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. 2020 Jan;122(2):209-220.
doi: 10.1038/s41416-019-0658-4. Epub 2019 Dec 10.

MFN1-dependent alteration of mitochondrial dynamics drives hepatocellular carcinoma metastasis by glucose metabolic reprogramming

Ze Zhang #  1 , Tian-En Li #  1 , Mo Chen #  1 , Da Xu  1 , Ying Zhu  1 , Bei-Yuan Hu  1 , Zhi-Fei Lin  1 , Jun-Jie Pan  1 , Xuan Wang  1 , Chao Wu  1 , Yan Zheng  1 , Lu Lu  1 , Hu-Liang Jia  1 , Song Gao  2 , Qiong-Zhu Dong  3   4 , Lun-Xiu Qin  5   6
Affiliations

MFN1-dependent alteration of mitochondrial dynamics drives hepatocellular carcinoma metastasis by glucose metabolic reprogramming

Ze Zhang et al. Br J Cancer. 2020 Jan.

Abstract

Background: Mitochondrial dynamics plays an important role in tumour progression. However, how these dynamics integrate tumour metabolism in hepatocellular carcinoma (HCC) metastasis is still unclear.

Methods: The mitochondrial fusion protein mitofusin-1 (MFN1) expression and its prognostic value are detected in HCC. The effects and underlying mechanisms of MFN1 on HCC metastasis and metabolic reprogramming are analysed both in vitro and in vivo.

Results: Mitochondrial dynamics, represented by constant fission and fusion, are found to be associated with HCC metastasis. High metastatic HCC displays excessive mitochondrial fission. Among genes involved in mitochondrial dynamics, MFN1 is identified as a leading downregulated candidate that is closely associated with HCC metastasis and poor prognosis. While promoting mitochondrial fusion, MFN1 inhibits cell proliferation, invasion and migration capacity both in vitro and in vivo. Mechanistically, disruption of mitochondrial dynamics by depletion of MFN1 triggers the epithelial-to-mesenchymal transition (EMT) of HCC. Moreover, MFN1 modulates HCC metastasis by metabolic shift from aerobic glycolysis to oxidative phosphorylation. Treatment with glycolytic inhibitor 2-Deoxy-D-glucose (2-DG) significantly suppresses the effects induced by depletion of MFN1.

