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Glucose-Mediated N-Glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth

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Glucose-Mediated N-Glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth Article Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth Graphical Abstract Authors Chunming Cheng, Peng Ru, Feng Geng, ..., Jianjie Ma, Arnab Chakravarti, Deliang Guo Correspondence [email protected] In Brief Cheng et al. show that glucose controls N-glycosylation of SCAP, which is essential for SREBP-dependent lipogenesis, and that activated growth factor receptors increase glucose intake and SREBP activation. Inhibition of SCAP N-glycosylation inhibits the growth of glioblastoma cells with hyperactived EGFR. Highlights d SCAP acts as a glucose-responsive protein, linking fuel supply to SREBP activation d N-glycosylation of SCAP facilitates SCAP/SREBP trafficking from ER to the Golgi d EGFR activates SCAP/SREBP by promoting glucose import and SCAP N-glycosylation d Targeting SCAP N-glycosylation is a promising strategy to treat malignancies Cheng et al., 2015, Cancer Cell 28, 569–581 November 9, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.ccell.2015.09.021 Cancer Cell Article Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth Chunming Cheng,1 Peng Ru,1 Feng Geng,1 Junfeng Liu,1,2 Ji Young Yoo,3 Xiaoning Wu,1 Xiang Cheng,1 Vanessa Euthine,4 Peng Hu,1 Jeffrey Yunhua Guo,1 Etienne Lefai,4 Balveen Kaur,3 Axel Nohturfft,5 Jianjie Ma,6 Arnab Chakravarti,1 and Deliang Guo1,* 1Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA 2College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China 3Department of Neurosurgery, The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA 4CarMeN Laboratory, INSERM U1060, INRA 1397, Faculte´ de Me´ decine Lyon Sud BP 12, Universite´ de Lyon, 69921 Oullins Cedex, France 5Vascular Biology Research Centre, St. George’s University of London, London SW17 0RE, UK 6Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2015.09.021 SUMMARY Tumorigenesis is associated with increased glucose consumption and lipogenesis, but how these pathways are interlinked is unclear. Here, we delineate a pathway in which EGFR signaling, by increasing glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an ER-bound transcription factor with central roles in lipid metabolism. Glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent proteolytic activation of SREBP. Xenograft studies reveal that blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth. Thus, SCAP acts as key glucose-responsive protein linking oncogenic signaling and fuel availability to SREBP-dependent lipogen- esis. Targeting SCAP N-glycosylation may provide a promising means of treating malignancies and meta- bolic diseases. INTRODUCTION scribed from different transcriptional start sites, mainly regulate the expression of genes required for fatty acid synthesis. Elevated lipogenesis is a common patho-physiological charac- SREBP-2 is encoded by SREBF2 and is responsible for the teristic of cancer and metabolic diseases (Guo et al., 2013; synthesis of cholesterol (Goldstein et al., 2006; Horton et al., Menendez and Lupu, 2007; Moon et al., 2012; Ru et al., 2013; 2002, 2003). Recent evidence shows that the nuclear form of Tang et al., 2011). In these processes, a critical regulatory role SREBP-1 is highly upregulated in a variety of malignancies (Et- is played by sterol regulatory element-binding proteins tinger et al., 2004; Guo et al., 2009b; Li et al., 2014). Targeting (SREBPs), a family of transcription factors that control the SREBP-1 has become a promising therapeutic strategy to treat expression of genes important for the uptake and synthesis of cancer and other metabolic syndromes (Griffiths et al., 2013; cholesterol, fatty acids, and phospholipids (Goldstein et al., Guo et al., 2014; Kamisuki et al., 2009; Tang et al., 2011). 2006; Jeon and Osborne, 2012; Nohturfft and Zhang, 2009). SREBP-1 and SREBP-2 are synthesized as inactive precur- There are two mammalian SREBP genes, SREBF1 and SREBF2. sors bound to the membrane of the ER through two trans- SREBP-1a and -1c, which are encoded by SREBF1 with different membrane domains (Goldstein et al., 2006). SREBP activation N terminus (20 amino acids) owing to their mRNAs being tran- requires proteolytic release of an N-terminal fragment that Significance Twenty years ago, Nobel Laureates Brown & Goldstein discovered SREBPs as key proteins that regulate lipid metabolism. They revealed that sterols modulate Insig interaction with SCAP/SREBP to inhibit SREBP function under physiological con- ditions. Our study identifies glucose as an indispensible component of SREBP function. Without glucose, reduction of ste- rols is insufficient to initiate SCAP/SREBP trafficking and SREBP activation. Glucose-mediated N-glycosylation of SCAP facilitates SCAP/SREBP movement from the ER to the Golgi and the subsequent lipogenic function of SREBP. We