A novel PTEN/mutant p53/c-Myc/Bcl-XL axis mediates context-dependent oncogenic effects of PTEN with implications for cancer prognosis and therapy

Abstract

Phosphatase and tensin homolog located on chromosome 10 (PTEN) is one of the most frequently mutated tumor suppressors in human cancer including in glioblastoma. Here, we show that PTEN exerts unconventional oncogenic effects in glioblastoma through a novel PTEN/mutant p53/c-Myc/Bcl-XL molecular and functional axis. Using a wide array of molecular, genetic, and functional approaches, we demonstrate that PTEN enhances a transcriptional complex containing gain-of-function mutant p53, CBP, and NFY in human glioblastoma cells and tumor tissues. The mutant p53/CBP/NFY complex transcriptionally activates the oncogenes c-Myc and Bcl-XL, leading to increased cell proliferation, survival, invasion, and clonogenicity. Disruption of the mutant p53/c-Myc/Bcl-XL axis or mutant p53/CBP/NFY complex reverses the transcriptional and oncogenic effects of PTEN and unmasks its tumor-suppressive function. Consistent with these data, we find that PTEN expression is associated with worse patient survival than PTEN loss in tumors harboring mutant p53 and that a small molecule modulator of p53 exerts greater antitumor effects in PTEN-expressing cancer cells. Altogether, our study describes a new signaling pathway that mediates context-dependent oncogenic/tumor-suppressive role of PTEN. The data also indicate that the combined mutational status of PTEN and p53 influences cancer prognosis and anticancer therapies that target PTEN and p53.

Figure 1

PTEN exerts oncogenic effects that are mediated by gain-of-function mut-p53. (A) MTS assay of PTEN-null/mut-p53 U373 and SNB19 glioblastoma cells with or without PTEN restoration with Ad-PTEN and/or with or without mut-p53 silencing with shRNA (sh-mp53). (B) Apoptosis assay of U373 and SNB19 cells with or without Ad-PTEN and/or with or without sh-mp53. (C) Immunoblot showing the levels of mut-p53 silencing and PTEN restoration and their effects on apoptotic regulators PARP and caspase-3 in glioblastoma cells. (D) Transwell invasion assay of U373 and SNB19 cells with or without Ad-PTEN and/or with or without sh-mp53. Con, control; *P < .05.

Figure 2

PTEN induces clonogenicity of glioblastoma cells through mut-p53/c-Myc/Bcl-XL. Colony formation assay of PTEN-null/mut-p53 U373 and SNB19 glioblastoma cells with or without PTEN restoration with Ad-PTEN and/or with or without mut-p53/c-Myc/Bcl-XL silencing with shRNA (sh-mp53/sh-c-Myc/sh-Bcl-XL). (A) Colony formation assay. (B) Quantification of A.

Figure 3

PTEN and mut-p53 silencing inhibits malignancy in primary glioblastoma GBM6 cells. (A) Immunoblot showing PTEN silencing with shRNA (sh-PTEN) in wt-PTEN/mut-p53 GBM6 cells. (B) Proliferation assay of GBM6 cells with or without sh-PTEN. (C) Apoptosis assay of GBM6 cells with or without sh-PTEN. (D) Phenotypic changes of GBM6 cells with or without sh-PTEN. (E) Transwell invasion assay of GBM6 cells with or without sh-PTEN. (F) Immunoblot showing mut-p53 silencing with shRNA (sh-mp53) in wt-PTEN/mut-p53 GBM6 cells. (G) Proliferation assay of GBM6 cells with or without sh-mp53. (H) Apoptosis assay of GBM6 cells with or without sh-mp53. (I) Phenotypic changes of GBM6 cells with or without sh-mp53. (J) Transwell invasion assay of GBM6 cells with or without sh-mp53.

Figure 4

PTEN induces the expressions of mut-p53 target genes c-Myc and Bcl-XL in cancer cells. (A) Immunoblots showing the expressions of Bcl-XL and c-Myc in U373 and SNB19 cells with or without PTEN restoration with Ad-PTEN. (B) Immunoblots showing the expressions of c-Myc and Bcl-XL in wt-PTEN/mut-p53 GBM6 primary glioblastoma cells with or without Ad-PTEN. (C) Immunoblots showing the expressions of Bcl-XL and c-Myc in U373 and SNB19 cells with or without Ad-PTEN and/or with or without mut-p53 silencing with shRNA (sh-mp53). (D) Immunoblots showing the expressions of Bcl-XL and c-Myc in PTEN-null/p53-null LNZ308 cells with or without Ad-PTEN and/or with or without mut-p53 restoration with adenovirus-encoding mut-p53 (Ad-mp53).

