Cholesterol biosynthesis supports the growth of ...

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Hepatology

Original article

Cholesterol biosynthesis supports the growth of hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans

Li Che,1,2 Wenna Chi,3,4 Yu Qiao,2,5 Jie Zhang,1,2 Xinhua Song,2 Ye Liu,3 Lei Li,6 Jiaoyuan Jia,2,7 Maria G Pilo,8 Jingxiao Wang,2,9 Antonio Cigliano,10 Zhilong Ma,3 Wenhua Kuang,3 Zefang Tang,11,12 Zemin Zhang,11,12 Guanghou Shui,13 Silvia Ribback,10 Frank Dombrowski,10 Matthias Evert,14 Rosa Maria Pascale,8 Carla Cossu,8 Giovanni Mario Pes,8 Timothy F Osborne,15 Diego F Calvisi,8 Xin Chen, 2 Ligong Chen3,4

Additional material is published online only. To view, please visit the journal online ( rg/10.1136/ gutjnl-2 018-317581). For numbered affiliations see end of article.

Correspondence to Dr Diego F Calvisi, Department of Medical, Surgical, and Experimental Sciences, University of Sassari, via Padre Manzella 4, 07100 Sassari, Italy; c alvisid@uniss.it, Dr Xin Chen, UCSF, 513 Parnassus Avenue, San Francisco, CA 94143, USA; x in.chen@ucsf.edu and Dr Ligong Chen, D410 Medical Science Building, School of Pharmaceutical Sciences, Tsinghua University, Beijing, China 100084; l igongchen@tsinghua.

Received 12 September 2018 Revised 14 March 2019 Accepted 15 March 2019

? Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Abstract ObjectiveIncreased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC). Design We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies. ResultsAblation of Fasn significantly delayed sgPten/cMet-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASNmediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture. Conclusion Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

To cite: Che L, Chi W, Qiao Y, et al. Gut Epub ahead of print: [please include Day Month Year]. doi:10.1136/ gutjnl-2018-317581

Introduction The global incidence of primary liver cancer has progressively increased over the last decades.

Significance of this study

What is already known on this subject? Aberrant activation of fatty acid synthase

(FASN) and related de novo lipogenesis is major metabolic event along hepatocellular carcinoma (HCC) development. Targeting FASN-mediated de novo lipogenesis is able to inhibit HCC growth in vitro and in vivo. Emerging evidence suggests the existence of additional mechanisms supporting HCC cell proliferation and survival in the absence of de novo lipogenesis.

What are the new findings? Loss of Fasn and its mediated de novo

lipogenesis delays but does not completely prevent oncogene-driven hepatocarcinogenesis in some mouse models. Tumour development eventually occurs and is orchestrated by the upregulation of Srebp2driven cholesterol biosynthesis in Fasn null mouse liver tumour samples. Similar phenotypes are found in human HCC cell lines when they are genetically depleted of FASN. These results unveil a novel biochemical crosstalk between de novo lipogenesis and cholesterol biosynthesis pathways along hepatocarcinogenesis Blocking cholesterol biosynthesis completely prevents tumour formation in liver-specific Fasn KO mice. Concomitant targeting de novo lipogenesis and cholesterol biosynthesis are highly detrimental for the growth of human HCC cells.

Among primary liver malignancies, hepatocellular carcinoma (HCC) is the predominant type.1 HCC has limited treatment options. The standard care of drugs for the treatment of advanced HCC are the multikinase inhibitors sorafenib and regorafenib, but they display limited efficacy.2 Thus, there is

Che L, et al. Gut 2019;0:1?10. doi:10.1136/gutjnl-2018-317581

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Hepatology

Significance of this study

How might it impact on clinical practice in the foreseeable future? Our findings strongly suggest that simultaneous targeting of

de novo lipogenesis and cholesterol biosynthesis may be a highly effective strategy for the treatment and prevention of HCC. Furthermore, the same molecular mechanisms might occur (and, thus, could be effectively targeted) in other tumour types as well.

an obvious unmet medical need for new drug targets for HCC treatment.

Metabolic reprogramming is now recognised as one of the cancer hallmarks, and a key event during tumour initiation and progression.3 4 Among these alterations, de novo lipogenesis consists of the synthesis of endogenous fatty acids (FAs) from acetyl-CoA. Increased de novo lipogenesis has been observed in multiple cancer types.5 In human HCC, data from our and other laboratories have documented the increased expression of major enzymes associated with de novo lipogenesis, including fatty acid synthase (FASN), in tumour lesions when compared with liver non-neoplastic counterparts.6 7 Ablation of Fasn completely inhibited hepatocarcinogenesis in mice overexpressing an activated form of AKT, either alone or in combination with c-Met.8 9 However, emerging evidence suggests the existence of additional mechanisms supporting HCC cell proliferation and survival in the absence of de novo lipogenesis. Indeed, silencing of FASN delays, but does not completely inhibit, in vitro growth of HCC cell lines.7 10 Importantly, in a liver-specific Pten and Tsc1 double KO mouse HCC model, silencing of Fasn via AAV-shFasn significantly decreased, but did not completely prevent, HCC formation.11

Dysregulated cholesterol biosynthesis is another metabolic event frequently observed in HCCs. HMG-CoA reductase (HMGCR) is the rate limiting enzyme in cholesterol biosynthesis and the target of cholesterol-lowering drugs statins. Previous findings indicate the upregulation of HMGCR expression in human HCC samples.7 12 Noticeably, the use of statins has been associated with reduced risk of HCC development in large epidemiological studies, suggesting a potential tumour supporting role of the cholesterol biosynthesis pathway in this disease.13 14 Statins have also been shown to inhibit human HCC cell proliferation in culture as well as c-Myc-driven HCC development in mice.15?17

In this manuscript, we investigated the requirement of Fasn in sgPten/c-Met mouse HCC.18 Our results unveil novel biochemical crosstalk between de novo lipogenesis and cholesterol biosynthesis pathways along hepatocarcinogenesis.

Materials and methods Hydrodynamic injection and mouse monitoring Fasnfl/fl mice in C57BL/6 background were described previously.19 AlbCre mice in C57BL/6 background20 were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). Fasnfl/fl mice were crossed with AlbCre mice to eventually generate liver-specific Fasn knockout mice (FasnLKO mice). The physiological phenotype of FasnLKO mice has been extensively characterised.21 Both male and female mice were used in the study, and no sex-dependent differences were detected. Hydrodynamic transfection was performed as described.22 Mice were monitored weekly,

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euthanised and liver tissues collected when they developed large abdominal masses or by 40?50 weeks postinjection. The detailed mouse experiment data are available in online supplementary table S1. All mice were housed, fed and monitored in accordance with protocols approved by the Committee for Animal Research at the University of California, San Francisco.

Human tissue samples Sixty-five frozen HCC and corresponding non-tumourous surrounding livers (SL) were used. Clinicopathological features of patients with liver cancer are summarised in online supplementary table S2. HCC specimens were collected at the Medical University of Greifswald (Greifswald, Germany). Informed consent was obtained from all individuals.

Statistical analysis Data were analysed using the Prism 6 software (GraphPad, San Diego, California, USA). Comparisons between two groups were performed with two-tailed unpaired t-test. Statistical differences among the various groups were assessed with the Tukey-Kramer's test. Kaplan-Meier method and log-rank test were used for survival analysis. All graphs are the mean?SEM P values 1or log2FoldChange ................
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