The following reagents and antibodies were purchased from the indicated companies: Rabbit polyclonal antibodies recognizing PFKP (cat. no. 12746, 1:1,000 for western blot analysis), phosphorylated (p-)AKT (pT308, cat. no. 4056, 1:1,000 for western blot analysis), p-AKT (pS473, cat. no. 4060, 1:1,000 for western blot analysis) and AKT (cat. no. 9272, 1:1,000 for western blot analysis) from Cell Signaling Technology, Inc.; mouse monoclonal antibodies for PFKL (A-6, cat. no. sc-393713, 1:500 for western blot analysis), PFKM (cat. no. sc-67028, 1:1,000 for western blot analysis), PFK2 (cat. no. sc-377416, 1:500 for western blot analysis), lactate dehydrogenase A (LDHA) (cat. no. sc-137243, 1:500 for western blot analysis) and β-catenin (E-5, cat. no. sc-7963, 1:200 for western blot analysis) from Santa Cruz Biotechnology, Inc.; mouse monoclonal antibody for tubulin (clone B-5-1-2, T6074, 1:5,000 for western blot analysis) from MilliporeSigma; rabbit polyclonal antibodies for EGFR (pY869, cat. no. 11229, 1:1,000 for western blot analysis) and PFKP [pS386, 1:1,000 for western blot analysis; customized as previously described (37)] from Signalway Antibody LLC; mouse monoclonal EGFR antibody (cat. no. 610016, 1:1,000 for western blot analysis) from Abcam; human recombinant EGF (E9644) and cycloheximide (CHX; 66-81-9) from MilliporeSigma; Wnt3A (5036-WN) from R&D Systems, Inc.; AG1478 (658552) from Calbiotech, Inc.; LY294002 (L-7988) from LC Laboratories; MK-2206 (S1078) from Selleck Chemicals; DAPI and Alexa Fluor 488 goat anti-rabbit antibody (A11008, 1:10,000 for immunofluorescence) from Molecular Probes; Thermo Fisher Scientific, Inc.; HyFect transfection reagents (E2650) from Denville Scientific, Inc.

A431 (21555) human epidermoid carcinoma cells and MDA-MB-231 (30026) human breast carcinoma cells were purchased from the Korean Cell Line Bank (KCLB). LN18 and LN229 GBM cells were kindly provided by Dr Hyunggee Kim (Korea University, Seoul, Korea). U251 GBM cells were kindly provided by Dr Kyu Heo (Dongnam Institute of Radiological and Medical Sciences, Busan, Korea). The human epidermal squamous carcinoma cell lines, SCC12 and SCC13, were kindly provided by Dr Tae-Jin Yoon (Gyeongsang National University and Hospital, Jinju, Korea). These cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% bovine calf serum (Capricorn Scientific GmbH) and 1% penicillin/streptomycin (Capricorn Scientific GmbH).

PCR-amplified human PFKP was cloned into the pcDNA3.1/hygro(+)-Flag vector. pcDNA3.1/hygro(+)-Flag PFKP S386A was created using the QuikChange site-directed mutagenesis kit (Stratagene; Agilent Technologies, Inc.). The pGIPZ shRNA vector (RHS4348) was purchased from GE Dharmacon, Inc. shRNA-resistant (r) PFKP contained the a448c, g450c, c453t and c456g mutations. The following pGIPZ shRNAs were used: Control shRNA oligonucleotide, 5-GCTTCTAACACCGGAGGTCTT-3; PFKP shRNA oligonucleotide, 5-AGGAACGGCCAGATCGATA-3; and β-catenin shRNA oligonucleotide, 5-TTACCACTCAGAGAAGGAG-3. Cells were plated at a density of 4×10⁵/60-mm dish 18 h prior to transfection and were directly transfected with the constructed vectors (1 µg/6-well plate) using HyFect transfection reagent (Denville Scientific, Inc.) according to the manufacturer's instructions. Transfected cells were stabilized for 1 day, and then the cells were selected for 1 week using 200 µg/ml hygromycin (400053; EMD Millipore) and 2 µg/ml puromycin (540222; EMD Millipore).

