Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: Erap1 (a, c, e) and Tap1 (b, d, e) expression in normal neural stem cells (NSCs, n=3), MP tumors (n=3), MG tumors (n=3), MG tumors overexpressing DNp53 (MG+P) (n=3) and MP tumors overexpressing GFI1 (MP+G) (n=3) were measured by qRT-PCR (a-d) and by western blotting (e); quantification of 3 independent experiments is shown below the western blot. Error bars represent means ± SD. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. p-values were determined by two-sided unpaired t-test. In (a), NS: p=0.21 (MG vs NSC), ** p=0.0019 (MG vs MP). In (b) NS, p=0.33 (MG vs NSC); *** p=1.9E-05. In (c), *** p=1E-04 (MP vs MG) and p=8E-05 (MG vs MG+P). In (d), *** p=2.3E-05 (MP vs MG), ** p=0.003 (MG vs MG+P, Tap1). In (e), * refers to p=0.002 (MP vs MG, Erap1), p=0.0052 (MG vs MG+P,Erap1), p=0.0097 (MP vs MG, Tap1), p=0.0049 (MG vs MG+P, Tap1). (f-g) Analysis of ERAP1 (f) and TAP1 (g) mRNA levels in human Sonic Hedgehog-associated MB with mutant (red) or wild-type (WT, blue)) TP53. Fragments Per Kilobase per Million mapped reads (FPKM) of ERAP1 and TAP1 are shown. p-values were determined by two-sided Wilcoxon rank sum test. Boxplot center lines show median, box limits indicate the 25th and 75th percentiles, lower and upper whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles, respectively. (h) Putative p53 binding sites in the mouse Erap1 and Tap1 promoters. (i-j) Chromatin Immunoprecipitation (ChIP) and PCR analysis of p53 binding site-containing regions in the Erap1 (i) and Tap1 (j) promoters. qPCR results for anti-p53 ChIP (IP p53) or isotype control ChIP (IP Ctl) in MG (n=3) and MP (n=3) tumor cells. p-values were determined by two-sided unpaired t-test * p=0.0046 (Erap1) and ** p=0.0011 (Tap1). (k) Putative p53 binding sites in the human ERAP1 and TAP1 promoters. (l-m) ChIP and PCR analysis of the p53 binding site-containing regions in the ERAP1 (l) and TAP1 (m) promoters. qPCR results for anti-p53 ChIP (IP p53) or isotype control ChIP (IP Ctl) in p53 mutant PDXs (RCMB18, Icb984, BT084) and p53 wild-type PDXs (Icb1299, RCMB40) (n=3 for each tumor type). p-values were determined by two-sided unpaired t-test. * p=0.026 (Icb1299, ERAP1), p=0.01 (Icb1299, TAP1), p=0.002 (RCMB40, TAP1) ; ** p=0.001 (RCMB40, ERAP1).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Control

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: MG (a, c, e, g) and MP (b, d, f, h) tumor cells were treated in vitro for 48h with IFNα (20 ng/mL), IFNγ (20 ng/mL), TNF (50 pg/mL) or LtβRag (1.6μg/mL). MHC-I expression in untreated cells (Ctl, black histograms) and cells treated with IFNα (red, a, b), IFNγ (red, c, d), TNF (green, e, f) or LTβRag (blue, g, h) were analyzed by FACS. Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below each histogram; data points represent MFIs for individual tumor samples. p-values were determined by two-sided unpaired t-test. p-values are indicated on the corresponding graphs. Erap1 (i) and Tap1 (j) mRNA expression in untreated MP tumor cells (Ctl) or MP tumor cells treated with TNF, LTβRag or IFNγ were determined by qRT-PCR (n=3). Data points represent expression values for individual tumor samples; error bars represent the mean ± SD. p-values were determined by two-sided unpaired t-test. In (i), NS, p=0.351;* p=0.006, ***p=0.0004. In (j), NS, p=0.453, * p=0.0086, ** p=0.003. (k) Tap1 and Erap1 protein levels were assayed by western blotting. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. Relative protein levels of Erap1 (l) and Tap1 (m) normalized to actin are shown for three independent samples. Error bars represent mean ± SD; data points represent expression values for individual tumor samples. p-values were determined by two-sided unpaired t-test. In (l), NS p=0.133, * p=0.0025, ** p=0.0004 ; In (m), NS = 0.378, * p=0.029, ** p=0.0116.

