Ether lipid generating enzyme AGPS alters the balance of structural and signaling lipids to fuel cancer pathogenicity Daniel I. Benjamina, Alyssa Cozzoa, Xiaodan Jib, Lindsay S. Robertsa, Sharon M. Louiea, Melinda M. Mulvihilla, Kunxin Luob, and Daniel K. Nomuraa,1 aProgram in Metabolic Biology, Department of Nutritional Sciences and Toxicology, and bDivision of Cell and Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 29, 2013 (received for review June 7, 2013) Aberrant lipid metabolism is an established hallmark of cancer metabolism to favor generation of oncogenic signaling lipids, such cells. In particular, ether lipid levels have been shown to be as LPAe, lyosphosphatidic acid (LPA), and eicosanoids, which fuel elevated in tumors, but their specific function in cancer remains aggressive and tumorigenic features of cancer (12, 13, 16). Our elusive. We show here that the metabolic enzyme alkylglycer- studies thus reveal a heretofore unrecognized role of AGPS and onephosphate synthase (AGPS), a critical step in the synthesis of ether lipids in shifting the balance of fatty acid utilization from ether lipids, is up-regulated across multiple types of aggressive structural membrane lipids toward generation of oncogenic sig- human cancer cells and primary tumors. We demonstrate that naling lipids. ablation of AGPS in cancer cells results in reduced cell survival, Results and Discussion cancer aggressiveness, and tumor growth through altering the Cancer Cells Exhibit Heightened AGPS Expression and Ether Lipid balance of ether lipid, fatty acid, eicosanoid, and fatty acid– Metabolism. AGPS converts acyl-glycerone-3-phosphate into al- derived glycerophospholipid metabolism, resulting in an over- kyl-glycerone-3-phosphate, which is a requisite step in the gen- all reduction in the levels of several oncogenic signaling lipids. eration of all ether lipids. We hypothesized that cancer cells and Taken together, our results reveal that AGPS, in addition to main- tumors, which possess elevated levels of ether lipids, would have taining ether lipids, also controls cellular utilization of fatty acids, heightened AGPS expression. Consistent with this premise, we favoring the generation of signaling lipids necessary for promot- find that AGPS is highly expressed across aggressive breast ing the aggressive features of cancer. (231MFP), melanoma (C8161), and prostate cancer (PC3) cells compared with less aggressive cancer cells (MCF7, MUM2C, cancer metabolism | metabolomics | lipid signaling lysophosphatidic acid | and LNCaP, respectively) (Fig. 1A; Fig. S1A). We have reported eicosanoids that these aggressive cancer cells have heightened migratory, invasive, and tumorigenic properties compared with less ag- – ancer cells have fundamentally altered metabolism that gressive cells (17 19). We also show that AGPS expression is Cdrives their pathogenic features (1, 2). One hallmark of elevated two- to fourfold in Nottingham grade I (low-grade), II cancer cells is a heightened de novo lipogenic signature that (intermediate-grade), and III (high-grade) primary human breast tumors (Fig. 1B), as well as in estrogen receptor-positive/pro- serves as a critical foundation for generating lipids required for gesterone receptor-positive [ER(+)/PR(+)] and ER-negative/ cell proliferation (3, 4). For nearly half a century, it has also been PR-negative [ER(−)/PR(−)] breast tumors (Fig. 1C) compared known that cancer cells possess dramatically higher levels of – with normal breast tissue, indicating that AGPS expression may ether lipids compared with normal cells (5 8). Ether lipids have be heightened early in breast cancer development. Accordingly, we an alkyl or alkenyl chain on one or more carbons of the glycerol find that the expression of the commonly dysregulated transforming backbone bonded through an ether or vinyl linkage, rather than Harvey-Rat sarcoma oncogene (HRAS) (20) is heightened in the usual ester linkage. The physiological roles of ether lipids are not well understood, but they have been implicated in maintaining Significance physicochemical properties of cell membranes, such as membrane fluidity, membrane fusion events, and lipid raft microdomains fi (9, 10). Certain ether lipids, such as lysophosphatidic acid-ether Ether lipid levels are higher in tumors, but their speci c func- (LPAe) or platelet-activating factor-ether (PAFe), are signaling tion in cancer has remained unclear. We show here that the molecules that have been shown to possess bioactive and even metabolic enzyme alkylglyceronephosphate synthase (AGPS), oncogenic properties through binding specific receptors (11–15). a critical step in the synthesis of ether lipids, is up-regulated In the late 1960s, Snyder and Wood first reported that rodent across multiple types of aggressive human cancer cells and fi and human tumors possess significantly higher levels of ether lipids primary tumors. Inactivation of AGPS leads to signi cant relative to normal tissue. Over the ensuing decades, dramatic ele- impairments in