1. Volume 59, Issue 6, Pages 904-916 (September 2015)
Truncated ERG Oncoproteins from TMPRSS2-ERG Fusions Are Resistant to SPOP- Mediated Proteasome Degradation 
Jian An, Shancheng Ren, Stephen J. Murphy, Sumiya Dalangood, Cunjie Chang, Xiaodong Pang, Yangyan Cui, Liguo Wang, Yunqian Pan, Xiaowei Zhang, Yasheng Zhu, Chenji Wang, Geoffrey C. Halling, Liang Cheng, William R. Sukov, R. Jeffrey Karnes, George Vasmatzis, Qing Zhang, Jun Zhang, John C. Cheville, Jun Yan, Yinghao Sun, Haojie Huang 
Molecular Cell 
Volume 59, Issue 6, Pages (September 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2015 59, 904-916DOI: (10.1016/j.molcel.2015.07.025)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
The SPOP-CUL3-RBX1 Complex Targets ERG for Polyubiquitination and Degradation
(A) Schematic showing the SPOP-CUL3-RBX1 E3 ubiquitin (Ub) ligase complex and two putative SPOP SBC motifs in human ERG.
(B) Immunoblot (IB) of whole-cell lysates (WCLs) and co-IP samples of immunoglobulin G (IgG) or indicated antibody obtained from 293T cells transfected with indicated plasmids and treated with 20 μM of MG132 for 8 hr.
(C) IB of co-IP samples of IgG or anti-ERG antibody obtained from the nuclear extracts of LNCaP cells treated with 20 μM of MG132 for 8 hr.
(D) IB of WCLs of indicated cells transfected with indicated small interfering RNAs (siRNAs). ERK2 was used as a loading control.
(E) IB of WCLs of LNCaP cells transfected with indicated siRNAs.
(F) IB of WCLs of 293T cells transfected with indicated plasmids.
(G) IB of WCLs from 293T cells transfected with indicated plasmids and treated with or without 20 μM of MG132 for 8 hr.
(H) IB of WCLs of 293T cells transfected with indicated plasmids.
(I) IB of the products of in vivo ubiquitination assay performed in 293T cells transfected with indicated plasmids.
(J) IB of the products of in vitro ubiquitination assay performed by incubating the reconstituted E3 Ub ligase complex (CUL3-RBX1, GST-SPOP) with E1, E2, Ub, and GST-ERG (N200, amino acids 1–200) at 30°C for 2 hr.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The Role of ERG in SPOP Inactivation-Induced Increase in Prostate Cancer Cell Invasion and Proliferation
(A–C) PC-3 and 22Rv1 cells were infected with lentivirus expressing indicated shRNAs for invasion assay (A and B) and for immunoblotting (C). All data shown are mean values ± SD (error bar) from three replicates. ∗p < 0.01.
(D) RT-qPCR assessment of mRNA expression of four ERG target genes PLAT, PLAU, ADAM19, and MMP3 in PC-3 and 22Rv1 cells. GAPDH was used for normalization. All data shown are mean values ± SD (error bar) from three independent experiments. ∗p < 0.01.
(E) MTS assay of C4-2 cells infected with lentivirus expressing indicated shRNAs. All data shown are mean values ± SD (error bar) from three independent experiments. ∗p < 0.01 in C4-2 cells at the time point of day 6.
(F) Screen shots of the University of California, Santa Cruz, Genome Browser showing co-localization of AR and ERG binding sites at loci of SPOP/AR co-regulated genes SGK1 and CAMKK2.
(G) RT-qPCR assessment of mRNA expression of five AR and SPOP co-regulated target genes SGK1, CAMKK2, ABCC4, HOMER, and SEPP1 in C4-2 cells infected with lentivirus expressing indicated shRNAs. GAPDH was used for normalization. All data shown are mean values ± SD (error bar) from three independent experiments.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 The 42ASSSS46 Motif in ERG Is a Degron Recognized by SPOP
(A) Diagram showing two putative SBC motifs located in the ERG N terminus (42ASSSS46) and the C terminus (423VTSSS427), respectively, and ERG mutants with internal deletion of 42ASSSS46 or 423VTSSS427 alone or both (2Δ). CAE, central-alternative-exons region; ETS, DNA-binding domain; NTD, N-terminal domain; PNT, pointed domain; TAD, transactivation domain.
