University of Birmingham Activation of SREBP1-mediated lipogenesis by the Epstein-Barr Virus-encoded LMP1 promotes cell proliferation and progression of nasopharyngeal carcinoma.

Nasopharyngeal Carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element-binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target: fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, siRNA knockdown of LMP1 in EBV-infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mTOR pathway, either through the use of mTOR inhibitors or siRNAs significantly reduced LMP1-mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1-mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells; effects which were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo . Our findings demonstrate that LMP1 activation of SREBP1-mediated lipogenesis promotes tumor cell growth and is involved in EBV-driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC.


Introduction
Non-keratinizing undifferentiated nasopharyngeal carcinoma (NPC) is a distinct type of cancer prevalent in southeast Asia and southern China. The unique feature of NPC is its strong association with Epstein-Barr virus (EBV) infection [1,2]. Among the EBV-encoded gene products expressed in NPC, latent membrane protein 1 (LMP1) is of particular interest as it displays oncogenic properties in vitro and in vivo. LMP1 is an integral membrane protein containing two signaling domains: CTAR1 and CTAR2. Through these two domains, LMP1 engages multiple signaling cascades that include the Ras-ERK-MAPK, PI3K-AKT, NF-κB, and p38-MAPK pathways, which modulate the expression of a variety of cellular targets that contribute to the transforming activities of LMP1 [1][2][3]. Previous studies have established a role for LMP1 in promoting cell proliferation, transformation, cell invasion and migration, aerobic glycolysis and metabolic reprogramming in nasopharyngeal epithelial cells [3][4][5].
These observations imply an essential role for LMP1 in the pathogenesis of NPC.
Deregulated lipid metabolism is an established hallmark of cancer. Cells obtain fatty acids either from the diet or through de novo lipid synthesis (lipogenesis). Normal cells rely primarily on dietary fatty acid for the synthesis of new structural lipids and lipogenesis is not universal. However, cancer cells extensively engage de novo lipogenesis to produce longchain fatty acids that are essential for the synthesis of glycerophospholipid membrane and membrane signal molecules during rapid cell proliferation (supplementary material, Figure   S5) Fatty acids are also necessary for energy storage as lipid droplets [6][7][8]. Lipogenesis is tightly regulated by sterol regulatory element-binding protein 1 (SREBP1), a transcription factor that regulates transcription of most genes involved in lipogenesis [9][10][11]. There are two SREBP1 isoforms (SREBP1a and SREBP1c) encoded from SREBF1. SREBF1a and SREBF1c are produced from different promoters, differing only in the length of their Nterminal transactivation domains. SREBPs are synthesized as precursor proteins bound to the ER membrane. After stimulation, the SREBP precursor undergoes proteolytic cleavage in the Golgi to release the transcriptionally active-N-terminal domain. Once mature, active SREBP1 translocates to the nucleus, where it binds to sterol regulatory element (SRE) within its promoter and those of its target genes [9][10][11]. mTOR complex 1 (mTORC1) and 2 (mTORC2) have been shown to regulate SREBP1 activity and lipogenesis. Activation of mTOR signaling by the Ras-ERK, and PI3K-AKT pathways increases expression of SREBPs and SREBP-mediated lipogenesis [12][13][14]. Numerous lipogenic genes, including fatty acid This article is protected by copyright. All rights reserved.
Accepted Article synthase (FASN), are upregulated in a variety of cancers [7;8;10]. In this study, we demonstrate that FASN expression is common in primary NPC tumors, with higher levels correlating with LMP1 expression. Furthermore, LMP1 activates de novo lipogenesis and LMP1 activation of SREBP1-mediated lipogenesis contributes to cancer cell growth and tumor progression. These implicate the involvement of LMP1-mediated lipogenesis in the pathogenesis of EBV-infected NPC.

RT-qPCR
All RT-qPCR products were amplified using the Power SYBR green PCR Master Mix Kit (Thermo Fisher Scientific). Details including primer sequences are provided in supplementary material, Supplementary materials and methods.

