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. 2018 Sep;12(9):1623-1638.
doi: 10.1002/1878-0261.12368. Epub 2018 Aug 29.

Heterogeneity of fatty acid metabolism in breast cancer cells underlies differential sensitivity to palmitate-induced apoptosis

Seher Balaban  1 , Lisa S Lee  1 , Bianca Varney  1 , Atqiya Aishah  1 , Quanqing Gao  2 , Robert F Shearer  3 , Darren N Saunders  4 , Thomas Grewal  2 , Andrew J Hoy  1
Affiliations

Heterogeneity of fatty acid metabolism in breast cancer cells underlies differential sensitivity to palmitate-induced apoptosis

Seher Balaban et al. Mol Oncol. 2018 Sep.

Abstract

Breast cancer (BrCa) metabolism is geared toward biomass synthesis and maintenance of reductive capacity. Changes in glucose and glutamine metabolism in BrCa have been widely reported, yet the contribution of fatty acids (FAs) in BrCa biology remains to be determined. We recently reported that adipocyte coculture alters MCF-7 and MDA-MB-231 cell metabolism and promotes proliferation and migration. Since adipocytes are FA-rich, and these FAs are transferred to BrCa cells, we sought to elucidate the FA metabolism of BrCa cells and their response to FA-rich environments. MCF-7 and MDA-MB-231 cells incubated in serum-containing media supplemented with FAs accumulate extracellular FAs as intracellular triacylglycerols (TAG) in a dose-dependent manner, with MDA-MB-231 cells accumulating more TAG. The differences in TAG levels were a consequence of distinct differences in intracellular partitioning of FAs, and not due to differences in the rate of FA uptake. Specifically, MCF-7 cells preferentially partition FAs into mitochondrial oxidation, whereas MDA-MB-231 cells partition FAs into TAG synthesis. These differences in intracellular FA handling underpin differences in the sensitivity to palmitate-induced lipotoxicity, with MDA-MB-231 cells being highly sensitive, whereas MCF-7 cells are partially protected. The attenuation of palmitate-induced lipotoxicity in MCF-7 cells was reversed by inhibition of FA oxidation. Pretreatment of MDA-MB-231 cells with FAs increased TAG synthesis and reduced palmitate-induced apoptosis. Our results provide novel insight into the potential influences of obesity on BrCa biology, highlighting distinct differences in FA metabolism in MCF-7 and MDA-MB-231 cells and how lipid-rich environments modulate these effects.

Keywords: DGAT; CPT1; breast cancer; fatty acid oxidation; oleate; triacylglycerols.

