Liang et al. Cell Death and Disease (2019) 10:830 https://doi.org/10.1038/s41419-019-2054-7 Cell Death & Disease ARTICLE Open Access ASPP2 inhibits tumor growth by repressing the mevalonate pathway in hepatocellular carcinoma Beibei Liang1,RuiChen2, Shaohua Song3,HaoWang4,GuoweiSun5, Hao Yang1,WeiJing6, Xuyu Zhou6,ZhirenFu3, Gang Huang1 and Jian Zhao1 Abstract Cancer is, fundamentally, a disorder of cell growth and proliferation, which requires adequate supplies of energy and nutrients. In this study, we report that the haplo-insufficient tumor suppressor ASPP2, a p53 activator, negatively regulates the mevalonate pathway to mediate its inhibitory effect on tumor growth in hepatocellular carcinoma (HCC). Gene expression profile analysis revealed that the expression of key enzymes in the mevalonate pathway were increased when ASPP2 was downregulated. HCC cells gained higher cholesterol levels and enhanced tumor-initiating capability in response to the depletion of ASPP2. Simvastatin, a mevalonate pathway inhibitor, efficiently abrogated ASPP2 depletion-induced anchorage-independent cell proliferation, resistance to chemotherapy drugs in vitro, and tumor growth in xenografted nude mice. Mechanistically, ASPP2 interacts with SREBP-2 in the nucleus and restricts the transcriptional activity of SREBP-2 on its target genes, which include key enzymes involved in the mevalonate pathway. Moreover, clinical data revealed better prognosis in patients with high levels of ASPP2 and low levels of the mevalonate pathway enzyme HMGCR. Our findings provide functional and mechanistic insights into the critical role of ASPP2 in the regulation of the mevalonate pathway and the importance of this pathway in tumor initiation and tumor growth, which may provide a new therapeutic opportunity for HCC. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction development4. The mevalonate pathway (MVA) is the Dysregulation of a wide range of metabolic pathways essential metabolic pathway that converts acetyl-CoA to can be involved in carcinogenesis1. For example, choles- sterols such as cholesterol and non-sterol isoprenoids. terol has a critical role in maintaining the stability and Clinical and experimental findings suggest that malignant architecture of the plasma membrane and is needed by cells and tissues need higher amounts of cholesterol and highly proliferative cancer cells2. Tumor cell membranes intermediates of the mevalonate pathway than their nor- have been found to be enriched in cholesterol, suggesting mal counterparts5,6. Some types of cancers, such as that enhanced cholesterol utilization is an important hepatocellular carcinoma (HCC), depend on mevalonate feature of malignant and perhaps metastatic tumors3. pathway for growth. For example, inhibition of mevalo- Mutational data from The Cancer Genome Atlas (TCGA) nate metabolism suppresses tumor initiation and growth project also implicates cholesterol metabolism in cancer in a myc-transgenic murine HCC model7, activation of the mevalonate pathway also plays an essential role in liver tumorigenesis caused by p53 loss in mice8. Correspondence: Gang Huang ([email protected])or The mevalonate pathway is tightly regulated by a series Jian Zhao ([email protected]) of enzymes. 3-hydroxy-3-methylglutaryl-CoA reductase 1Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine and Health Sciences, 201318 Shanghai, China (HMGCR) is the rate-limiting enzyme, and can be blocked 2Angecon Biotechnology Limited, 201318 Shanghai, China by statins9; indeed, such inhibition has antitumor effects Full list of author information is available at the end of the article. in multiple tumor types9. Clinical observations have These authors contributed equally: Beibei Liang, Rui Chen, Shaohua Song, Hao Wang Edited by: A. Finazzi-Agrò © The Author(s) 2019 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/. Official journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2019) 10:830 Page 2 of 11 shown a negative association between the use of statins However, it remains unknown whether ASPP2 is involved and the risk of developing liver cancer10. in the regulation of mevalonate metabolism, which may Transcriptional activation of key rate limiting enzymes, contribute to its inhibitory effect on tumor progression. In like HMGCR, in mevalonate pathway is controlled