Probing the metabolic phenotype of breast cancer cells by multiple tracer stable isotope resolved metabolomics

Abstract

Breast cancers vary by their origin and specific set of genetic lesions, which gives rise to distinct phenotypes and differential response to targeted and untargeted chemotherapies. To explore the functional differences of different breast cell types, we performed Stable Isotope Resolved Metabolomics (SIRM) studies of one primary breast (HMEC) and three breast cancer cells (MCF-7, MDAMB-231, and ZR75-1) having distinct genotypes and growth characteristics, using ¹³C₆-glucose, ¹³C-1+2-glucose, ¹³C₅,¹⁵N₂-Gln, ¹³C₃-glycerol, and ¹³C₈-octanoate as tracers. These tracers were designed to probe the central energy producing and anabolic pathways (glycolysis, pentose phosphate pathway, Krebs Cycle, glutaminolysis, nucleotide synthesis and lipid turnover). We found that glycolysis was not associated with the rate of breast cancer cell proliferation, glutaminolysis did not support lipid synthesis in primary breast or breast cancer cells, but was a major contributor to pyrimidine ring synthesis in all cell types; anaplerotic pyruvate carboxylation was activated in breast cancer versus primary cells. We also found that glucose metabolism in individual breast cancer cell lines differed between in vitro cultures and tumor xenografts, but not the metabolic distinctions between cell lines, which may reflect the influence of tumor architecture/microenvironment.

Keywords: (13)C(3)-glycerol; (13)C(3)-glycerol (PubChem CID:753); (13)C(5); (13)C(5),(15)N(2)-Glutamine (PubChem CID:5961); (13)C(6)-glucose; (13)C(6)-glucose (PubChem CID:5793); (13)C(8)-octanoate; (13)C(8)-octanoate (PubChem CID:11939); (15)N(2)-Gln; (PubChem CID:5927); 1D/2D NMR; ATP; Breast cancer metabolism; FT-ICR-MS; Mouse xenografts; Stable isotope-resolved metabolomics; UTP(PubChem CID:6133); alanine (PubChem CID:5950); aspartate (PubChem CID:5460541); glutamate (PubChem CID:5128032); lactate (PubChem CID:91435).

Figure 1. Carbon fates in central metabolism

Observed fates of glucose and glutamine-derived carbon in breast cancer cells. The inner dashed box represents mitochondrial reactions. 1 glucose transporter; 2 hexokinase; 3 phosphoglucomutase; 4 aldolase; 5 lactate dehydrogenase; 6 alanine amino transferase; 7 glucose/hexosamine pathway; 8 pentose phosphate pathway: oxidative branch; 9 pentose phosphate pathway: non-oxidative branch ; 10, pyrimidine nucleotide synthesis; 11 hexosamine pathway; 12 glycogen synthesis; 13 phosphoglycerate dehydrogenase; 14 glycerol-3-phosphate dehydrogenase 15 pyruvate dehydrogenase; 16 pyruvate carboxylase; 17 citrate synthase; 18, 18′ malate dehydrogenase; 19,19′ aspartate aminotransferase; 20 malic enzyme; 21 glutaminase; 22 ATP-dependent citrate lyase; 23 fatty acid synthesis; 24 purine biosynthesis.

Figure 2. 2D ¹H TOCSY Spectra of four breast cell lines grown in the presence of ¹³C₆-glucose show differential capacity for ribose incorporation into nucleotides

TCA extracts were prepared from cells grown for 24 h in the presence of ¹³C₆-glucose. 2D TOCSY contour maps were shown along with the corresponding 1D high-resolution ¹H spectra. Dashed boxes delineate ¹³C satellite cross-peaks for the H1′ (x-axis) to H2′ and H3′ (y-axis) in the ribosyl unit of adenine (AXP) and H1′ to H2/H3′ in the ribosyl unit of uracil (UXP) nucleotides. Such ¹³C labeling patterns are consistent with the presence of ¹³C₅-ribose subunits in both types of nucleotides, which in turn suggests the operation of PPP via the oxidative and/or non-oxidative branches. Values in parenthesis denote fractional ¹³C enrichment in the ribosyl unit of AXP or UXP at the 1′ position. A. HMEC; B. MCF-7; C. ZR-75-1; D. MDA-MB-231