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: In Vitro, Expressing, Fluorescence, Quantitative RT-PCR, Western Blot

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: (a-d) MG tumor cells were transduced with control shRNA (shCtl) or shRNAs targeting Erap1 (shErap1#1, shErap1#2). Knockdown efficiency was determined by western blotting in three independent tumor samples (a). MHC-I expression was determined by FACS in control cells (shCtl, black) and Erap1 knockdown cells (shErap1, red). Western blots are cropped at the molecular weights for Erap1 (120 kDa) and β-actin (42 kDa); original blots are available in Source Data. (b). Quantification of the mean fluorescence intensity (MFI) values for three independent tumors is shown below each histogram; data points represent MFIs for individual tumor samples. p-values, determined by two-sided unpaired t-test, are indicated on each bar graph. (c-d) Erap1 knockdown cells were transplanted into aB6 mice. Bioluminescence imaging of representative mice (c) and survival curves (n=6) (d) are shown. p-values for the difference in survival between shErap1 and shCtl were determined using the two-sided log-rank (Mantel-Cox) test. * p=0.024; ** p= 0.0075. (e-i) MP tumor cells were transduced with empty vector (vect) or vectors encoding Erap1, Tap1 or both. Erap1 (e) and Tap1 (f) expression levels were assessed by western blotting in three independent tumor samples. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. (g) Analysis of MHC-I expression by FACS in control cells (vect, black) and Erap1 + Tap1 overexpressing cells (pink). Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below the histogram; data points represent MFIs for individual tumor samples. The p-value was determined by two-sided unpaired t-test. (h, i) MP tumor cells transduced with control vector (vect, black), Tap1 (green), Erap1 (blue) or Erap1 + Tap1 (pink) were orthotopically transplanted into aB6 mice. Bioluminescence imaging of representative mice (h) and survival curves (n=6 per group) (i) are shown. p-values were determined by the two-sided log-rank (Mantel-Cox) test. NS (Ctl vs. Tap1), p=0.79; * (Ctl vs. Erap1) p=0.0047; ** (Ctl vs. Erap1+Tap1) p=0.0005. Mice carrying MP tumors expressing Erap1 + Tap1 survive significantly longer than mice carrying tumors expressing Erap1 alone (p=0.0016 for Erap1 vs. Erap1 + Tap1) or Tap1 alone (p = 0.0005 for Tap1 vs. Erap1 + Tap1).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Transduction, Control, shRNA, Knockdown, Western Blot, Expressing, Fluorescence, Imaging, Plasmid Preparation