(B) IB of WCLs and co-IP samples of IgG or indicated antibody obtained from 293T cells transfected with indicated plasmids and treated with 20 μM of MG132 for 8 hr.
(C) Schematic diagram showing four GST-ERG recombinant proteins.
(D) IB of SPOP protein in WCLs of LNCaP cells pulled down by GST-ERG recombinant proteins purified from bacteria.
(E) IB of WCLs of 293T cells transfected with indicated plasmids.
(F) IB of the products of in vivo ubiquitination assay of 293T cells transfected with indicated plasmids.
(G and H) IB of WCLs of 293T cells transfected with indicated plasmids and treated with 50 μg/ml of cycloheximide (CHX) and harvested at different time points (G). At each time point, the intensity of ERG was normalized to the intensity of ERK2 (loading control) first and then to the value at the 0-hr time point (H) Similar results were obtained from three independent experiments.
(I) IB of WCLs of 293T cells transfected with indicated plasmids.
(J–L) BPH-1 cells were transfected with indicated plasmids for immunoblotting (J) and for invasion assay (K and L). Representative images are shown in (L) and quantitative data are shown in (K). All data shown are mean values ± SD (error bar) from three replicates. ∗p < 0.01.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Prostate Cancer-Associated SPOP Mutants Cannot Bind to and Promote ERG Ubiquitination
(A–C) Computational modeling of structures of the wild-type (WT) (A) and F102C-mutated (B) and F133V-mutated (C) SPOP MATH domain in complex with the ERG SBC motif 42ASSSS46. The MATH domain is shown in gray cartoon with its key substrate-interacting residues in ball and sticks. The ERG SBC 42ASSSS46 is shown in green sticks. The hydrogen bonds between ERG SBC and SPOP MATH domain are indicated as dashed lines. Oxygen and nitrogen atoms are colored in red and blue, respectively.
(D) IB of WCLs and co-IP samples of IgG or indicated antibody obtained from 293T cells transfected with indicated plasmids and treated with 20 μM of MG132 for 8 hr.
(E) IB of the products of in vivo ubiquitination assay of 293T cells transfected with indicated plasmids.
(F) IB of WCLs of C4-2 cells transfected with WT or SPOP mutants as indicated. The density of ERG was determined by normalizing to ERK2 (loading control) first and then to the normalized value in cells without transfection of Myc-SPOP.
(G) Quantitative data of invasion assay performed in BPH-1 cells transfected with indicated plasmids. All data shown are mean values ± SD from three replicates. p < 0.01.
(H) Diagram showing 155 TMPRSS2-ERG fusion-negative prostate cancers, including 19 (approximately 12%) ERG IHC staining-positive cases, 8 of which (approximately 5%) harbored SPOP mutations.
(I and J) Representative FISH image (I) and ERG IHC staining (J) of a TMPRSS2-ERG rearrangement-negative case that harbored a SPOP mutation. ∗ERG staining-positive endothelial cells of blood vessel. The scale bar represents 50 μm.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Oncogenic TMPRSS2-ERG Fusions Are Resistant to SPOP-Mediated Degradation
(A) Schematic diagram showing exons of the ERG gene and wild-type and truncated ERG proteins produced by TMPRSS2-ERG fusion isoforms T1-E1/2 (occurred infrequently) and T1/E4 and T1/E5 (the majority of fusions occurred in prostate cancers), respectively. The ERG coding exons are in yellow. The ERG SBC motif 42ASSSS46 is red.
(B) IB of WCLs and co-IP samples of IgG or indicated antibody obtained from 293T cells transfected with indicated plasmids and treated with 20 μM of MG132 for 8 hr.