show that SCAP acts as a key glucose-responsible protein to integrate oncogenic signaling and fuel availability for control of lipid metabolism and tumor growth. Cancer Cell 28, 569–581, November 9, 2015 ª2015 Elsevier Inc. 569 constitutes a basic helix-loop-helix transcription factor (Gold- the expression of SREBP-1 or SREBP-2 regulated genes stein et al., 2006; Wang et al., 1994). Currently, post-translational involved in lipid metabolism (Figure S1C). Using confocal fluo- activation of SREBPs is best understood in the context of cellular rescence imaging, we found that stimulation of U87 cells with cholesterol homeostasis (Radhakrishnan et al., 2008; Sun et al., glucose promoted nuclear translocation of transgenic GFP- 2007). When cholesterol level is high, it binds to SREBP-cleav- SREBP-1 (Figures 1D and S1D). Translocation of endogenous age activating protein (SCAP), inducing a conformational change SREBP-1 into the nucleus in response to glucose stimulation that promotes binding to ER-anchored insulin induced gene pro- was demonstrated by immunofluorescence (Figures 1E and tein (Insig), thus preventing Golgi transport and activation of S1E). In contrast, under glucose-deprived conditions, even with SREBPs (Adams et al., 2004; Sun et al., 2007; Yang et al., removal of sterols, SREBP-1 was still retained in the ER mem- 2002). When the cholesterol concentration drops, the SCAP/ brane as shown by its co-staining with protein disulfide isom- SREBP complex dissociates from Insig, allowing vesicular trans- erase (PDI), an ER membrane protein (Figures 1E and S1E) port to the Golgi where SREBPs are exposed to proteases that (Uehara et al., 2006). These data suggest that glucose is required release the transcriptionally active N-terminal fragment (Gold- for SREBP activation under conditions of sterol deprivation. stein et al., 2006; Nohturfft et al., 2000; Sun et al., 2007). SREBP stability, transport to the Golgi, and cleavage require Glucose is a major resource for de novo lipid synthesis. In can- formation of a complex between SREBP and SCAP (Nohturfft cer cells, elevated glucose consumption is often accompanied et al., 2000; Rawson et al., 1999; Sakai et al., 1998). We found by increased lipogenesis (Guo et al., 2013; Menendez and that glucose-induced cleavages of SREBP-1 and SREBP-2 Lupu, 2007), and the link between glucose supply and SREBP-1 were accompanied by an increase of SCAP protein levels in activation seems common in both physiological and patho- GBM cells (Figures 1F, S1A, and S1B). Similar results were physiological conditions (Guillet-Deniau et al., 2004; Hasty observed in various other cancer cell lines in which both et al., 2000; Horton et al., 1998; Kaplan et al., 2008). In a previous SREBP-1 and -2 were activated (see Figure S1F). HEK293T cells study, we showed that epidermal growth factor receptor (EGFR) expressing GFP-SCAP and full length FLAG-SREBP-1a, -1c, via PI3K/Akt signaling activated SREBP-1 in glioblastoma (GBM) or HA-SREBP-2 displayed glucose-dependent upregulation of cells (Guo et al., 2009b, 2011). While a number of studies have GFP-SCAP and activation of all three SREBP isoforms, which demonstrated that elevated EGFR signaling is coupled with were detected by increased N-terminal fragment of epitope- enhanced glucose uptake and lipogenesis in tumorigenesis (Ba- tagged SREBP in the nuclear fraction under conditions of bic et al., 2013; Cloughesy et al., 2014; Guo et al., 2009a, 2009b), glucose supplement (Figures 1G and S1G). We found that the molecular mechanisms that underlie the crosstalk between knock down of SCAP reduced glucose-mediated activation of the altered glucose and lipid metabolism in tumorigenesis SREBP-1 and SREBP-2 (Figure 1H). remain largely unknown. In this study, we test the hypothesis Because liver X receptor (LXR), a member of the nuclear re- that SCAP acts as a key glucose-responsive protein to integrate ceptor family of transcription factors, promotes the expression oncogenic signaling and fuel availability for modulation of of SREBP-1c upon activation (Repa et al., 2000), and glucose SREBP-dependent lipogenesis. is reported to activate LXR in hepatocytes (Mitro et al., 2007), we sought to examine whether LXR possibly involves in RESULTS glucose-activated SCAP/SREBP signaling. As shown in Figures S1A–S1C, our data show that both the protein and mRNA levels Glucose Activates SREBPs via Upregulation of SCAP of ATP-binding cassette activating proteins ABCA1 and ABCG1, To investigate how glucose and sterol signaling are integrated which are major downstream targets of LXR (Zelcer and Tonto- during SREBP activation, human GBM U87 cells that have noz, 2006), were not significantly upregulated by glucose sup- been shown to contain a high SREBP-1 activity (Guo et al., plement in U87 cells within 12 hr, demonstrating that LXR was 2009b, 2011) were grown in the absence or presence of glucose not strongly activated by glucose in GBM cells.
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