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. 2004 May 4;101(18):6993-8.
doi: 10.1073/pnas.0400921101. Epub 2004 Apr 26.

PIKE-A is amplified in human cancers and prevents apoptosis by up-regulating Akt

Jee-Yin Ahn  1 , Yuanxin Hu, Todd G Kroll, Paulette Allard, Keqiang Ye
Affiliations

PIKE-A is amplified in human cancers and prevents apoptosis by up-regulating Akt

Jee-Yin Ahn et al. Proc Natl Acad Sci U S A. .

Abstract

PIKE-A (PIKE-activating Akt), an isoform of PIKE GTPase that enhances phosphatidylinositol 3-kinase (PI3-kinase) activity, specifically binds to active Akt but not PI3-kinase. PIKE-A stimulates Akt activity in a GTP-dependent manner and promotes invasiveness of cancer cell lines. Here, we show that PIKE-A is amplified in a variety of human cancers and that amplified PIKE-A directly stimulates Akt and inhibits apoptosis compared to cells with normal PIKE-A copy number. Overexpression of PIKE-A wild-type but not dominant-negative mutant stimulates Akt activity and prevents apoptosis. Moreover, knockdown of PIKE-A diminishes Akt activity and increases apoptosis. Our findings suggest that PIKE-A amplification contributes to cancer cell survival and progression by inhibiting apoptosis through up-regulating Akt.

