The Mir-30A-5P/CLCF1 Axis Regulates Sorafenib Resistance and Aerobic Glycolysis in Hepatocellular Carcinoma

Zhang et al. Cell Death and Disease (2020) 11:902 https://doi.org/10.1038/s41419-020-03123-3 Cell Death & Disease

ARTICLE Open Access The miR-30a-5p/CLCF1 axis regulates sorafenib resistance and aerobic glycolysis in hepatocellular carcinoma Zhongqiang Zhang1,2,XiaoTan3,JingLuo1, Hongliang Yao 4, Zhongzhou Si1 and Jing-Shan Tong5

Abstract HCC (hepatocellular carcinoma) is a major health threat for the Chinese population and has poor prognosis because of strong resistance to chemotherapy in patients. For instance, a considerable challenge for the treatment of HCC is sorafenib resistance. The aberrant glucose metabolism in cancer cells aerobic glycolysis is associated with resistance to chemotherapeutic agents. Drug-resistance cells and tumors were exposed to sorafenib to establish sorafenib- resistance cell lines and tumors. Western blotting and real-time PCR or IHC staining were used to analyze the level of CLCF1 in the sorafenib resistance cell lines or tumors. The aerobic glycolysis was analyzed by ECAR assay. The mechanism mediating the high expression of CLCF1 in sorafenib-resistant cells and its relationships with miR-130-5p was determined by bioinformatic analysis, dual luciferase reporter assays, real-time PCR, and western blotting. The in vivo effect was evaluated by xenografted with nude mice. The relation of CLCF1 and miR-30a-5p was determined in patients’ samples. In this study, we report the relationship between sorafenib resistance and increased glycolysis in HCC cells. We also show the vital role of CLCF1 in promoting glycolysis by activating PI3K/AKT signaling and its downstream genes, thus participating in glycolysis in sorafenib-resistant HCC cells. Furthermore, we also show that miR-30a-5p directly targets CLCF1 and that sorafenib-mediated suppression of miR-30a-5p results in the upregulation 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; of CLCF1 in HCC cells resistant to sorafenib. We also found that when a cholesterol modiﬁed agomiR-30a-5p was delivered systemically to mice harboring sorafenib-resistant HCC tumors, tumor growth decreased signiﬁcantly. There is an uncharacterized mechanism of biochemical resistance to hormone therapies orchestrated by the miR-30a-5p/ CLCF1 axis to mediate sorafenib resistance and aerobic glycolysis in HCC. Therefore, this study indicates that targeting the miR-30a-5p/CLCF1 axis may hold promise for therapeutic intervention in HCC sorafenib resistance patients.

Introduction including resection, ablation, and transplantation, are only On a global scale, HCC (hepatocellular carcinoma) feasible in patients diagnosed in the early stages1,2. The ranks sixth among cancers and is the third leading cause manifestation of HCC is more frequent in cirrhosis and of death due to cancer1. The incidence of this deadly liver dysfunction patients, which limits the interventions disease is still on the rise, and curative treatments, with systemic cytotoxic drugs3,4. HCC at the advanced stage is not amenable to surgery, and the only treatment strategy is chemotherapy5,6. Correspondence: Zhongzhou Si ([email protected]) or Jing- For HCC treatment, conventional chemotherapeutics Shan Tong ([email protected]) fl fi 1 such as doxorubicin and uorouracil are not ef cacious Department of Liver Transplantation, The Second Xiangya Hospital of Central 7,8 South University, 410011 Changsha, Hunan Province, P.R. China and exhibit potent side effects . Significant progress in 2Department of Surgery, University of Pittsburgh School of Medicine, cancer treatment has been achieved with the advent of University of Pittsburgh Medical Center Presbyterian Hospital, Pittsburgh, PA targeted drug therapy and small molecule kinase inhibi- 15213, USA 9,10 Full list of author information is available at the end of the article tors . The only clinical solution currently available for Edited by I. Amelio

