Page 75 - D. Cancer biology

Phosphofructokinase 1 Platelet Isoform Promotes β-Catenin Transactivation

for Tumor Development

Jong-Ho Lee 1,2

1 Department of Health Sciences, The Graduate School of Dong-A University

2 Department of Biological Science, Dong-A University, Busan 49315, South Korea

ABSTRACT RESULTS

Metabolism plays a critical role in direct regulation of a

variety of cellular activities via metabolic enzymes and

metabolites. Here, we demonstrate that phosphofructokinase

1 platelet isoform (PFKP), which catalyzes a rate-limiting

reaction in glycolysis, promotes EGFR activation-induced

nuclear translocation and activation of β-catenin, thereby

enhancing the expression of its downstream genes CCND1

and MYC in human glioblastoma cells. Importantly, we

showed that EGFR-phosphorylated PFKP Y64 has a critical

role in AKT activation and AKT-mediated β-catenin S552

phosphorylation and subsequent β-catenin transactivation

and promotion of tumor cell glycolysis, migration, invasion,

proliferation, and brain tumor growth. These findings Figure 1. PFKP expression is required for EGFR activation-induced nuclear translocation and Figure 4. PFKPY64 phosphorylation-induced β-catenin transactivation promotes brain tumor growth.

transactivation of β-catenin. (A,B) A total of 5 × 105 PFKP-depleted U87/EGFRvIII cells with reconstituted expression of the indicated
highlight a novel mechanism underlying a glycolytic enzyme- (A) Serum-starved U87/EGFR cells with the indicated shRNAs (left panel) were treated with or without EGF proteins was intracranially injected into athymic nude mice. After 2 weeks, the mice were euthanized and

(100 ng/ml) for 9 h. The cells were harvested for the isolation of nuclear fractions (right panel). examined for tumor growth. Hematoxylin-and-eosin–stained coronal brain sections show representative

mediated β-catenin transactivation and underscore the Immunoblotting analyses were performed with the indicated antibodies. (B) U87/EGFRvIII cells were stably tumor xenografts (A). Tumor volumes were measured (B). Data represent the means ± SD of seven mice
(bottom panel). *P < 0.001, #P < 0.001, based on the one-way ANOVA; n.s., not significant. Scale bar, 2 mm.
expressed with control shRNA or PFKP shRNA. Immunofluorescent staining was performed with an anti-β-
catenin antibody. (C) U87/EGFR cells with or without PFKP depletion were transfected with TOP-FLASH or (C) EGFR-phosphorylated PFKP Y64 promotes AKT activation-mediated nuclear translocation of β-catenin,
integrated and reciprocal regulation of metabolism and gene FOP-FLASH, which was followed by EGF treatment for 6 h. Luciferase activity was measured. The relative β-catenin transactivation, TCF/LEF-induced transcription of CCND1 and MYC, leading to tumor
development.
levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase
activity in the Renilla control plasmid. Data represent the means ± SD of three independent experiments. *P
expression, which are two fundamental biological processes in < 0.001, based on the Student's t-test. (D,E) Serum-starved U87/EGFR cells with or without depleted PFKP

were treated with or without EGF for the indicated periods of time. The mRNA expression levels (D) and the
tumor development. protein expression levels (E) of CCND1 and MYC in U87/EGFR cells were determined by real-time PCR and
immunoblotting analyses with the indicated primers and antibodies, respectively. Data represent the means ±
SD of three independent experiments. *P < 0.001, based on the Student's t-test.

CONCLUSIONS

PFKP promotes EGFR activation-induced nuclear
INTRODUCTION translocation and activation of β-catenin in a PFKP Y64

phosphorylation-dependent manner, thereby enhancing the

A. Increased transcriptional activity of β-catenin, which is essential expression of β-catenin downstream genes CCND1 and

for cell proliferation, migration, invasion, and survival (1, 2), has MYC. Thus, PFKP Y64 phosphorylation enhances tumor

been detected in many types of human cancer (3–6). cell migration, invasion, proliferation, and brain tumor

growth. These findings highlight the potential to target

B. β-catenin transactivation, which is activated not only by Wnt Y64-phosphorylation of PFKP for GBM treatment.

ligands but also by receptor tyrosine kinases, such as epidermal

growth factor receptor (EGFR), leads to enhanced T-cell factor

(TCF)/lymphoid enhancer factor (LEF)-driven transcription of genes, Figure 2. PFKP Y64 phosphorylation by EGFR enhances AKT-mediated β-catenin S552 phosphorylation

and nuclear translocation of β-catenin and expression of its downstream genes.

such as CCND1 (encoding cyclin D1) and MYC (encoding c-Myc) (A) Nuclear fractions of U87/EGFRvIII cells with or without PFKP depletion and with or without reconstituted
expression of WT Flag-rPFKP or Flag-rPFKP Y64F mutant were prepared Immunoblotting analyses were
performed with the indicated antibodies. (B) U87/EGFRvIII cells with or without PFKP depletion and with or
(7–9). without reconstituted expression of WT Flag-rPFKP or Flag-rPFKP Y64F mutant in the presence or absence of

CA β-catenin expression were transfected with TOP-FLASH or FOP-FLASH. The relative levels of luciferase
activity were normalized to the levels of luciferase activity of Renilla control plasmid. Data represent the
means ± SD of three independent experiments. *P < 0.001, based on the one-way ANOVA. (C,D) The mRNA
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