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

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.