Luciferase Reporter Assay
Ten thousand HeLa or 293 cells grown in 96-well plate were co-transfected with 20 ng of the luciferase reporter construct together with increasing amounts of an LMP1 expression vector (pCDNA3-LMP1) as indicated in the Figure. Renilla pRL-SV40 vector was transfected as an internal control to correct for transfection efficiency. Two days post-transfection, cells were lysed in reporter lysis buffer and then assayed for luciferase and Renilla activities with the Dual-Luciferase Reporter Assay System (Promega).

Immunofluorescence Staining
Immunofluorescence staining was performed as previously described [16] and as further

Immunohistochemical Staining
Immunohistochemical staining was performed as described previously [17]. Information regarding normal and tumor specimens, staining and intensity score methods are outlined in supplementary material, Supplementary materials and methods.

Cell Proliferation Assay
Cell proliferation assays were performed with cell proliferation reagent CCK-8 (Dojindo Molecular Technologies, Rockville, MD, USA and as described in supplementary material, Supplementary materials and methods.

De Novo Lipogenesis Assay
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Accepted Article
For the lipogenesis assay, cells were incubated in serum-free medium containing 2.5 μCi/ml

Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.0 GraphPad Software Inc, La Jolla, CA, USA). P-value was calculated using either Fisher's exact test or unpaired twotailed Student's t-test. The IC 50 values of inhibitors were calculated by applying the fourparameter logistic equation to generate the Sigmoidal dose-response (variable slope) curves.
Survival curves were analyzed using the Kaplan-Meier method, and were compared with a log-rank test. P-value <0.05 was considered to be statistically significant.

LMP1 increases SREBP1 expression and activity
To examine the impact of LMP1 on SREBP1-mediated lipogenesis, increasing amounts of an LMP1 expression vector was transfected into the nasopharyngeal epithelial cell line, NP69.
Under serum-deprived conditions, a dose-dependent induction of SREBF1a and SREBF1c mRNA was observed ( Figure 1A). Similarly, LMP1 increased the levels of both precursor and mature forms of SREBP1 protein ( Figure 1C). SREBP1 transactivates target genes by binding to SRE elements within the promoter region. Using a pGL2-3xSRE reporter construct containing 3 tandem copies of an SRE/SP1 element [18], we found that LMP1 increased SREBP1 transcriptional activity ( Figure 1B). FASN is a transcriptional target of This article is protected by copyright. All rights reserved. Accepted Article SREBP1. FASN promoter activity was also strongly enhanced in response to LMP1 expression ( Figure 1B). The expression of FASN mRNA and protein were also induced by LMP1 ( Figure 1A & 1C). These findings suggest that FASN induction by LMP1 is mediated primarily through modulation of the expression and activity of SREBP1. Both the mTORC1 and mTORC2 pathways are involved in regulating SREBP1 activity [12][13][14]. LMP1 has been reported to activate the mTOR signaling pathway [18][19][20]. Here, we further confirmed that LMP1 induced phosphorylation of 4E-BP1 and p70S6K, two established downstream targets of mTOR signaling ( Figure 1C).
In nasopharyngeal epithelial cells stably expressing LMP1 we also observed increased activities of the SREBP1 and mTOR pathways ( Figure 1D). Immunofluorescence staining revealed overexpression of FASN in LMP1-expressing nasopharyngeal epithelial cells ( Figure 1E). To determine whether LMP1 promotes de novo lipogenesis, LMP1-expressing cells were labelled with 14 C-acetate for 8-12 h under serum-deprived conditions. The 14 C labelled lipid fraction was then extracted for quantification. As shown in Figure 1F, LMP1expressing cells produced more newly synthesized lipid than control cells. Furthermore, Nile Red staining revealed more intracellular lipid droplets in LMP1-expressing cells ( Figure 1G, H). Overall, these data indicate a role for LMP1 in promoting lipid biosynthesis.

