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Hypoxia induces a lipogenic cancer cell phenotype via HIF1α-dependent and -independent pathways.

Valli A, Rodriguez M, Moutsianas L, Fischer R, Fedele V, Huang HL, Van Stiphout R, Jones D, Mccarthy M, Vinaxia M, Igarashi K, Sato M, Soga T, Buffa F, Mccullagh J, Yanes O, Harris A, Kessler B - Oncotarget (2015)

Bottom Line: To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites.Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner.Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

ABSTRACT
The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

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Platelet activating factor C16 (PAFC16) is regulated in hypoxia independently of HIF1α(a) Heat map of organic extract molecular features showing the detection of the m/z=524.3736 by nanoflow LC/MS positive mode. (b) PAF biosynthesis via de novo pathway and via remodeling pathways. Hypoxia, favoring cell membrane remodeling releases PC the substrate used for PAF biosynthesis. Abbreviations: LPCAT, acetyltransferase; PLA2, phospholipase A2; CMP, Cytidine monophosphate; Pi, phosphate inorganic; CDP-choline, Cytidine-diphosphocholine. (c) Molecular structure of PAFC16. In hypoxia PC provides the skeleton of PAFC16 (glycerol and phosphocholine); the characteristic saturated hexadecil moiety (16:0) is a derivative of palmitate reduction. Acetyl deriving from acetyl-CoA completes the structure of PAFC16. (d) PAFC16 identification was performed by LC/MS QTOF nanoflow using mass matching and retention time comparison. (e) Tandem mass (MS/MS) spectra performed by LC/MS QTOF nanoflow of experimental detection of m/z=524.3736 [M+H]+ and comparison matching with METLIN database was the third parameter used for PAFC16 identification. (f) Intracellular PAFC16 concentrations reported as femtomol/106 cells data are shown as mean ±sd, intensities were quantified by LC/MS Q Exactive (n=3). Concentration was calculated interpolating a linear range standard curve with the unknown quantified relative intensities.
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Figure 7: Platelet activating factor C16 (PAFC16) is regulated in hypoxia independently of HIF1α(a) Heat map of organic extract molecular features showing the detection of the m/z=524.3736 by nanoflow LC/MS positive mode. (b) PAF biosynthesis via de novo pathway and via remodeling pathways. Hypoxia, favoring cell membrane remodeling releases PC the substrate used for PAF biosynthesis. Abbreviations: LPCAT, acetyltransferase; PLA2, phospholipase A2; CMP, Cytidine monophosphate; Pi, phosphate inorganic; CDP-choline, Cytidine-diphosphocholine. (c) Molecular structure of PAFC16. In hypoxia PC provides the skeleton of PAFC16 (glycerol and phosphocholine); the characteristic saturated hexadecil moiety (16:0) is a derivative of palmitate reduction. Acetyl deriving from acetyl-CoA completes the structure of PAFC16. (d) PAFC16 identification was performed by LC/MS QTOF nanoflow using mass matching and retention time comparison. (e) Tandem mass (MS/MS) spectra performed by LC/MS QTOF nanoflow of experimental detection of m/z=524.3736 [M+H]+ and comparison matching with METLIN database was the third parameter used for PAFC16 identification. (f) Intracellular PAFC16 concentrations reported as femtomol/106 cells data are shown as mean ±sd, intensities were quantified by LC/MS Q Exactive (n=3). Concentration was calculated interpolating a linear range standard curve with the unknown quantified relative intensities.

Mentions: To extend the pool of identified molecular features with an altered profile in hypoxia (Figure 1d and 7a), we performed METLIN database searches, and observed an accurate mass match for m/z 524.3736 [M+H]+ corresponding to platelet activating factor 16 (PAFC16), a C16:0 monoalkylglycerol ether-derivative, esterified with an acetyl group in C2 and condensed with a Cho polar head in C3 (Figure 7b and c). PAF is a lipid synthesized through the (i) de novo pathway where a transferase adds PC to the sn-3 site of the 1-O-alkyl-2-acetyl-sn-glycerol-3-P and (ii) remodeling pathway where PC is converted to lyso-PAF through a phospholipase D mediated loss of an acyl group in sn-2 and subsequently re-acetylated (Figure 7b). PAFC16 identity was confirmed by matching the m/z observed in biological experiments with the calculated mass (Δppm <5) and by comparing PAFC16 LC retention times between biological experiments and standard. Finally, PAFC16 MS/MS spectra were matched between biological samples and the MS/MS spectrum reported in the METLIN data base, demonstrating identical profiles of fragmented ions (Figure 7d and e; table 3). Wild type and hif1α−/− hypoxic cells showed both a clear HIF1α-independent accumulation of this bioactive lipid (Figure 7f). PAFC18 and PAF catabolism products Lyso-PAFC16 and Lyso-PAFC18 did not show any significant difference in our experiments (data not shown). HIF1α-independent PAFC16 accumulation in hypoxia was confirmed in hif1αKD DLD-1 and SW1222 colorectal cancer cells (figure S1). Also, a HIF-independent accumulation was observed in hif2αKD and hif1/2αKD DLD-1 cell lines after 24 hours of hypoxia (figure S2). Normoxic basal levels of PAFC16 were in the range of 3.8 to 13.3 femtomol/106 cells. Under hypoxia, levels increased to the range of 21.7 to 59 femtomol/106 cells. No statistical differences were observed when we compared PAFC16 levels within the different parental cell lines (wild type, hif1α−/−, hif1αKD, hif2αKD and hif1/2αKD) in normoxia and in hypoxia (table S3).