© The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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the treatment of HCC in patients who cannot undergo anticancer therapies based on miRNA are actively being surgery is sorafenib. As an inhibitor of Raf kinase, sor- developed27,28. Additionally, miRNAs also act as impor- afenib inhibits tumor growth by decreasing the activities tant regulators in the Warburg effect (reprogramming of of the B-RAF serine/threonine kinase and RAF-111,12; cellular energy metabolism) and in drug resistance in concurrently, sorafenib can also inhibit tumor growth by cancers29,30. Therefore, miRNA targeting may be a pro- decreasing tumor angiogenesis via the tyrosine kinase mising treatment strategy to interrupt aerobic glycolysis activities of platelet-derived growth factor (PDGF), FMS- in cancer cells as well as in sorafenib-resistant HCC. like tyrosine kinase 3 (FLT-3), Vascular Endothelial In the present study, we identified CLCF1 as a novel Growth Factor 2 (VEGF-2), and -3 receptors13,14. Never- regulator in sustaining sorafenib resistance in HCC theless, HCC exhibits notable resistance to sorafenib, and cells. We further found that CLCF1 acts as a key only one-third of patients with HCC at an advanced stage mediator to orchestrate sorafenib resistance and aero- respond to treatment11,15. Furthermore, the results of bic glycolysis in HCC cells. Mechanistically, upregula- multiple random clinical cases were systematically ana- tion of CLCF1 boosted PI3K/AKT signaling and thus lyzed and revealed an increase in the survival time by only activated its downstream glycolytic genes in sorafenib- 3 months, indicating the urgent need to discover more resistant HCC cells. We further identified CLCF1 as a robust molecular targeted drugs against drug-resistant direct target of miR-30a-5p and revealed that sorafenib tumors and to further conduct in-depth evaluation of the upregulated CLCF1 by downregulating miR-30a-5p. HCC resistance mechanism and improve the sensitivity of Functional studies showed that the miR-30a-5p/CLCF1 HCC to sorafenib. axis plays an important role in regulating both sorafenib Cardiotrophin-like cytokine factor 1 (CLCF1, also resistance and aerobic glycolysis in sorafenib-resistant referred to as BSF-3 or NNT-1) is a cytokine of the IL-6 cells. Taken together, our findings reveal that the miR- family that is secreted in the form of a soluble CRLF1 30a-5p/CLCF1/PI3K/AKT regulatory cascade is part of (cytokine receptor cytokine-like factor 1) complex16,17. an important mechanism of sorafenib resistance and CLCF1 activates the tripartite CNTFR (ciliary neuro- aerobic glycolysis in HCC cells, suggesting that miR- trophic factor receptor), which consists of LIFRβ, gp130, 30a-5p is a promising therapeutic target to reverse and CNTFRα18. The common signaling pathways shared sorafenib resistance in HCC. by CLCF1 and CNTF indicate that the exhaustive eva- luations conducted preclinically and clinically on CNTF Materials and methods may pave a way for the curative ability of CLCF119.In Cell culture vitro studies and animal models of neurodegenerative The human HCC cell lines HepG2 and Huh7 were diseases have demonstrated the significant neurotrophic obtained from American Type Culture Collection (ATCC, activities of CNTF and have encouraged clinical trials in Manassas, VA, USA) and maintained in DMEM (Dul- patients with amyotrophic lateral sclerosis, Huntington’s becco’s modified Eagle’s medium) with streptomycin/ disease, and cases of retinal degeneration20. In addition, penicillin (1%) and fetal bovine serum (FBS, 10%) in a when administered systemically, CNTF leads to remark- humidified atmosphere of 5% CO2 at 37 °C. HepG2-S-R able, unexpected weight loss, thus leading to preclinical and Huh7-S-R, the sorafenib-resistant sublines, were assessments of CNTF in metabolic syndrome models21,22. derived from HepG2 and Huh7 cells as previously When injections of CNTF were given daily, persistent described31. anorexic effects were observed in leptin-deficient, diet- induced obese mice23. The conditions of hyper- HCC specimens and Immunohistochemistry insulinemia and hyperglycemia were reduced due to HCC tumor specimens and paired normal adjacent CNTF, indicating that type II diabetes could benefit and tissues were obtained from Proteo Genex (CA, USA). that leptin resistance could be overcome in obese indivi- EnVision™ Flex target retrieval solution was employed to duals24,25. CNTF is believed to exert both systemic and deparaffinize and unmask epitopes in the samples. central effects because CNTFR is expressed in the region Endogenous peroxidase was blocked by adding 0.3% H2O2 of the brain directing energy balance, in skeletal muscle or hydrogen peroxide for 5 min. This was followed by the and in adipose tissues26. However, there is no report on addition of primary antibody at 4 °C overnight. Subse- the role of CLCF1 in sorafenib resistance in HCC. Hence, quently, the following treatments were done: 15 min with we studied the relationship between CLCF1 and sorafenib EnVision™ Flex linker rabbit, 30 min with EnVision™ resistance in hepatocellular carcinoma. Flex/HRP enzyme, and 10 min with 3′-3-diaminobenzi- MicroRNAs (miRNAs) are small noncoding RNAs that dine tetrahydrochloride. Hematoxylin was added as a posttranscriptionally regulate eukaryotic gene expression. counterstain followed by dehydration and mounting on a miRNAs have been shown to have crucial roles in the Richard-Allan Scientific Cyto seal XYL (Thermo advancement and progression of cancers, and new Scientific, USA).

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RNA oligonucleotides, plasmid constructs, and antibodies instructions. Briefly, the indicated cells were treated and The lentiviral vector pLVX-3×Flag-Puro was used to clone stained using the apoptosis detection kit Annexin V- the cDNA of human CLCF1 to obtain the pLVX-CLCF1- FITC/PI from KeyGen (China) according to the manu- 3×Flag-Puro plasmid. The pmirGLO dual-luciferase repor- facturer’s instructions. Finally, apoptosis was detected by ter from Promega was used to clone the human CLCF1 3′- flow cytometry. Analysis of more than 1 × 106 cells was UTR to obtain the wild-type pmirGLO-CLCF1-3′-UTR performed per measurement. reporter. In the mutant reporter pmirGLO-CLCF1-3′-UTR- mut, mutations of seven nucleotides in the CLCF1 3′-UTR Metabolic parameters of glucose utilization and that correspond to the miR-30a-5p seed sequences were production of lactate performed. DNA sequencing was performed to confirm all The assays were performed using a glucose assay kit constructs. The agomiR-30a-5p, miR-30a-5p mimics, from Sigma and a lactate assay kit from BioVision as per CLCF1 siRNA and their respective control RNAs and anti- the provided protocols. The cell number was used to miR-30a-5p reagents were procured (RiboBio, China). normalize lactate production and glucose consumption rates. Transfection of cells and viral transduction Cell transfection was performed with Lipofectamine Determination of cellular ATP 2000 (Invitrogen) according to the manufacturer’s The level of ATP in cells was measured using a instructions. Specifically, for each transfection, RNA oli- luminometer and a CellTiter-Glo 2.0 Assay kit from gonucleotide (50 pmol) or plasmid DNA (2 μg) was used Promega as per instructions. The total protein in the in a 6-well plate. The pLVX-CLCF1-3×Flag-Puro lenti- corresponding cell lysate was used to normalize the virus or control vector was packaged into a pseudovirus relative intracellular ATP level (measured as with the corresponding packaging plasmids as per stan- luminescence). dard protocol. Centrifugation of the supernatant was performed at 5 × 104 g for 1.5 h at 4 °C, and lentivirus at Measurement of ECAR (extracellular acidification rate) high titer (>1 × 108 transduction units/mL) was prepared. The mitochondrial functions and capacity for glycolysis Lentiviruses expressing CLCF1 shRNA or miR-30a-5p at were assessed by Seahorse Bioscience XF96 Extracellular high titer were procured from GeneChem (Shanghai, Flux Analyzer, as instructed by the Seahorse XF Cell Mito China). Lentiviruses were incubated with target cells in Stress Test Kit or Glycolysis Stress Test Kit from Seahorse media supplemented with polybrene (8 μg/mL) for Bioscience (MA, USA). Cells (initial density of 4 × 104 24 hours. After transduction, puromycin (1 μg/mL) was cells/well) were plated in Cell Culture Microplates XF96 added for the selection. (Seahorse Bioscience) the day before measurement. Next, the addition of oligomycin (1 μM), 2-deoxy-glucose Luciferase reporter assay (100 mM) and glucose (10 mM) was performed to deter- Luciferase reporter assay was performed as previously mine the ECAR value. Cell numbers were used to nor- described29, luciferase reporter assays were carried out. malize the final ECAR values. Briefly, cotransfection of HepG2 cells with Renilla luciferase reporter vectors, siRNAs or protein expression Real-time PCR vectors, or/and miRNA mimics was performed in 24-well After extracting total RNA with TRIzol reagent plates. The Promega (USA) supplied Dual-Luciferase obtained from Invitrogen (USA), reverse transcription Reporter Assay System was used to assess luciferase (RT) was performed with PrimeScript RT reagent from activity, and the internal control was the firefly luciferase TaKaRa (Dalian, China) to obtain cDNA. Quantitative plasmid pGL3, with luciferase activity normalized to the real-time PCR was performed on an ABI 7900HT Real- corresponding controls. Time PCR System from Applied Biosystems (USA) to evaluate the expression of specific genes and β-actin. Each −ΔΔ Cell viability reaction was run independently in triplicate. The 2 CT Viability assays were conducted as previously descri- method was used to calculate the mRNA levels. The bed32,33. Briefly, viable cells in equal number were seeded primers are list in Table 1. in DMEM with sorafenib or regorafenib at varying con- centrations in 96-well plates. Cell viability was determined Western blotting by MTT assay after 3 days of incubation. Western blotting was performed as previously study34,35. Briefly, cells were lysed using RIPA (Abmole, Apoptosis assay USA) buffer. The supernatant was collected after lysate Apoptosis was assessed using the Fluorometric TUNEL centrifugation at 12,000 rpm for 30 min at 4 °C. The System from Promega, as per the manufacturer’s protein concentration was measured using the BCA

