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LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer

Nature Cancer volume 2, pages 201–217 (2021)Cite this article

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Abstract

The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer.

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Fig. 1: Sorafenib activates LXR signaling in HCC.
Fig. 2: Sorafenib-mediated induction of LXRα signaling contributes to its therapeutic efficacy.
Fig. 3: Raf counteracts the therapeutic efficacy of LXR activation in HCC.
Fig. 4: Therapy comprising LXRα activation and Raf inhibition is effective in therapy-resistant mouse HCCs in vivo.
Fig. 5: LXRα activation and Raf inhibition result in a toxic accumulation of saturated fatty acids in liver cancer cells.
Fig. 6: Raf-1 influences intracellular lipid composition in HCC cells by stabilizing SCD1 protein levels.
Fig. 7: DFG-out Raf inhibitors disrupt the Raf-1–SCD1 protein complex in HCC.
Fig. 8: Pharmacologically induced lipotoxicity is effective in human HCC.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. For the e:Med-HCC-1 clinical study (NCT02372162), all data can be requested for research purposes from Michael Bitzer (michael.bitzer@uni-tuebingen.de). Deep sequencing (RNA-seq) data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE162322 and GSE162324. For the molecular modeling studies, all data are deposited in the Zenodo repository (https://doi.org/10.5281/zenodo.3253878, under accession code 3253878) and can also be requested for research purposes from Antti Poso (antti.poso@uef.fi). Associated raw data for Figs. 1b,c and 2b and Extended Data Fig. 3d are provided as supplementary tables. The gating strategy for flow cytometry is depicted in Extended Data Figs. 2a,b and 6f–h. Source data are provided with this paper.

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Acknowledgements

We thank E. Rist, P. Schiemann, A. Heinrich, E. Enzinger, O. Hihn, W. Albrecht, L. Herrmann, P. Wenk, F. Iglauer, S. Gerold, M. A. Jarboui, U. Koppenhoefer, S. Poths, F. J. Hilke, D. Quetting, M. Riebold and all members of the Zender department for fruitful discussions and/or technical assistance. Furthermore, we thank M. Eilers and J. Zuber for providing vectors, the c.ATG facility of Tuebingen University for sequencing analyses and the CSC-IT Center for Science, Finland, for computational resources. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (FOR2314 (project number 267467939; D.D., L.Z., A.S., N.P., B.P., S.L., M.T.R., A.P., N.P.M.), SFB-TR209-314905040 (D.D., L.Z., N.P.M., M.B.), SFB-TR240 (L.Z., B.P.), Gottfried Wilhelm Leibniz Program (L.Z.)), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s excellence strategy EXC 2180-390900677 (Image Guided and Functionally Instructed Tumor Therapies (iFIT) (L.Z., B.P., D.D., S.L., N.P., M.B., A.P., N.P.M.)) and the German Ministry for Education and Research (BMBF) (eMed/Multiscale HCC (L.Z., B.P., N.P.M., M.B.), Neuroallianz (S.L.)). Further funding was provided by the Landesstiftung Baden-Wuerttemberg (Improve CRC (D.D., S.L., L.Z.)), the European Research Council (CholangioConcept (L.Z.)), the German Center for Translational Cancer Research (DKTK (S.L., L.Z.)) and the Ministry of Science, Research and Arts of Baden-Wuerttemberg (MWK-BW (S.L., M.B.)). Furthermore, S.L. is supported by the Robert Bosch Foundation and the Interfaculty Centre for Pharmacogenomics and Pharma Research (ICEPHA), and B.P. is supported by the Werner Siemens Foundation and the DFG (project number 403462768).

Author information

Author notes

  1. Marteinn T. Snaebjornsson & Almut Schulze

    Present address: German Cancer Research Center (DKFZ), Heidelberg, Germany

Authors and Affiliations

  1. Department of Medical Oncology and Pneumology, University Hospital Tuebingen, Tuebingen, Germany

    Ramona Rudalska, Jule Harbig, Sabrina Klotz, Stefan Zwirner, Liudmyla Taranets, Florian Heinzmann, Thales Kronenberger, Wei Cui, Luana D’Artista, Elias Einig, Martina Hinterleitner, Agata Dylawerska, Tae-Won Kang, Antti Poso, Nikita Popov, Lars Zender & Daniel Dauch

