HSF2 regulates aerobic glycolysis by suppression of FBP1 in hepatocellular carcinoma

Abstract

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. Recent years have witnessed the progress in uncovering the importance of HSFs in cancer cell oncogenesis, progression and metastasis. However, their roles in hepatocellular carcinoma (HCC) proliferation and the underlying mechanism have seldom been discussed. The present study aims to uncover the two important HSFs members HSF1 and HSF2 in hepatocellular carcinoma (HCC). By using the Cancer Genome Atlas (TCGA) dataset analysis, we investigated the prognosis value of HSF1 and HSF2 in HCC and identified HSF2 as a prediction factor of overall survival of HCC. In vitro cell line studies demonstrated that silencing HSF2 expression could decrease the proliferation in HCC cells. In depth mechanism analysis demonstrated that HSF2 promoted cell proliferation via positive regulation of aerobic glycolysis, and HSF2 interacted with euchromatic histone lysine methyltransferase 2 (EHMT2) to epigenetically silence fructose-bisphosphatase 1 (FBP1), which is a tumor suppressor and negative regulator of aerobic glycolysis in HCC. HSF2 expression indicated unfavorable prognosis of HCC patients and it could regulate aerobic glycolysis by suppression of FBP1 to support uncontrolled proliferation of HCC cells.

Keywords: Hepatocellular carcinoma; aerobic glycolysis; euchromatic histone lysine methyltransferase 2; fructose-bisphosphatase 1; heat shock factor 2.

Figure 1

HSF2 predicts prognosis and regulates proliferation of HCC cancer cells. A. Patients with higher levels of HSF2 displayed worse prognosis as revealed by TCGA-LIHC cohort analysis. B. HSF2 expression in liver cancer cell lines was assayed by western blot analysis. C. Real-time PCR results confirmed that HSF2 was silenced in Huh7 and SMMC7721 cells. D. Western blot analysis further confirmed that HSF2 was effectively silenced in Huh7 and SMMC7721 cells. E and F. CCK8 assay results indicated that knockdown of HSF2 inhibited cell variability of Huh7 and SMMC7721 cells. G and H. Silencing HSF2 expression inhibited colony formation capacity of liver cancer cells.

Figure 2

HSF2 regulates aerobic glycolysis in HCC cancer cells. A and B. Silencing HSF2 expression inhibited ECAR that reflected by Seahorse extracellular flux analyzer in Huh7 and SMMC7721 cells. C and D. Knockdown of HSF2 decreased glycolysis and glycolytic capacity in Huh7 and SMMC7721 cells. E and F. Decreasing HSF2 expression increased OCR values in Huh7 and SMMC7721 cells. G and H. Silencing HSF2 increase mitochondrial respiration as reflected by ATP production and maximal respiration.

Figure 3

HSF2 regulated GLUT1, HK2 and LDHA expression, which could also reflect prognosis of HCC patients. A and B. Silencing HSF2 expression decreased expression of glycolytic genes including GLUT1, HK2 and LDHA in Hun7 and SMMC7721 cells. C-E. Expression of SLC2A1, HK2 and LDHA predicted prognosis and patients with higher levels of these genes displayed worse overall survival respectively. F. HSF2 positively correlated with SLC2A1 expression in TCGA-LIHC cohort. G. HSF2 positively correlated with HK2 expression in TCGA-LIHC cohort. H. No significant correlation between HSF2 with LDHA was observed.

Figure 4

FBP1 was a downstream target gene of HSF2. A. Silencing HSF2 expression increased FBP1 expression in mRNA levels. B. Knockdown of HSF2 increased FBP1 protein levels in Huh7 and SMMC7721 cells. C. HSF2 suppressed FBP1 promoter activity in a dose-dependent manner. D. We mutated HSE on FBP1 promoter from TTCTGAACTTC to TTCTGTTCTTC to generate the HSE^Mut construct and luciferase assay results demonstrated that HSF2 lost its suppressive impact of HSE^Mut. E. HSF2 occupied the HSE in FBP1 promoter region. F. Patients with low levels of FBP1 expression have worse prognosis. G. HSF2 negatively correlated with FBP1 expression in TCGA-LIHC cohort.

Figure 5

EHMT2 interacts with HSF2 and regulates proliferation of liver cancer cells. A. HSF2 interacted with EHMT2 in Huh7 and SMMC7721 cells as reflected by co-immunoprecipitation assay. B and C. EHMT2 was effectively silenced in Huh7 and SMMC7721 cells as validated by real-time PCR and western blot. D. Knockdown of EHMT2 expression suppressed cell variability that measured by CCK-8 assay. E and F. Knockdown of EHMT2 decreased colony formation capacity of Huh7 and SMMC7721 cells. G. Patients with higher levels of EMHT2 had shorter overall survival.

Figure 6

EHMT2 regulates aerobic glycolysis in liver cancer cells. A and B. Silencing EHMT2 expression decreased ECAR values. C and D. Knockdown of EHMT2 decreased glycolysis and glycolytic capacity of Huh7 and SMMC7721 cells. E and F. Decreasing EHMT2 expression increased OCR that measured by Seahorse extracellular flux analyzer. G and H. Knockdown of EHMT2 increased ATP production and maximal respiration in Huh7 and SMMC7721 cells.

Figure 7

EHMT2 negatively regulated FBP1 expression in liver cancer cells. A. Silencing of EHMT2 expression increased FBP1 expression in mRNA levels. B. The protein levels of FBP1 increased in EHMT2-silenced Huh7 and SMMC7721 cells. C and D. EHMT2 suppressed promoter activity of FBP1 in a dose-dependent manner. E. EHMT2 occupied the HSE in FBP1 promoter region that demonstrated by ChIP assay. F. EHMT2 negatively correlated with FBP1 expression in TCGA-LIHC cohort patients.

Figure 8

HSF2 interacted with EHMT2 to epigenetically silence FBP1 expression. A. Co-transfection of HSF2 and EHMT2 could suppress FBP1 promoter activity to a more extent. B. ReChIP assay results demonstrated that EHT2 and HSF2 co-occupied the same HSE region in FBP1 promoter. C and D. Silencing HSF2 expression decreased occupancy of EHMT2 and H3K9Me3 in FBP1 promoter and increased euchromatin marker AcH3. E. Schematic representation of the working model.