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Table 1 Primers for real-time PCR.

Gene Forward Reverse

CLCF1 5′-CTTAGCTGGGACCTACCTGAA-3′ 5′-CCACACTTCCAAGTTGACCGT-3′ CCL2 5′-AGGTCCCTGTCATGCTTCTG-3′ 5′-TCTGGACCCATTCCTTCTTG-3′ IL-6 5′-TAGTCCTTCCTACCCCAATTTCC-3′ 5′-TTGGTCCTTAGCCACTCCTTC-3′ CCL7 5′-CTGCTCTCCAGCGCTCTCA-3′ 5′-GTAAGAAAAGCAGCAGGCGG-3′ TGFB1 5′-TATCGACATGGAGCTGGTGA-3′ 5′-CAGCTTGGACAGGATCTGGC-3′ IL-10 5′-TGCAGGACTTTAAGGGTTAC-3′ 5′-GGCCTTGTAGACACCTTGGTC-3′ CNTF 5′-GGAAGATTCGTTCAGACCTGAC-3′ 5′-CCCATCAGCCTCATTTTCAGGG-3′ IL-1α 5′-ATCAGTACCTCACGGCTGCT-3′ 5′-TGGGTATCTCAGGCATCTCC-3′ NGF 5′-ACAGGAGCAAGCCCTCCT-3′ 5′-GCCTGCTTGCCGTCCAT-3′ CD40 5′-TCGGCTTCTTCTCCAATCAGT-3′ 5′-TGAAATTTGGTGTCTACTGT-3′ CXCL14 5′-CCAAGATTCGCTATAGCGAC-3′ 5′-CCTGCGCTTCTCGTTCCAGG-3′ LIF 5′-CTCTTCCCATCACCCCTGTA-3′ 5′-TGGTCTTCTCTGTCCCGTTG-3′ OSM 5′-CCAGACTTCCTCCTTTCCGTG-3′ 5′-ACACCCTGCCGCTGTTACAG-3′ IL-19 5′-GGTCTGGTTGGATCCCAATG-3′ 5′-CCCATCCTTGATCAGCTTCCT-3′ IFNAR1 5′-ATTTACACCATTTCGCAAAGCTC-3′ 5′-TCCAAAGCCCACATAACACTA-3′ BAFF 5′-TGCCTTGGAGGAGAAAGAGA-3′ 5′-GGCAGTGTTTTGGGCATATT-3′ IFNAR2 5′-TGAGCAGGATGCGTTCAC-3′ 5′-TCTCTAGGCTCGCAGACACC-3′ ALK-7 5′-ATAGCGGACTTAGGGTTG-3′ 5′-CTGGTTTGGGATACTTGG-3′ CCL1 5′-GCTCCAATGAGGGCTTAATC-3′ 5′-ATTTTTCAGTGCCTCTGAACC-3′ β-actin 5′-TGTGTGACGACGAAGTCGCC-3′ 5′-TGGTCGTGAAGGTGTAACCA-3′ miR-30a-5p 5′-AACGAGACGACGACAGAC-3′ 5′-TGTAAACATCCTCGACTGGAAG-3′ U6 5′-GTGCTCGCTTCGGCAGCA-3′ 5′-CAAAATATGGAACGCTTC-3′ GLUT3 5′-TTCTGGTCGGAATGCTCTTC-3′ 5′-AATGTCCTCGAAAGTCCTGC-3′ HK2 5′-CTTGCGAATATGGTTGCCTCA-3′ 5′-CCTCCCAATGCCTGATCTGAT-3′ PDK1 5′-TTACGGATTGCCCATCTCACG-3′ 5′-CCCGGTCACTCATCTTCACAGT-3′