LMP1 Promotes SREBP1-Mediated Lipogenesis In EBV-Infected NPC Cells
In NPC xenografts (C17, X2117, and C15), western blotting analysis revealed higher levels of the precursor and mature forms of SREBP1 and FASN in LMP1-positive X2117 and C15 compared to the LMP1 negative C17 xenograft and EBV negative non-malignant NP69 nasopharyngeal epithelial cell line ( Figure 2A). In NPC cell lines, the levels of mature SREBP1 and FASN proteins were higher in EBV-infected HK1-EBV and C666-1 cells compared to HK-1 cells ( Figure 2B). Similarly, levels of the phosphorylated forms of 4E-BP1 and p70S6K were elevated in HK1-EBV and C666-1 cells ( Figure 2B). A role for LMP1 in these effects was established, as siRNA silencing of LMP1 in EBV-infected cells resulted in reducing the expression of mature SREBP1 and FASN as well as the signaling activity of mTOR ( Figure 2C). Furthermore, LMP1 silencing in C666-1 cells resulted in a decrease in FASN promoter activity (supplementary material, Figure S1) as well as lipogenesis, particularly under serum-deprived condition ( Figure 2D). These data suggest that SREPB1mediated lipogenesis in EBV-infected cells is induced by LMP1 through the mTOR signaling pathway.
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LMP1 induces SREBP1-mediated lipogenesis through the mTOR signaling pathway
To investigate whether LMP1-induced lipogenesis was dependent on mTOR signaling, NP69-pLNSX and NP69-LMP1 cells were treated with the mTOR inhibitors, Torin 1 and Torin 2. Torin 1 is documented to inhibit both mTORC1 and mTORC2 complexes, while Torin 2 inhibits the mTORC1 complex [21,22]. 14 C-acetate incorporation assays revealed that these two mTOR inhibitors could suppress lipogenesis in both cell lines, although the effects were more profound in LMP1-expressing cells. As shown in Figure 3A, NP69-LMP1 cells exhibited 53-72% reduction in lipogenesis, while only a 15-39% reduction was observed in NP69-pLNSX cells. To further determine whether LMP1 induction of mTOR signaling promoted SREBP1-mediated lipogenesis, NP69 cells were transiently transfected with an LMP1 expression vector together with siRNAs targeting Raptor to inhibit mTORC1 signaling, Rictor to inhibit mTORC2 signaling, or mTOR to inhibit both mTORC1 and mTORC2 signaling pathways. As shown in Figure 3B, inhibition of either mTORC1, mTORC2 or both, reduced LMP1-induced FASN expression and SREBP1 maturation. Also, NP69-LMP1 cells exhibited 20-35% reduction in lipogenesis, while a 14-27% reduction was observed in control NP69-pLNSX cells ( Figure 3C). These finding indicated that mTOR signaling contributes to LMP1-induced lipogenesis.

Expression of FASN correlates with LMP1 expression in NPCs and poor prognosis in NPC patients
To examine the expression of FASN in NPC, we performed immunohistochemical staining for FASN and LMP1 in 38 NPC primary tumors. The intensity of FASN staining was scored, and a graph of the statistical dot-plot of FASN staining intensity against LMP1 expression was generated ( Figure 4B). Immunohistochemical staining revealed a negative or low expression of FASN (immunoreactivity score <3) in normal nasopharyngeal epithelium  Table S1). Also, Kaplan-Meier survival analysis revealed that elevated FASN expression significantly correlated to poor survival (p = 0.02) ( Figure 4C). Overall, these findings indicate that FASN overexpression is common in NPC and correlated with LMP1 expression. Moreover, the elevated FASN is associated with aggressive disease and poor prognosis in NPC patients. These also imply a role of LMP1 in up-regulating FASN for NPC progression.

LMP1 induction of lipid synthesis contributes to cell growth
Next, we examine whether LMP1 induction of lipogenesis contributed to cell growth. NP69 and HK1 cells expressing pLNSX control or LMP1 were treated with luteolin, or fatostatin.
To determine whether lipogenesis conferred a growth advantage to LMP1-expressing cells, pLNSX control and LMP1 expressing NP69 and HK-1 cells were grown in culture medium supplemented with 1% serum together with increasing doses of luteolin or fatostatin for 1-4 days, and the effects on cell growth examined. As shown in Figures 5D-5G as an essential mechanism in facilitating cell proliferation.