  2. Theodor Boveri Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany

    Marteinn T. Snaebjornsson, Werner Schmitz & Almut Schulze

  3. Department of Pharmaceutical Chemistry, University of Tuebingen, Tuebingen, Germany

    Michael Forster & Stefan Laufer

  4. German Cancer Research Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany

    Michael Forster, Tae-Won Kang, Lars Zender & Daniel Dauch

  5. School of Pharmacy, University of Eastern Finland, Kuopio, Finland

    Antti Poso

  6. iFIT Cluster of Excellence EXC 2180 ‘Image-Guided and Functionally Instructed Tumor Therapies’, University of Tuebingen, Tuebingen, Germany

    Antti Poso, Nisar P. Malek, Michael Bitzer, Stefan Laufer, Bernd J. Pichler, Nikita Popov, Lars Zender & Daniel Dauch

  7. Institute of Pathology, University Hospital Wuerzburg, Wuerzburg, Germany

    Mathias T. Rosenfeldt

  8. Department of Gastroenterology, Hepatology and Infectiology, University Hospital Tuebingen, Tuebingen, Germany

    Nisar P. Malek & Michael Bitzer

  9. Tuebingen Center of Academic Drug Discovery (TüCAD2), Tuebingen, Germany

    Stefan Laufer

  10. Werner Siemens Imaging Center, University of Tuebingen, Tuebingen, Germany

    Bernd J. Pichler

  11. Comprehensive Cancer Center (CCCM), University Hospital Wuerzburg, Wuerzburg, Germany

    Almut Schulze

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  1. Ramona Rudalska
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Contributions

The study was designed by D.D. and L.Z. The research was conducted by R.R., J.H., M.T.S., S.K., S.Z., L.T., F.H., M.F., W.C., L.d’A., E.E., M.H., W.S., A.D., T.-W.K. and D.D. Histopathological analyses were performed by M.T.R. Human HCC samples were collected by M.B. Molecular modeling was conducted by T.K. and A.P. N.P.M., S.L., B.P., N.P. and A.S. contributed to research design and supervised experiments. D.D. and L.Z. supervised all research. The manuscript was written by D.D. and L.Z.

Corresponding authors

Correspondence to Lars Zender or Daniel Dauch.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The treatment response of sorafenib in MycOE;NrasG12V-driven HCCs is dependent on LXRα activation.

a,b, Treatment of murine, MycOE;NrasG12V-driven HCCs with sorafenib. Representative pictures (a) and H+E staining (b) of MycOE;NrasG12V HCCs in wildtype mice upon a 5-week therapy with sorafenib or the corresponding carrier (n = 3 mice per group). Scale bars, 1 cm, 100 µm. c, Survival of mice with MycOE;NrasG12V HCCs under sorafenib or carrier treatment (Kaplan-Meier curve, statistical significance was calculated using a log-rank test, n = 6 mice per group). d, Overview of LXRß structure with the coactivator peptide from NCOA1 (dark blue) in the AF2 region and sorafenib structure, docked in the ligand-binding pocket (orange). e, Suggested binding mode of sorafenib proposed by molecular modelling into the ligand-binding pocket of LXRß (PDB ID: 1PQ6). Residues contributing towards the interaction are colored by property, hydrophobic side chains are purple and hydrogen bonds are cyan. f, Schematic outline of an LXRα-binding saturation assay. Pulldown of LXRα was performed upon incubation with different concentrations of 3H-sorafenib. This experiment was side-by-side conducted in the presence of 320 µM unlabeled sorafenib to determine unspecific binding. g, Representative western blot analysis of LXRα in individual MycOE;NrasG12V;gN1h3 tumors (cropped blot images, MycOE;NrasG12V;gNC control: n = 3 mice, the western blot was repeated twice with similar results). α-tubulin was used as a loading control. Unprocessed images of the blots and numerical source data are provided as source data files.