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: (a, b) FACS analysis of MHC-I expression in MP tumor cells that were untreated (Ctl, black histograms) or treated for 48h with TNFR1 agonist (0.5μg/mL, purple histogram, a) or TNFR2 agonist (1.67nM, red histogram, b). (c) MP tumor cells generated from TNFR2 knockout (TNFR2 KO) mice were treated for 48h with no stimulus (Ctl, black histogram) or with TNF (50 pg/mL, green histogram) and then analyzed by FACS. Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below each histogram; data points represent MFIs for individual tumor samples. p-values were determined by two-sided unpaired t-test. p-values are indicated on the corresponding graphs. (d-f) MP tumor cells were untreated (NT) or treated with TNF for 15’, 30’ or 1h. Expression of RelA and RelB protein were assessed by western blotting in nuclear extracts (d) and in total cellular protein extracts (e). Histone H3 and GAPDH were used as controls for nuclear and total extracts respectively. Western blots are cropped at the molecular weights for RelA (65 kDa), RelB (68kDa), Histone H3 (17kDa) and GAPDH (37 kDa); original blots are available in Source Data. (f) Quantification of Western blots for RelA (black) and RelB (blue) protein levels in nucleus relative to levels in total extract at 0’, 15’, 30’ and 60’ for three independent experiments. p-values were determined by two-sided unpaired t-test. For RelA, * p=0.049 (15’) ; ***p=0.0032 (30’) ; **p=0.0043 (1h); for RelB: NS, p=0.088 (15’) ; *p=0.037 (30’) ; NS p=0.073 (1h). (g, i) Putative binding sites for the RelA-p50 heterodimer in the mouse Erap1 and Tap1 promoters. (h, j) MP tumor cells were treated for 1h with no stimulus (ctl), TNF or LtβRag. Chromatin Immunoprecipitation (ChIP) was performed using antibodies specific for RelA (h) or p50 (j), and precipitates were analyzed by qPCR for the presence of the RelA-p50 binding site-containing regions of the Erap1 and Tap1 promoters. Data represent the mean fold-induction ± SD. p values were determined by two-sided unpaired t test. In (h), * p=0.0034, **p=0.003, ***p=2.6E-06 (TNF, Erap1), p=1E-04 (TNF, Tap1). (k) MP tumor cells were transduced with control shRNA (shCtl) or shRNAs targeting RelA (shRelA#832, shRelA#833), p50 (shp50#484, shp50#485) or RelB (shRelB#494, shRelB#495). MHC-I expression was determined by FACS in control cells (shCtl) and RelA, p50 or RelB knockdown cells treated in vitro for 48h with vehicle (DMSO, ctl), TNF or LtβRag. Data shown represent quantification of mean fluorescence intensity (MFI) for three independent experiments; data points represent MFIs for individual tumor samples. p values were determined by two-sided unpaired t test. shCtl : ** p=0.0019, *** p=1.1E-05; shRelA (#832): NS, Not significant p=0.063 (TNF) and p=0.098 (LtβRag); shRelA (#833): *p=0.035, **p=0.009; shp50 (#484): NS, Not significant p=0.341 (TNF), p=0.125 (LtβRag); shp50 (#485): NS, p=0.601, *p=0.049 ; shRelB (#494) *** p=1.8E-05 (TNF), p=2.3E-04 (LtβRag); shRelB (#495): *** p=9E-06 (TNF), p=1.6E-05 (LtβRag).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Expressing, Generated, Knock-Out, Fluorescence, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Transduction, Control, shRNA, Knockdown, In Vitro

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 positively regulates the Hh pathway at postreceptor level. a Luciferase activity of NIH3T3 Shh-Light II cells treated for 24 h with SAG and increasing amounts of Leu-SH or DTT as control. b , c Quantitative real-time PCR (qRT-PCR) ( b ) and representative immunoblotting ( c ) analyses of Gli1 expression in the NIH3T3 murine fibroblasts transduced with lentiviral vectors encoding either control shRNA (shCTRL) or ERAP1 shRNA (shERAP1#1 and shERAP1#2) and treated with SAG or DMSO for either 24 or 48 h. In c ERAP1 expression was also evaluated and actin was used as loading control. d , f qRT-PCR analysis of Hh target genes expression in Ptch −/− ( d ) and SuFu −/− MEFs ( f ) both treated with Leu-SH (30 μM) or DTT as control. e Representative model of the constitutive activation of Smo or Gli1 in Ptch −/− and SuFu −/− MEFs, respectively. g , h qRT-PCR analysis of Hh target genes expression in Ptch −/− ( g ) and SuFu −/− MEFs ( h ) transduced with shCTRL or shERAP1 constructs. Data in b , d , f , g , and h are normalized to endogenous GAPDH and HPRT controls and expressed as the fold change respect to the control sample value. All data represent the mean of three independent experiments. Mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001 calculated with two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transduction, shRNA, Activation Assay, Construct

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 activates Hh signaling by impairing βTrCP protein expression. a – f Representative immunoblotting analyses of the indicated proteins in MEFs transfected with increasing amounts of vector encoding ERAP1 ( a , d ) or treated for 24 h with Leu-SH at the indicated concentration ( b , e ), or SAG (200 nM) and Leu-SH (30 μM) ( c ), or DTT as control. In f MEFs were transduced with shCTRL or shERAP1 and transfected with a vector encoding ERAP1. Actin ( a – c , f ) and tubulin ( d , e ) were used as loading controls. g ERAP1, Gli1 and βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 in the presence of small interfering RNAs (siRNAs) to a non-relevant mRNA (siCTRL) or murine βTrCP mRNA (siβTrCP). h βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 and treated with cycloheximide (CHX, 100 µg/mL) at different time points. Densitometry analysis of actin-normalized βTrCP values of three independent experiments is shown (right panel). i , j Endogenous βTrCP was immunoprecipitated from MEFs expressing the indicated proteins and treated with MG132 (50 μM) for 4 h ( i ) or increasing doses of Leu-SH for 24 h ( j ), followed by immunoblotting with an anti-HA antibody to detect conjugated HA-Ub. Blots were both reprobed with a βTrCP antibody. Bottom ERAP1 and βTrCP protein levels in total cell lysate. Actin was used as loading control. k Immunoblotting (upper panel) and densitometric analysis (lower panel) of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs and treated after 24 h with increasing amount of Leu-SH for 24 h. l Immunoblotting analysis of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs transduced with shCTRL or shERAP1. Actin was used as loading control

