Non-canonical splice variant of PFKFB4 in hepatocellular carcinoma activates AKT through direct interaction

Abstract

Background & aims: The glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) has emerged as a vital oncogene in many cancer types as a result of its metabolic function and signaling pathways. However, the effects of alternative splicing variants of PFKFB4 remain largely unexplored.

Methods: Clinical hepatocellular carcinoma (HCC) specimens were used to established the clinical implications of PFKFB4 variants. HCC cell lines and xenograft models were applied to characterize the function of PFKFB4 variants. The underlying mechanisms involved were investigated through the use of human proteome phosphate arrays, in vitro kinase assays, and immunoprecipitation.

Results: We identified a previously uncharacterized splicing variant of PFKFB4 in human HCC. This variant displayed a skipping exon 6 (ΔEx6), resulting in an in-frame 19-amino acid deletion. Functionally, PFKFB4-ΔEx6 demonstrated more pronounced effects compared with its canonical PFKFB4-full length (FL) counterpart on HCC cell proliferation, migration, and in vivo tumorigenicity (n = 8, p <0.01). Mechanistically, Human Phospho-Kinase Array profiling revealed the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway as the major downstream effector of PFKFB4-ΔEx6. Protein immunoprecipitation and in vitro kinase assays revealed that PFKFB4-ΔEx6 could trigger AKT activation through direct binding to the kinase domain, whereas such effects were not detected for PFKFB4-FL. Moreover, the presence of PFKFB4-ΔEx6 notably sensitized HCC cells to everolimus both in vitro and in subcutaneous models (n = 8, p <0.01). Clinically, common upregulation of PFKFB4-ΔEx6 in HCC tumors correlated with poorer overall survival of patients (n = 116, p = 0.001).

Conclusions: Our study highlights the oncogenic characteristics of the novel PFKFB4-ΔEx6 variant in HCC. The direct effect of PFKFB4-ΔEx6 on AKT activation underscores its role in HCC progression and as a therapeutic biomarker for mTOR/AKT inhibitors.

Impact and implications: Our study identified a non-canonical variant of PFKFB4 as a crucial activator of the AKT/mTOR signaling pathway in HCC. This discovery reveals a novel oncogenic mechanism that extends beyond the conventional role of PFKFB4 in metabolism. The PFKFB4-ΔEx6 variant enhances tumor progression and renders HCC cells sensitive to everolimus, indicating its potential as both a prognostic biomarker and a therapeutic target. Given the clinical importance of mTOR/AKT inhibitors, PFKFB4-ΔEx6 could act as a predictive marker for treatment response, paving the way for personalized therapeutic strategies in HCC. These findings provide new insights into the functional diversity of glycolytic enzymes in cancer biology and targeted therapy.

Keywords: AKT; Hepatocellular carcinoma; PFKFB4; Spliced variant.

Graphical abstract

Fig. 1

Identification of a non-canonical PFKFB4-ΔEx6 variant in HCC. (A) Gel electrophoresis showing two PFKFB4 isoforms detected in primary HCC tumors. RNA extracted from primary human HCC T and adjacent NT tissues was reverse transcribed to cDNA and used for PCR. Primers F1/R1 amplified the complete coding region of PFKFB4. (B) Sanger sequencing revealing that the non-canonical PFKFB4 variant exhibited exon 6 skipping. Red solid lines represent the junction of Exon 6–7 or Exon 5–7 for FL or ΔEx6, respectively. (C) Exon composition of PFKFB4 variants encoding PFKFB4-FL and PFKFB4-ΔEx6. (D) Representative images of semi-quantitative RT-PCR analysis showing the expression and ratio of PFKFB4 spliced isoforms in primary human HCC T and adjacent NT tissues. Forward and reverse primers flanking exon 5 and exon 7 were used to amplify PFKFB4 variants. The expected amplicon sizes for PFKFB4-FL and -ΔEx6 were 198 and 141 base pairs, respectively. (E) Low backbone RMSDs from molecular dynamics simulations confirmed the structural stability of PFKFB4-FL and PFKFB4-ΔEx6. FL, full length; HCC, hepatocellular carcinoma; NT, non-tumor; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4; RMSD, root mean square deviation; RT, reverse transcription; T, tumor.