Figure 5

c-Myc partially mediates the oncogenic effects of PTEN in mut-p53 glioblastoma cells. (A) Proliferation assay of U373 and SNB19 cells with or without PTEN restoration with Ad-PTEN and/or with or without c-Myc silencing with shRNA (sh-c-Myc). (B) Apoptosis assay of U373 and SNB19 cells with or without Ad-PTEN and/or with or without sh-c-Myc. (C) Immunoblot showing the levels of c-Myc silencing and PTEN restoration and their effects on apoptotic regulators PARP and caspase-3 in glioblastoma cells. (D) Transwell invasion assay of U373 and SNB19 cells with or without Ad-PTEN and/or with or without sh-c-Myc. Con, control; *P < .05.

Figure 6

Bcl-XL partially mediates the oncogenic effects of PTEN in mut-p53 glioblastoma cells. (A) Proliferation assay of U373 and SNB19 cells with or without PTEN restoration with Ad-PTEN and/or with or without Bcl-XL silencing with shRNA (sh-Bcl-XL). (B) Apoptosis assay of U373 and SNB19 cells with or without Ad-PTEN and/or with or without sh-Bcl-XL. (C) Immunoblot showing the levels of Bcl-XL silencing and PTEN restoration and their effects on apoptotic regulators PARP and caspase-3 in glioblastoma cells. (D) Transwell invasion assay of U373 and SNB19 cells with or without Ad-PTEN and/or with or without sh-Bcl-XL. Con, control; *P < .05.

Figure 7

There exists a mut-p53/CBP/NFYA complex in glioblastoma cells. (A) IP experiments showing the interaction between two of the three proteins, mut-p53, CBP, and NFYA, in U373 and SNB19 cells. C, control samples immunoprecipitated with control IgG; E, experimental samples immunoprecipitated with relevant antibody; WB, immunoblot. (B) ID experiments to define the relationship between the three proteins mut-p53, CBP, and NFYA in U373 and SNB19 cells. HC, heavy chain; PC, positive control. (C and D) IP and ID experiments showing the relationship between the three proteins mut-p53, CBP, and NFYA in human glioblastoma (GBM) tissue samples. C, control samples immunoprecipitated with control IgG; 3, 4, 6 are distinct GBM samples. (E) Immunoblots (WB) showing the expressions of mut-p53 or NFYA in glioblastoma cells with or without mut-p53 and NFYA silencing with specific shRNA, sh-mp53, or sh-NFYA. (F) IP experiments showing the changes in the interaction between mut-p53/NFYA and CBP with or without NFYA/mut-p53 silencing with sh-NFYA or sh-mp53. C1, C2, control samples immunoprecipitated with control IgG.

Figure 8

PTEN regulates mut-p53/CBP/NFYA binding to mut-p53 target genes c-Myc and Bcl-XL promoters. (A) Immunofluorescence assays showing the interaction between PTEN and mut-p53/CBP in mut-p53 glioblastoma cells. (B) IP experiments showing the interaction between mut-p53/NFYA and CBP in mut-p53 glioblastoma cells in response to PTEN restoration with Ad-PTEN. C, control samples immunoprecipitated with control IgG. (C and D) ChIP experiments showing the binding of mut-p53, acetyl-CBP, and NFYA to the promoter regions of Bcl-XL and c-Myc with or without Ad-PTEN. PC, positive control; Ab, antibody. (E) Immunoblot showing PTEN expression in wt-PTEN/mut-p53 GBM6 cells with or without silencing with specific shRNA (sh-PTEN). (F and G) ChIP experiments showing the binding status of mut-p53, acetyl-CBP, and NFYA to the promoter regions of Bcl-XL and c-Myc in GBM6 cells with or without sh-PTEN.

Figure 9

Prognostic and experimental therapeutic implications of the PTEN/mut-p53 oncogenic effects. (A) All p53 exons were sequenced in 38 human glioblastoma samples and PTEN protein expression was determined by immunohistochemistry in the same tumor specimens. The combined PTEN/p53 mutational status was plotted against patient survival. (B) Immunoblot showing the expressions of wt-p53 target genes p21 and Mdm2 in mut-p53 U373 and wt-p53 U87 cells in response to PRIMA-1 treatment (left panel). Apoptosis assay of U373 and U87 cells in response to PRIMA-1 treatment (right panel). (C) Proliferation assay of U373 cells with or without PTEN restoration with Ad-PTEN and/or with or without PRIMA-1 treatment. (D) Apoptosis assay of U373 cells with or without PTEN restoration with Ad-PTEN and/or with or without PRIMA-1 treatment. (E) Schematic representation of the mechanisms underlying the dual oncogenic/tumor-suppressive effects of PTEN in mut-p53 cancer cells. PTEN enhances a transcriptional complex containing mut-p53, NFY, and CBP. The mut-p53/CBP/NFY complex binds to the promoter of the oncogenes c-Myc and Bcl-XL and induces their transcription, leading to increased cell proliferation, survival, invasion, and clonogenicity. Disruption of the mut-p53/c-Myc/Bcl-XL axis unmasks the hidden tumor-suppressive effects of PTEN in mut-p53 cancers.