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Concentration Assay, Transduction, Immunoprecipitation

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 promotes βTrCP ubiquitylation by interacting with USP47. a – d MEFs were transfected with ERAP1 and/or Flag-USP47. Interaction between USP47 and ERAP1 was detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. e MEFs transfected with ERAP1 were stained with anti-ERAP1 and anti-USP47 antibodies. Green and red, USP47 and ERAP1 expressing cells, respectively. Nuclei were counter stained with Hoechst (Blue). Magnification ×60; Bars: 5 μm. Representative images from three independent experiments. f βTrCP and Gli steady state in USP47 +/+ and USP47 −/− MEFs. g βTrCP protein level in USP47 +/+ , USP47 −/− and USP47 −/− Flag-USP47 transfected MEFs. h βTrCP half-life in USP47 +/+ vs. USP47 −/− MEFs treated with CHX (100 µg/mL) at the indicated times. i βTrCP protein levels in MEFs transfected with empty vector as control or Flag-USP47 and treated with CHX (100 µg/mL) at different time points. j Gli1 protein levels in Ptch −/− MEFs transfected with empty vector as control or Flag-USP47 and treated after 24 h with CHX (100 µg/mL) for different time points. In g – j densitometric analysis of βTrCP and Gli1 protein levels of three independent experiments are shown (right panels). k MEFs were transfected with HA-Ub and increasing amount of ERAP1 in the presence or absence of Flag-USP47. Endogenous βTrCP was immunoprecipitated with an anti-βTrCP antibody and the ubiquitylated forms were revealed with an anti-HA antibody (upper panel). The blot was reprobed with an anti-βTrCP antibody. Flag-USP47, ERAP1 and βTrCP total protein levels are shown (lower panel). l MEFs were transfected with Flag-USP47 and increasing amount of ERAP1. Interaction between Flag-USP47 and endogenous βTrCP was assessed by immunoprecipitation and immunoblotting with the indicated antibodies. Actin was used as loading control. m MEFs were transfected with Flag-USP47 and treated for 24 h with Leu-SH at the indicated concentration. Interaction between Flag-USP47 and endogenous βTrCP was detected as described in l . Densitometric analysis of the Flag-USP47/βTrCP binding ratio representative of three independent experiments is shown (right panel). n MEFs were transduced with shCTRL or shERAP1 and transfected with Flag-USP47. Interaction between Flag-USP47 and endogenous βTrCP was assessed as described in l

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Expressing, Plasmid Preparation, Concentration Assay, Binding Assay, Transduction

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 impairs Hh-dependent growth of cerebellar granule cell progenitors. a H&E and immunohistochemical staining of Ki67, ERAP1 and Gli1 in the outer EGL during mouse cerebellum development. Magnification ×40. Scale bars represent 50 μm. b , c GCPs were isolated from 4-day-old mice and treated with either SAG alone or in combination with increasing doses of Leu-SH for 24 h. BrdU uptake ( b ) and mRNA levels of Gli1 ( c ) are shown. d , i GCPs isolated from 4-day-old mice were infected with lentiviral particles encoding for shERAP1 ( d – f ) or ERAP1 ( g – i ) and the corresponding controls, respectively. The percentage of BrdU uptake ( d , g ), mRNA ( e , h ), and protein levels ( f , i ) of Gli1 are shown. Results in c , e , h were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. legend, and represent the mean of three independent experiments. In f and i, actin was used as loading control. Mean ± S.D. * P < 0.05; ** P < 0.01 determined with two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Immunohistochemical staining, Staining, Isolation, Infection

