1. Volume 147, Issue 2, Pages 382-395 (October 2011)
In Vivo Identification of Tumor- Suppressive PTEN ceRNAs in an Oncogenic BRAF- Induced Mouse Model of Melanoma 
Florian A. Karreth, Yvonne Tay, Daniele Perna, Ugo Ala, Shen Mynn Tan, Alistair G. Rust, Gina DeNicola, Kaitlyn A. Webster, Dror Weiss, Pedro A. Perez-Mancera, Michael Krauthammer, Ruth Halaban, Paolo Provero, David J. Adams, David A. Tuveson, Pier Paolo Pandolfi 
Cell 
Volume 147, Issue 2, Pages (October 2011)
DOI: /j.cell
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Identification of Putative PTEN ceRNAs
(A) Schematic outline of our hypothesis: oncogenic BRAF mediates the transformation from melanocytes to nevi, and additional loss of PTEN promotes progression to melanoma. PTEN expression is regulated by PTEN ceRNAs, which sequester PTEN-targeting miRNAs. Transposon insertion into ceRNA gene loci suppresses their expression, which results in increased availability of PTEN-targeting miRNAs and a reduction of PTEN expression.
(B) Bioinformatics approach to identify putative PTEN ceRNAs among Sleeping Beauty CIS. MREs predicted by TargetScan in the murine PTEN 3′UTR were utilized for a mutually targeted MRE enrichment (MuTaME) analysis to determine MRE overlap between PTEN and Sleeping Beauty CIS. CIS that share seven or more MREs with PTEN were considered putative PTEN ceRNAs.
(C) Enrichment of putative PTEN ceRNAs among Sleeping Beauty CIS. The Sleeping Beauty screen identified 320 CIS, 33 of which are putative PTEN ceRNAs. This enrichment is highly significant (p = 7.76 × 10−11).
See also Table S1 and Figure S1.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 2 Putative PTEN ceRNAs Modulate PTEN Expression Levels
(A) List of candidate PTEN ceRNAs identified by Sleeping Beauty. The 33 putative PTEN ceRNAs are ranked by their similarity score, which is based on the identity and number of MREs, as well as the length of the CIS 3′UTRs. The number of MREs shared between PTEN and the individual CIS is also shown.
(B) Western blot (top) and quantification (bottom) of siRNA-mediated knockdowns of putative PTEN ceRNAs in WM35 human melanoma cells. HSP90 expression is shown as a loading control, and PTEN expression levels were normalized to HSP90.
(C) Transposon insertion does not increase expression of the ZEB2 3′UTR (left) or PTEN 3′UTR (right). qRT-PCR depicting the ratio between ZEB2 5′UTR and 3′UTR expression levels in melanomas with transposon insertions in CDKN2A, PTEN, or ZEB2 is shown.
(D) Melanomas with ZEB2 transposon insertions express less PTEN. Western blot showing expression of PTEN and ZEB2 in melanomas isolated from LSL-B-RafV619E; TyrCreERt2 mice (control) or LSL-B-RafV619E; TyrCreERt2; LSL-SB; T2Onc mice with a transposon insertion in either PTEN (CIS: PTEN) or ZEB2 (CIS: ZEB2). Quantification of the western analysis is shown in the bottom panel. Note that PTEN and ZEB2 expression is diminished in tumors with either PTEN or ZEB2 insertions.
NC, negative control (nontargeting siRNA pool). Data are represented as mean ± SEM. See also Figure S2.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. Figure 3 ZEB2 Depletion Downregulates PTEN
(A) ZEB2 silencing lowers PTEN protein levels in murine melanoma cells TB Western analysis for PTEN and ZEB2 expression and HSP90 as loading control is shown in the left panel. Quantification of three independent western analyses is shown in the right panel.
(B) AKT activation kinetics in TB13602 cells following ZEB2 depletion. Cells were starved overnight and stimulated with 10% FCS-containing media for the indicated time points. Western blots for phosphorylated AKT (pAKT), total AKT, ZEB2, and PTEN are shown in the left panel; quantification of pAKT is shown in the right panel.
(C) ZEB2 silencing in human melanoma cell lines lowers PTEN expression and activates AKT. A375 and WM35 melanoma cells lines were transfected with siRNA pools against PTEN or ZEB2. Western analysis for PTEN, ZEB2, and phospho-AKT is shown. HSP90 and total AKT were used as loading controls.