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Figures

Figure 1
Figure 1
Effect of extracellular lipid availability on intracellular TAG content. (A) MCF‐7 and MDA‐MB‐231 (MM231) cells were treated with a concentration range (0–450 μm) of a lipid cocktail (1 : 2 : 1 palmitate/oleate/linoleate, P/O/L) or oleate alone for 24 h and then assayed for TAG content. (B) Schematic of the pathways mapped by radiotracing of oleate metabolism. 14C‐oleate (C) uptake, (D) oxidation, and (E) incorporation into triacylglycerol (TAG) in MCF‐7 and MDA‐MB‐231 cells. *P < 0.05 compared to MCF‐7 by unpaired Student's t‐test. (F) MCF‐7 and MDA‐MB‐231 cells were treated with 1 μCi/mL [1‐14C]‐oleate and 80 μm or 450 μm cold oleate for 24 h and were subsequently treated with 10 μm triacsin C for 4 h to measure the release of TAG‐derived FAs into lipid‐free media. (G) In another cohort, MCF‐7 and MDA‐MB‐231 cells were incubated with 100 mm carnitine for 4 h and 14 CO 2 production assessed. (H) Representative immunoblots and densitometric quantitation of ATGL in MCF‐7 and MDA‐MB‐231 cells with or without prior overnight incubation with oleate. Data show means ± SEM of three independent experiments performed in triplicate. (A) †P ≤ 0.05 main effect for cells by three‐way ANOVA. (C–E) *P < 0.05 compared to MCF‐7 by unpaired Student's t‐test. (F–H) †P ≤ 0.05 main effect for cells; *P ≤ 0.05 vs. – oleate; #P ≤ 0.05 vs. MCF‐7 cells – oleate by two‐way ANOVA followed by Tukey's multiple comparisons test.
Figure 2
Figure 2
The MCF‐7 cells are not sensitive to palmitate‐induced apoptosis. MTT assays of MCF‐7 cells treated with 250 μm palmitate for 4 days with or without prior overnight incubation with (A) oleate or (B) 1 : 2 : 1 mixture of palmitate/oleate/linoleate (FA mix). MTT results are presented as percentages of MTT absorbance at indicated time points relative to that at Day 0 for each group (five independent experiments performed in quadruplicate). (C) Cell number of MCF‐7 cells treated with 250 μm palmitate for 4 days with or without prior overnight incubation with oleate. The dashed line represents the number of cells present at Day 0 (three independent experiments performed in triplicate). Data are presented as mean ± SEM. *P ≤ 0.05 vs. palmitate; #P ≤ 0.05 vs. control by two‐way ANOVA (A and B) or one‐way ANOVA (D) followed by Tukey's multiple comparisons test.
Figure 3
Figure 3
The MDA‐MB‐231 cells are sensitive to palmitate‐induced apoptosis and lipid‐loading protects MDA‐MB‐231 cells from palmitate‐induced apoptosis. MTT assays of MDA‐MB‐231 cells treated with 250 μm palmitate for 4 days with or without prior overnight incubation with (A) oleate or (B) 1 : 2 : 1 mixture of palmitate/oleate/linoleate (FA mix). MTT results are presented as percentages of MTT absorbance at indicated time points relative to that at Day 0 for each group. (five independent experiments performed in quadruplicate). (C) Representative immunoblots of cPARP and ATF4 levels of MDA‐MB‐231 cells treated with 250 μm palmitate for 1 day with or without prior overnight incubation with oleate or 1 : 2 : 1 mixture of palmitate/oleate/linoleate (FA mix; representative of three independent experiments performed in triplicate). (D) Cell number of MDA‐MB‐231 cells treated with 250 μm palmitate for 4 days with or without prior overnight incubation with oleate. The dashed line represents the number of cells present at Day 0 (three independent experiments performed in duplicate). Cell protein content of MDA‐MB‐231 cells treated with 250 μm palmitate for 4 days with or without prior overnight incubation with (E) oleate or (F) 1 : 2 : 1 mixture of palmitate/oleate/linoleate (FA mix) (three independent experiments performed in duplicate). (G) MTT assays of MDA‐MB‐231 cells serum‐starved for 4 days with or without prior overnight incubation with oleate. (H) Palmitate sensitivity of MCF‐7, BT‐474, MDA‐MB‐175, MDA‐MB‐231, BT‐549, and MDA‐MB‐468 cells expressed as the difference in MTT signal between Day 0 (D0) and Day 4 (D4) for cells cultured in serum‐containing media supplemented with 250 μm. Data are presented as mean ± SEM. *P ≤ 0.05 vs. palmitate; #P ≤ 0.05 vs. control by two‐way ANOVA (A and B) or one‐way ANOVA (D–F) followed by Tukey's multiple comparisons test. *P ≤ 0.05 vs. Day 0 MTT signal by Student's t‐test (H).