Figure 3. ¹³C atom tracing of ¹³C-1+¹³C-2-glucose transformations via glycolysis, PPP, and nucleotide (NT) biosynthesis

TCA extracts were prepared from cells grown for 24 h in the presence of equimolar ¹³C-1- and ¹³C-2-glucose. ● denotes ¹²C while , represent ¹³C derived from OxPPP and NOxPPP, respectively. Ox PPP and NOx PPP: oxidative and non-oxidative branches of the pentose phosphate pathway, respectively; Glc: glucose; F6P: fructose-6-phosphate; GAP: glyceraldehyde-3-phosphate; Pyr: pyruvate; Lac: lactate; R5P: ribose-5-phosphate; X5P: xylulose-5-phosphate; Sed7P: sedoheptulose-7-phosphate; NXP: nucleotides; TK: transketolase; TA: transaldolase

Figure 4. 2D ¹H TOCSY Spectra of three breast cell lines grown in the presence of ¹³C-1-+¹³C-2-glucose reveal differential capacity for oxidative and non-oxidative branches of the pentose phosphate pathway

TCA extracts were prepared from cells grown for 24 h in the presence of ¹³C-1-+¹³C-2 glucose. ¹³C labeling patterns in the ribosyl subunits of nucleotides. These labeling pattern showed that both oxidative and non-oxidative branches of the PPP were active. Dashed lines indicated ¹³C satellite cross-peaks at given atomic positions of metabolites.

Figure 5

¹³C tracing from ¹³C₆-Glc or ¹³C₅,¹⁵N₂-Gln into uracil ring. The two sets of atom tracing depict the transformation of ¹³C₆-Glc (A) or ¹³C₅,¹⁵N₂-Gln (B) into Asp via the first turn of the Krebs cycle, and then into the uracil ring of UMP via the pyrimidine synthesis pathway. ●: ¹²C; : ¹³C from pyruvate dehydrogenase-initiated Krebs cycle in A or from glutaminolysis+Krebs cycle in B; : ¹³C from pyruvate carboxylase-initiated Krebs cycle ; : ¹³C from glutaminolysis+Krebs cycle+malic enzyme (Le, Lane et al. 2012); DHO : dihydroorotate.

Figure 6. ¹³C incorporation from ¹³C₆-glucose versus ¹³C₅,¹⁵N₂-Gln into pyrimidine nucleotides in three breast cell lines

Cells were grown for 24 h in the presence of 5 mM each labeled glutamine (A) or glucose (B) tracer. 2D TOCSY spectra were recorded at 600 or 800 MHz using a mixing time of 50 ms along. The 2D TOCSY contour maps were shown along with the 1D high-resolution ¹H spectra. Dashed boxes depicted the ¹³C satellites of C5 to C6 cross-peaks of the uracil ring in UXP with horizontal pairs, vertical pairs, and 4-corner satellites representing ¹³C labeling at C5, C6, and C5,6 of uracil, respectively.