(bicinchoninic acid) method, and the proteins were elec- GLUT3 (ab41525, Abcam), HK2 (ab209147, Abcam), trophoresed on a 10% SDS-PAGE gel. The separated PDK1 (#5662, Cell Signaling Technology). proteins were transferred onto a PVDF membrane using a Trans-Blot Transfer Slot (both from Bio-Rad), and the Xenograft assays membrane was blocked using milk (fat-free; 5%) for The Ethics Committee of University of Pittsburgh 120 min at RT (room temperature). Then, the primary approved all experiments with animals in compliance with antibody was added and incubated overnight at 4 °C, fol- the Care and Use of Laboratory Animals Guide. To carry lowed by the addition of a secondary antibody and out assays on tumor xenografts, suspensions of cells (5 × another incubation for 60 min at RT. The immunoblot 105) were made in Matrigel from BD Biosciences and was developed with an ECL reagent. β-Actin was used as subcutaneously injected into athymic nude BALB/c mice the internal reference. The primary antibodies are list as (female; n = 5; 6–8 weeks of age). For the sorafenib assay, follows: CLCF1 (HPA042444, Sigma), p-AKT (#9611, Cell mice were randomly divided into two groups and no Signaling Technology), AKT (#9272, Cell Signaling blinding was done, and the sorafenib treatment group was Technology), p-mTOR (#5536, Cell Signaling Technol- administered sorafenib (25 mg/kg) through oral gavage ﬁve ogy), mTOR (#2972, Cell Signaling Technology), β-actin times per week until week 7. Sorafenib was dissolved in (A5441, Sigma), Ki-67 (#9449, Cell Signaling Technology), ethanol Cremephor EL/95% (50:50) to prepare a stock

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solution (4×) and was diluted in sterile water to obtain the sorafenib resistance cells compared with the parental final concentration prior to use. For treatment using miR- cells, and found CLCF1 upregulated in the resistance cells 30a-5p, agomiR (chemically modified) along with the neu- by real-time PCR (Figs. 1c and S1C). Our findings also tral, lipid-based delivery reagent “In Vivo RNA-LANCEr II” showed a remarkable enhancement of CLCF1 levels in (BIOO Scientific) was formulated and injected (1.5 mg/kg cells resistant to sorafenib using western blotting (Fig. 1d). mice body weight) into mouse tail veins for five cycles of We also observed that PI3K/AKT signaling and its one injection per week for seven weeks. All mice were downstream effector mTOR were activated noticeably in euthanized at the end of the seventh week, and surgical Huh7-S-R and HepG2-S-R cells (Fig. 1e). Hence, CLCF1 resection of tumors was performed for further analyses. is upregulated, and PI3K/AKT signaling is activated in Once per week for seven weeks, tumor volumes were HCC cells resistance to sorafenib. determined and assessed for tumor width (W) and length We then evaluated whether a similar effect of CLCF1 is (L) and volume was calculated using the formula LW2/2. observed in HCC tumors resistant to sorafenib. For this, subcutaneous injection of HepG2 cells into female nude In situ hybridization mice was administered, and mice were treated with low- In situ hybridization kits were purchased from Boster dose sorafenib one-week post-implantation (Fig. 1f). Indeed, Sci corp, Wuhan, China to conduct In situ hybridization tumor growth was significantly inhibited after sorafenib by excellent probes (locked nucleic acid (LNA)-modified treatment for the first four weeks but was ineffective later antisense oligonucleotides). 5′-CTTCCAGTCGAG- (Fig. 1f). At the end of treatment, tumors of similar sizes GATGTTTACA-3′ was the nucleotides sequence of the were observed in the treatment and control groups (Fig. 1f). LNA modified probe (miR-30a-5p). In short, the tissues Thus, sorafenib resistance was developed in due course in sections (deparaffinized) were incubated with 25 μg/mL xenografted tumors in the sorafenib treatment group. After proteinase-K (at 37 °C for 8 min). The Scrambled LNA™ sacrificing all mice and harvesting xenografted tumors at and miR-30a-5p (labeled with DIG) probes were treated at week seven for further analyses, IHC staining showed sig- 30 nM concentration with Exiqon hybridization buffer for nificantly increased CLCF1 levels in HepG2 tumors in the 60 min (57 °C). The probes were visualized at 30 °C by sorafenib-treated group, in contrast to the controls (Fig. 1f). using anti-DIG-alkaline phosphatase for 60 min, treated Therefore, there is a likely association between CLCF1 and with the fresh substrate (NBT-BCIP) comprising of leva- sorafenib resistance in HCC. misole with 0.2 nM concentration at 30 °C. Maintenance of resistance to sorafenib in HCC cells Statistical analyses requires CLCF1 The mean ± SD (standard deviation) was used to pre- Next, we evaluated whether CLCF1 is required for sent all results. The variations among the control and resistance to sorafenib in HCC cells and discovered that treated groups were assessed using Student’s t test. The knockdown of CLCF1 led to re-sensitization of HepG2-S- cell viability and tumor growth data were analyzed using R cells to sorafenib (Fig. 2a) along with potentially one-way ANOVA. The P-values (***: P < 0.001, **: P < increased apoptosis (Fig. S2A). Moreover, knockdown of 0.01; *: P < 0.05) denoted different levels of statistical CLCF1 led to significantly reduced HepG2-S-R cell pro- significance. The correlation between control and treated liferation (Fig. 2b), as confirmed by western blotting groups as well as regression were calculated by Pearson’s indicating a decline in Ki-67, a cell proliferation marker correlation analyses. (Fig. 2c), as well as p-AKT and p-mTOR in cells transfected with CLCF1 siRNA (Fig. 2d). The similar results Results were obtained in Huh7 and Huh7-S-R cells (Fig. S2B– Upregulation of CLCF1 in sorafenib resistance HCC cells S2F). Conversely, an effective impairment of the inhibi- To investigate the mechanisms of sorafenib resistance in tory effect of sorafenib and better survival of cells was HCC, we generated Huh7-S-R and HepG2-S-R sorafenib- found after ectopic expression of CLCF1 in the parental resistant human HCC cell lines by exposing Huh7 and HepG2 and Huh7 cells (Figs. 2e, f and S2J). Likewise, HepG2 cells, respectively, to sorafenib at increasing con- overexpression of CLCF1 enhanced p-mTOR and p-AKT centrations. The Huh7-S-R and HepG2-S-R cells were levels in HepG2 and Huh7 cells (Figs. 2g and S2K). resistant to sorafenib cytotoxicity even at higher con- Therefore, our results indicate that CLCF1 is important centrations, as per the cell viability assay (Fig. 1a, b). for sorafenib resistance in HCC cells. Interestingly, both cells were also regorafenib resistant (Fig. S1A, S1B). Proinflammatory cytokines in the tumor Enhanced glycolysis in sorafenib-resistant HCC cells microenvironment can promote tumor growth, yet their requires CLCF1 value in sorafenib resistance remains underexploited. We Several cancers have been associated with aerobic gly- next analyzed a series of proinflammatory cytokines in colysis and chemo-resistance36. We next assessed whether

