Supporting data for “The Regulation and Functional Significance of the Hypoxia Inducible Bifunctional Glycolytic Enzyme, PFKFB4, in Hepatocellular Carcinoma”

Liver cancer is currently the sixth most common malignancy and the second leading cause of cancer-related mortalities worldwide. Hepatocellular carcinoma (HCC), the predominant type of primary liver cancer, is particularly prevalent in East Asia, South East Asia, and Sub-Saharan Africa, due to chronic hepatitis B endemic in these regions. However, therapeutic options for advanced stage HCC are extremely limited and often provide unsatisfactory survival benefits. Activation of the HIF-1 pathway by tumor hypoxia is a major factor contributing to the aggressive and resilient nature of HCC. Metabolic reprogramming is recognized as a cancer hallmark which is intimately linked to tumor hypoxia. Not only do the rewired metabolic pathways support the rapid growth of the tumor and the adaptation to the hostile microenvironment, but they also mitigate the consequential oxidative stress. PFK2, a family of bifunctional glycolytic enzymes, plays a pivotal role in regulating glucose metabolism by maintaining a delicate balance between glycolysis and the pentose phosphate pathway. Our laboratory has identified one of the four isoforms of PFK2, PFKFB4, as a potential hypoxia-inducible oncogene in HCC by a transcriptomics-based bioinformatics analysis. In this study, I aim to characterize

the clinical relevance, transcriptional regulation, functional significance, and the oncogenic mechanism of PFKFB4 in the development of HCC. In the examination of 100 paired HBV-associated HCCs, PFKFB4 expression was found to be upregulated in 54% of cases. PFKFB4 overexpression positively correlated with TP53 and TSC2 loss-of-function mutations, and was associated with younger patient age, more aggressive tumor behavior with metastatic features and advanced tumor staging. PFKFB4 was also verified to be hypoxia-inducible and is located under the HIF1 axis. CRISPR/Cas9-mediated PFKFB4 knockout significantly impaired in vivo HCC development. Elimination of PFKFB4 reduced the tumor incidence rate by approximately 50% in orthotopic xenotransplantation model. This also decreased the average tumor mass by 70% and suppressed the overall tumor growth rate in subcutaneous xenotransplantation model. Furthermore, ectopic expression of PFKFB4 conferred growth advantage and sorafenib resistance in HCC. Targeted metabolomics analysis demonstrated that the ablation of PFKFB4 in HCC cells caused an increase of PFK1 downstream glycolytic metabolites but diminished pyruvate and lactate production. It also resulted in the accumulation of intermediate metabolites in the pentose phosphate pathway and an increase in the total proteogenic amino acids. Unbiased transcriptomics analysis showed that the abolishment of PFKFB4 in HCC xenografts induced the expression of hypoxia-responsive genes involved in glycolysis and reactive oxygen species (ROS) detoxification. Finally, the loss of PFKFB4 was found to increase intracellular ROS level by flow cytometry analysis.

Taken together, my findings suggest that TP53 and TSC2 loss-of-function mutations as well as tumor hypoxia, can upregulate the expression of PFKFB4, which plays an oncogenic role in HCC development, likely by restraining the PFK1-mediated committed step in glycolysis and redirecting glucose into the pentose phosphate pathway for maintaining redox homeostasis. My study highlights the pivotal role of PFKFB4 in mediating the adaptational process of HCC cells to the microenvironment and suggest that PFKFB4 might serve as a possible therapeutic target for HCC treatment.