Total RNA was prepared from tumor cells using an RNeasy Mini kit (Qiagen, Inc.) according to the manufacturer's instructions, and cDNA was synthesized from 2 µg of total RNA by reverse transcriptase (Superscript II Preamplification System, Gibco; Thermo Fisher Scientific, Inc.). Quantitative PCR (qPCR) was performed on an ABI Prism 7500 sequence detection system using a SYBR^®-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and following the manufacturer's protocols. The ABI 7500 sequence detector was programmed with the following PCR conditions: 40 cycles of 15-sec denaturation at 95°C and 1-min amplification at 60°C. All reactions were run in triplicate and normalized to the housekeeping gene β-actin. The evaluation of relative differences of PCR results was calculated using the 2^−ΔΔCq method (38). The following primer pairs were used for RT-qPCR: Human PFKP forward, 5′-CGGAAGTTCCTGGAGCACCTCTC-3′ and reverse, 5′-AAGTACACCTTGGCCCCCACGTA-3′; human PFKL forward, 5′-GGCATTTATGTGGGTGCCAAAGTC-3′ and reverse, 5′-CAGTTGGCCTGCTTGATGTTCTCA-3′; human PFKM forward, 5′-GAGTGACTTGTTGAGTGACCTCCAGAAA-3′ and reverse, 5′-CACAATGTTCAGGTAGCTGGACTTCG-3′; human PFKPB1 forward, 5′-AGAAGGGGCTCATCCATACCC-3′ and reverse, 5′-CTCTCGTCGATACTGGCCTAA-3′; human PFKFB2 forward, 5′-AGTCCTACGACTTCTTTCGGC-3′ and reverse, 5′-TCTCCTCAGTGAGATACGCCT-3′; human PFKFB3 forward, 5′-ATTGCGGTTTTCGATGCCAC-3′ and reverse, 5′-GCCACAACTGTAGGGTCGT-3′; human PFKFB4 forward, 5′-CAACATCGTGCAAGTGAAACTG-3′ and reverse, 5′-GACTCGTAGGAGTTCTCATAGCA-3′; and human β-actin forward, 5′-ATGGATGATGATATCGCCGCGC-3′ and reverse, 5′-GCAGCACGGGGTGCTCCTCG-3′.

Protein extraction from the cultured cancer cells was performed using a lysis buffer [50 mM Tris-HCl (pH 7.5), 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 100 µM sodium orthovanadate, 100 µM sodium pyrophosphate, 1 mM sodium fluoride, and proteinase inhibitor cocktail]. The cell extracts were centrifuged at 12,000 × g (at 4°C for 15 min) and protein concentrations of cell lysates were determined using the DC protein assay kit (5000116, Bio-Rad Laboratories, Inc.). Equal amounts of lysates (2 mg/ml protein) were resolved by 8–10% SDS-PAGE and were then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBST at room temperature for 1 h and then incubated with the indicated antibodies (as indicated above in Reagents and antibodies) at 4°C overnight. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit (NA934V; Cytiva) or anti-mouse (NA931V; Cytiva) at a dilution of 1:3,000) at room temperature for 2 h and were visualized using a chemiluminescence technique (Amersham Biosciences). Band intensity was quantified using ImageJ 1.53e software (National Institutes of Health). Each experiment was repeated at least three times.

A431 cells were fixed with 4% paraformaldehyde for 20 min and permeabilized using 0.5% Triton X-100 at room temperature for 10 min. The cells were blocked with 5% normal serum at 37°C for 30 min and incubated with an anti-PFKP antibody at a dilution of 1:100 at 4°C overnight, subsequently, an Alexa Fluor dye-conjugated secondary antibody at 37°C for 1 h, and in the end, stained with DAPI at room temperature for 10 min. The cells were examined using a deconvolution microscope (Zeiss AG) with a 63-Å oil-immersion objective. Axio Vision software from Zeiss AG was used to deconvolute the Z-series images.

The A431 cells were seeded in culture dishes, and the medium was changed after 6 h with non-serum DMEM. The cells were then incubated at 37°C for 48 h, and the culture medium was collected for the measurement of glucose and lactate concentrations. Glucose levels were determined using a glucose (GO) assay kit (GAGP20, Sigma-Aldrich; Merck KGaA). Glucose consumption was calculated as the difference in glucose concentration between the collected culture medium and DMEM. The absorbance was recorded at 540 nm at room temperature in a 96-well plate. Lactate levels were determined using a lactate assay kit (1200051002, Eton Bioscience, Inc.). The absorbance was recorded at 570 nm using a microplate reader (SpectraMax^® ABS; Molecular Devices, LLC) at room temperature in a 96-well plate. All results were normalized to the final cell number.