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 impinges Hh-dependent tumor cell growth in vitro. a – f Primary cell cultures from Math1-cre/Ptc C/C mice MBs were treated with different amounts of Leu-SH. a , b Cells were counted with trypan blue at the indicated time points to evaluate the growth rate of viable cells ( a ) and the percentage of cell death ( b ). c Cleaved Caspase-3 protein levels in cells treated with Leu-SH at the indicated concentration for 24 h. d – f Percentage of BrdU uptake ( d ) and Gli1 mRNA ( e ), and protein ( f ) expression in MB cells treated with Leu-SH at the indicated concentrations for 24 h. g MB Stem-Like Cells (MB-SLCs) from Math1-cre/Ptc C/C mice were treated with Leu-SH as in (a) and counted with trypan blue at the indicated time points. h MB-SLCs were dissociated and treated with the indicated concentrations of Leu-SH or DTT as control. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was evaluated. The self-renewal MB-SLCs capability is expressed as percentage of neurosphere-forming cells (right). Representative bright field images of tumor neurospheres after Leu-SH treatment are shown (left). Scale bar 100 µM. i , j mRNA and protein expression levels of Hh target genes of MB-SLCs treated with the indicated concentrations of Leu-SH for 24 h. Actin was used as loading control. Results in e , i were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. legend. All data are representative of three independent experiments. Mean ± S.D. * P < 0.05; ** P < 0.01 calculated using two-tailed Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: In Vitro, Concentration Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 inhibition impairs Hh-dependent tumor growth in vivo. a – f NSG mice were grafted with spontaneous primary MB from Math1-cre/Ptc C/C mice. Tumor masses (150 mm 3 ) were intratumorally injected with Leu-SH. a Tumor growth was monitored. b Representative flank allograft average volumes (lower panel) and quantification of tumor explants (upper panel). c , d Ki67, NeuN, and cleaved Caspase-3 (Cl.Casp-3) immunohistochemical stainings of allograft tumor samples. d Quantification of immunohistochemical stainings shown in c . Scale bar 100 μm. e mRNA and f protein expression levels of Hh targets from tumors assayed in b . g Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor in NSG mice after i.p. injection of Leu-SH. Scale bars, 500 μm and 200 μm (upper and lower panels, respectively). h Representative average volume of orthotopic tumor. i – j NSG mice were grafted with spontaneous primary MB from Math1-cre/Ptc C/C mice genetically silenced for ERAP1 expression. i Representative images of mice and the explanted tumor masses. j Quantification of the flank allograft average tumor volume. ERAP1 protein expression is shown below. In f , j , actin was used as loading control. k H&E and representative Masson’s trichrome staining of tumors. Scale bar 100 μm. l mRNA levels of the indicated Hh target genes. m Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor genetically interfered for ERAP1 before the injection in NSG mice cerebella. Scale bars, 500 and 200 μm (upper and lower panels, respectively). n – p ERAP1 accelerates Hh-MB formation. n Tumor volume of mice subcutaneously transplanted with GCPs from tumor-prone Math1-cre/Ptc C/C animals overexpressing ERAP1. o Representative flank allograft average volumes (lower panel) and quantification of the explanted tumor masses (upper panel). p mRNA expression of Hh target genes from the tumor masses assayed in o . q Survival curves of Math-cre/Ptc C/C mice treated with Leu-SH or vehicle. Results in e , l , p were normalized to endogenous GAPDH and HPRT controls and expressed as in Fig. . All data represent the mean of three independent experiments. Mean ± S.D. of tumor ( n = 6) for each treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 calculated by two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Inhibition, In Vivo, Injection, Immunohistochemical staining, Expressing, Derivative Assay, Staining

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: A representative model showing the role of ERAP1 in Hh-dependent tumorigenesis. ERAP1 promotes ubiquitylation and proteasomal degradation of βTrCP by sequestering USP47. This event leads to increase of Gli1 and Gli2 protein levels and decrease of Gli3R, thus triggering the Hh pathway and favoring cell growth and tumorigenesis. In the absence of ERAP1, USP47 binds and stabilizes βTrCP, which, in turn, promotes ubiquitylation and proteasomal degradation of Gli1 and Gli2, and ubiquitylation and proteolytic cleavage of Gli3 into the repressor form Gli3R. These events lead to the repression of the Hh pathway and inhibition of cell proliferation and tumor growth

