Published OnlineFirst November 5, 2019; DOI: 10.1158/0008-5472.CAN-19-1023 CANCER RESEARCH | TRANSLATIONAL SCIENCE Inactivation of the AMPK–GATA3–ECHS1 Pathway Induces Fatty Acid Synthesis That Promotes Clear Cell Renal Cell Carcinoma Growth A C Yuan-Yuan Qu1,2,3, Rui Zhao1, Hai-Liang Zhang1,3, Qian Zhou1, Fu-Jiang Xu1,3, Xuan Zhang2, Wen-Hao Xu1,3, Ning Shao1,3, Shu-Xian Zhou1,2, Bo Dai1,3, Yao Zhu1,3, Guo-Hai Shi1,3, Yi-Jun Shen1,3, Yi-Ping Zhu1,3, Cheng-Tao Han1,3, Kun Chang1,3, Yan Lin1,2,4, Wei-Dong Zang5, Wei Xu1,2,4, Ding-Wei Ye1,3, Shi-Min Zhao1,2,4, and Jian-Yuan Zhao1,2,4 ABSTRACT ◥ The tumorigenic role and underlying mechanisms of lipid accu- expression of GATA3, a transcriptional activator of ECHS1. BCAA mulation, commonly observed in many cancers, remain insuffi- accumulation induced activation of mTORC1 and de novo FA ciently understood. In this study, we identified an AMP-activated synthesis, and promoted cell proliferation. Furthermore, GATA3 protein kinase (AMPK)–GATA-binding protein 3 (GATA3)– expression phenocopied ECHS1 in predicting ccRCC progression enoyl-CoA hydratase short-chain 1 (ECHS1) pathway that induces and patient survival. The AMPK–GATA3–ECHS1 pathway may lipid accumulation and promotes cell proliferation in clear cell renal offer new therapeutic approaches and prognostic assessment for cell carcinoma (ccRCC). Decreased expression of ECHS1, which is ccRCC in the clinic. responsible for inactivation of fatty acid (FA) oxidation and acti- vation of de novo FA synthesis, positively associated with ccRCC Significance: These findings uncover molecular mechanisms progression and predicted poor patient survival. Mechanistically, underlying lipid accumulation in ccRCC, suggesting the AMPK– ECHS1 downregulation induced FA and branched-chain amino GATA3–ECHS1 pathway as a potential therapeutic target and acid (BCAA) accumulation, which inhibited AMPK-promoted prognostic biomarker. Introduction obesity is a well-established independent risk factor of ccRCC (6–9). Weight gain is also associated with increased ccRCC risk (6), as are Recently, dysregulated fatty acid (FA) metabolism has been increased body mass index (7) and high dietary intake of saturated fat, observed in many types of cancers, including renal cell carcinoma, animal fat, or oleic acid (10). Excess lipids in cancer cells are stored in breast cancer, prostate cancer, and lung cancer (1–5). The relevance of lipid droplets, and high levels of lipid droplets are currently considered FA metabolism to cancer cell functioning, alongside that of perturbed a hallmark of cancer aggressiveness (11–14). The histologic appear- glucose metabolism—known as the Warburg effect—and altered ance of ccRCC cells, i.e., their clear cytoplasm, is due to lipid accu- amino acid metabolism, which is represented by glutamine metabo- mulation (15) and suggests that metabolic reprogramming may occur lism, is becoming increasingly recognized. during ccRCC development. Furthermore, gene expression profiling Clear cell renal cell carcinoma (ccRCC), which accounts for approx- has revealed that the expression of FA synthesis genes, such as acetyl- imately 80% of diagnosed RCCs, exhibits intracellular lipid droplet CoA carboxylase (ACC) and fatty acid synthase (FASN; refs. 3, 16), is accumulation and is closely related to aberrant FA metabolism. In fact, significantly increased in ccRCC, indicating enhanced de novo FA synthesis in ccRCC. In addition, AMP-activated protein kinase 1Department of Urology, Fudan University Shanghai Cancer Center, the Obstet- (AMPK), the master sensor of cellular energy balance (17, 18), inhibits rics and Gynecology Hospital of Fudan University, State Key Lab of Genetic de novo FA synthesis and lipid accumulation through inhibitory Engineering and School of Life Sciences, Fudan University, Shanghai, P.R. China. phosphorylation of ACC, thus maintaining cellular energy homeo- 2Key Laboratory of Reproduction Regulation of NPFPC, Institutes of Biomedical stasis and protecting cells from metabolic stress (19). Loss of AMPK Sciences and Collaborative Innovation Center of Genetics and Development, activity, which is frequently observed in ccRCC (3, 20), is correlated 3 Fudan University, Shanghai, P.R. China. Department of Oncology, Shanghai with enhanced de novo FA synthesis and lipid accumulation (20). Medical College, Fudan University, Shanghai, P.R. China. 4Collaborative Inno- vation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Although evidence suggests a causative role of FA accumulation in P.R. China. 