A total of 2×10⁴ A431 cells were plated and incubated for 5 days after seeding in DMEM with 0.5% bovine calf serum. The cells were trypsinized and counted using a hemocytometer following staining with trypan blue at room temperature for 1 min (EBT-001; NanoEnTek, Inc.). The data represent the mean ± SD of three independent experiments.

PFK activity was assessed in A431 cells with the use of the PFK activity colorimetric assay kit (K776-100, BioVision, Inc.). The reaction was performed using cell lysate (3 µg) in 100 µl of reaction buffer, which was prepared according to the instructions provided with the kit. The absorbance was recorded at 450 nm using a microplate reader (SpectraMax^® ABS) at 37°C in a 96-well plate.

LDH activity was assessed in A431 cells using a lactate dehydrogenase activity colorimetric assay kit (K726-500, BioVision, Inc.). The reaction was performed using cell lysate (1 µg) in 100 µl of reaction buffer, which was prepared according to the instructions provided with the kit. The absorbance was recorded at 450 nm using a microplate reader (SpectraMax^® ABS) at 37°C in a 96-well plate.

A431 cells (1.5×10⁵ cells/well) were seeded into 12-well plates and cultured at 100% confluence, followed by starvation in serum-free DMEM for 24 h. A sterile 200 µl pipette tip was used to create a scratch in the monolayer in the middle of the well. At the 0 and 18-h incubation at 37°C time points, cells were photographed using a light microscope (×200 magnification) and the migration distance was measured using ImageJ 1.53e software (National Institutes of Health). The data represent the mean ± SD of three independent experiments.

A431 cells (1×10³ cells/well) were seeded into 6-well plates and cultured in DMEM supplemented with 10% bovine calf serum with or without Wnt3A (20 ng/ml). Colony formation assay was performed for 7 or 12 days. The cells were fixed with 10% formalin (F8775, Sigma-Aldrich; Merck KGaA) for 10 min and stained with 0.1% crystal violet (V5265, Sigma-Aldrich) at room temperature for 1 h. The residual dye was washed off with water and the plate was air-dried.

All quantitative data are presented as the mean ± SD of at least three independent experiments. A 2-group comparison was conducted using the 2-sided, 2-sample Student's t-test. A simultaneous comparison of >2 groups was conducted using one-way ANOVA followed by Tukey's post hoc tests. The SPSS statistical package (version 12; SPSS Inc.) was used for the analyses. Values of P<0.05 were considered to indicate statistically significant differences.

Wnt3A was used to stimulate A431, SCC12, and SCC13 human epidermal carcinoma cells, U251, LN229 and LN18 human GBM cells, and MDA-MB-231 human breast carcinoma cells, in order to determine whether the Wnt-induced EGFR transactivation has effects on tumor development or not. As presented in Fig. 1A, Wnt3A stimulation significantly induced EGFR phosphorylation levels at 30, 60 and 90 min, as compared with the unstimulated control A431 cell EGFR levels. Similar results were also observed in other types of cancer cells: MDA-MB-231, LN229, U251 and LN18 (Fig. 1B), and SCC12 and SCC13 (Fig. S1). Pre-treatment with the specific EGFR tyrosine kinase inhibitor, AG1478, following the successful blocking of Wnt3A-induced EGFR phosphorylation (Fig. 1C), markedly abrogated both basal and Wnt3A-enhanced glucose consumption (Fig. 1D), lactate production (Fig. 1E), proliferation (Fig. 1F), colony-forming ability (Fig. 1G) and migration (Fig. 1H) in the A431 cells. These results indicated that Wnt signaling successfully may transactivate EGFR in cancer cells, which is required for the Wnt-induced the Warburg effect, as well as proliferation, colony formation and cancer cell migration.