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Inhibition

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Highly heterogeneous constitutive and IFN-γ-inducible ERAP1/ERAP2 expression in melanoma cells. a The constitutive mRNA expression levels of ERAP1 and ERAP2 in primary melanocytes and representative members of the panel of melanoma cell lines as indicated on the x-axis were determined by qRT-PCR using ERAP1- and ERAP2-specific primer sets as described in “Materials and methods” and supplementary Table 1. The relative mRNA expression levels were normalized to GAPDH serving as a control. In addition, the ERAP/GAPDH ratio of the primary melanocytes were set to 1. For the determination of IFN-γ inducibility of ERAP at the transcriptional and translational level, representative qRT-PCR (b) and Western blot analyses (c) were performed with a set of four selected melanoma cell lines (Buf1287, Colo857, FM6 and FM81), which were either left untreated or treated with IFN-γ (48 h) as described in “Materials and methods”. Western blots were performed using the set of murine anti-ERAP1-, anti-ERAP2- and β-actin-specific antibodies. In addition, immunostainings targeting HLA class I HC served as positive controls for the IFN-γ treatment

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Effects of ERAP siRNA transfection on the relative ERAP1 mRNA and the corresponding HLA class I surface expression levels. The siRNA-mediated reduction of ERAP1 expression was monitored in a panel of melanoma cell lines (UKRV, Buf1287 and Buf1379) by qRT-PCR (a). The results are expressed as ratios of ERAP1 mRNA expression levels normalized to GAPDH mRNA expression levels as described in “Materials and methods”. The corresponding expression levels determined with the non-targeting (nt) control siRNA were set to 100%, respectively. All ERAP1-targeting siRNA transfectants show decreased ERAP1 transcription rates. b The HLA class I surface antigen expression was determined by flow cytometry as described in “Materials and methods” and is represented as the mean fluorescence intensity (MFI), the HLA class I surface expression of cells transfected with the non-sense control siRNA (nt) was set as 100%. The siRNA-mediated reduction of the ERAP1 mRNA levels caused slightly increased HLA class I expression levels in each of the analysed cell lines. The cell lines correspond to the one in a

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Transfection, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Heterogeneous ERAP promoter activity in melanoma cells. a The wt ERAP1 promoter and the pGL3 enhancer vector, which served as a control, were transiently transfected into a series of melanoma cells as indicated on the x-axis. Cells were left untreated or treated for 24 h with IFN-γ before the given ERAP1 (a) promoter activities were determined as described in “Materials and methods”. All results are expressed as relative luciferase activity (RLU) normalized to the corresponding β-galactosidase activity. Grey bars represent untreated cells; black bars IFN-γ-treated cells. b The relative activities of the wt ERAP1 (E1_WT) and mutated ERAP1 promoter (E1_1066) constructs as well as of the corresponding mock control (pGL3 enhancer vector) were transfected in the melanoma cell line Colo857 and the keratinocyte cell line HaCaT serving as a control. The relative promoter activities are expressed as RLU as outlined above in a. The mutation located at nt position 1066 leads to a markedly reduced transcriptional activity, independent from the cellular background. c Altered aminopeptidase activities of ERAP1 variants. The relative enzymatic activities of various ERAP1 constructs in the microsome fractions were determined as described in “Materials and methods”. The ERAP1-negative cell line Buf1182 was either transfected with the empty vector (mock), the wtERAP1 (E1_WT), the novel mut349ERAP1 (E1_349) or with the previously described enzymatically almost inactive ERAP1 variant mut320ERAP1 (E1_320). The relative enzymatic activities are expressed as RLU normalized to the relative ERAP1 enrichment within the indicated microsomal fractions as determined by ERAP1-targeting Western blot analyses. The newly characterized variant with the amino acid substitution M→V at position 349, which is located near the active site, slightly enhances the enzymatic activity of ERAP1

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Activity Assay, Plasmid Preparation, Control, Transfection, Luciferase, Construct, Mutagenesis, Variant Assay, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: Heterogeneous mRNA and/or protein expression of ER-resident aminopeptidases, IRF1 and HLA class I in human melanoma cell lines

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma

doi: 10.1007/s00262-010-0856-7

Figure Lengend Snippet: SNPs within the ERAP1 coding region

Article Snippet: For the determination of the ERAP1 promoter activity, 5 × 10 3 cells/well were transiently transfected with 0.3 μg/well of ERAP1 luc promoter construct using effectene (Qiagen) as recently described [ 31 ].

Techniques: