ZEB1 enhances Warburg effect to facilitate tumorigenesis and metastasis of HCC by transcriptionally activating PFKM

Abstract

Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.

Parathyroid Tumors: Molecular Signatures

“…They found, in two unrelated patients with PCs, the recurrent p.Ile482Met mutation of the ADCK1 gene, which encodes a putative kinase with unknown molecular function. AKAP9 and ZEB1 were found mutated in 3 cases, the first encoding a regulator of protein kinase A and presumably involved in multiple signal transduction pathways, and the second encoding a zinc finger transcription factor that acts as a transcriptional repressor and is shown to facilitate tumorigenesis and promote tumor metastasis [46,47]. Two different somatic truncating loss-of-function mutations of the FAT3 gene were identified in two sporadic PCs, suggesting this gene as a novel candidate tumor suppressor gene in parathyroid carcinogenesis, possibly regulating the Wnt signaling.…”

lncRNA CYTOR promotes aberrant glycolysis and mitochondrial respiration via HNRNPC-mediated ZEB1 stabilization in oral squamous cell carcinoma

“…Importantly, highly metastatic cancer cell subpopulations can exploit mitochondrial function by inducing elevated oxidative stress via increased reactive oxygen species (ROS) production [ 8 ]. Moreover, the “Warburg effect” provides evidence that glycolysis concurrently serves a supporting factor in cancer cell metastasis [ 12 , 13 ]. Finding potential targets that regulate mitochondrial activity and glycolysis could therefore provide a sound strategy for treating cancer metastasis.…”

“…ZEB1, a major transcription factor controlling the EMT, has long been recognized as an important regulator of OSCC metastasis [ 14 ]. Recent studies have demonstrated that ZEB1 also plays essential roles in regulating processes related to aerobic glycolysis [ 12 , 13 ]. However, it remains unclear whether and how ZEB1 contributes to regulating mitochondrial reprogramming and aerobic glycolysis in OSCC.…”

Long Non-Coding RNA LINC01572 Promotes Hepatocellular Carcinoma Progression via Sponging miR-195-5p to Enhance PFKFB4-Mediated Glycolysis and PI3K/AKT Activation

“…In actual fact, the carcinogenic role of PFKFB4 has been documented in a variety of malignant tumors, including in HCC ( Cai et al, 2021 ; Shen et al, 2021 ; Wang et al, 2021 ). Considering that in cancers, PFKFB4 is critically involved in enhanced aerobic glycolysis, a phenomenon termed the Warburg effect ( Chen et al, 2021 ; Zhou et al, 2021 ), and we reckoned LINC01572 might exert its oncogenic effect via PFKFB4-enhanced glycolysis. This postulation was corroborated in our study as the influence of LINC01572 on both glycolysis and HCC malignant behaviors was dependent on the PFKFB4 level.…”