Fig. 2

PFKFB4-ΔEx6 conferred growth advantages to HCC cells. (A–C) Effect of PFKFB4-FL or -ΔEx6 knockdown on (A) proliferation, (B) colony formation, and (C) migration of HKCI-10 and PLC/PRF/5 cells. PFKFB4-ΔEx6 significantly suppressed the oncogenicity of HCC cells, whereas there was no apparent effect of PFKFB4-FL knockdown. Data are presented as mean ± SD of n ≥3 biological replicates; ns, not significant; ∗p <0.05; ∗∗p <0.01; ∗∗∗∗p <0.0001 vs. shCtrl by two-way (B) or one-way (C,D) ANOVA. (D) TUNEL staining demonstrated that knockdown of PFKFB4-ΔEx6 induced marked cell death in HKCI-10 and PLC/PRF/5 cells, whereas knockdown of PFKFB4-FL had a less potent effect. Data are presented as mean ± SD, n = 5 biological replicates; ns, not significant; ∗p <0.05; ∗∗∗∗p <0.0001 vs. shCtrl by one-way ANOVA. (E) PFKFB4-ΔEx6 significantly promoted proliferation of Hep3B and HKCI–C2 cells. Data are presented as mean ± SD from n = 3 biological replicates; ns, not significant; ∗∗p <0.01 vs. EV by two-way ANOVA. (F) Representative images and quantification of migrated cells in Hep3B and HKCI–C2 cells expressing EV, PFKFB4-FL, or PFKFB4-ΔEx6. PFKFB4-ΔEx6 promoted cell migration in Hep3B and HKCI–C2 cells. Data are presented as mean ± SD from n = 5 biological replicates; ns, not significant; ∗∗p <0.01; ∗∗∗p <0.001 vs. EV by one-way ANOVA. (G) PFKFB4-ΔEx6 promoted the growth of Hep3B-derived subcutaneous xenografts. Representative picture and growth curves of xenografts from EV, PFKFB4-FL, or PFKFB4-ΔEx6 groups. Data are presented as mean ± SEM from n = 8 mice per group; ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 vs. EV by two-way ANOVA. (H) Ki-67 staining showed that xenografts from the PFKFB4-ΔEx6-overexpressing group were highly proliferative compared with the EV and PFKFB4-FL groups, as evidenced by the increased percentage of Ki-67-positive cells. Data are presented as mean ± SD from at least 15 different fields of five tissue sections; ∗∗∗∗p <0.0001 vs. EV by one-way ANOVA. Scale bars: 200 μm (F), 100 μm (C,D), 0.5 cm (G), 5 μm (H). EV, empty vector; FL, full length; HCC, hepatocellular carcinoma; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4; shCTRL, short hairpin control; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling.

Fig. 3

PFKFB4-ΔEx6 activates AKT/mTOR signaling. (A) Hallmark gene set enrichment revealed that the PI3K/AKT/mTOR pathway was the most significantly dysregulated pathway following knockdown of PFKFB4-ΔEx6. (B) Array blots showed that phosphorylation of AKT at Ser473 was reduced upon knockdown of PFKFB4-ΔEx6. (C) Immunoblots showed that knockdown of PFKFB4-ΔEx6 diminished phospho-Akt (Ser473) level and induced the activation of apoptosis mediators in HKCI-10 and PLC/PRF/5 cells, whereas there was no evident effect of PFKFB4-FL knockdown. (D) The insulin-induced AKT/mTOR pathway was enhanced in PFKFB4-ΔEx6-overexpressing cells. Hep3B and HKCI–C2 cells expressing vector control, PFKFB4-FL, or -ΔEx6 were treated with 0, 1, 10, or 100 nM of insulin for 15 min after 16 h of serum starvation. An equal amount of protein was subjected to immunoblotting. (E,F) Expression of (E) PFKFB4-ΔEx6, but not (F) PFKFB4-FL positively correlated with phospho-Akt (Ser 473) level in 20 HCC primary tumor specimens. Data are analyzed using Spearman’s correlation; p values are shown in each figure. AKT, protein kinase B; HCC, hepatocellular carcinoma; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; Ser, serine.