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Extended Data Fig. 2 Inhibition of Raf improves the therapeutic effect of LXRα activation in liver cancer.

a,b, Gating strategy to quantify LXR activity in HCC cells. FACS analysis of 10 µM sorafenib-treated MycOE;NrasG12V;Cdkn2aARF–/–;p-LXRE-GFP cells. c, LXRE reporter assay in MycOE;NrasG12V;Cdkn2aARF–/–;p-LXRE-GFP cells that were treated for 3 days with different concentrations of sorafenib or T0901317 (GFP measurements in individual cells, values represent mean ± SD, n = 3 independent experiments, nonlinear regression curves, variable slope). d, Reporter assay to compare LXR activation by sorafenib or T0901317 in vivo. Representative photographs of tumor sections stained for native GFP and native RFP (+DAPI) after intrahepatic delivery of pT-CaM-LXRE-G, pT-CaRIN and pSB13 and treatment with carrier, sorafenib or T0901317 (3 d, n = 4 mice per group). Scale bar, 100 µm. e, Establishing Nr1h3 or Nr1h2 gene knockout in MycOE;NrasG12V;Cdkn2aARF–/– HCC cells by transient transfection of cells with pX-458-GFP expressing the corresponding gRNAs and generation of single cell clones. f,g, Representative western blot analysis of LXRα (f) and LXRβ (g) in MycOE;NrasG12V;Cdkn2aARF–/– single cell clones upon transient transfection with pX-458-GFP expressing the corresponding gRNAs (single cell clones, cropped blot images, n = 2 independent experiments). Vinculin and Histone H3 were used as loading controls. h, Treatment of MycOE;NrasG12V;Cdkn2aARF–/–gNr1h3, MycOE;NrasG12V;Cdkn2aARF–/–gNr1h2 or MycOE;NrasG12V;Cdkn2aARF–/–gNC cells for 4 days with T0901317/sorafenib or carrier (quantification of viable cells by crystal violet staining, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). i, Treatment of MycOE;NrasG12V;Cdkn2aARF–/– cells for 4 days with sorafenib and/or 24-S hydroxycholesterol, GW3965 or LXR-623 (quantification of viable cells by crystal violet staining, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). Unprocessed images of the blots and numerical source data are provided as source data files.

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Extended Data Fig. 3 Sorafenib resistant, MycOE;Akt1Myr-driven murine HCCs exert reduced LXRα activity.

a, Co-delivery of a transposon construct encoding for oncogenic Myc and myristoylated Akt1 (Akt1Myr) and pSB13 into C57BL/6 wildtype mice via hydrodynamic tail vein injection. b, Representative western blot analysis of P-Mek1/2 (S217/221) and Mek1/2 in MycOE;Akt1Myr tumors 6 h after treatment with sorafenib or BI-882370 (cropped blot images, n = 2 mice per condition). α-tubulin was used as a loading control. c, Survival of mice with MycOE;Akt1Myr HCCs under sorafenib or carrier treatment (Kaplan-Meier curve, statistical significance was calculated using a log-rank test, n = 6 mice per group). d, mRNA levels of Nr1h3, Abcg1, Abcg5, Abcg8 and Apoe in MycOE;Akt1Myr tumors (compared to MycOE;NrasG12V HCCs, mRNAseq data, n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test). e, Representative western blot analysis of Akt-1 and LXRα in MycOE;Akt1Myr;Cdkn2aARF–/– and MycOE;NrasG12V;Cdkn2aARF–/– cells (cropped blot images, n = 3 independent experiments). β-actin was used as a loading control. f, LXRE reporter assay in MycOE;Akt1Myr;Cdkn2aARF–/–;p-LXRE-GFP and MycOE;NrasG12V;Cdkn2aARF–/–;p-LXRE-GFP cells (GFP measurements in individual cells, values represent mean ± SD, n = 5 independent experiments, statistical significance was calculated using two-tailed Student´s t test). g, mRNA levels of Nfe2l2 in MycOE;Akt1Myr tumors (compared to MycOE;NrasG12V HCCs, mRNAseq data, n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test). h, Representative western blot analysis of Nrf2 in MycOE;Akt1Myr;Cdkn2aARF–/– and MycOE;NrasG12V;Cdkn2aARF–/– cells (cropped blot images, n = 3 independent experiments). α-tubulin was used as a loading control. i, Representative western blot analysis of Nrf2 in MycOE;Akt1Myr;Cdkn2aARF–/–shNfe2l2 or MycOE;Akt1Myr;Cdkn2aARF–/–shNC cells upon 4 days of treatment with doxycycline (cropped blot images, n = 3 independent experiments). α-tubulin was used as a loading control. j, LXRE reporter assay in MycOE;Akt1Myr;Cdkn2aARF–/–;p-LXRE-GFP cells 4 days after transient transfection with pT-CaR-shNfe2l2.2406, pT-CaR-shNfe2l2.2272, or pT-CaR-shNC (GFP measurements in RFP positive, individual cells, values represent mean ± SD, n = 5 independent experiments, statistical significance was calculated using two-tailed Student´s t test). k, LXRE reporter assay in MycOE;Akt1Myr;Cdkn2aARF–/–;p-LXRE-GFP cells upon 3 days of treatment with sorafenib or T0901317 (GFP measurements in individual cells, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). Unprocessed images of the blots and numerical source data are provided as source data files.

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Extended Data Fig. 4 A combination of LXR activation and Raf inhibition blocks MycOE;Akt-1Myr-driven HCC development.

a, Quantification of MycOE;Akt1Myr tumor nodules from mice that were treated for 7 weeks with sorafenib, BI-882370 or carrier +/− T0901317 (values represent mean ± SD, n = 4 mice per group, statistical significance was calculated using two-tailed Student´s t test). b-d, Treatment of MycOE;Akt1Myr;gNr1h3 or MycOE;Akt1Myr;gNC HCCs with T0901317/sorafenib or carrier. Representative pictures of livers upon a 6-week therapy (c, n = 3 mice per group) and survival of mice under treatment (d, Kaplan-Meier curve, statistical significance was calculated using a log-rank test, n = 6 mice per group). Scale bars, 1 cm. Numerical source data are provided as source data files.

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Extended Data Fig. 5 A T0901317/sorafenib combinatorial therapy influences lipid metabolism in liver cancer.

a, Representative pictures of murine Yap1;NrasG12V;Trp53-/- tumors that were treated for 2 weeks with T0901317/sorafenib, carrier, T0901317 or sorafenib and stained with H+E (n = 3 mice per group). Scale bars, 100 µm. b, Representative pictures of MycOE;NrasG12V tumors that were treated for 3 weeks with T0901317/sorafenib, carrier, T0901317 or sorafenib and stained with Oil red O (n = 3 mice per group). Scale bars, 50 µm. c, Representative western blot analysis of Srebp1c and Acetyl-CoA Carboxylase1 (ACACA) in MycOE;NrasG12V;Cdkn2aARF–/– cells upon 3 days of treatment with sorafenib, T0901317 or a combination thereof (cropped blot images, n = 2 independent experiments). α-tubulin was used as a loading control. d, Treatment of MycOE;NrasG12V;Cdkn2aARF–/– cells with the ACACA inhibitor ND-630 (quantification of viable cells by crystal violet staining, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). e, Treatment of MycOE;NrasG12V;Cdkn2aARF–/– cells with T0901317/sorafenib +/− cholesterol/methyl-β-cyclodextrin (cholesterol/mbc) (quantification of viable cells by crystal violet staining, values represent mean, n = 6 cell independent experiments, statistical significance was calculated using two-tailed Student´s t test). f, Schematic overview describing the role of Raf in fatty acid metabolism in liver cancer. Unprocessed images of the blots and numerical source data are provided as source data files.

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Extended Data Fig. 6 A T0901317/sorafenib therapy results in lipid peroxidation, an ER stress response and macrophage activation in HCCs.