PFK is critical for glycolysis (33), and its activity and expression is important for tumor development (37,39,40). Thus, the effect of Wnt signaling on PFK enzyme activity was examined in the present study. As shown in Fig. 2A, Wnt3A stimulation markedly induced PFK enzyme activity, which was significantly inhibited by treatment with AG1478 EGFR inhibitor, suggesting that Wnt signaling induced PFK activity through EGFR transactivation. Subsequently, the effect of Wnt signaling on PFK expression was determined. PFK includes PFK1 and PFK2 isoforms (33). Additionally, PFK1 includes PFKP, PFKM and PFKL isoforms. Furthermore, PFK2 [also known as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)], a bi-functional enzyme encoded by four different genes (PFKFB1, PFKFB2, PFKFB3 and PFKFB4) with both a kinase and a phosphatase domain, is responsible for the production and degradation of fructose-2,6-bisphosphate (41). The aforementioned enzymes and their corresponding isoforms have been originally detected in different tissues: PFKFB1 in the liver and skeletal muscle, PFKFB2 in the heart tissue, PFKFB3 in the brain and PFKFB4 in the testis (42). The analyses of the isoform expression profiles using RT-qPCR and western blot analysis revealed that Wnt3A treatment did not markedly alter the PFK1 (Fig. 2B) and PFK2 (Fig. 2C) isoform mRNA expression levels and in A431 cells, whereas the PFKP protein expression levels, but not those of PFKL, PFKM and PFK2, were upregulated in the A431 cells (Fig. 2D), and the LN18, MDA-MB-231, SCC12 and SCC13 cells (Fig. S2) in response to Wnt3A stimulation. Consistent with these findings, immunofluorescence analyses with anti-PFKP antibody revealed that Wnt3A treatment enhanced PFKP expression in A431 cells (Fig. 2E). Taken together, it was observed that Wnt signaling increased PFK activity and PFKP expression. In order to determine the role of PFKP expression in Wnt-induced PFK activation, PFKP expression was depleted with the use of shRNA. It was then demonstrated that a reduction in PFKP expression significantly decreased the Wnt3A-induced total PFK enzyme activity in A431 cells (Fig. 2F), suggesting a crucial role of PFKP expression in Wnt signaling-induced PFK activation.

The A431 cells were pre-treated with CHX to block protein synthesis, in order to determine whether PFKP protein expression is regulated by Wnt signaling. CHX treatment exerted a limited effect on Wnt3A-induced PFKP expression (Fig. 3A). These results suggest that Wnt signaling enhances PFKP expression primarily by enhancing PFKP stability. Of note, β-catenin depletion in A431 cells revealed that a reduction in β-catenin expression did not affect Wnt3A-induced PFKP protein expression (Fig. 3B), suggesting that Wnt signaling induces PFKP expression in the canonical Wnt-independent manner in cancer cells.

It has been previously reported by the authors that AKT phosphorylates PFKP at S386, which results in the inhibition of TRIM21 E3 ligase binding to PFKP and the subsequent TRIM21-mediated polyubiquitylation and degradation of PFKP, ultimately resulting in an increased PFKP expression (37). Wnt3A stimulation successfully induced AKT phosphorylation, which was blocked by treatment with AG1478 EGFR inhibitor (Fig. 3C). In addition, Wnt3A induced PFKP S386 phosphorylation in A431 cells (Fig. 3D; first and second lanes). As expected, treatment of the A431 cells with the PI3K inhibitor, LY294002, or the AKT inhibitor, MK-2206, successfully blocked Wnt3A-induced AKT T308/S473 and PFKP S386 phosphorylation (Fig. 3D). Furthermore, the Wnt3A-induced half-life of endogenous PFKP (Fig. 3E and F) and PFKP expression (Fig. 3G) were markedly decreased following treatment with the AG1478 EGFR inhibitor or MK-2206 AKT inhibitor, respectively. In line with this finding, the Wnt3A-induced half-life of the PFKP S386A mutant was much shorter than that of its wild-type (WT) counterpart (Fig. 3H). These results indicated that EGFR/PI3K/AKT activation-induced PFKP S386 phosphorylation is required for Wnt-enhanced PFKP protein expression.

To investigate the role of PFKP S386 phosphorylation in the Wnt signaling-induced Warburg effect, cell proliferation, colony formation and migration, endogenous PFKP was depleted in A431 cells and the expression of RNAi-resistant (r) WT Flag-rPFKP or Flag-rPFKP S386A was reconstituted in these cells. The depletion of PFKP (Fig. 4A) markedly impaired Wnt3A-induced glucose consumption (Fig. 4B), lactate production (Fig. 4C), proliferation (Fig. 4D), colony formation ability (Fig. 4E) and cell migration (Fig. 4F); this inhibition was reversed by the expression of WT rPFKP, but not by the expression of rPFKP S386A mutant in A431 cells (Fig. 4A-F). Of note, Wnt3A stimulation or PFKP depletion did not affect LDHA enzyme expression and activity in A431 cells (Fig. S3). These results suggested that PFKP S386 phosphorylation may be important for the Wnt-induced the Warburg effect, as well as for proliferation, colony formation and cancer cell migration.