(C) Representative structure of the SPOP MATH domain in complex with the ERG SBC motif 42ASSSS46 and its N-terminal-adjacent 11 amino acids (31–41). The SPOP MATH domain is shown in gray surface, while the backbone of ERG SBC is shown as a green tube. The heavy atoms of the side chains of 42ASSSS46 are also displayed.
(D) IB of WCLs and co-IP samples of IgG or indicated antibody obtained from 293T cells transfected with indicated plasmids and treated with 20 μM of MG132 for 8 hr.
(E) IB of WCL of 293T cells transfected with indicated plasmids.
(F) IB of the products of in vivo ubiquitination assay in 293T cells transfected with indicated plasmids.
(G) IB of WCLs and co-IP samples of IgG or indicated antibody obtained from VCaP cells transfected with Myc-SPOP and treated with 20 μM of MG132 for 8 hr. FL, full-length.
(H) IB of WCLs of VCaP cells infected with indicated lentivirus-based shRNAs.
(I) Luciferase assay in C4-2 cells transfected with indicated plasmids. All data shown are mean values ± SD (error bar) from three independent experiments. p < 0.01.
(J–L) BPH-1 cells were transfected with indicated plasmids for immunoblotting with indicated antibodies (J) and for invasion assay (K and L). Quantitative data are shown in (K), and the representative images are shown in (L). All data shown are mean values ± SD (error bar) from three replicates. ∗p < 0.01.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Detection of Expression of SPOP-Sensitive and SPOP-Resistant TMPRSS2-ERG Fusion Proteins in Human Prostate Cancer Specimens
(A) Mapping of DNA breakpoints of T1-E5, T2-E5, T1-E4, T1-E1, and T1-E2 fusions in human prostate cancers identified by MP-NGS and Sanger sequencing.
(B) Gleason pattern and score of tumor specimens in (A). Tumor groups with low to high Gleason pattern and score: (1) G3_GS6/IND, Gleason score 6 indolent (IND) tumors with majority of tissues in Gleason pattern 3; (2) G3_GS6/LV, Gleason score 6 large volume (LV) tumors with majority of tissues in Gleason pattern 3; (3) G3_GS7, Gleason score 7 tumors with majority of tissues in Gleason pattern 3; (4) G3/G4_GS7, Gleason score 7 tumors with approximately equivalent percentage of tissues in Gleason pattern 3 or 4; (5) G4_GS7, Gleason score 7 tumors with majority of tissues in Gleason pattern 4; (6) G5_GS9, Gleason score 9 tumors with majority of tissues in Gleason pattern 5. Cases with higher Gleason pattern and score (groups 4–6) are highlighted in purple.
(C) Heatmap showing the IHC staining intensities (increased from 1 to 3+) of ERG proteins in T1/2-E5, T1-E4, and T1-E1/2 prostate tumors. See detailed scoring method in Supplemental Information.
(D) Representative images of ERG IHC in T1-E2 (left), T1-E4 (middle), and T1-E5 (right) prostate tumors. ∗Positive staining of ERG in endothelial cells of blood vessel. Arrows indicate malignant prostatic tissues with moderate ERG staining; arrowhead indicates benign prostatic tissues with no ERG staining.
(E and F) Anchorage-independent cell growth assay of BPH-1 cells infected with lentivirus-expressing PTEN shRNA in combination with different ERG fusions. Representative images are shown in (E), and quantitative data are shown in (F). The scale bar represents 200 μm. All data shown are mean values ± SD from three replicates. p < 0.01.
See also Figures S6 and S7.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Working Model
A model depicting ERG resistance to SPOP-mediated proteasome degradation due to either SPOP mutations or degron-impaired TMPRSS2-ERG fusions (e.g., T1-E4 and T1-E5) detected most frequently in human prostate cancers. ETS, DNA binding domain of ERG. TA, transactivation domain.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions