shRNA, short hairpin RNA. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
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Figure 7 Intracellular distribution of TRAIL-R2 and HMGA2 in PDAC tissues. (A) Tumor (AIII, AIV) and non-neoplastic tissues (AI, AII) from PDAC specimens were stained with anti–TRAIL-R2 (AI, AIII) and anti-HMGA2 (AII, AIV) antibodies. Magnifications corresponding to the rectangles in the large pictures are shown in the small windows. Arrows indicate exemplary nuclear staining. Scale bars: 50 μm. (B and C) Results are shown as numbers (num) of cases as well as percentages of samples with particular staining intensity in relation to histologic specification (tumor vs non-neoplastic) and for cytoplasmic vs nuclear staining. (D and E) Kaplan–Meier analyses of PDAC patient survival after curative tumor resection (R0) in relation to TRAIL-R2 and HMGA2 nuclear staining. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2014 AGA Institute Terms and Conditions