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Fig. 1 Increased CLCF1 expression in sorafenib-resistant HCC cells. a Huh7 and Huh7-S-R cells were treated with increasing dose of sorafenib for 3 days. Relative cell viability was analyzed by MTT assay. The significance of the difference in IC50 values was analyzed by extra sum-of-squares F test. b HepG2 and HepG2-S-R cells were treated with increasing dose of sorafenib for 3 days. Relative cell viability was analyzed by MTT assay. The significance of the difference in IC50 values was analyzed by extra sum-of-squares F test. c mRNA level of CLCF1 in Huh7-S-R, HepG2 and their parental cells was analyzed by real-time PCR. d Protein level of CLCF1 in Huh7-S-R, HepG2 and their parental cells was analyzed by Western blotting. e Indicated protein level in Huh7-S-R, HepG2 and their parental cells was analyzed by Western blotting. f HepG2 tumors resistant to sorafenib were generated in nude mice. Left, schematics of the experimental design. Post 14 days of HepG2 cells implantation subcutaneously, treatment of mice was done with a control vehicle (non-therapeutic) or with sorafenib five times per week across week seven; bottom left, tumor growth assessed through the time course. Right, HepG2 tumor sections from sorafenib or vehicle-treated mice were immunohistochemically stained for CLCF1. Scale bar: 25 µm. All data are mean ± SD of three separate experiments. ** P < 0.01. metabolism in HCC cells is altered by sorafenib resistance S-R cells with an inhibitor of AKT, called MK-2206, and and observed a significant increase in lactate and ATP found a strong decrease in lactate production, glucose production, glucose consumption (Fig. S3A–S3C), and consumption, and cellular ATP levels (Fig. S3E–S3G). ECAR (Fig. S3D) in HepG2-S-R cells in comparison to the Previous studies have shown that PI3K/AKT signaling parental HepG2 cells. Therefore, sorafenib resistance mediates the activation of several glycolytic genes, such as leads to an increase in glycolysis in HCC cells. The above GLUT3 (glucose transporter type 3), HK2 (hexokinase 2), observations of CLCF1 upregulation in HCC cells resis- and PDK1 (pyruvate dehydrogenase kinase 1). Accord- tant to sorafenib, along with the participation of PI3K/ ingly, upregulation of each of these genes was observed in AKT signaling in the regulation of aerobic glycolysis in HepG2-S-R cells (Fig. S3H), whereas a significant decline HCC cells, prompted us to speculate that CLCF1 con- in their levels was observed after treatment with MK-2206 tributes to the enhanced rate of glycolysis in HCC cells (Fig. S3H). We also found that the xenografted HepG2-S- resistant to sorafenib. To test this, we first treated HepG2- R tumor growth in nude mice was remarkably inhibited

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Fig. 2 HCC cells require CLCF1 to sustain sorafenib resistance. a HepG2 cells transfected with si CLCF1 were treated with increasing dose of sorafenib for 72 h. Relative cell viability was analyzed by MTT. The significance of the difference in IC50 values was analyzed by extra sum-of-squares F test. b Cell proliferation was analyzed in the HepG2 cells with si CLCF1 transfection. c HepG2 cells were transfected with si CLCF1. Indicated protein level was analyzed by Western blotting. d HepG2 cells were transfected with si CLCF1. Indicated protein level was analyzed by Western blotting. e HepG2 cells transfected with CLCF1 overexpression plasmid were treated with increasing dose of sorafenib for 72 hours. Relative cell viability was analyzed by MTT. The significance of the difference in IC50 values was analyzed by extra sum-of-squares F test. f CLCF1 transfection efficiency was analyzed by Western blotting. g HepG2 cells were transfected with CLCF1 overexpression plasmid. Indicated protein level was analyzed by Western blotting. All data are mean ± SD of three separate experiments. ** P < 0.01.

by systemic delivery of MK-2206 (Fig. S3I), thus sup- CLCF1 siRNA (Fig. 3d). In an alternate assay, our ﬁndings porting the important role of AKT/PI3K signaling in indicated that overexpression of ectopic Flag-CLCF1 glucose metabolism regulation in HCC cells resistant to enhanced the levels of HK2, GLUT3, and PDK1 (Fig. sorafenib. 3e), as well as glycolysis was remarkably promoted due to Next, the role of CLCF1 in the increased rate of gly- the expression of ectopic Flag-CLCF1 in HepG2 and colysis in HCC cells resistant to sorafenib was assessed. A Huh7 parental cells (Fig. 3f–h). Thus, aerobic glycolysis is signiﬁcant decrease in lactate production, glucose con- promoted in HCC cells resistant to sorafenib by CLCF1. sumption, and intracellular ATP levels was observed in As the functions of aerobic glycolysis in cancer cell pro- HepG2-S-R and Huh7 cells with CLCF1 knockdown (Fig. liferation and survival are well known, our above results 3a–c). In addition, HK2, GLUT3, and PDK1 protein levels indicate that CLCF1 may enhance glycolysis to confer were decreased in HepG2-S-R cells transfected with sorafenib resistance in HCC cells.

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Fig. 3 Enhanced glycolysis requires CLCF1 in HCC cells resistant to sorafenib. a Glucose consumption in HepG2-S-R and Huh7-S-R cells transfected with siRNA against CLCF1. b Lactate production in HepG2-S-R and Huh7-S-R cells transfected with siRNA against CLCF1. c Relative ATP luminescence in HepG2-S-R and Huh7-S-R cells transfected with siRNA against CLCF1. d Western blotting of indicated protein level in HepG2-S-R and Huh7-S-R cells transfected with siRNA against CLCF1. e Western blotting of indicated protein in HepG2 and Huh7 cells were transfected with CLCF1 overexpression plasmid. f Glucose consumption in HepG2 and Huh7 cells transfected with CLCF1 overexpression plasmid. g Lactate production in HepG2 and Huh7 cells transfected with CLCF1 overexpression plasmid. h Relative ATP luminescence in HepG2 and Huh7 cells transfected with CLCF1 overexpression plasmid. All data are mean ± SD of three separate experiments. ** P < 0.01.