a, Quantification of lipid peroxidation in MycOE;NrasG12V;Cdkn2aARF–/– cells, upon 3 days of treatment with sorafenib, T0901317 or a combination thereof (by analysing the ratio of green and red fluorescence, values represent mean, ± SD, n = 3 cell cultures per condition, statistical significance was calculated using two-tailed Student´s t test, the analysis was repeated twice with similar results). b, Representative western blot analysis of uncleaved or cleaved Atf6, BiP/HSPA5, P-IRE1α (S724) and Xbp-1s in MycOE;NrasG12V;Cdkn2aARF–/– cells after 3 days of treatment with T0901317, sorafenib or a combination thereof (cropped blot images, n = 3 independent experiments). α-tubulin was used as a loading control. c, Representative western blot analysis of eIF2α, P-eIF2α (S51), GADD34, BiP/HSPA5 and P-IRE1α (S724) in MycOE;NrasG12V;Cdkn2aARF–/– cells after 3 days of treatment with T0901317, BI-882370 or a combination thereof (cropped blot images, n = 3 independent experiments). α-tubulin and Vinculin were used as loading controls. d, Representative western blot analysis of MLKL, P-MLKL (S345), RIPK3, P-RIPK3 (S232) and AIF (nuclear and cytoplasmic fraction) in MycOE;NrasG12V;Cdkn2aARF–/– cells after 3 days of treatment with T0901317, sorafenib or a combination thereof (cropped blot images, n = 3 independent experiments). Vinculin and Histone H3 were used as loading controls. e, FACS based analysis of myeloid cells in MycOE;NrasG12V HCCs upon treatment with T0901317/sorafenib or the corresponding monotherapies (3 d). f-h, Flow cytometry gating strategy. The following populations are identified: immature myeloid cells (iMC: CD11b+,Ly6G+/High,F4/80−, fraction 1), monocyte-macrophages (Mac-mono: CD11b+,Ly6G+,F4/80High, fraction 2) and Kupffer cells (Ku: CD11b+,Ly6G+,F4/80High, fraction 3). Monocyte and macrophages are further distinguished/labeled as Ly6C+ (fraction 2a) or Ly6C− (fraction 2b). Samples were gated on viable leukocytes by DAPI exclusion and doublets were excluded using height versus area dot plots. (shown are representative pseudocolor plots, n = 3 mice per group). i,j, Results of FACS analysis in MycOE;NrasG12V HCCs upon 3 days of treatment with T0901317/sorafenib, T0901317, sorafenib or carrier. Charts show the percentage mean ± SD of monocyte-macrophages (Mac−mono: CD11b+,Ly6G+,F/480+, i), Kupffer cells (Ku: CD11b+,Ly6G+,F4/80High, i) and immature myeloid cells (iMC: CD11b+, j) (n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test, the experiment was repeated with similar results). k, Treatment of MycOE;NrasG12V;Cdkn2aARF–/– cells for 4 days with T0901317 and/or the Bcl-2 inhibitor ABT-199 (quantification of viable cells by crystal violet staining, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). Unprocessed images of the blots and numerical source data are provided as source data files.

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Extended Data Fig. 7 Raf-1 stabilizes SCD1 protein in liver cancer.

a, Quantification of western blot analysis (shown in Fig. 6b) to quantify relative SCD1 expression in MycOE;NrasG12V;Cdkn2aARF–/– cells upon 3 days of treatment with sorafenib, T0901317 or a combination thereof (quantified with ImageJ, only samples from the same blot were compared, values represent mean ± SD, n = 6 independent experiments, statistical significance was calculated using two-tailed Student´s t test, α-tubulin was used as a loading control). b, Quantification of western blot analysis (shown in Fig. 6c) to quantify relative SCD1 expression in MycOE;NrasG12V;Cdkn2aARF–/– cells upon treatment with T0901317 (2 d) and sorafenib (1-3 h) (quantified with ImageJ, only samples from the same blot were compared, values represent mean ± SD, n = 6 independent experiments, statistical significance was calculated using two-tailed Student´s t test, α-tubulin was used as a loading control). c, Representative western blot analysis of SCD1 in HLE cells upon treatment with T0901317 (2 d) and sorafenib (1-3 h) (cropped blot images, n = 3 independent experiments). α-tubulin was used as a loading control. d-f, Representative western blot analysis of SCD1 in MycOE;NrasG12V;Cdkn2aARF–/– cells upon treatment with T0901317 (2 d) and the DFG-out RAF inhibitors RAF-265 (d) and RAF-709 (e) or the DFG-in DFG-in RAF inhibitor SB590885 (f) for 1 to 3 h (cropped blot images, n = 2 independent experiments). α-tubulin was used as a loading control. g, Representative western blot analysis of MEK and p-MEK1/2 (Ser217/221) in MycOE;NrasG12V;Cdkn2aARF–/– cells upon 6 h of treatment with sorafenib, BI-882370, dabrafenib or SB590885 (cropped blot images, n = 2 independent experiments). α-tubulin was used as a loading control. h, Representative western blot analysis of SCD1 in MycOE;NrasG12V;Cdkn2aARF–/–;Raf1WT cells upon treatment with T0901317 (2 d) and sorafenib for 1 to 3 h (cropped blot images, n = 2 independent experiments). α-tubulin was used as a loading control. i, Representative western blot analysis of Raf-1 in MycOE;NrasG12V;Cdkn2aARF–/–;shRaf1 or MycOE;NrasG12V;Cdkn2aARF–/–;shNC cells, 4 days upon treatment with doxycycline (cropped blot images, n = 2 independent experiments). j, Treatment of MycOE;NrasG12V;Cdkn2aARF–/– cells with carrier, T0901317, the MEK1/2 inhibitor PD0325901 or a combination thereof (quantification of viable cells by crystal violet staining, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). Unprocessed images of the blots and numerical source data are provided as source data files.