cancer pathogenicity through not only lower- vations in ether lipid content have been confirmed for a wide range ing the levels of cellular ether lipids, but also by altering fatty of cancer cells and primary tumors from several tissues of origin and acid, eicosanoid, and glycerophospholipid metabolism, result- have been correlated with the proliferative capacity and tumorigenic ing in an overall reduction in the levels of several oncogenic potential of cancer cells (5–8). Nonetheless, whether elevated ether signaling lipids. lipids are causally linked, or merely associated with, cancer patho- Author contributions: D.I.B., L.S.R., S.M.L., K.L., and D.K.N. designed research; D.I.B., A.C., genicity has remained unclear. X.J., L.S.R., S.M.L., M.M.M., and D.K.N. performed research; D.I.B., K.L., and D.K.N. con- In this study, we show that inactivation of the critical enzyme tributed new reagents/analytic tools; D.I.B., A.C., X.J., L.S.R., S.M.L., M.M.M., and D.K.N. for ether lipid synthesis, alkylglycerone phosphate synthase (AGPS), analyzed data; and D.I.B., K.L., and D.K.N. wrote the paper. lowers ether lipid levels and impairs cancer pathogenicity, whereas The authors declare no conflict of interest. AGPS overexpression elevates ether lipid levels and increases This article is a PNAS Direct Submission. cancer cell motility, survival, and tumor growth. We also show that 1To whom correspondence should be addressed. E-mail: [email protected]. AGPS has a larger role beyond generating ether lipids to include This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. controlling fatty acid, eicosanoid, and acylglycerophospholipid 1073/pnas.1310894110/-/DCSupplemental. 14912–14917 | PNAS | September 10, 2013 | vol. 110 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1310894110 Downloaded by guest on September 25, 2021 HRAS transformation of MCF10A mammary epithelial cells (21) A AGPS expression in B AGPS expression in C AGPS expression in human cancer cells primary human tumors primary human tumors induces up-regulation of AGPS expression (Fig. 1F). We thus show breast cancer melanoma that transformation of cells by HRAS is one regulatory route through 25 5 15 * 20 * * * 20 4 * 15 which cells may up-regulate AGPS expression. We also show that the ion 10 * 15 3 10 aggressive human breast 231MFP, melanoma C8161, and prostate 10 press 2 5 expression ex expression 5 5 1 expression PC3 cancer cells, as well as HRAS-transformed MCF10A cells, 0 0 0 0 possess significantly higher ether lipid levels compared with their MCF7 C8161 less aggressive MCF7, MUM2C, and LNCaP counterparts, or grade 3 231MFP MUM2C grade 1/2 G B–F ER(-)/PR(-) ER(+)/PR(+) MCF10A control cells (Fig. 1 ; Fig. S1 ; Dataset S1). Height- normal tissue normal tissue breast tumorsbreast tumors breast tumorsbreast tumors ened lipid species include phosphatidic acid-ether (PAe), LPAe, D correlation between E F HRAS and AGPS expression HRAS expression in AGPS expression upon phosphatidyl inositol-ether (PIe), phosphatidylcholine-ether (PCe), in primary breast tumors human cancer cells RAS transformation lysophosphatidylcholine-ether (LPCe), phosphatidylserine-ether HRAS expression primary breast tumors breast cancer melanoma AGPS expression (PSe), phosphatidylglycerol-ether (PGe), lysophopshatidylglycerol- in primary breast tumors 4 4 5 * ether(LPGe)lipids,andplasmalogen ether lipids, such as 15 15 r = 0.59 * * * 4 G p<0.0001 3 3 phosphatidylethanolamine-plasmalogen (PEp) (Fig. 1 ; Fig. S1 10 * 10 3 2 2 B–F fi 2 ). We annotate ether lipids in this study by the speci c ether (e) pression 5 5 x expression expression e 1 1 expression 1 or plasmalogen (p) species and alkyl chain length and type (carbon 0 HRAS expression 0 0 0 0 number:degree of unsaturation—e.g., C16:0e is a palmityl-ether 0 5 10 15 G B–F AGPS expression MCF7 C8161 linkage with no unsaturation) in Fig. 1 and Fig. S1 . Our results MUM2C grade 3 231MFP MCF10A grade 1/2 HRAS-10A show that AGPS expression and ether lipid levels are heightened in normal tissue breast tumorsbreast tumors multiple types of aggressive human cancer cells and upon RAS ether lipid levels in human cancer cells G transformation and that AGPS expression is increased in primary breast cancer melanoma RAS transformation breast tumors. AGPS Is a Critical Enzyme in Cancer Pathogenicity. We next sought to MCF7 231MFP MUM2C C8161 MCF10AHRAS-10A C16:0e/18:1 PAe C16:0e/18:1 PAe C16:0e/18:1 PAe determine the extent to which AGPS was necessary for maintaining C16:0e/20:4 PAe C16:0e/20:4 PAe C16:0e/20:4 PAe C16:0e LPAe C16:0e LPAe C16:0e LPAe the pathogenic features of cancer cells. We generated two inde- C16:0e/18:1 PIe C16:0e/18:1 PIe C16:0e/18:1 PCe C16:0e/20:4 PIe C16:0e/20:4 PIe C16:0e/20:4 PCe pendent stable short hairpin knockdown lines of AGPS (shAGPS-1 C16:0e/18:1 PCe C16:0e/18:1 PCe C16:0e LPCe and shAGPS-2) with greater than 90% knockdown in breast C16:0e/20:4 PCe C16:0e/20:4 PCe C16:0e/18:1 PEe C16:0e LPCe C16:0e LPCe C18:0e/C18:1 PEe 231MFP and melanoma C8161 cancer cells (Fig.