miR-30a-5p targets CLCF1, and sorafenib upregulates decrease in miR-30a-5p was observed in cultured HepG2 CLCF1 through miR-30a-5p downregulation cells treated with sorafenib (Fig. S4C), along with It is well known that miRNAs play a role in the pro- increased CLCF1 levels (Fig. S4D). These data thus indi- gression of cancer and in the development of drug resis- cate that miR-30a-5p downregulation may play a part in tance37. Hence, we investigated if CLCF1 expression is the upregulation of CLCF1 induced by sorafenib in HCC controlled by any miRNA(s) in HepG2-S-R cells. We used cells. bioinformatic tools to search for miRNAs targeting the To experimentally conﬁrm the above observation, human CLCF1 3′-UTR and discovered potential miR-30a- luciferase reporters containing the wild-type CLCF1 3′- 5p binding sites (Fig. 4a). Curiously, a marked down- UTR or the mutant miR-30a-5p seed with site mutations regulation of miR-30a-5p was found by real-time PCR were constructed (Fig. 4a), and HepG2 cells were assay in HepG2-S-R cells in comparison to the parental cotransfected with reporters and miR-30a-5p mimics or HepG2 cells (Fig. S4A). We also found signiﬁcantly negative control RNA. We observed noticeable inhibition reduced miR-30a-5p expression in sorafenib-treated of the wild-type vector, but not the mutant construct, by HepG2 tumors (Fig. S4B). Likewise, a time-dependent miR-30a-5p (Fig. 4b). Furthermore, a decrease in CLCF1

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Fig. 4 CLCF1 is targeted directly by miR-30a-5p. a Prediction of CLCF1 as a target of miR-30a-5p. miR-30a-5p binding site, as predicted in CLCF1 3′-UTR. b Assay using dual-luciferase reporters. Normalization of activity of renilla luciferase was done with ﬁreﬂy luciferase activity. c, d CLCF1 expression in miR-30a-5p mimic-transfected HepG2-S-R cells was assessed through real-time PCR or Western blotting. e, f CLCF1 expression in miR- 30a-5p inhibitor-transfected HepG2 cells transfected with anti-miR-30a-5p was analyzed by real-time PCR and Western blotting. All data are mean ± SD of three separate experiments. ** P < 0.01.

expression in HepG2-S-R and Huh7-S-R cells due to and survival in HepG2-S-R cells overexpressing miR-30a- overexpression of miR-30a-5p was conﬁrmed by western 5p (Figs. 5a, S5A, and S5B). We also observed AKT/PI3K blotting and real-time PCR (Figs. 4c, d and S4E, S4F), signaling inhibition in sorafenib-resistant HCC cells by whereas anti-miR-30a-5p-mediated inhibition of miR- miR-30a-5p targeting CLCF1 (Fig. 5b). Thus, sorafenib 30a-5p led to enhanced expression of CLCF1 in HepG2 resistance and cell growth are suppressed by miR-30a-5p and Huh7 cells at the transcript and protein levels (Figs. by targeting CLCF1. 4e, f and S4G, S4H). Taken together, the results demon- Next, the activity of the miR-30a-5p/CLCF1 axis in strate that CLCF1 is a direct target of miR-30a-5p, and a sorafenib-resistant HCC cell tumorigenesis was tested. role of the miR-30a-5p/CLCF1 regulatory axis is indicated HepG2-S-R cells were infected with pseudoviruses in HCC cells resistant to sorafenib. expressing miR-30a-5p and CLCF1, and nude mice were implanted with these infected cells. To maintain sorafenib Sorafenib resistance and tumorigenesis is regulated by the resistance in HepG2-S-R tumors, the mice bearing tumors miR-30a-5p/CLCF1 axis were treated systematically with sorafenib. Over- Our next obvious step was to determine whether this expression of miR-30a-5p greatly inhibited the growth of miR-30a-5p/CLCF1 axis plays a role in regulating resis- HepG2-S-R tumors, which was effectively rescued by tance to sorafenib in HCC cells. Indeed, HepG2-S-R cells coexpression of the Flag-CLCF1 protein (Figs. 5c and were re-sensitized to sorafenib (Fig. 5a) upon increased S5C), supporting the functional importance of the miR- expression of miR-30a-5p, indicating the suppressive 30a-5p/CLCF1 axis in the regulation of tumorigenesis in action of miR-30a-5p on sorafenib resistance in HCC sorafenib-resistant HCC cells. In addition, the AKT/ cells. Furthermore, apoptosis was signiﬁcantly enhanced mTOR signaling pathway was inhibited in miR-30a-5p in HepG2-S-R cells overexpressing miR-30a-5p (Fig. transfected tumors, which was attenuated by CLCF1 S5A), in addition to a remarkable reduction in the pro- overexpression (Fig. S5D). Furthermore, miR-30a-5p liferation of HepG2-S-R cells (Fig. S5B). To investigate overexpression resulted in a decrease in the level of Ki- whether miR-30a-5p exerts the abovementioned effects 67 protein, but an increase in the cell apoptosis marker on sorafenib resistance by targeting CLCF1, ectopic cleaved caspase-3 protein was observed by IHC staining in expression of CLCF1 was achieved through p3xFlag- HepG2-S-R tumors and in comparison to the controls CLCF1, which did not encode the CLCF1 3′-UTR, making (Fig. 5d). Additionally, restoring CLCF1 expression sub- this construct resistant to miR-30a-5p. There was an stantially negated the action of miR-30a-5p on the effective recovery of sorafenib resistance, cell proliferation expression of both cleaved caspase-3 and Ki-67 (Fig. 5d).

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Fig. 5 The miR-30a-5p/CLCF1 axis regulates tumorigenesis and sorafenib resistance in HCC cells resistant to sorafenib. a HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection were treated with increasing dose sorafenib for 72 h. Relative cell viability was analyzed by MTT. The signiﬁcance of the difference in IC50 values was analyzed by extra sum-of-squares F test. b Western blotting of indicated proteins in HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection. c The growth of HepG2-S-R tumors in mice is controlled by the miR-30a-5p/CLCF1 axis. d Parafﬁn-embedded sections of HepG2-S-R tumor tissues from mice treated as in (a) were analyzed by IHC staining for Ki-67, or IF staining for cleaved caspase 3. Scale bars: 25 µm.

