Volume 143, Issue 5, Pages 1341-1351 (November 2012) Inhibition of MKK7–JNK by the TOR Signaling Pathway Regulator-Like Protein Contributes to Resistance of HCC Cells to TRAIL-Induced Apoptosis  In Sung Song, Soo Young Jun, Hee–Jun Na, Hyun–Taek Kim, So Young Jung, Ga Hee Ha, Young–Ho Park, Liang Zhe Long, Dae–Yeul Yu, Jin–Man Kim, Joo Heon Kim, Jeong–Heon Ko, Cheol–Hee Kim, Nam–Soon Kim  Gastroenterology  Volume 143, Issue 5, Pages 1341-1351 (November 2012) DOI: 10.1053/j.gastro.2012.07.103 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Up-regulation of TIPRL in HCC. (A) The relative messenger RNA (mRNA) level of TIPRL was analyzed in 20 pairs of cancerous and noncancerous tissues prepared from the same liver cancer patients at the indicated stages using quantitative reverse-transcription polymerase chain reaction. The data reflect mean ± standard error (n = 3; *P < .05, **P < .01, ***P < .001). (B) Immunoblotting with anti-TIPRL antibody was performed using 14 of the total paired tissues taken from the same liver cancer patients with HBV (P1–P7) and without HBV (P8–P14). T represents tumor, and N stands for normal tissue, in the liver. Protein loading was monitored by α-tubulin. (C) Normal (i and ii) and HCC (iii and iv) tissues prepared from the liver cancer patients were stained using anti-TIPRL antibody and pictured at 200× (left) and then at 400× (right) for high magnification. Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 TIPRL knockdown increases TRAIL-induced apoptotic cell death. (A, top right) For comparison of TIPRL levels, cell lysates were prepared from Huh7, Sk-hep-1 cells, and human primary hepatocytes and were then subjected to immunoblotting with anti-TIPRL antibody. Cells were transfected with small interfering RNA (siRNA)-mediating silencing of TIPRL (siTIPRL) or nontargeting siRNAs (siCont) (50 nmol/L) for 72 hours and were then treated with TRAIL (100 ng/mL). (A, bottom) The cell death percentage represents annexin V(+)/PI(+)-stained cells. (B) Morphologic changes of transfected cells were observed by Hoechst 33342 staining (top), and the apoptotic cell death percentage corresponds to annexin V(+)/PI(−) (bottom). The enlarged insert (right) is representative of a nucleus scored for apoptosis as compared with a normal nucleus. Each percentage scoring for apoptotic nuclei (arrowheads) was established by counting 300 cells for each condition. Cell lysates prepared from siTIPRL and/or TRAIL-treated Huh7 cells were subjected to (C) immunoblotting with the indicated antibodies or (D) were separated into a cytosol and mitochondrial fraction and were then subjected to immunoblotting with anti-cytochrome c antibody. Fraction purity and protein loading were assessed by prx III and α-tubulin, respectively. Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 MKK7/JNK axis mediates siTIPRL/TRAIL-induced apoptotic cell death. (A) Seventy-two hours after transfection with siTIPRL, the Huh7 cells were treated with TRAIL (100 mg/mL) for the indicated times and then subjected to immunoblotting with the indicated antibodies. TIPRL and α-tubulin antibodies were used for verification of siTIPRL activity and loading control, respectively. Huh7 cells were transfected with (B) siTIPRL and/or siRNA against MKK7 (siMKK7) in the presence or absence of TRAIL for the indicated time points or (C) were treated with siTIPRL and/or TRAIL in the presence or absence of 10 μmol/L of SP600125 (JNK specific inhibitor). Cell death percentage representing annexin V(+)/PI(+)-stained cells, which were analyzed by flow cytometry. The data represent the mean ± standard error (n = 3; **P < .01; ***P < .001 comparing siTIPRL with siTIPRL/siMKK7 or siTIPRL/SP600125). (D) MKK7 was immunoprecipitated from cell lysates using anti-MKK7 antibody. The resulting precipitates were either assayed for kinase activity (KA) with GST-JNK1 (upper panel) or GST-c-Jun (lower panel) as substrates or were subjected to immunoblot analysis using anti-MKK7 and anti-GST for loading control. Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Neither p53 activation nor induction of TRAIL receptors mediates siTIPRL/TRAIL-induced apoptotic cell death. (A) Immunoblotting was performed using cell lysates prepared from Huh7 cells treated with siTIPRL and/or TRAIL. (B) HepG2 (p53+/+) and Hep3B (p53−/−) cells were treated with siTIPRL and/or TRAIL (100 ng/mL) for the indicated time points. The cell death percentage representing annexin V(+)/PI(+) cells was analyzed by fluorescence-activated cell sorter analysis. The data reflect the mean ± standard error (n = 3; ***P < .001 comparing siTIPRL with siCont at 4 hours of TRAIL treatment) (top). Western blot using the indicated antibodies was performed (bottom). Anti-TIPRL, anti-p53, and anti-α-tubulin antibodies were used for verification of small interfering RNA (siRNA) activity and of cell types and protein loading control. (C) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed for death receptors (DRs) as well as TIPRL using Huh7 cells transfected with siTIPRL and/or TRAIL. (D) The levels of TRAIL receptors were determined by qRT-PCR analysis using 20 pairs of tissues from human liver cancer patients. The transcriptional level of each sample was normalized against the β-2-microglobulin of the sample after qRT-PCR. (n = 3; *P < .05; **P < .01; ***P < .001 comparing tumorous with nontumorous tissue). Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 TIPRL mediates the association of MKK7 and PP2Ac. (A) Huh7 and (B) Sk-hep-1 cells were treated with siTIPRL and/or TRAIL (100 ng/mL). PP2Ac, MKK7, and IgG were then immunoprecipitated with anti-PP2Ac or anti-MKK7 or a normal rabbit IgG antibody from cell lysates. (C) Huh7 cells were transfected with siTIPRL for 72 hours and were then stained with the indicated antibodies. For nucleus staining, 4',6-diamidino-2-phenylindole (DAPI) was used. (D) Various deletion fragments of TIPRL were cloned into the pCGN-HA vector (upper panel), and MKK7 fragments were inserted in-frame into the pEBG-GST vector (bottom panel). The protein-protein interactions (E) between the protein fragments of TIPRL and MKK7 and (F) between TIPRL and the protein fragments of MKK7 were investigated using deletion constructs in HEK293T cells. Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 siTIPRL/TRAIL suppresses the growth of Huh7 tumor xenografts.For determination of siTIPRL/TRAIL effects on the kinetics of tumor growth, 4 mice were placed in each group (siCont, siCont/TRAIL, siTIPRL, and siTIPRL/TRAIL). Approximately 2 × 106 Huh7 cells were injected into the mice, and then the mice were incubated until the tumor mass grew to 50 to 100 mm3 over a period of 12 days. siTIPRL was injected into the tumor mass on days 0, 4, 6, 8, and 10; and TRAIL was administrated on days 1, 5, 7, 9, and 11. The mice were killed on day 12, and the tumor mass was calculated based on the measured length (l) and width (w). (A) Images of the tumor masses taken from the mice from each group are presented. (B) The data represent the mean value of tumor volumes recorded at 2-day intervals. The data reflect the mean ± standard error (n = 4; **P < .01; ***P < .001 comparing the tumor mass of siCont with that of siTIPRL/TRAIL. #P < .05 comparing the tumor mass of siCont with that of siTIPRL). Two independent experiments were performed. Tumor samples from each mouse were subjected to TUNEL staining (C) and Western blot analysis using anti-caspase-8 antibody (D). Anti-TIPRL and anti-α-tubulin antibodies were used for verification of siTIPRL activity and loading control. Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Proposed mechanism for TIPRL-mediating resistant to TRAIL in cancer. In TRAIL-resistance cancer, TRAIL alone fails to reach a critical threshold to induce apoptosis because of the dephosphorylation event of MKK7 by PP2Ac via TIPRL, resulting in the negative regulating of the MKK7/JNK pathway and inhibiting of caspase cascades and cell death (black lines). Conversely, when TIPRL is depleted TRAIL induces the heterodimerization of DR4/5, transmitting a signal inside of cells, and then MKK7 is phosphorylated and activated, which in turn activates JNK, eventually leading to the activation of a caspase cascade and cell death (red lines). Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Gastroenterology 2012 143, 1341-1351DOI: (10.1053/j.gastro.2012.07.103) Copyright © 2012 AGA Institute Terms and Conditions