Inhibitors of lipogenesis suppress NPC growth and induce apoptosis in vivo
Next, we examined whether blocking lipogenesis inhibited the growth of NPC tumor in vivo. In animals treated with luteolin or fatostatin, substantially lower tumor weights and smaller tumor size were observed (p<0.0001) ( Figure 6B,C). Surprisingly, no noticeable weight loss was observed in treated animals with either drug (supplementary material, Figure S3).
Immunohistochemical staining analysis of NPC tumors from these two groups of mice indicated that both luteolin and fatostatin effectively inhibit FASN expression ( Figure 6D & supplementary material, Figure S4). In addition, histological investigations of tumor tissues revealed more pronounced necrosis in both luteolin (35%) and fatostatin (43%) treated groups compared to the control (5%) group ( Figure 6D). An examination of cell proliferation by IHC staining for Ki67; and apoptosis by staining for cleaved capase-3 (CC3), revealed that both drugs significantly inhibited cell proliferation and increased apoptosis. As shown in Figure 6D, the amount of Ki67 positive cells was markedly lower in the tumor tissues of luteolin (56%) and fatostatin (45%) treated groups compared to the control group (70%).
Western blotting analysis further confirmed induction of CC3 and cleaved PARP in luteolin and fatostatin treated tumors (supplementary material, Figure S4). These findings indicate that inhibition of lipogenesis by luteolin and fatostatin causes cell apoptosis and necrosis, leading to tumor growth suppression.

Discussion
The oncogenic LMP1 protein is frequently found to be expressed in NPC tumors. While, LMP1 promotes cell growth, transformation, invasion and migration, it also plays a role in This article is protected by copyright. All rights reserved.
Accepted Article modulating host cell metabolic pathways [1,3,26]. In previous studies, we have demonstrated an ability of LMP1 to promote aerobic glycolysis through constitutive activation of FGF2/FGFR1 signaling pathway and upregulation of c-Myc and HIF-1α, two primary regulators of aerobic glycolysis [5]. LMP1 also inactivates LKB1-AMPK to promote proliferation and anchorage-independent growth [4]. Here, we report a novel function of LMP1 in promoting SREBP1-mediated de novo lipogenesis, an effect that facilitates cell growth and tumor development (supplementary material, Figure S5).
In this study, we show that LMP1 increases SREBP1 expression and activity in nasopharyngeal epithelial cells. Luciferase promoter reporter assays and RT-qPCR analysis demonstrate that LMP1 upregulates SREBP1 at the transcriptional level ( Figures 1A & 1B), while western blotting analysis revealed that LMP1 promotes SREBP1 maturation and the expression of its downstream target, FASN ( Figures 1C,D). Using 14 C-acetate incorporation assay, we demonstrate an ability of LMP1 to promote lipogenesis ( Figure 1F). Interestingly, silencing LMP1 in EBV-infected NPC cells resulted in a reduction in FASN expression and lipogenesis ( Figures 2C,D). Furthermore, suppressing lipogenesis by inhibitors significantly reduced LMP1-induced proliferation ( Figure 5). Collectively, these findings demonstrate that LMP1 induction of lipogenesis in NPC contributes to cell proliferation.
SREBPs tightly regulate lipogenesis. However, the mechanism underlying the expression and activity of SREBPs is still unclear. Hypoxia, glucose and insulin have been found to increase the expression of SREBPs [10]. Interestingly, we and others have reported a function of LMP1 in promoting HIF-1 expression and glucose uptake [5;26]. mTORC1 and mTORC2 have been shown to regulate SREBP activity and lipogenesis through multiple inputs [27][28][29][30]. Here, in line with other studies [18][19][20], we demonstrate that LMP1 increases the phosphorylation of 4E-BP1 and p70S6K, two established targets of mTOR, in nasopharyngeal epithelial cells (Figures 1-3). Using siRNAs targeting Raptor or Rictor, we show that both mTORC1 and mTORC2 signaling pathways are involved in LMP1-mediated lipogenesis ( Figure 3). The mTOR signaling pathway is complicated as it contains both positive and negative feedback loops, and its downstream signaling interconnects with other cell growth and survival pathways [28][29][30][31]. mTORC2 is activated by ribosomes and PI3K signaling and is negatively regulated by a mTORC1-induced p70S6 kinase, which dampens PI3K signaling by inhibiting IRS-1 [28][29][30][31]. mTORC1 activity is induced by PI3K-AKT, This article is protected by copyright. All rights reserved.
Overexpression of FASN has been documented in oral hairy leukoplakia (OHL), an EBVassociated benign lesion associated with robust EBV lytic replication [32]. FASN is induced by the EBV-encoded lytic protein, BRLF-1 through the p38-MAPK pathway and required for lytic viral gene expression [32]. In a similar vein, HBV and HCV lytic infection in liver cancer cells is associated with the induction of fatty acid synthesis for the formation of viral envelopes [33][34][35]. Another γ-herpesvirus, KSHV, has been shown to induce FASN expression and fatty acid synthesis for the survival of latently infected PEL cells [36]. Here,  Table S1) [37]. These findings suggest that LMP1 induction of FASN and lipogenesis is involved in NPC progression. Of the LMP1-negative NPC tumors examined, 9/25 (36%) displayed a moderate or high level of FASN ( Figure 4B). Our previous genetic analyses indicated that somatic mutations and/or aberrant expression of signaling proteins (LTBR, PIK3CA, p50, RelB, Bcl3, EGFR and RAS), or signaling regulators (INPP4B, TRAF3, TRAF2, A20, NFKBIA, TNFAIP3, and CYLD) are common in NPC, resulting in constitutive activation of the ERK1/2, NFκB and PI3K/AKT pathways [38][39][40]. Therefore, the genetic background of NPC tumors, in addition to LMP1 expression, is likely to contribute to FASN upregulation. This article is protected by copyright. All rights reserved.