(D) Quantification of PTEN expression in A375 and WM35 cells in three independent western analyses. PTEN expression was normalized to HSP90 levels.
(E) Quantification of AKT activation in A375 and WM35 cells in three independent western analyses. Phospho-AKT expression was normalized to total AKT levels.
(F) qRT-PCR analysis of PTEN and ZEB2 expression in TB13602 cells.
(G and H) qRT-PCR analyses of PTEN and ZEB2 expression in A375 (G) and WM35 (H) cells. Mouse and human siRNA pools against PTEN and ZEB2 efficiently reduce PTEN and ZEB2 mRNA, respectively. Note the reciprocal effect of ZEB2 knockdown on PTEN expression levels and vice versa in WM35 cells.
NC, negative control (nontargeting siRNA pool). Data are represented as mean ± SEM. See also Figure S3.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 4 Regulation of PTEN by ZEB2 Is 3′UTR and miRNA Dependent
(A) Effect of ZEB2 depletion on luciferase activity of a luciferase-PTEN3′UTR reporter. Knockdown of PTEN (positive control) and ZEB2 reduces luciferase activity measured in relative light units (RLU).
(B) ZEB2 depletion lowers PTEN expression in wild-type HCT116 colon cancer cells, but not in Dicer-deficient HCT116 cells. Western blot showing expression of PTEN and HSP90 as loading control in wild-type and Dicer null HCT116 cells (top). (Bottom) Quantification of three independent western blot analyses.
(C) ZEB2 expression is efficiently reduced following treatment of HCT116 cells with ZEB2 siRNA. qRT-PCR analysis of HCT116 cells treated with siRNA for expression of PTEN and ZEB2.
(D) Activation of AKT following ZEB2 depletion is miRNA dependent. Western blot for pAKT and total AKT in WT and Dicer null HCT116 cells is shown (top). (Bottom) Quantification of three independent western blot analyses.
(E and F) Overexpression of ZEB 3′UTR increases PTEN 3′UTR reporter activity. A375 (E) or TB13602 (F) melanoma cells were cotransfected with the luciferase-PTEN3′UTR reporter and expression plasmids containing either the PTEN 3′UTR or ZEB2 3′UTR, followed by luciferase activity measurement 3 days later. The graph depicts the fold increase in RLU when PTEN 3′UTR or ZEB2 3′UTR was expressed compared to an empty control vector.
(G) Overexpression of ZEB2 3′UTR or PTEN 3′UTR increases PTEN levels in A375 cells. Western blot for PTEN and HSP90 as a loading control is shown (top). (Bottom) Quantification of PTEN expression in three independent experiments.
(H) Western analyses showing PTEN expression in response to ZEB2 3′UTR overexpression in WT and Dicer null HCT116 cells (top). (Bottom) Quantification of three independent western blot analyses.
(I) Effect of overexpression of the ZEB2 3′UTR on the activity of a luciferase-PTEN 3′UTR reporter in WT and Dicer null HCT116 cells is shown.
Data are represented as mean ± SEM. NC, negative control (nontargeting siRNA pool). See also Figure S4.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 5 Characterization of miRNAs that Are Common to PTEN and ZEB2
(A) miRNA response elements (MREs) shared by PTEN and ZEB2. This table depicts the nine miRNAs that the 3′UTRs of PTEN and ZEB2 have in common, as well as the number of sites for each miRNA.
(B) Multiple miRNAs target both PTEN and ZEB2. Western analysis of PTEN and ZEB expression in TB13602 melanoma cells treated with the indicated miRNAs is shown. Actin expression is shown as a loading control. Quantification of the western blot is shown in the bottom panel.
(C) Expression of common miRNAs in A375, WM35, and TB13602 melanoma cells as determined by qRT-PCR. n.d., not detectable.
(D) MS2-RIP followed by qRT-PCR to identify endogenous miRNAs associated with the PTEN and ZEB2 3′UTRs.
(E) MS2-RIP followed by qRT-PCR to identify miRNA binding to the PTEN 3′UTR in cells depleted for PTEN or ZEB2.
NC, negative control (nontargeting siRNA pool). Data are represented as mean ± SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