These results further support the role of the miR-30a-5p/ We next investigated whether miR-30a-5p targets CLCF1 axis in regulating tumorigenesis and sorafenib CLCF1 to inhibit aerobic glycolysis. We observed that resistance in sorafenib-resistant HCC cells. Flag-CLCF1 protein coexpression remarkably abrogated the inhibitory action of miR-30a-5p on glucose metabo- Aerobic glycolysis in HCC cells resistant to sorafenib is lism in HepG2-S-R cells (Fig. 6a–d), which followed the regulated by the miR-30a-5p/CLCF1 axis restoration of GLUT3, HK2 and PDK1 levels (Figs. 6e, 6f After observing the requirement of CLCF1 for aerobic and S6A, S6B). Therefore, the miR-30a-5p/CLCF1 axis glycolysis in HCC cells resistant to sorafenib, we next plays an important role in the regulation of aerobic gly- tested the role of the miR-30a-5p/CLCF1 axis in reg- colysis in HCC cells and tumors resistant to sorafenib. ulating glucose metabolism in these cells. As expected, We next investigated whether the restoration of miR- overexpression of miR-30a-5p led to a remarkable 30a-5p expression may exert a therapeutic effect on HCC reduction in lactate production, glucose consumption, cells resistant to sorafenib. To assess this, we performed a ECAR, and intracellular ATP levels in HepG2-S-R cells treatment study by injecting 2′-O-methyl-modiﬁed miR- (Fig. 6a–d), as well as suppressed the glycolytic genes 30a-5p (agomiR-30a-5p) conjugated with cholesterol into HK2, GLUT3, and PDK1 (Figs. 6e, 6f and S6A, S6B). In an the tail veins of nude mice and enveloped them in delivery alternate experiment, an increase in glucose utilization, vehicles made of natural lipids two weeks after HepG2-S- intracellular ATP levels and lactate production in HepG2 R cell implantation. The injections were given for ﬁve cells following anti-miR-30a-5p treatment mediated cycles (n = 5), one every week. Control groups received inhibition of miR-30a-5p (Fig. S6C–S6E). Thus, miR-30a- agomiR-NC treatments. We found that HepG2-S-R 5p functions as an aerobic glycolysis repressor in tumor growth was effectively suppressed compared with sorafenib-resistant HCC cells. agomiR-NC when agomiR-30a-5p was systemically

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Fig. 6 The miR-30a-5p/CLCF1 axis is important in regulating metabolism of glucose in HCC cells resistant to sorafenib. a Glucose consumption in HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection. b Lactate production in HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection. c Relative ATP luminescence in HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection. d Left, real-time measurement of extracellular acidiﬁcation rate (ECAR) in HepG2-S-R cells transfected with miR-30a- 5p with or without CLCF1 cotransfection. The value of glycolysis (Middle) and glycolytic capacity (Right) in the indicated cells. e Relative mRNA level of GLUT3, HK2, and PDK1 in HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection. f Relative protein level of GLUT3, HK2, and PDK1 in HepG2-S-R cells transfected with miR-30a-5p with or without CLCF1 cotransfection. All data are mean ± SD of three separate experiments. ** P < 0.01.

delivered (Fig. S6F). Hence, our findings demonstrated the CLCF1 transcript levels in 20 primary HCC and paired therapeutic efficacy of agomiR-30a-5p against sorafenib- adjacent normal tissue samples were evaluated by real- resistant HCC. time PCR (Table 2). A significant decrease in the expression of miR-30a-5p was observed in HCC com- Inverse relationship between levels of miR-30a-5p and pared to normal tissues (Fig. 7a), while there was a CLCF1 in human HCC specimens remarkable increase in CLCF1 levels (Fig. 7b). Impor- Next, the relevance of the above findings was clinically tantly, an inverse relationship was observed between assessed in HCC patients. Paired specimens from HCC CLCF1 mRNA levels and miR-30a-5p in tumors (Fig. 7c). patients are difficult to obtain before initiating sorafenib Additionally, IHC staining of tumor sections showed that treatment and post-development of sorafenib resistance HCC with a low miR-30a-5p level produced stronger due to ethical issues; alternatively, miR-30a-5p and CLCF1 staining (Fig. 7d), while tumors with a high miR-

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30a-5p level had weaker CLCF1 signal (Fig. 7d). In addi- Discussion tion, the data from the TCGA dataset indicated that HCC The first and the primary targeted therapy for advanced patients with high CLCF1 had poor survival (Fig. 7e). HCC is sorafenib38,39. However, patients that acquire sorafenib resistance are deprived of this drug’s bene- fits15,40. Sorafenib is the most approved and widely used Table 2 Patient characteristics and univariate P value. systemic drug and occupies a unique position in the international effort against HCC41. Nevertheless, there are P Characteristics N (%) frequent cases of drug resistance in HCC patients post Gender 0.41 sorafenib treatment and can be categorized into primary or intrinsic resistance and secondary or acquired resis- Male 12 (60) tance. However, the mechanisms of sorafenib resistance Female 8 (40) remain unclear, and it becomes imperative to assess the Age (years) 0.03 inherent mechanism of the acquired resistance to sor- <60 18 (90) afenib. Here, we found that sorafenib potentiates glucose metabolism in HCC cells because enhanced glycolysis was ≥ 60 2 (10) observed in HCC cells as a result of sorafenib resistance. Size of tumor (diameter/cm) 0.24 Furthermore, an important regulatory pathway of the <5 16 (80) miR-30a-5p/CLCF1 axis was found to modulate sorafenib ≥5 4 (20) resistance and aerobic glycolysis in HCC cells. A ubiquitous increase in CLCF1 has been observed in fi TNM classi cation 0.33 several cancers, indicating an oncogenic function of I–II 13 (65) CLCF142. However, the exact activity of CLCF1 in sor- III–IV 7 (35) afenib resistance in HCC remains unexplored. As per the Child–Pugh classification 0.63 data of this study, resistance to sorafenib increased CLCF1 expression, which is crucial for sustaining sorafenib A 11 (55) resistance in HCC cells. The development of resistance to B 9 (45) sorafenib in HCC may be facilitated by PI3K/AKT signaling, which is a yet unexplored mechanism. In this

Fig. 7 Clinical relevance of miR-30a-5p/CLCF1 expression in human HCC. a Comparison of mRNA level of miR-30a-5p in primary human HCC tumors and normal paired adjacent tissues. b Comparison of mRNA level of CLCF1 in primary human HCC tumors and normal paired adjacent tissues. c A negative correlation was seen by Pearson’s correlation analyses in above comparison in human HCC tumor samples. r = −0.78, p < 0.001. d Human HCC tumor sections were IHC stained and representative images of CLCF1 were acquired with low or high miR-30a-5p expression. Scale bars, 25 μm. e TCAG dataset shows that High CLCF1 expression predicate a poor survival in HCC. All data are mean ± SD of three separate experiments. ** P < 0.01.