Accepted Article
Given that FASN is commonly increased in virus-associated cancers, lipogenesis appears to be essential for viral infection and cancer progression [35]. As lipogenesis is not common in normal cells, targeting lipogenesis (or lipogenic pathways) might selectively inhibit the growth of virus-infected cells as well as highly proliferative cancer cells in the early stage of cancer development. In this study, we examined the effects of fatostatin and luteolin on cell growth and tumor development in relation to NPC. Fatostatin is a small molecule, which specifically blocks proteolytic activation of SREBPs [24]. Luteolin is a plant flavonoid, which possesses inhibitory effects on lipogenesis [23]. Both fatostatin and luteolin significantly inhibit SREBP1 activity, lipid synthesis and cell proliferation induced by LMP1 ( Figure 5 & Supplementary Figure S2). Interestingly, luteolin has been reported to inhibit PI3K-AKT, MAPK-ERK and mTOR signaling activities [41], the major pathways required for SREBP-mediated lipogenesis by LMP1. Moreover, fatostatin and luteolin significantly inhibit the proliferation and tumorigenic growth of C666-1 cells. In nude mice, fatostatin and luteolin at a doses which effectively downregulates FASN expression, brought about a marked reduction in tumor growth (Figures 6 & supplementary material, Figure S4). Surprisingly, no significant weight loss was observed in animals treated with luteolin or fatostatin (supplementary material, Figure S3). The promising effects of these inhibitors in suppressing NPC tumor growth with low-level toxicity suggest a possible clinical utility in the treatment of NPC. Luteolin is a dietary flavonoid found in vegetables, fruits and herbs. In addition to its inhibitory effect on lipogenesis, luteolin also suppresses inflammation, angiogenesis, cell proliferation, and metastasis, all of which associated with cancer development [41]. Interestingly, luteolin has been shown to induce G1 arrest and inhibit EBV reactivation in NPC cells [42,43]. Given that luteolin is a natural polyphenolic compound and its toxicity is barely highlighted, it may prove to be a promising compound for the inhibition of EBV infection and the treatment of EBV-associated cancers. The potential of luteolin for the treatment of EBV-associated malignancies is worth to be further evaluated, particularly in clinical trials.