Simultaneous treatment with sorafenib and glucose restriction inhibits hepatocellular carcinoma in vitro and in vivo by impairing SIAH1-mediated mitophagy.

Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical significance to explore the synergistic effect of TACE with antiangiogenic inhibitors and the molecular mechanisms involved. This study determined that glucose, but not other analyzed nutrients, offered significant protection against cell death induced by sorafenib, as indicated by glucose deprivation sensitizing cells to sorafenib-induced cell death. Next, this synergistic effect was found to be specific to sorafenib, not to lenvatinib or the chemotherapeutic drugs cisplatin and doxorubicin. Mechanistically, sorafenib-induced mitophagy, as indicated by PINK1 accumulation, increased the phospho-poly-ubiquitination modification, accelerated mitochondrial membrane protein and mitochondrial DNA degradation, and increased the amount of mitochondrion-localized mKeima-Red engulfed by lysosomes. Among several E3 ubiquitin ligases tested, SIAH1 was found to be essential for inducing mitophagy; that is, SIAH1 silencing markedly repressed mitophagy and sensitized cells to sorafenib-induced death. Notably, the combined treatment of glucose restriction and sorafenib abolished ATP generation and mitophagy, which led to a high cell death rate. Oligomycin and antimycin, inhibitors of electron transport chain complexes, mimicked the synergistic effect of sorafenib with glucose restriction to promote cell death mediated via mitophagy inhibition. Finally, inhibition of the glucose transporter by canagliflozin (a clinically available drug used for type-II diabetes) effectively synergized with sorafenib to induce HCC cell death in vitro and to inhibit xenograft tumor growth in vivo. This study demonstrates that simultaneous treatment with sorafenib and glucose restriction is an effective approach to treat HCC, suggesting a promising combination strategy such as transarterial sorafenib-embolization (TASE) for the treatment of unresectable HCC.

ACSL4 is a predictive biomarker of sorafenib sensitivity in hepatocellular carcinoma.

Sorafenib is the first-line treatment of advanced hepatocellular carcinoma (HCC). However, there is a lack of validated biomarkers to predict sorafenib sensitivity. In this study we investigated the role of ACSL4, a positive-activating enzyme of ferroptosis, in sorafenib-induced cell death and HCC patient outcome. We showed that ACSL4 protein expression was negatively associated with IC50 values of sorafenib in a panel of HCC cell lines (R = -0.952, P < 0.001). Knockdown of ACSL4 expression by specific siRNA/sgRNA significantly attenuated sorafenib-induced lipid peroxidation and ferroptosis in Huh7 cells, and also rescued sorafenib-induced inhibition of xenograft tumor growth in vivo. We selected 29 HCC patients with surgery as primary treatment and sorafenib as postoperative adjunct therapy from a hospital-based cohort. A high proportion (66.7%) of HCC patients who had complete or partial responses to sorafenib treatment (according to the revised RECIST guideline) had higher ACSL4 expression in the pretreated HCC tissues, compared with those who had stable or progressed tumor growth (23.5%, P = 0.029). Since ACSL4 expression was independent of sorafenib treatment, it could serve as a useful predictive biomarker. Taken together, this study demonstrates that ACSL4 is essential for sorafenib-induced ferroptosis and useful for predicting sorafenib sensitivity in HCC. This study may have important translational impacts in precise treatment of HCC.

Identification of Key Genes in Purine Metabolism as Prognostic Biomarker for Hepatocellular Carcinoma.

BACKGROUND: Deregulated purine metabolism is critical for fast-growing tumor cells by providing nucleotide building blocks and cofactors. Importantly, purine antimetabolites belong to the earliest developed anticancer drugs and are still prescribed in clinics today. However, these antimetabolites can inhibit non-tumor cells and cause undesired side effects. As liver has the highest concentration of purines, it makes liver cancer a good model to study important nodes of dysregulated purine metabolism for better patient selection and precisive cancer treatment. METHODS: By using a training dataset from TCGA, we investigated the differentially expressed genes (DEG) of purine metabolism pathway (hsa00230) in hepatocellular carcinoma (HCC) and determined their clinical correlations to patient survival. A prognosis model was established by Lasso-penalized Cox regression analysis, and then validated through multiple examinations including Cox regression analysis, stratified analysis, and nomogram using another ICGC test dataset. We next treated HCC cells using chemical drugs of the key enzymes in vitro to determine targetable candidates in HCC. RESULTS: The DEG analysis found 43 up-regulated and 2 down-regulated genes in the purine metabolism pathway. Among them, 10 were markedly associated with HCC patient survival. A prognostic correlation model including five genes (PPAT, DCK, ATIC, IMPDH1, RRM2) was established and then validated using the ICGC test dataset. Multivariate Cox regression analysis found that both prognostic risk model (HR = 4.703 or 3.977) and TNM stage (HR = 2.303 or 2.957) independently predicted HCC patient survival in the two datasets respectively. The up-regulations of the five genes were further validated by comparing between 10 pairs of HCC tissues and neighboring non-tumor tissues. In vitro cellular experiments further confirmed that inhibition of IMPDH1 significantly repressed HCC cell proliferation. CONCLUSION: In summary, this study suggests that purine metabolism is deregulated in HCC. The prognostic gene correlation model based on the five purine metabolic genes may be useful in predicting HCC prognosis and patient selection. Moreover, the deregulated genes are targetable by specific inhibitors.

Histone deacetylases up-regulate C/EBPα expression through reduction of miR-124-3p and miR-25 in hepatocellular carcinoma.

BACKGROUND: CCAAT enhancer binding protein α (C/EBPα), as an important transcription factor involved in cell proliferation, differentiation and metabolism, was up-regulated in primary hepatocellular carcinoma (HCC) and predicted poorer prognosis. In this study, we explored how histone deacetylases (HDACs) up-regulated C/EBPα in HCC. METHODS: The protein expressions of HDAC1, HDAC2 were associated with C/EBPα by immunohistochemistry staining in a HCC tissue microarray. HCC cells were then treated with HDAC inhibitors or siRNAs to determine the roles of miR-124-3p and miR-25 in the regulation of C/EBPα mRNA expression. RESULTS: Both HDAC1 and HDAC2 proteins were significantly associated with C/EBPα. Inhibition of HDAC by either pharmacological inhibitors or siRNAs decreased C/EBPα mRNA expression in dose-dependent manners in HCC cells. HDAC inhibitors reduced C/EBPα mRNA stability as shown by pmiRGLO luciferase reporter assays. HDAC inhibition consistently induced miR-124-3p and miR-25 expression. Conversely, blockage of miR-124-3p and/or miR-25 by treatment with specific synthetic inhibitors abolished C/EBPα reduction. More importantly, C/EBPα mRNA stability could be rescued by site-directed mutations of miR-124-3p or miR-25 recognition sites in the C/EBPα 3'UTR sequence. In summary, HDAC may up-regulate C/EBPα expression through miR-124-3p and miR-25 in HCC.