PMC

Lactate, the endogenous agonist of HCA₁, is an indicator for increased rates of glycolysis. Excess acetyl-CoA is converted to ketone bodies, one of which is 3HB - the endogenous agonist of HCA₂ and 3HO, agonist of HCA₃ is an intermediate of FAO. FFA: free fatty acid.

(A) Crystal violet staining of BT-474, HCC1954 MCF12A and HEK293T cells transfected with siRNA directed against HCA₁, HCA₂ and HCA₃ versus scrambled negative control (siNC) after 48h. Cell viability of siHCA₁ (B), siHCA₂ (C) and siHCA₃ (D) versus siNC transfected BT-474, HCC1954 (both: 24h: n = 3, 48h: n = 4, 72h: n = 6), HCC38 (24h, 48h, 72h: each n = 3) MCF12A and HEK293T (both 72h: n = 3) cells. All data is shown as mean ± SEM. p-values were determined using a two-tailed unpaired t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

(A) Caspase 3/7 activity in siHCA₁ versus siNC transfected BT-474, HCC1954 and HCC38 cells. (B) Z-VAD-FMK blocks siHCA₁ induced apoptosis in HCC1954 and HCC38 cells. (C) siHCA₃ transfection induces caspase 3/7 activity in all three breast cancer cell lines. (D) siHCA₃ induced caspase 3/7 activity is diminished in the presence of 30 μM Z-VAD-FMK (24h after transfection: BT-474, HCC38; 48h post-transfection: HCC1954). All data is shown as mean ± SEM of three independent experiments carried out in triplicates. p-values were determined using a two-tailed unpaired t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

(A-C) HCA agonist-mediated inhibition of forskolin-induced cAMP production in BT-474 cells was determined using the AlphaScreen® cAMP Assay Kit. The cyclic AMP level of cells stimulated with 10 μM forskolin in absence of agonist was set 1. EC₅₀ values were determined from concentration-response curves of agonists using GraphPad Prism (n = 3). (D-F) Agonist-induced decrease in intracellular cAMP levels is pertussis toxin sensitive (n = 4). 3,5 DHB: 3,5-dihydroxybenzoic acid, MMF: mono-methyl fumarate, IPBT-5CA (IBC-293): 1-(1-Methylethyl)-1H-benzotriazole-5-carboxylic acid. All data is shown as mean ± SEM. p-values were determined using a two-tailed unpaired t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

(A) Metabolites differing in medium of siHCA₃ versus siNC transfected BT-474 cells point towards an increased FAO rate in cells with knocked-down HCA₃. Total siRNA concentration in each well was 60 nM. Concentration of HCA₃-specific siRNA is specified on the x-axis inside the parentheses. FAO intermediates were determined using LC-MS (n = 2) and normalized to viable cells (shown in ). The data shown reflects relative metabolite quantity produced per metabolically active cells. The value determined for medium of siNC transfected BT-474 was set 100%. Peak height of compounds relative to siNC transfected cells are stated in . BT-474 and HCC1954 cell viability with knocked-down HCA₃ is rescued upon co-administration of (B) 30 μM etomoxir (n = 4) or (C) 2.5 μM perhexiline (n = 3), both inhibitors of FAO. All data is shown as mean ± SEM. ^# P ≤ 0.1* P ≤ 0.05; ** P ≤ 0.01.

(A-C) Expression of HCAs in breast cancer (n = 9) versus normal (n = 3) patient tissue (two-tailed unpaired t-test, Welch's correction). (D-F) Expression of HCAs in primary human breast cancer cells (n = 3) versus non-tumorigenic epithelia breast cells MCF12A (two-tailed unpaired t-test, Welch's correction). (G-I) HCA expression in breast cancer cells BT-474, HCC1954 and HCC38 versus non-tumorigenic epithelia breast cells MCF12A (n = 6 for all except for HCC38 n = 3, ordinary One-Way ANOVA, Dunnett's multiple comparisons test). (J-L) mRNA expression of HCA in two leukemia cell lines (CEM, HL60), two lung cancer cell lines (DMS53, A549) and one prostate cancer cell line (LNCaP) versus non-tumorigenic epithelia breast cells MCF12A (n = 3 except for MCF12A n = 6, ordinary One-Way ANOVA, Dunnett's multiple comparisons test). Data is shown as mean ± SEM. ^# P ≤ 0.1* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.