In vivo identification of tumor- suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma

Abstract

We recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). However, the functional role of ceRNAs in cancer remains unknown. Loss of PTEN, a tumor suppressor regulated by ceRNA activity, frequently occurs in melanoma. Here, we report the discovery of significant enrichment of putative PTEN ceRNAs among genes whose loss accelerates tumorigenesis following Sleeping Beauty insertional mutagenesis in a mouse model of melanoma. We validated several putative PTEN ceRNAs and further characterized one, the ZEB2 transcript. We show that ZEB2 modulates PTEN protein levels in a microRNA-dependent, protein coding-independent manner. Attenuation of ZEB2 expression activates the PI3K/AKT pathway, enhances cell transformation, and commonly occurs in human melanomas and other cancers expressing low PTEN levels. Our study genetically identifies multiple putative microRNA decoys for PTEN, validates ZEB2 mRNA as a bona fide PTEN ceRNA, and demonstrates that abrogated ZEB2 expression cooperates with BRAF(V600E) to promote melanomagenesis.

Figure 1

(related Table S1 and Figure S1): 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 7 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).

Figure 2

(related to Figure S2): 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 (upper panel) and quantification (lower panel) of siRNA-mediated knock-downs 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 panel) or PTEN 3′UTR (right panel). 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 B-Raf^V619E; TyrCreERt2 mice (control) or B-Raf^V619E; TyrCreERt2; SB13; 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 (non-targeting siRNA pool). Data are represented as mean +/- SEM.

Figure 3

(related to Figure S3): ZEB2 depletion downregulates PTEN. (A) ZEB2 silencing lowers PTEN protein levels in murine melanoma cells TB13602. Western analysis for PTEN and ZEB2 expression and HSP90 as loading control is shown in the left panel. Quantification of 3 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 3 independent Western analyses. PTEN expression was normalized to HSP90 levels. (E) Quantification of AKT activation in A375 and WM35 cells in 3 independent Western analyses. Phospho-AKT expression was normalized to total AKT levels. (F) Real-time PCR analysis of PTEN and ZEB2 expression in TB13602 cells. (G,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 reciprocal effect of ZEB2 knock-down on PTEN expression levels and vice versa in WM35 cells. Data are represented as mean +/- SEM.

Figure 4

(related to Figure S4): 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. Knock-down 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 (upper panel). Lower panel shows quantification of 3 independent Western blot analyses. (C) ZEB2 expression is efficiently reduced following treatment of HCT116 cells with ZEB2 siRNA. Real-time 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 (upper panel). Lower panel shows quantification of 3 independent Western blot analyses. (E, F) Overexpression of ZEB 3′UTR increases PTEN 3′UTR reporter activity. A375 (E) or TB13602 (F) melanoma cells were co-transfected with the Luciferase-PTEN3′UTR reporter and expression plasmids containing either the PTEN 3′UTR or ZEB2 3′UTR, followed by Luciferase activity measurement three 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 in the upper panel. Lower panel depicts quantification of PTEN expression in 3 independent experiments. (H) Western analyses showing PTEN expression in response to ZEB2 3′UTR overexpression in WT and Dicer null HCT116 cells (upper panel). Lower panel shows quantification of 3 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 (non-targeting siRNA pool).

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 9 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 lower panel. (C) Expression of common miRNAs in A375, WM35 and TB13602 melanoma cells as determined by qRT-PCR. (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. Data are represented as mean +/- SEM.

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 is 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 (upper panel). Lower panel depicts 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 (non-targeting siRNA pool).

Figure 7

(related to Figure S5): Functional cross-talk 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, where 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 datasets showing reduced PTEN and ZEB2 mRNA levels in melanoma (E), colon carcinoma (F), and glioblastoma (G) compared to normal tissue. Data are represented as mean +/- SEM.