by this study, we provide evidence that downregulation of sterol-regulated cleavage of the membrane-bound tran- ASPP2 enhances tumor-initiating capabilities and tumor scription factor SREBP-2 (sterol regulated element bind- growth by promoting mevalonate metabolism in HCC. – ing protein-2)11 13. Depletion of intracellular sterols enhances SREBP-2 translocation from the endoplasmic Results reticulum (ER) to the Golgi apparatus, leading to sub- Downregulation of ASPP2 enhances mevalonate pathway sequent cleavage to its active mature form and nuclear gene expression and cholesterol biosynthesis in HCC cells translocation14. SREBP-2 binds to sterol regulatory ele- To explore the mechanisms driving the inhibitory ments (SREs) in its target gene promoters to activate their effects of ASPP2 on tumor growth and progression, we transcription. performed a microarray analysis to identify the potential ASPP2 is a member of the ankyrin-repeat, SH3-domain, down-stream targets of ASPP2 by comparing gene and proline-rich region-containing protein (ASPP) family, expression in ASPP2-depleted HCC-LM3 cells and parent and is a haplo-insufficient tumor suppressor15,16. Aber- cells. Interestingly, GO analysis revealed a significant rant expression of ASPP2 has been found in various negative correlation between ASPP2 expression and ter- human cancers17. Our previous study found that ASPP2 is penoid backbone biosynthesis and steroid biosynthesis downregulated by DNA methylation in HCC18. ASPP2 (Fig. 1a). Expression of genes involved in mevalonate inhibits tumor growth and metastasis through regulation pathway, such as Hmgcs1, Hmgcr, Mvk, Mvd, and Idi1, – of apoptosis, autophagy, and epithelial plasticity16,19 21. appeared to be significantly enriched (P < 0.05, FDR < Fig. 1 Downregulation of ASPP2 enhances mevalonate pathway gene expression and cholesterol biosynthesis in HCC cells. a Top: KEGG Pathway analysis of ASPP2 knockdown show significant differential changes (P < 0.05) in canonical metabolic pathways. The P value for each pathway is indicated by the bar and is expressed as −1 times the log of the P value. Arrows are used to indicated the two pathways closely related with mevalonate metabolism. Bottom: Gene Ontology (GO) analysis revealed genes were most significantly upregulated and enriched by ASPP2 knockdown in the terpenoid backbone biosynthesis pathway (P < 0.05, FDR < 0.25). b Schematic of the mevalonate pathway with asterisk (*) marking upregulated genes by ASPP2 knockdown in panel (a) bottom. c qRT-PCR of Hmgcs1, Hmgcr mRNA expression in HCC-LM3 and HepG2 cells infected with lentivirus encoding shRNA was indicated for 72 h. β-actin was used as a control. d Corresponding western-blot of HMGCS1 and HMGCR in HCC- LM3 and HepG2 cells. e Total cholesterol and free cholesterol were measured by GC-MS methods, using the internal standard strategy as methods described. Each symbol represents the mean ± SD of three wells. Asterisk (*) indicates P < 0.05 Official journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2019) 10:830 Page 3 of 11 Fig. 2 Mevalonate metabolism is essential for maintaining tumor-initiating capability in ASPP2-depleted HCC cells. a Top: Representative images of spheroid formation in HCC-LM3 cells treated with 0.01 μM simvastatin or DMSO after knock down of ASPP2. Bottom: the number of spheres formation per 1000 HCC-LM3 cells.Scale bar, 100 mm. b qRT-PCR gene expression of Aspp2, Oct-4, Ck-19, and Abcg2 in HCC-LM3 and HepG2 cells transfected with the indicated lentivirus and treated with 10 μM simvastatin or DMSO. c Immunofluorescence stainings of HCC-LM3 spheres formed from cells transfected with the indicated lentivirus and treated with 10 μM simvastatin or DMSO. Green staining represented EpCAM protein. DAPI was used stain nuclear(blue staining) Scale bar, 40 mm d Cell viability of the infected HCC-LM3 and HepG2 cell treated with 10 μM simvastatin or DMSO and 5-FU(0.6 mM,2.5 mM or 10 mM) was analyzed by MTS assay. Asterisk (*) indicates P < 0.05, asterisks (**) indicates P < 0.01 0.25) in ASPP2-depleted HCC-LM3 cells (Fig. 1b). To cell-like properties27. To verify the importance of meva- validate this finding, we examined the expression of lonate metabolism in ASPP2-depletion induced tumor- HMG-CoA reductase (HMGCR) and HMG-CoA syn- initiating capability in HCC cells,