5School of Basic Medical Sciences, Zhengzhou University, ccRCC occurrence and development, the underlying mechanisms Zhengzhou, P.R. China. remain unclear. Most importantly, besides abnormal FA synthesis, Note: Supplementary data for this article are available at Cancer Research the relevance of FA oxidation (FAO) in FA accumulation and cancer Online (http://cancerres.aacrjournals.org/). cell function is unknown. Aberrant activation of mTORC1, which can be induced by intracellular elevation of branched-chain amino acids Y.-Y. Qu, R. Zhao, and H.-L. Zhang contributed equally to this article. (BCAA), especially leucine, is frequently observed in ccRCC (21–23). Corresponding Authors: Jian-Yuan Zhao, Fudan University, 2005 Songhu Road, Our previous study revealed the existence of cross-talk between BCAA Shanghai 200433, China. Phone: 86-21-31246782; Fax: 86-21-31246782; E-mail: [email protected]; Ding-Wei Ye, [email protected]; and Shi-Min Zhao, metabolism and FAO via a reaction catalyzed by enoyl-CoA hydratase [email protected] short-chain 1 (ECHS1; ref. 24). Therefore, dysregulated FAO can affect FA and protein synthesis simultaneously. Cancer Res 2020;80:319–33 In this study, we found that ECHS1 expression is nearly absent in doi: 10.1158/0008-5472.CAN-19-1023 ccRCC tumors, and its absence predicted poor patient survival. In Ó2019 American Association for Cancer Research. clinical samples, animal models, and cultured cells, we validated that AACRJournals.org | 319 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst November 5, 2019; DOI: 10.1158/0008-5472.CAN-19-1023 Qu et al. ECHS1 is regulated by the AMPK–GATA-binding protein 3 (GATA3) each of the groups. The heterozygous Echs1 KO mice were gener- pathway, and ECHS1 downregulation could inhibit AMPK via FA ated using the CRISPR-Cas 9 system, and their genotype was accumulation. Analysis of the pathologic mechanism revealed that loss confirmed by PCR. Kidneys were removed, and whole-cell homo- of ECHS1 results in FAO block BCAA-mediated mTORC1 activation genates were generated with 0.5% NP-40 buffer for Western blotting and mTORC1 activation–induced de novo FA synthesis, thus pro- and homogenated with 80% methanol for BCAA analyses. Forma- moting cancer cell proliferation. lin-fixed, paraffin-embedded tissue blocks were created using kid- ney tissues from wild-type and Echs1 KO mice, respectively, for IHC. For Oil Red O staining, kidney tissues were placed in optimal Materials and Methods cutting temperature (OCT) compound (Tissue Tek 4583) in a peel- Reagents and antibodies away mold and frozen at À80C for further analysis. Samples were AICAR (#A9978) and rapamycin (#553210) were purchased from processed blindly during the experiments and outcome assessment. Sigma-Aldrich. Palmitic acid (1-13C, 99%, CAS#57677-53-9) and glucose (U-13C6, CAS#110187-42-3) were purchased from RNA extraction and quantitative real-time PCR Cambridge Isotope Laboratories. Primary antibodies used in this study Total RNA was extracted from human ccRCC and patient-matched include b-actin (cat. no. A00702, Genscript), a-ECHS1 (cat. no. normal tissue samples preserved in RNAlater and then converted to H00001892-D01P, Abnova), a-GATA3 (cat. no. #5852, Cell Signaling cDNA using random hexamers, oligo (dT) primers, and Moloney Technology), a-AMPKa (cat. no. #2532, Cell Signaling Technology), murine leukemia virus reverse transcriptase (TaKaRa). The ECHS1, a-pT172-AMPK (cat. no. #2535, Cell Signaling Technology), GATA3, and AMPK mRNA levels were measured by quantitative real- a-4E-BP1 (cat. no. #9644, Cell Signaling Technology), a-phospho- time PCR using the ABI Prism 7900 sequence detection system 4E-BP1 (Thr37/46; cat. no. #2855, Cell Signaling Technology), a-p70 (Applied Biosystems), with actin as an internal reference gene. Each S6 kinase (cat. no. #9202, Cell Signaling Technology), a-S6K1 reaction was performed in triplicate. The primers used were listed in (phospho T389þT412; cat. no. ab60948, Abcam), a-SREBP1 (PA1- Supplementary Table S1. 337, Thermo Fisher), a-ACC (cat. no. ab45174, Abcam), a-FASN (cat. no. ab128870, Abcam), a-ATGL (cat. no. #2138, Cell Signaling RNA sequencing Technology), and a-LCAD (cat. no. ab82853, Abcam). Among them, Total RNA was extracted from human ccRCC and patient-matched antibodies of sterol regulatory element-binding protein (SREBP1; normal tissues with TRIzol/CHCl3 (Life Technologies) according to Supplementary Fig. S1A), ECHS1 (Supplementary Fig. S1B), and the manufacturer's protocol. RNA quality was examined by gel GATA3 (Supplementary Fig. S1C) were validated through IHC in electrophoresis, and only paired RNA of high quality was used for xenograft tumors that grew from either wild-type or candidate gene RNA sequencing. RNA sequencing libraries were prepared according knockout (KO) cells. to the manufacturer's instructions and then sequenced with the Illumina HiSeq 2000 at Genergy