Conclusions: Our results reveal a critical involvement of mitochondrial dynamics in HCC metastasis via modulating glucose metabolic reprogramming. MFN1 may serve as a novel potential therapeutic target for HCC.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Mitochondrial dynamics in HCC cell lines with different metastatic potential.
Mitochondria fusion is less frequent in HCC cells of higher metastatic capacity and correlated to MFN1. a Representative immunofluorescence images under a confocal microscope of mitochondrial morphology in HepG2 (G2), Hep3B (3B), SMMC-7721 (7721), MHCC97-L (97L), MHCC97-H (97H) and HCC-LM3 (LM3) cell lines stained with MitoTracker Deep Red. Scale bars: 10 µm. High metastatic HCC cells like MHCC97-H and LM3 intended to have more fragmented and less fused mitochondria, while low metastatic HCC cells like HepG2 exhibited an opposite morphology. b, c Quantified analysis on mitochondrial length in different HCC cells and ratio analysis based on quantified mitochondria length confirmed the morphologic alterations in IF images. n = 3, mean ± SEM. d, e qRT-PCR (n = 6, mean ± SEM) and western blot analysis for mRNA and protein levels of mitochondrial dynamic mediators. Mitochondria fusion mediator MFN1 showed most significant changes in protein level among HCC cells with different metastatic potential, consistent with mRNA levels.
Fig. 2
Fig. 2. Low MFN1 level in human HCC was correlated with vascular invasion and poor prognosis.
a qRT-PCR analysis for MFN1 expression levels in 34 HCC samples between tumour and peritumour mesenchyme tissues indicated that MFN1 expression was greatly inhibited in tumour tissues compared with para-tumour tissues. n = 6, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. b qRT-PCR analysis for MFN1 expression levels in 20 HCC patients with and without portal vein tumour thrombosis (PVTT) indicated that for patients with PVTT MFN1 expression decreased. n = 6, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. c Representative immunohistochemical (IHC) staining images of MFN1 in HCC tissue microarrays. Scale bar: 100 µm. d, e Kaplan–Meier analysis of overall survival (OS) and disease-free survival (DFS) in HCC patients classified by expression levels of MFN1 showed that MFN1 low expression contributes to poor prognosis.
Fig. 3
Fig. 3. Increased mitochondrial fusion mediated by MFN1 inhibits migration and invasion of HCC cells in vitro.
a, b Representative immunofluorescence images under a confocal microscope of mitochondrial morphology of OE-MFN1 97H cells and HepG2 cells. Scale bars: 10 μm. MFN1 overexpression leads to increased mitochondria fusion, while knockdown of MFN1 results in mitochondria fragmentation. Quantified analysis on mitochondrial length and ratio analysis based on quantified mitochondria length OE-MFN1 97H cells and sh-MFN1 HepG2 cells confirmed the morphologic changes in IF. n = 3, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. c, d Cell proliferation assay was assessed by CCK-8 assay in OE-MFN1 97 H cells and sh-MFN1 HepG2 cells. Overexpression of MFN1 inhibited 97H cell proliferation, while knockdown of MFN1 promoted HepG2 cells proliferation. OD, absorbance degrees. n = 3, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. e Images on plate clone formation assay in OE-MFN1 97H cells and sh-MFN1 HepG2 cells. The results were consistent with cell proliferation assay. f, g Images and quantified column figures on Transwell assay in OE-MFN1 97H cells and sh-MFN1 HepG2 cells. Overexpression of MFN1 inhibited migration and invasion of 97H cells, while knockdown of MFN1 promoted migration and invasion of HepG2 cells. n = 3, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05.
Fig. 4
Fig. 4. Increased mitochondrial fusion mediated by MFN1 inhibits proliferation and metastasis of HCC cells in vivo.
a–f The effects of MFN1 gain- or loss of function on the dynamic change of tumour volume in subcutaneous xenograft models (a, d). Quantitative analysis (b, c, e, f) demonstrated that overexpression of MFN1 inhibited subcutaneous tumour growth, while knockdown of MFN1 promoted subcutaneous tumour growth. n = 5, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. g, h The effects of MFN1 gain- or loss of function on the dynamic change of the spontaneous lung metastasis in orthotopic xenograft models. Representative H&E staining images of lung tissues (left) and the percentage of nude mice with lung metastasis (right) from five mice per group were shown. MFN1 overexpression inhibited lung metastasis of 97H cells, while knockdown of MFN1 promoted lung metastasis of HepG2 cells. n = 5, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. i IHC staining image of MFN1, E-cad and Ki67 in xenografted tumour from nude mice. MFN1 staining validated the efficiencies of MFN1 overexpression in 97 H cells and MFN1 knockdown in HepG2 cells. E-cadherin staining confirmed positive correlation between E-cadherin and MFN1. Ki67 staining verified that MFN1 negatively modulates proliferation of HCC cells. Scale bar: 100 μm.
Fig. 5
Fig. 5. Metabolic reprogramming in HCC cells mediated by MFN1.
a–f Sea-horse metabolism analysis on overexpression and knockdown of MFN1 HCC cells. The ratio of OCR/ECAR increased significantly in OE-MFN1 97H cells, stating increased O2 consumption compared with less lactic acid production, while the ratio of OCR/ECAR decreased significantly in knockdown HepG2 cells. n = 3, mean ± SEM, ***P < 0.001. g–j Aerobic glycolysis-related enzymes were detected using qRT-PCR in OE-MFN1 97H cells, sh-MFN1 HepG2 cells and the xenograft tumours. Most enzymes involved in aerobic glycolysis were downregulated in OE-MFN1 97 H cells (g) and their xenograft tumour (h) and upregulated in sh-MFN1 G2 cells (i) and the xenografts (j). n = 6, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05.
Fig. 6
Fig. 6. MFN1 influences HCC cells proliferation, invasion and migration via metabolic reprogramming.
a–c Proliferation assay (a) and plate clone formation assay (c) showed that 2-DG inhibited the proliferation of sh-MFN1 HepG2 cells. But 2-DG did not affect proliferation capacity in OE-MFN1 97H cells (b). n = 3, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05. d–i Transwell assay (d, e, f, g) and wound-healing assays (h, i) confirmed that 2-DG could reverse the effect of sh-MFN1 to retrain migration and invasion of HepG2 cells. n = 3, mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05.
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