Fig. 4

PFKFB4-ΔEx6 directly interacts with AKT1 at its kinase domain. (A) Co-IP validated the direct interaction between the PFKFB4-ΔEx6 isoform and AKT1 in 293FT cells. (B) Phosphorylation inactivation of PFKFB4-ΔEx6 did not disrupt its interaction with AKT1. Flag-tagged PFKFB4-ΔEx6-wild type (WT) and -T140A mutant constructs were co-transfected with AKT1 in 293FT cells. Co-IP assays demonstrated that the T140A mutant retained its interaction with AKT1. Co-IP was performed with n = 3 biological replicates. (C) Schematic workflow of the in vitro kinase assay. (D) In vitro kinase assays revealed that PFKFB4-ΔEx6 directly phosphorylates AKT1. MAPKAP was used as a positive Ctrl. Purified flag-tagged PFKFB4 isoforms were incubated with Flag-tagged AKT1 or mutant AKT1-Ser473A protein in the presence of ATP. Data are presented as mean ± SD of n = 3 biological replicates; ns, not significant; ∗∗∗∗p <0.0001 by two-way ANOVA. (E) Lysates from in vitro kinase assays were subjected to immunoblotting. Phosphorylated AKT was only detected in the presence of PFKFB4-ΔEx6, whereas AKT1-Ser473A was not phosphorylated by PFKFB4-ΔEx6. (F) Schematic showing myc-AKT1 expression constructs encoding three truncation mutants of AKT1. Flag-PFKFB4-ΔEx6 was co-expressed with myc-AKT1 truncation mutant (a), (b), or (c) in 293FT cells. (G) Co-IP result showed that PFKFB4-ΔEx6 directly interacts with AKT1 at its kinase domain. AKT, protein kinase B; Co-IP, co-immunoprecipitation; Ctrl, control; MAPKAP, MAPK-activated protein kinase 2; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4; Ser, serine; WT, wild type. (C) created with BioRender (biorender.com).

Fig. 5

PFKFB4-ΔEx6 renders HCC cells susceptible to the mTOR/AKT inhibitor everolimus. (A,B) Dose–response curves to everolimus showed that PFKFB4-ΔEx6-overexpressing (A) Hep3B and (B) HKCI–C2 cells are more sensitive to everolimus compared with vector control and PFKFB4-FL-overexpressing cells. Data are presented as mean ± SD of n = 3 biological replicates; ns, not significant; ∗∗∗∗p <0.0001 by Student's t test. (C) qPCR showing that three HCC cell lines exhibit similar levels of PFKFB4-FL, but different levels of PFKFB4-ΔEx6. Data are presented as mean ± SD of n ≥3 biological replicates; ns, not significant; ∗∗p <0.01; ∗∗∗∗p <0.0001 by one-way ANOVA. (D) IC₅₀ and dose–response curves showing that PFKFB4-ΔEx6 renders HCC cells sensitive to everolimus in a dose-dependent manner. Data are presented as mean ± SEM from n = 3 independent experiments; ∗p <0.05; ∗∗∗p <0.001; ∗∗∗∗p <0.0001 by one-way ANOVA. (E) Growth curves and representative images of subcutaneous xenografts showing that 10 mg/kg/day of everolimus significantly suppressed the growth of Hep3B-PFKFB4-ΔEx6-derived subcutaneous xenografts, whereas its effects on the EV and PFKFB4-FL groups were not significant. Data are presented as mean ± SEM of n = 9 mice per group; ns, not significant; ∗∗p <0.01 by Student's t test. AKT, protein kinase B; HCC, hepatocellular carcinoma; mTOR, mammalian target of rapamycin; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4.

Fig. 6

PFKFB4-ΔEx6 is upregulated in HCC and correlates with poor patient survival. (A) Expression of PFKFB4-FL and PFKFB4-ΔEx6 in adjacent NT liver tissues and HCC T tissues were examined by using SYBR green-based real-time qPCR. Data were analysised by paired t test. (B) PFKFB4-ΔEx6 was overexpressed (T/NT ≥1) in 73.2% of HCC cases. In total, 116 pairs of HCC T and paired NT tissues were assessed. (C–F) Kaplan-Meier survival plots of PFKFB4-FL and -ΔEx6 expression vs. (C,D) overall survival and (E,F) disease-free survival in 116 patients with HCC. A significant correlation was only observed between PFKFB4-ΔEx6 expression and patient survival. A fold change (T/NT) of 4 was set as a cutoff value to stratify patients with HCC into PFKFB4-FL or -ΔEx6 high- and low-expression groups. (G,H) Multivariate Cox regression analysis of PFKFB4 variants and clinicopathological parameters in 116 patients with HCC for (G) overall survival and (H) disease-free survival, adjusting for American Type Culture Collection T stage, neoadjuvant therapy, adjuvant therapy, microvascular invasion, age, and gender. Data are presented as hazard ratios with 95% CIs. HCC, hepatocellular carcinoma; HR, hazard ratio; NT, non-tumor; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4; qPCR, quantitative PCR; T, tumor.