Figure 7

MDAMB-231 cells synthesize less lipids from glucose than other breast cell lines despite its higher proliferation rate. HMEC (A), MCF-7 (B) and MDA-MB-231 (C) cells were grown for 24 h in the presence of ¹³C₆-glucose and extracted as described in the Methods. The lipid extracts were dissolved in d₄-methanol, and TOCSY spectra were recorded at 14.1 T with an isotropic mixing time of 50 ms at 25 °C. Both HMEC (A) and MCF-7 (B) cells showed extensive ¹³C enrichment (as ¹³C satellite cross-peaks, green boxes) in the glycerol subunit and fatty acyl chains, which was less in extent for the glycerol subunit and absent for the fatty acyl chains in MDAMB-231 cells (C). Blue boxes denote covalent linkages of protons attached to ¹²C of the glycerol backbone and fatty acyl chains. 1/3, 2: ¹³C satellites, ¹²C-attached unsaturated protons, respectively; 3, 4: ¹²C-attached, ¹³C satellite of H2 of glycerol backbone, respectively; 5, 6/7: ¹³C satellite,¹²C-attached H1 of glycerol backbone, respectively; 8/10, 9: ¹³C satellites, ¹²C-attached H3 of glycerol backbone, respectively; 11, 12: : ¹³C satellites, ¹²C-attached H2 of fatty acyl chains, respectively; 13, 14: ¹²C-attached, ¹³C satellite of H11 of fatty acyl chains, respectively; 15, 16: ¹²C-attached, ¹³C satellite of H3 of fatty acyl chains, respectively.

Figure 8. Differential incorporation of carbon from different sources into phospholipids of ZR-75-1 cells

Cells were grown in the presence of 20% oxygen and ¹³C₆-glucose (A), ¹³C₅,¹⁵N₂-glutamine (B), ¹³C₃-glycerol (C) or ¹³C₈-octanoate (D) for 72 h, and extracted for phospholipids (PL) with methanol. The TOCSY spectra were recorded at 18.8 T, 20 °C. Blue and green boxes respectively denote ¹H connectives between ¹²C-attached protons and ¹³C satellites of ¹³C-attached protons. See Fig. 7 for the assignment of each numbered peaks.

Figure 9. ¹³C incorporation from ¹³C₈-octonate into fatty acyl chains of lipids in ZR75-1 cells via beta-oxidation and malic enzyme activity

The ¹³C atom in ¹³C₈-octanoate is traced into lipids via the reactions of β-oxidation, Krebs cycle, malic enzyme (ME), pyruvate dehydrogenase (PDH), ATP-citrate lyase (ACL), acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS). Double dashed lines delineate mitochondria from the cytoplasm. OAA: oxaloacetate.

Figure 10. FT-ICR-MS analysis of lipids extracted from three breast cell lines reveals distinct capacity of ¹³C₆-glucose in fueling de novo lipid synthesis

Lipids were extracted from cells grown in the presence of ¹³C₆-glucose and analyzed by FT-ICR-MS as described in the Methods. A. the m/z range of MDAMB-231, MCF-7, and HMEC extracts dominated by diacylglycerophospholipids; B the expanded m/z range dominated respectively by is PC 32:1+H labeled in all 3 cell types. The prominent, labeled peaks are the isotopologues (blue box from A); C. expansion red box of panel A: assignments are as follows: MDA is PS-pmg 40:7 +H (820.548398 m/z for m0 ; MCF7 is PE 40:7+Na -m0 (812.513658 m/z) is at the base of the prominent peak (unlabeled). HMEC is PC-pmg 40:7 +H. The m0 (818.602868 m/z) was not detected.

Figure 11. ¹³C₆-glucose transformations in ZR75-1 and MDAMB-231 cells as mouse xenografts show similar metabolic distinctions as observed in vitro

Orthotopic tumors were grown from ZR75-1 or MDAMB-231 cells as described in the Methods. Mice were treated with 20 mg each ¹³C₆-glucose via three tail vein injections at a 15-min interval (Lane, Yan et al. 2015). The tumors were harvested, polar metabolites extracted, and analyzed by 1D ¹H{¹³C}-HSQC at 14.1 T and 20 °C in A. ZR75-1 and MDAMB231 cells in B were cultured in DMEM medium containing 0.1% ¹³C₆-glucose for 24 h before extraction of polar metabolites with 10% trichloroacetic acid (MDAMB231) or 60% acetonitrile (ZR75-1) and 1D HSQC analysis as described in Methods. Some of the metabolites in B such as lactate (Lac), Ala, Glu, Asp, and adenine nucleotides (AXP) displayed different chemical shifts between the extracts of the two cell lines due to their pH difference.