Figure 4
Figure 4
The MCF‐7 and MDA‐MB‐231 cells metabolize palmitate differently and this is selectively altered by pretreatment with oleate. 14C‐palmitate (A) uptake, (B) oxidation, (C) incorporation into triacylglycerol (TAG), (D) intracellular partitioning of 14C‐palmitate expressed as the ratio of 14C‐palmitate incorporation into triacylglycerol (storage) vs. 14C‐palmitate oxidation, 14C‐palmitate incorporation into (E) ceramide in MCF‐7 and MDA‐MB‐231 cells with or without prior overnight incubation with 150 μm oleate. Data are presented as mean ± SEM of three independent experiments performed in triplicate. †P ≤ 0.05 main effect for cells; *P ≤ 0.05 vs. – oleate; #P ≤ 0.05 vs. MCF‐7 cells – oleate by two‐way ANOVA followed by Tukey's multiple comparisons test.
Figure 5
Figure 5
Inhibition of fatty acid oxidation in MCF‐7 cells sensitizes cells to palmitate‐induced apoptosis. (A) 14C‐palmitate oxidation in MCF‐7 cells that were treated with or without 100 μM etomoxir (Eto) (five independent experiments performed in triplicate). (B) MTT assays of MCF‐7 cells treated with 250 μm palmitate (Palm), 100 μm etomoxir (Eto), or a combination for 4 days. MTT results are presented as percentages of MTT absorbance at indicated time points relative to that at Day 0 for each group (MTT: six independent experiments performed in quadruplicate). (C) Cell number and (D) protein amount of MCF‐7 cells treated with 250 μm palmitate (Palm), 100 μm etomoxir (Eto), or a combination for 4 days. The dashed line represents the number of cells present at Day 0 (three independent experiments performed in triplicate). (E) Representative immunoblots of cPARP of MCF‐7 cells treated with 250 μm palmitate, 100 μm etomoxir, or a combination for 1 day (three independent experiments performed in triplicate). Data are presented as mean ± SEM. (A) *P ≤ 0.05 vs. control by unpaired Student's t‐test. (B) *P ≤ 0.05 vs. palmitate; #P ≤ 0.05 vs. etomoxir by two‐way ANOVA followed by Tukey's multiple comparisons test. (C and D) *P ≤ 0.05 vs. palmitate; #P ≤ 0.05 vs. etomoxir by one‐way ANOVA followed by Tukey's multiple comparisons test.
Figure 6
Figure 6
Inhibition of TAG synthesis in MDA‐MB‐231 cells blunts the protective effects of prior oleate treatment to palmitate‐induced apoptosis. (A) MDA‐MB‐231 cell triacylglycerol (TAG) levels in cells treated with 300 μm oleate with or without 0.6 nm DGAT inhibitor AZD3988 (iDGAT) for 24 h (four independent experiments performed in duplicate). (B) 14C‐oleate incorporation into TAG in MDA‐MB‐231 cells that were treated with or without DGAT inhibitor (iDGAT; three independent experiments performed in duplicate). (C) MTT assays of MDA‐MB‐231 cells treated with 250 μm palmitate (Palm) for 4 days following prior incubation with 300 μM oleate (Ol) with or without 0.6 nm DGAT inhibitor AZD3988 (iDGAT). MTT results are presented as percentages of MTT absorbance at indicated time points relative to that at Day 0 for each group (four independent experiments performed in quadruplicate). (D) Representative immunoblots of cPARP of MDA‐MB‐231 cells treated with 250 μm palmitate for 1 days following prior incubation with oleate with or without DGAT1 inhibitor (three independent experiments performed in triplicate). (E) Protein amount of MDA‐MB‐231 cells treated with 250 μm palmitate for 4 days following prior incubation with 300 μM oleate with or without DGAT inhibitor (iDGAT; three independent experiments performed in triplicate). (F) Differential overall survival among breast cancer cases parsed by DGAT1 amplification. (G) Schematic of the mechanisms by which oleate pretreatment prevents palmitate‐induced apoptosis. Data are presented as mean ± SEM. (A) *P ≤ 0.05 vs. control; #P ≤ 0.05 vs. oleate by one‐way ANOVA followed by Tukey's multiple comparisons test. (B) #P ≤ 0.05 vs. oleate by unpaired Student's t‐test. (C) *P ≤ 0.05 vs. palmitate; #P ≤ 0.05 vs. control; $P ≤ 0.05 vs. palm + oleate by two‐way ANOVA followed by Tukey's multiple comparisons test. (E) *P ≤ 0.05 vs. palmitate; #P ≤ 0.05 vs. control; $P ≤ 0.05 vs. palm + oleate by one‐way ANOVA followed by Tukey's multiple comparisons test.
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