Aberrant lipid metabolism is an established hallmark of cancer cells. In particular, ether lipid levels have been shown to be elevated in tumors, but their specific function in cancer remains elusive. We show here that the metabolic enzyme alkylglyceronephosphate synthase (AGPS), a critical step in the synthesis of ether lipids, is up-regulated across multiple types of aggressive human cancer cells and primary tumors. We demonstrate that ablation of AGPS in cancer cells results in reduced cell survival, cancer aggressiveness, and tumor growth through altering the balance of ether lipid, fatty acid, eicosanoid, and fatty acidderived glycerophospholipid metabolism, resulting in an overall reduction in the levels of several oncogenic signaling lipids. Taken together, our results reveal that AGPS, in addition to maintaining ether lipids, also controls cellular utilization of fatty acids, favoring the generation of signaling lipids necessary for promoting the aggressive features of cancer.cancer metabolism | metabolomics | lipid signaling lysophosphatidic acid | eicosanoids

Summary Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but thought to be primarily diet-derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity that influence acyl-chain diversity in the lipidome.

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.

De novo lipogenesis is considered the primary source of fatty acids for lipid synthesis in cancer cells, even in the presence of exogenous fatty acids. Here, we have used an isotopic fatty acid labeling strategy coupled with metabolomic profiling platforms to comprehensively map palmitic acid incorporation into complex lipids in cancer cells. We show that cancer cells and tumors robustly incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids, and ether lipids. We also find that fatty acid incorporation into oxidative pathways is reduced in aggressive human cancer cells, and instead shunted into pathways for generating structural and signaling lipids. Our results demonstrate that cancer cells do not solely rely on de novo lipogenesis, but also utilize exogenous fatty acids for generating lipids required for proliferation and protumorigenic lipid signaling.