Hypoxia induces a lipogenic cancer cell phenotype via HIF1α-dependent and -independent pathways.

Valli A, Rodriguez M, Moutsianas L, Fischer R, Fedele V, Huang HL, Van Stiphout R, Jones D, Mccarthy M, Vinaxia M, Igarashi K, Sato M, Soga T, Buffa F, Mccullagh J, Yanes O, Harris A, Kessler B - Oncotarget (2015)

Platelet activating factor C16 (PAFC16) is regulated in hypoxia independently of HIF1α(a) Heat map of organic extract molecular features showing the detection of the m/z=524.3736 by nanoflow LC/MS positive mode. (b) PAF biosynthesis via de novo pathway and via remodeling pathways. Hypoxia, favoring cell membrane remodeling releases PC the substrate used for PAF biosynthesis. Abbreviations: LPCAT, acetyltransferase; PLA2, phospholipase A2; CMP, Cytidine monophosphate; Pi, phosphate inorganic; CDP-choline, Cytidine-diphosphocholine. (c) Molecular structure of PAFC16. In hypoxia PC provides the skeleton of PAFC16 (glycerol and phosphocholine); the characteristic saturated hexadecil moiety (16:0) is a derivative of palmitate reduction. Acetyl deriving from acetyl-CoA completes the structure of PAFC16. (d) PAFC16 identification was performed by LC/MS QTOF nanoflow using mass matching and retention time comparison. (e) Tandem mass (MS/MS) spectra performed by LC/MS QTOF nanoflow of experimental detection of m/z=524.3736 [M+H]+ and comparison matching with METLIN database was the third parameter used for PAFC16 identification. (f) Intracellular PAFC16 concentrations reported as femtomol/106 cells data are shown as mean ±sd, intensities were quantified by LC/MS Q Exactive (n=3). Concentration was calculated interpolating a linear range standard curve with the unknown quantified relative intensities.
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Figure 7: Platelet activating factor C16 (PAFC16) is regulated in hypoxia independently of HIF1α(a) Heat map of organic extract molecular features showing the detection of the m/z=524.3736 by nanoflow LC/MS positive mode. (b) PAF biosynthesis via de novo pathway and via remodeling pathways. Hypoxia, favoring cell membrane remodeling releases PC the substrate used for PAF biosynthesis. Abbreviations: LPCAT, acetyltransferase; PLA2, phospholipase A2; CMP, Cytidine monophosphate; Pi, phosphate inorganic; CDP-choline, Cytidine-diphosphocholine. (c) Molecular structure of PAFC16. In hypoxia PC provides the skeleton of PAFC16 (glycerol and phosphocholine); the characteristic saturated hexadecil moiety (16:0) is a derivative of palmitate reduction. Acetyl deriving from acetyl-CoA completes the structure of PAFC16. (d) PAFC16 identification was performed by LC/MS QTOF nanoflow using mass matching and retention time comparison. (e) Tandem mass (MS/MS) spectra performed by LC/MS QTOF nanoflow of experimental detection of m/z=524.3736 [M+H]+ and comparison matching with METLIN database was the third parameter used for PAFC16 identification. (f) Intracellular PAFC16 concentrations reported as femtomol/106 cells data are shown as mean ±sd, intensities were quantified by LC/MS Q Exactive (n=3). Concentration was calculated interpolating a linear range standard curve with the unknown quantified relative intensities.
Mentions: To extend the pool of identified molecular features with an altered profile in hypoxia (Figure 1d and 7a), we performed METLIN database searches, and observed an accurate mass match for m/z 524.3736 [M+H]+ corresponding to platelet activating factor 16 (PAFC16), a C16:0 monoalkylglycerol ether-derivative, esterified with an acetyl group in C2 and condensed with a Cho polar head in C3 (Figure 7b and c). PAF is a lipid synthesized through the (i) de novo pathway where a transferase adds PC to the sn-3 site of the 1-O-alkyl-2-acetyl-sn-glycerol-3-P and (ii) remodeling pathway where PC is converted to lyso-PAF through a phospholipase D mediated loss of an acyl group in sn-2 and subsequently re-acetylated (Figure 7b). PAFC16 identity was confirmed by matching the m/z observed in biological experiments with the calculated mass (Δppm <5) and by comparing PAFC16 LC retention times between biological experiments and standard. Finally, PAFC16 MS/MS spectra were matched between biological samples and the MS/MS spectrum reported in the METLIN data base, demonstrating identical profiles of fragmented ions (Figure 7d and e; table 3). Wild type and hif1α−/− hypoxic cells showed both a clear HIF1α-independent accumulation of this bioactive lipid (Figure 7f). PAFC18 and PAF catabolism products Lyso-PAFC16 and Lyso-PAFC18 did not show any significant difference in our experiments (data not shown). HIF1α-independent PAFC16 accumulation in hypoxia was confirmed in hif1αKD DLD-1 and SW1222 colorectal cancer cells (figure S1). Also, a HIF-independent accumulation was observed in hif2αKD and hif1/2αKD DLD-1 cell lines after 24 hours of hypoxia (figure S2). Normoxic basal levels of PAFC16 were in the range of 3.8 to 13.3 femtomol/106 cells. Under hypoxia, levels increased to the range of 21.7 to 59 femtomol/106 cells. No statistical differences were observed when we compared PAFC16 levels within the different parental cell lines (wild type, hif1α−/−, hif1αKD, hif2αKD and hif1/2αKD) in normoxia and in hypoxia (table S3).

Bottom Line: To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites.Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner.Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

ABSTRACT
The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

Show MeSH
Related in: MedlinePlus