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Extended Data Fig. 8 A lipotoxic therapy did not induce adverse metabolic effects in murine livers.

a, Analysing potential liver damage in C57BL/6 wildtype mice upon a 3-week T0901317/sorafenib treatment. b, Liver-to-body ratio of mice upon a 3-week treatment with T0901317/sorafenib or carrier (values represent mean ± SD, n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test). c, Representative pictures of murine livers upon a 3-week treatment with T0901317/sorafenib or carrier that were stained with H+E, Oil red O, Ki67, cleaved caspase 3, TUNEL and Sirius red (n = 3 mice per group). TUNEL and cleaved caspase 3 positive control = Intraperitoneal injection of Jo2 antibody. Sirius red positive control = Bi-weekly intraperitoneal injections of CCl4 for 6 weeks. Ki67 positive control = MycOE;NrasG12V HCC. Scale bars, 100 µm. d, Analysing potential liver damage in C57BL/6 wildtype mice upon 6 months of T0901317/sorafenib treatment. e, Liver-to-body ratio of mice upon a 6-month treatment with T0901317/sorafenib or carrier (values represent mean ± SD, n = 6 mice per group, statistical significance was calculated using two-tailed Student´s t test). f, Representative pictures of murine livers upon a 6-month treatment with T0901317/sorafenib or carrier that were stained with H + E, Oil red O, Ki67, cleaved caspase 3, TUNEL and Sirius red (n = 6 mice per group). Scale bars, 100 µm. g,h, Weight development of mice during 2 weeks (g) or 6 months (h) of treatment with T0901317/sorafenib or carrier (values represent mean ± SD, n = 3 (g) or 6 (h) mice per group, statistical significance was calculated using two-tailed Student´s t test). Numerical source data are provided as source data files.

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Extended Data Fig. 9 A lipotoxic therapy reduced NASH triggered HCC development and did not induce adverse metabolic effects in NASH livers.

a, Weight development of wildtype mice that were fed with choline deficient high fat diet (CD-HFD) or normal diet (ND) (values represent mean ± SD, n = 8 mice per group, statistical significance was calculated using two-tailed Student´s t test). b, Analysing tumor development and potential liver damage in NASH mice upon a 3-week T0901317/sorafenib treatment. c, Quantification of tumor nodules in mice that were fed for 12 months with CD-HFD and treated for 3 weeks with T0901317/sorafenib, sorafenib or carrier (values represent mean ± SD, n = 7 mice per group, statistical significance was calculated using two-tailed Student´s t test). d, Liver-to-body ratio of NASH mice upon a 3-week treatment with T0901317/sorafenib or carrier (values represent mean ± SD, n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test). e, Representative pictures of murine NASH livers upon a 3-week treatment with T0901317/sorafenib or carrier that were stained with H+E, Oil red O, Ki67, cleaved caspase 3, TUNEL and Sirius red (n = 3 mice per group). Scale bars, 100 µm. f, Weight development of NASH mice during treatment with T0901317/sorafenib or carrier (values represent mean ± SD, n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test). Numerical source data are provided as source data files.

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Extended Data Fig. 10 Comparison of human HCC cells and weight development of tumor bearing mice.

a, Representative western blot analysis of Nrf2, Akt-1 and LXRα in different human HCC cells (cropped blot images, n = 3 independent experiments). β-actin was used as a loading control. b, LXRE reporter assay in different human HCC cells upon lentiviral infection of pL-LXRE-GFP (GFP measurements in individual cells; values represent mean ± SD, n = 5 independent experiments, statistical significance was calculated using two-tailed Student´s t test). c, Weight development of CB17.Cg-PrkdcscidLystbg/Crl mice upon subcapsular injection of HuH7 cells into the liver and treatment with T0901317/sorafenib or carrier (values represent mean ± SD, n = 6 mice per group statistical significance was calculated using two-tailed Student´s t test). Unprocessed images of the blots and numerical source data are provided as source data files.

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Supplementary information

Reporting Summary

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Rudalska, R., Harbig, J., Snaebjornsson, M.T. et al. LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer. Nat Cancer 2, 201–217 (2021). https://doi.org/10.1038/s43018-020-00168-3

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