8. Figure 6 ZEB2 Displays Tumor-Suppressive Properties in Melanoma Cells
(A–C) Proliferation curves of TB13602 (A), WM35 (B), and A375 (C) melanoma cells treated with siPTEN, siZEB2, and NC are shown.
(D–F) Anchorage-independent growth in soft agar. Representative pictures of TB13602 (D), WM35 (E), and A375 (F) are shown on the right, and quantifications are shown on the left.
(G) Western blot showing ZEB2 and PTEN expression in TB13602 cells infected with shZEB2 and shGFP pLKO.1 lentiviruses (top). (Bottom) Quantification of western analysis.
(H) Proliferation curves of cells shown in (G).
(I) Quantification of xenograft tumor volume of TB13602 cells infected with shZEB2 and shGFP pLKO.1 lentiviruses.
Data are represented as mean ± SEM. NC, negative control (nontargeting siRNA pool).
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

9. Figure 7 Functional Crosstalk of PTEN and ZEB2 in Human Cancer
(A) mRNA expression of PTEN and ZEB2 correlates in human melanoma. Graph showing mRNA expression levels for PTEN and ZEB2 in human melanoma samples, in which each dot corresponds to one tumor sample. Pearson score shows a positive and significant correlation.
(B) ZEB2 mRNA expression is significantly lower in human melanomas with reduced PTEN expression when compared to normal melanocytes. Box plot depicting mRNA expression levels for PTEN and ZEB2 in normal melanocytes and “PTEN low” tumors (melanomas with PTEN mRNA expression below the average).
(C) Correlation of PTEN and ZEB2 mRNA expression in human prostate cancer samples. Similar to (A), each dot represents one tumor sample. Pearson score shows a positive and significant correlation.
(D) ZEB2 expression is significantly reduced in prostate tumors with low PTEN mRNA expression levels. Box plot showing PTEN and ZEB2 mRNA expression levels in normal adjacent prostate and primary prostate adenocarcinoma.
(E–G) Additional mRNA expression data sets showing reduced PTEN and ZEB2 mRNA levels in melanoma (E), colon carcinoma (F), and glioblastoma (G) compared to normal tissue.
In the box plots, the top and bottom boxes represent the 75th and 25th percentile, respectively, and the whiskers represent the minimum and maximum of all data points. See also Figure S5.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

10. Figure S1 Identification of Putative PTEN ceRNAs, Related to Figure 1
Schematic outline of Sleeping Beauty insertional mutagenesis screen. Heterozygous LSL-B-RafV619E (B) mice were intercrossed with triple heterozygous TyrCreErt2; LSL-SB; T2Onc (C; S; T2) mice to generate quadruple heterozygous mutant mice. Experimental and control mice were topically treated with 4-Hydroxytamofixen (4-OHT) at 3 weeks of age and monitored for melanoma development. Tumor genomic DNA was used to identify common insertion sites (CIS) by 454 sequencing.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