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study, we also showed that upregulation of CLCF1 led to miR-30a-5p regulates both glycolysis and sorafenib resis- PI3K/AKT signaling activation in sorafenib-resistant tance in HCC cells and tumors by targeting CLCF1 HCC cells. Hence, the findings of this study combined directly. In particular, miR-30a-5p, the cognate miRNA with those of previous studies firmly support the reg- from the opposite arm of pre-miR-30a-5p, was down- ulatory cascade of CLCF1/PI3K/AKT underlying sor- regulated by several chemotherapy drugs and acts to afenib resistance development in HCC. improve chemosensitivity in several kinds of tumors. miR- A major reason for relapse and mortality in HCC is 30a-5p further attenuates metabolism in cancer cells by resistance to sorafenib43. The regulatory activity of miR- downregulating HIF-1α and GLUT1, in accordance with NAs in HCC growth and progression is well known44. our findings that miR-30a-5p is a key regulator of resis- miRNAs may be regulated during carcinogenesis and can tance to anticancer therapeutics and aerobic glycolysis in function as tumor suppressors or oncogenes depending HCC cells. on the characteristics of their target genes45. Aberrant expression of miRNAs is a universal feature of solid Conclusions tumors and HCC46. In this study, we observed a direct In summary, this is the first study to report that the binding of miR-30a-5p to the 3′-UTR of CLCF1 mRNA to miR-30a-5p/CLCF1/PI3K/AKT regulatory cascade is a repress its protein expression. pathway linking sorafenib resistance to metabolism in The cytokine CLCF1 has been suggested to be involved HCC cells. More importantly, agomiR-30a-5p has a in the pathogenesis of multiple myelomas, but its role in therapeutic effect on sorafenib-resistant HCC, further HCC has not yet been reported47. The signaling of CLCF1 corroborating the potential of miRNAs as targets for the and IL-6 occurs via multimeric complexes, including the treatment of sorafenib-resistant HCC. β-subunit/gp130 receptor, and consequently activates a signaling cascade moderated by the JAK-STAT, PI3K/ Acknowledgements 42 This work was supported by the National Natural Science Foundation of China AKT, and MAPK pathways . Specific cellular responses (81900371), Natural Science Foundation of Hunan Province (2019JJ50899), by these cytokines are managed by the different α-subunit China Scholarship Council (201506370112) to Z.Z. receptor expression patterns, IL-6R for IL-6, and CNTFR Author details for CLCF1, which can independently be switched on by 1Department of Liver Transplantation, The Second Xiangya Hospital of Central 16,42 each cytokine . In addition, a previous study showed South University, 410011 Changsha, Hunan Province, P.R. China. 2Department of Surgery, University of Pittsburgh School of Medicine, University of Pittsburgh that CLCF1-CNTFR axis inhibition may be a potential 3 19 Medical Center Presbyterian Hospital, Pittsburgh, PA 15213, USA. Department approach for the treatment of cancer . of Oncology, Xiangya Hospital, Central South University, 410008 Changsha, As an aberrant trait of cancer cell metabolism, aerobic Hunan Province, P.R. China. 4Department of General Surgery, The Second glycolysis has been associated with resistance to che- Xiangya Hospital of Central South University, 410011 Changsha, Hunan Province, P.R. China. 5Department of Pharmacology and Chemical Biology, motherapies in several human cancers, although the University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA mechanism of how resistance to sorafenib is attributed to cancer cell metabolism is still unclear48. Cancer cell sur- Author contributions Z.Z., Z.S., and J.S.T. developed the hypothesis and designed the experiments. Z. vival and proliferation requires aerobic glycolysis, which Z., X.T., J.L., and H.Y. performed the statistical analyses. Z.Z., X.T., Z.S., and J.S.T. also favors cancer cell metastasis and invasion. Interest- conducted experiments and wrote the main manuscript. ingly, significantly enhanced glycolysis was found in HCC cells and tumors resistant to sorafenib, indicating that Conflict of interest The authors declare that they have no conflict of interest. glucose metabolism is associated with sorafenib resistance in HCC cells. We further observed enhanced glycolysis by Publisher’s note CLCF1 via PI3K/AKT signaling activation and the glyco- Springer Nature remains neutral with regard to jurisdictional claims in fi lytic genes GLUT3, HK2,andPDK1 functioning down- published maps and institutional af liations. stream in sorafenib-resistant HCC cells. Concurrent with Supplementary Information accompanies this paper at (https://doi.org/ the observations of this study, previous reports also found 10.1038/s41419-020-03123-3). that HCC cells become sensitized to sorafenib after targeting PDK1, thus supporting the requirement of aerobic Received: 18 May 2020 Revised: 29 September 2020 Accepted: 30 September 2020 glycolysis for the maintenance of sorafenib resistance in HCC49. In an earlier study, sorafenib was shown to inhibit oxygen consumption through inhibition of complex I of mitochondria, ultimately affecting glycolysis in HCC References cells50. We also demonstrated that the CLCF1/PI3K/AKT 1. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2019. CA Cancer J. Clin. 69, regulatory cascade governs aerobic glycolysis and sorafenib 7–34 (2019). 2.Yang,J.D.etal.Aglobalviewofhepatocellular carcinoma: trends, risk, resistance in HCC cells. Additionally, in accordance with prevention and management. Nat. Rev. Gastroenterol. 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