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Figures

Fig. 1.
Fig. 1.
PIKE-A is amplified in human glioblastoma cells. (A and B) Fluorescent in situ hybridization staining of glioblastoma TP366 (A) and SF188 (B) cells. Red dot stains for the centromere of human chromosome 12, whereas the green stains for PIKE-A with the amplified PIKE (arrowhead) versus normal PIKE (arrow). Amplified PIKE-A translocates from chromosome 12 to another chromosome in TP366 cells (A). Amplified PIKE-A also forms double minute chromosomes in SF188 cells (B). (C) RT-PCR analysis of PIKE-A and -L mRNA expression in human cancer cell lines. PIKE-A mRNA is robustly expressed in NGP-127, CRL-2061, TP366, LN-Z308, and SF188 cells, but not in LN229 or U87MG cells (Top, left lane). By contrast, PIKE-L is weakly expressed in U87MG, SF188, and TP366 cells (left lane, Middle). Equal levels of GAPDH were monitored as control (Bottom, left lane). The relative gene amplification folds are labeled at the bottom. The primers for PIKE-A and -L used in RT-PCR analysis and the antigen used to raise anti-PIKE antibody are indicated on the right. (D) Western blotting analysis of PIKE-A protein expression. Cell lysate (50 μg) of a variety of human glioblastoma cell lines was used for immunoblotting analysis with rabbit polyclonal anti-PIKE and mouse monoclonal CDK4 antibodies.
Fig. 2.
Fig. 2.
PIKE-A interacts with Akt via its N terminus. (A) Diagram of PIKE-A fragments. (B and C) The N terminus of PIKE-A is sufficient to bind to Akt. (B) In vitro binding assay: HEK293 cells were transfected with hemagglutinin (HA)-Akt. The transfected cells were serum-starved overnight, then stimulated with EGF for 5 min. Purified GST-PIKE-A recombinant proteins were conjugated to glutathione beads, and incubated 293 cell lysate at 4°C for 3 h. The glutathione bead-associated proteins were analyzed with anti-HA antibody (B Upper). GST-recombinant proteins were verified with anti-GST–HRP (B Lower). (C) In vivo coimmunoprecipitation. Cell lysate (1 mg) of TP366 cells was incubated with 25 μl of Protein A/G conjugated beads and 2 μg of rabbit polyclonal anti-PIKE antibody in the presence of GST–PIKE-A-1–356 (lane 2) or GST alone (lane 1). The coimmunoprecipitated proteins were analyzed with anti-Akt antibody. The input in lane 3 is whole cell lysate. Akt specifically binds to PIKE-A in the absence of its N-terminal fusion protein (lane 1). (D) PIKE-A stimulates Akt kinase activity. Purified recombinant GST–PIKE-A fragments incubated with in vitro kinase assay mixture containing purified Akt (1 μg) and GST–GSK3. GST alone was used as a negative control. The phosphorylation status was monitored by anti-phospho-GSK3-specific antibody. (E) Western blotting analysis of Akt, phospho-Akt, phospho-Bad, and α-tubulin in various cancer cells.
Fig. 3.
Fig. 3.
Cancer cells with PIKE-A amplification resist to apoptosis. (A) Apoptotic assay. Various human cancers were treated with 250 nM staurosporine for 16 h, and followed by chromatin condensation and fragmentation analysis with DAPI staining. PIKE-A amplified cells display lower apoptotic activity compared to cells with normal copy. (B) Western blotting analysis of Lamin A/C and Caspase-3 cleavage during apoptosis.
Fig. 4.
Fig. 4.
Overexpression of PIKE-A prevents apoptosis by stimulating Akt. (A) Apoptotic assay. Glioblastoma cells (SF767 and LN229) were infected with control adenovirus or adenovirus expressing wild-type PIKE-A or dominant-negative PIKE-A-DN, and treated with staurosporine for 16 h, and followed by chromatin condensation and fragmentation analysis with DAPI staining. Wild-type PIKE-A markedly prevents apoptosis compared to control cells (*, P < 0.005, Student's t test). Conversely, dominant-negative PIKE-A-DN increases the sensitivity of the infected cells to apoptosis (#, P < 0.01, Student's t test). (B) Western blotting analysis of Lamin A/C cleavage during apoptosis. Cell lysate (50 μg) from adenovirus-infected cells was separated by SDS/10% PAGE and analyzed by Western blotting with anti-lamin A/C antibody. Wild-type PIKE-A noticeably prevents lamin A/C cleavage compared to control cells. In contrast, dominant-negative PIKE-A-DN enhances lamin A/C cleavage. (C) PIKE-A stimulates Akt activity in glioblastoma cells. SF767 and LN229 cells were infected with adenovirus expressing PIKE-A-WT and PIKE-A-DN, respectively; after 24-h infection, the cell lysate was analyzed with phospho-serine-473 antibodies. Robust phosphorylation on S473 of Akt was observed in wild-type PIKE-A but not control adenovirus-infected cells. By contrast, the phosphorylation was completely inhibited by dominant-negative PIKE-A-DN (Upper). The in vitro Akt kinase assay was performed with the immunoprecipitated endogenous Akt, and GST–GSK3 was used as a substrate. GSK3 phosphorylation was analyzed by Western blotting with anti-phospho-GSK3-specific antibody (Lower).
Fig. 5.
Fig. 5.
PIKE-A knockdown increase apoptosis by inactivating Akt. (A) Apoptotic assay. Serum-starved cells were treated with Penetratin 1-conjugated antisense and sense oligonucleotides of PIKE-A. After 6 h, the cells were treated with staurosporine for 16 h, and followed by chromatin condensation and fragmentation analysis with DAPI staining (Upper). PIKE-A expression is selectively decreased by antisense but not control sense oligonucleotide. By contrast, tubulin expression level is not changed (Lower). (B) MTT cell viability assay. Serum-starved cells were treated with Penetratin 1-conjugated antisense and sense oligonucleotides of PIKE-A. After 6 h, the cells were treated with staurosporine for various time points, and followed by MTT assay. (C and D) Western blotting analysis of Caspase-3 and Lamin A/C cleavage in sense and antisense oligonucleotide treated cells during apoptosis. (E) PIKE-A knockdown inhibits Akt activity. Western blotting analysis of Akt and Bad phosphorylation in sense and antisense oligonucleotide-treated cells. Consistent with PIKE-A knockdown, Akt phosphorylation is diminished in antisense but not sense oligonucleotide treated cells (Upper). The phosphorylation of Akt physiological substrate, Bad, in sense and antisense oligonucleotide-treated cells (Lower).
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