11. Figure S2
Silencing of PTEN ceRNAs in Human Melanoma Cells, Related to Figure 2
(A) qRT-PCR analysis to determine PTEN expression in response to knock-down of AFF1, JARID2, DCBLD2, TNRC6a, TNRC6b, MBNL1, RBM9 and ZEB2 in WM35 cells.
(B) qRT-PCR to determine knock-down efficiency of the putative ceRNAs AFF1, JARID2, DCBLD2, TNRC6a, TNRC6b, MBNL1, RBM9 and ZEB2 in WM35 cells. Note reciprocal effect of PTEN ceRNA knock-down on PTEN expression levels and vice versa.
(C) Effect of silencing of putative PTEN ceRNAs on PTEN protein levels. Western blot (upper panel) and quantification (lower panel) of siRNA-mediated knock-downs of putative PTEN ceRNAs in A375 human melanoma cells. HSP90 expression is shown as a loading control and PTEN expression levels were normalized to HSP90.
(D) qRT-PCR analysis to determine PTEN expression in response to knock-down of AFF1, JARID2, DCBLD2, TNRC6a, TNRC6b, MBNL1, RBM9 and ZEB2 in A375 cells.
(E) qRT-PCR analysis to determine knock-down efficiency of the putative ceRNAs AFF1, JARID2, DCBLD2, TNRC6a, TNRC6b, MBNL1, RBM9 and ZEB2 in A375 cells. NC, negative control (nontargeting, scrambled siRNA pool). Data are represented as ± SEM.
(F) Schematic outlines of the genomic loci of PTEN, CNOT6L and the putative PTEN ceRNAs analyzed in Figure 2. The depicted orientation of the genes is in accordance with their orientation in the mouse genome. Transposon insertion sites are depicted by red and green arrowheads. The orientation of the arrowheads indicates the orientation of the inserted transposon. Red arrowheads represent the transposon orientation that results in repression of transcription. Green arrowheads represent the transposon orientation that represses transcription but that may also lead to transcription of the gene portion downstream of the transposon.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

12. Figure S3 Silencing of ZEB2 in Melanoma Cells, Related to Figure 3
(A) ZEB2 depletion in 451Lu and WM278 cell lines lowers PTEN expression and activates AKT. Cell lines were transfected with siRNA pools against PTEN or ZEB2. Western analysis for PTEN, ZEB2, and phospho-AKT is shown (left panel). HSP90 and total AKT were used as loading controls. (middle panel) Quantification of PTEN expression in 451Lu and WM278 cells in 3 independent Western analyses. PTEN expression was normalized to HSP90 levels. (right panel) Quantification of AKT activation in 451Lu and WM278 cells in 3 independent Western analyses. Phospho-AKT expression was normalized to total AKT levels.
(B) Knock-down with individual siRNAs against ZEB2 (TB13602 and WM35) or shZEB2-pLKO.1 (A375). Some of the siRNAs of the ZEB2 siRNA pools contain 6 nucleotide stretches that could serve as seeds and that are also found in the 3′UTR of PTEN. To ensure that decreased PTEN is due to the ceRNA activity of ZEB2 and not a potential off-target effect of the siRNA pools we used 2 siRNAs and 1 shRNA without seed matches with PTEN and show that these efficiently silence ZEB2, thereby leading to PTEN reduction. Western blots for ZEB2, PTEN and actin are shown (upper panels). Quantification of Western blots is shown in lower panels.
NC, negative control (nontargeting siRNA pool). Data are represented as mean ± SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

13. Figure S4
Ectopic Expression of the ZEB2 Coding Sequence in Wild-Type and Dicer Null HCT116 Cells, Related to Figure 5
The upper panel displays Western blot analysis for ZEB2 and PTEN expression, while the lower panel shows the quantification of the Western blot. HSP90 was used as a loading control.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions

14. Figure S5
ZEB2 Expression Correlates with PTEN Expression in Human Melanoma, Prostate Cancer, and Multiple Normal Tissues, Related to Figure 7
(A) Box plot showing PTEN and ZEB2 mRNA expression in normal melanocytes, melanoma, and “PTEN high” and “PTEN low” melanomas.
(B) Box plot showing PTEN and ZEB2 mRNA expression in normal prostate, primary prostate cancer (CaP), and “PTEN high” and “PTEN low” primary prostate cancer (CaP). Note that ZEB2 is downregulated in primary CaP, and further reduced in “PTEN low” CaP. In the box plots, the top and bottom boxes represent the 75th and 25th percentile, respectively, and the whiskers represent the minimum and maximum of all data points.
(C) Table depicting human tissues in which PTEN and ZEB2 mRNA expression correlates.
Cell  , DOI: ( /j.cell )
Copyright © 2011 Elsevier Inc. Terms and Conditions