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. 2011 Jul;13(7):736-47.
doi: 10.1093/neuonc/nor036. Epub 2011 Jun 3.

The Wnt Inhibitory Factor 1 (WIF1) Is Targeted in Glioblastoma and Has a Tumor Suppressing Function Potentially by Induction of Senescence

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Free PMC article

The Wnt Inhibitory Factor 1 (WIF1) Is Targeted in Glioblastoma and Has a Tumor Suppressing Function Potentially by Induction of Senescence

Wanyu L Lambiv et al. Neuro Oncol. .
Free PMC article

Abstract

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Figures

Fig. 1.
Fig. 1.
Gene expression–based prediction of copy number aberrations (CNAs) in glioblastoma. (A) The maximum (red) and mean (blue) amplification probabilities on chromosome 12q14–12q15 were estimated from glioblastoma gene expression data by a hidden Markov model (HMM). The interrogated region flanked by CDK4 and MDM2 encompasses a ∼11 Mb window. (B) The respective mean DNA copy number of this chromosomal region was determined by array comparative genomic hybridization (aCGH, Humarray 3.0 and 3.1 of the University of California at San Francisco; manuscript in preparation). The bacterial artificial chromosome (BAC) probes are ordered by their genomic positions. The BACs corresponding to CDK4 and MDM2 BACs are shown in red. (C) The heat map visualizes the structure of the aCGH data shown in (B) for the genomic region encompassing 12q13 to 12q15 from 68 glioblastomas. The BACs are ordered by their genomic position, while the glioblastomas on the x-axis are ordered by similarity using Sorting Points into Neighborhood software. Blue depicts deletion; red, amplification; and white, missing data. The color scale is truncated to [−1, 1] for presentation. The BAC corresponding to CDK4 is GS-561N1; for MDM2, CTB-136O14 (red); and for WIF1, RP11-18B8 (blue). (D) WIF1 expression (Affymetrix probe set 204712_at) in glioblastoma is significantly lower than in nonneoplastic brain tissues (P = .001), as determined in our gene expression data set. (E) Low WIF1 expression was confirmed in 5 independent glioblastoma data sets (Freije et al., red; Rich et al., violet; Phillips et al., orange; Sun et al., dark blue; Horvath et al., green; our data set, Murat et al., light blue). WIF1 expression values are median centered within each data set independently.
Fig. 2.
Fig. 2.
Promoter methylation of WIF1 in glioblastoma. Methylation-specific PCR (MSP) for WIF1 was performed via a nested approach using published primer sequences. DNA isolated from peripheral blood lymphocytes (PBLs) and the colon cancer cell line SW48 served as controls for unmethylated (U) and methylated (M) WIF1 promoter status, respectively. Glioblastomas #2445, #2447, and #2448 contain a methylated WIF1 promoter, whereas the others harbor only an unmethylated gene promoter.
Fig. 3.
Fig. 3.
Alteration of WIF1 expression modulates Wnt signaling. (A) Glioblastoma cell lines were treated with the DNA demethylating agent 5-aza-cytidine for 4 days. All 3 WIF1 nonexpressing cell lines with a methylated WIF1 promoter re-expressed WIF1 mRNA in response to 5-aza-cytidine treatment (LN319, LN401, U87). Only LN992 was completely unmethylated. The lower panel shows mRNA expression of the POLR2A gene used to control for mRNA quality. The methylation status of the cell lines displayed was determined by MSP. (B) Wnt signaling activity was measured with the TCF luciferase reporter (TOP5/FOP5; ratio >1 Wnt pathway active). The non–small cell lung carcinoma cell line A549 served as positive control. LN235 and LN992 show no significant activity. (C) Wnt signaling (TOP5/FOP5) after cotransfection of increasing amounts (0 μg, 0.5 μg, and 1 μg) of the WIF1 expression vector into glioblastoma cell lines U87, LN319, LN401, and A549. The DNA quantity was adjusted with the control vector. (D). LN235 and LN992, negative for Wnt signaling activity, were transfected with siWIF1 and the siScrambled as control. Knockdown of WIF1 induced Wnt pathway activity (TOP5/FOP5) in both LN992 and LN235. WIF1 expression measured after transfection of siWIF1 or the respective control siRNAs is documented in Supplementary Fig. S2. Results are marked with 1 asterisk (*) if P< .05 and 2 (**) if P < .01.
Fig. 4.
Fig. 4.
LN319 cell clones stably transfected with WIF1 show reduced Wnt pathway signaling. Two stably transfected LN319 clones were analyzed for WIF1 expression by qRT-PCR (normalized to the control cells transfected with the empty vector, pcDNA3.1) (A) and WIF1 secretion by enzyme-linked immunosorbent assay (ELISA) (B). Wnt pathway signaling was measured both with the TCF luciferase reporter (TOP5/FOP5) and normalized to the control cells (C) and by measuring AXIN2 mRNA expression (D). The specificity of the WIF1-induced effects in the 2 clones was controlled by transfection of specific siRNAs against WIF1 or a respective scrambled control. Wnt pathway signaling was measured using the TCF reporter (E) and WIF1 secretion using ELISA (F). Results are marked with 1 asterisk (*) if P< .05 and 2 (**) if P < .01.
Fig. 5.
Fig. 5.
WIF1 overexpression reduces the growth potential of LN319 cells in vitro and in vivo. Growth of WIF1-transduced LN319 clones and the respective empty vector control (pcDNA3.1) was followed over 3 days in culture (A). Anchorage-independent growth was evaluated in soft agar; representative images are shown (B). The average number of colonies counted in 10 randomly chosen fields is reported (C). Tumor growth kinetics of nude mouse xenografts, after subcutaneous injection of WIF1-overexpressing clones and the corresponding empty vector control cells are displayed. The WIF1-overexpressing clones did not form any measurable tumors. The average tumor volume per group (5 mice) is reported (D). Results are marked with 1 asterisk (*) if P < .05 and 2 (**) if P < .01.
Fig. 6.
Fig. 6.
WIF1 expression and detection of senescence-like cells. Cell morphology of the different LN319 clones was analyzed by fluorescence-activated cell sorting (FACS) (A). Senescence-like cells were defined as highly granulated cells, high side scatter (SS) (blue rectangle), and big cells, high forward scatter (FS) (red rectangle). (B) Quantification of FACS analysis, % of highly granulated cells (SS high, blue), and % of both highly granulated and big cells (FS and SS high, red). Quantification of LN319 (C) and LN229 (E) cells positive for SA-β-galactosidase activity scored in 10 different randomly chosen fields. (D, F) Representative images of clones stained for SA-β-galactosidase and DAPI are shown (200×). Large, SA-β-galactosidase positive (blue) and multinucleated cells are highlighted with red arrows. Results are marked with 1 asterisk (*) if P < .05 and 2 (**) if P < .01.
Fig. 7.
Fig. 7.
Model for mechanisms implicated in deregulation of both tumor suppressing and proto-oncogenic pathways on chromosome 12. The model suggests that deletion at the WIF1 locus, if concomitant with amplification of the proto-oncongenes CDK4 and MDM2, can subvert the important tumor suppressing pathways regulated by RB1 and TP53 while simultaneously activating proto-oncogenic Wnt signaling.

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