Pla2g16 phospholipase mediates gain-of-function activities of mutant p53

Abstract

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis.

Keywords: fatty acid metabolism; mammary tumor.

Fig. 1.

Increased Pla2g16 expression in p53^R172H/+ osteosarcomas. (A) Heat map of Affymetrix array. (B) Validation of Pla2g16 expression in osteosarcoma tumors was determined by real-time quantitative PCR (*P < 0.05). (C) Representative immunohistochemical staining of Pla2g16 in p53^+/− and p53^R172H/+ primary and metastatic osteosarcoma samples. (Scale bar, 50 µm.) Tumors were considered to be positive when 10% or more of dysplastic/tumor cells were stained.

Fig. 2.

Pla2g16 contributes to the metastatic potential of p53^R172H/+ osteosarcomas. (A) shRNA knockdown of Pla2g16 in p53^R172H/+ osteosarcoma cell lines H76 and H318-1 was determined by qRT-PCR (Upper) and Western blot analysis (Lower). Migration (B) and invasion (C) assays were performed using Pla2g16 knockdown cells for 16 h and 40 h, respectively. (D) Colony formation assays were performed for Pla2g16 knockdown cells. (E) Number of mice with or without metastasis after injection with control and Pla2g16 knockdown cells. (F) Overexpression of Pla2g16 in p53^–/o osteosarcoma cells increased invasion. Pla, Pla2g16; Flag, Flag-tag; Vin, vinculin; Ctrl, shRNA of EGFP; shPla, shRNA of Pla2g16; vec, pBabe-puro; *P < 0.05 and ***P < 0.0005.

Fig. 3.

Higher expression of Pla2g16 in human osteosarcoma cells contributes to higher metastatic potential. (A) Expression of Pla2g16 in LM7 osteosarcoma cells was determined by Western blot analysis. (B) Two shRNAs were used to knock down Pla2g16 in LM7 cells which showed reduced migration (C), invasion (D), and colony formation (E). (F) Western blot of Pla2g16 overexpression in Saos2 cells affects colony formation (G). (H) Representative immunohistochemical staining of human osteosarcoma samples with a Pla2g16 antibody. (Scale bar, 50 µm.) *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 4.

Up-regulation of Pla2g16 by p53 mutants. (A) Western blots of H318-1 cells with and without p53R172H knockdown. (B) Western blots of p53^−/− MEFs and p53^–/o osteosarcoma cell line (#222) with addition of p53R172H. (C) Western blots for p53R175H overexpression in Saos2 cells with Pla2g16 shRNA knockdown. (D) shRNA knockdown of Pla2g16 in p53R175H expressing Saos2 cells inhibited colony formation. shp53, shRNA for p53; shpla, shRNA for Pla2g16; Vec, pBabe vector for overexpression of p53R175H. **P < 0.005, ***P < 0.0005.

Fig. 5.

ETS2 mediated up-regulation of Pla2g16 by mutant p53. (A) Western blot of Saos2 cells and (B) ChIP assays in Saos2 cells engineered to express different p53 mutants. (Upper) Relative occupancy of binding site 1 (BS1) and binding site 2 (BS2) in the Pla2g16 promoter; (Lower) levels of p53 mutants from the same extracts used in ChIP assays loaded on one gel. (C) Knockdown of ETS2 reduced Pla2g16 expression in mutant p53 overexpressing cells. (Upper) Relative expression levels of Pla2g16 in siRNA knockdown cells determined by qRT-PCR; (Lower) Western blots of ETS1 and ETS2 in siRNA knockdown cells. GFP: pWZL vector for overexpression of GFP and p53 mutations, R175H, H179R, G245S, R248Q, R273H. Ctr, Control.

Fig. 6.

Pla2g16 contributes to p53^R172H/+ mammary tumor metastatic potential. (A) Kaplan–Meier tumor-free survival curves for p53^R172H/+ mice with or without radiation. (B) Pla2g16 expression levels in p53^R172H/+ metastatic and nonmetastatic p53^+/− primary mammary tumors by qRT-PCR. shRNA knockdown of Pla2g16 in the metastatic mammary tumor cell line 4T1 was measured by qRT-PCR (C) and Western blot analysis (D). Pla2g16 shRNA treated 4T1 cells had reduced migration (E), and invasion (F). (G) Western blots with mutant p53 knockdown in breast cancer cells. (H) Flag-tagged Pla2g16 overexpression in human MDA-MB231 breast cancer cells was measured by Western blotting. Proliferation (I) and colony formation (J) assays were performed in Pla2g16 overexpressing cells. *P < 0.05, **P < 0.005, ***P < 0.0005.