Ketohexokinase-mediated fructose metabolism is lost in hepatocellular carcinoma and can be leveraged for metabolic imaging

Abstract

The ability to break down fructose is dependent on ketohexokinase (KHK) that phosphorylates fructose to fructose-1-phosphate (F1P). We show that KHK expression is tightly controlled and limited to a small number of organs and is down-regulated in liver and intestinal cancer cells. Loss of fructose metabolism is also apparent in hepatocellular adenoma and carcinoma (HCC) patient samples. KHK overexpression in liver cancer cells results in decreased fructose flux through glycolysis. We then developed a strategy to detect this metabolic switch in vivo using hyperpolarized magnetic resonance spectroscopy. Uniformly deuterating [2-¹³C]-fructose and dissolving in D₂O increased its spin-lattice relaxation time (T₁) fivefold, enabling detection of F1P and its loss in models of HCC. In summary, we posit that in the liver, fructolysis to F1P is lost in the development of cancer and can be used as a biomarker of tissue function in the clinic using metabolic imaging.

Fig. 1.. KHK expression is limited spatially and temporally and lost in cancer.

(A) Immunohistochemistry (IHC) of embryonic mouse tissue demonstrates lack of expression on E9 with positive staining evident from E14 across to E16. (B) Adult human tissues demonstrate staining in the liver, intestines, and kidney with no staining in other tissues. (C) Murine liver sections of normal BL6 and models of liver cancer. T represents tumor, while B represents regions of the liver. (D) Representative images in normal and diseased liver. Scale bars, 500 μm. (E) Semiquantitative scoring of normal liver (n = 9) with a mean ± SD value of 241.1 ± 49.6, steatosis (n = 3) 133.3 ± 57.5, adenoma (n = 7) 135.7 ± 63.3, and HCC (n = 8) 70.0 ± 23.9. (F) Western blots showing loss of proteins associated with fructose metabolism in cancer compared to normal with associated (G) densitometric analysis. ns, not significant; GAPDH, glyceraldehyde phosphate dehydrogenase. (H) Cells incubated with 10 μM of the fluorescent fructose analog, 1-NBDF. Fluorescence intensity displayed statistically significant reduction of 1-NBDF uptake in HCC cells compared to HepG2. AU, arbitrary units. (I) Representative ¹³C NMR spectra of cells supplemented with [2-¹³C]-fructose. Fructose consumption in media (J) of HepG2 cells increased over 24 hours, with 2.8 ± 0.9 mM fructose consumed compared to 0.8 ± 0.2 mM in Huh7. F1P levels in HepG2 cells (K) peaked 4 hours after labeling at 0.19 ± 0.06 mM while undetectable in Huh7. (L) HepG2 cells grown in 25 mM fructose proliferated at similar rates to control, while Huh7 cells were unable to grow at the same rate. All P values were calculated using a Student’s t test with *P < 0.05. DMEMHG, Dulbecco’s modified Eagle’s medium high glucose.

Fig. 2.. KHK overexpression shunts ¹³C fructose through F1P and reduced glycolysis.

(A) Fluorescent micrographs of Huh7 cells transiently transfected with plasmid expression KHKC-2A-GFP. A progressive loss of fluorescent cells was observed in the presence of DMEM supplemented with 12.5 mM fructose, indicating negative selection of HCC cells that express KHK. (B) Similar results were quantified with transfected Hep3B cells, with only 12.5 ± 1.2% fluorescent cells remaining after 6 days of culture in the presence of fructose (n = 3 biological replicates are used per group, and statistics were calculated using a Student’s t test with a *P < 0.05). (C) Representative Western blot demonstrating increased expression of KHK for stable HepG2 and Huh7 cell lines as compared to control virus. (D) Representative ¹³C NMR data showing increased generation of intracellular [2-¹³C] fructopyraonse-1-phosphate after incubation with [2-¹³C]fructose for 4 hours in KHK overexpression cells as compared to control vector. (E) Quantitation of ¹³C F1P generated in 4 hours (means ± SD, n = 3 biological replicates). (F) Metabolic scheme highlighting the isotope labeling of glycolytic intermediates and the TCA cycle when cells are rapidly exposed to [U-¹³C]fructose. ADP, adenosine diphosphate; NADH, reduced form of NAD⁺; DHAP, glyceraldehyde and dihydroxyacetone phosphate; CoA, coenzyme A; GA, glyceraldyhyde; GA3P, glyceraldehyde 3-phosphate; alphaKG, α-Ketoglutaric acid. (G) Key enriched intermediates from [U-¹³C]fructose are reduced with KHK overexpression in Huh7 cells (means ± SD, n = 3 biological replicates). N.D., not determined. (H) Total ATP and (I) AMP pools as well as the (J) ATP/AMP ratio are significantly reduced with KHK overexpression in Huh7 cells (means ± SD, n = 3 biological replicates). All P values were calculated using a Student’s t test with *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 3.. Metabolic imaging of liver disease using Hyperpolarized [2-¹³C]fructose.

(A) Synthesis of deuterated [2-¹³C]fructose 3. RT, room temperature. (B) Dynamic spectra of HP [2-¹³C]fructose dissolved in H₂O (black) and HP 3 dissolved in D₂O (red) on a 1-T ¹³C NMR spectrometer shows prolonged hyperpolarized signal as a result of substrate deuteration and dissolution in D₂O. (C) Quantification of T₁ values measured using the area under the curve obtained from dynamic spectra. (D) Calculated T₁ values were found to be 18.9 ± 0.1 s for HP [2-¹³C]fructose dissolved in H₂O and 92.5 ± 17.2 s for HP 3 dissolved in D₂O. (E) Schematic of [2-¹³C]fructose injection in the autochthonous model of liver cancer, at 10 weeks of age. The autochthonous mouse model was imaged using hyperpolarized (HP) [2-¹³C]fructose MRI at 10 weeks of age against age-matched controls (F). (G) Healthy livers produce F1P as shown by HP MRI, whereas disease state livers exhibit decreased production of F1P. Typical spectra of healthy and disease state livers obtained from HP MRI infusions showing decreased production of F1P in liver disease. (H) The observed ratio of F1P to (fructose + F1P) using HP fructose imaging can be used to diagnosis liver cancer. (I) The observed ratio of the HP furanose peak to total carbon signal is significantly increased in liver disease and can be used to diagnosis liver cancer (means ± SD, n = 4 normal and n = 3 10-week tumor biological replicates). All P values were calculated using a Student’s t test with *P < 0.05 and **P < 0.01.

Fig. 4.. Isotope tracing of fructose metabolism in liver cancer.

(A) Scheme of the rapid isotope tracing experiment. (B) Western blot analysis showing progress loss of KHK with age. (C) Immunohistochemistry confirming loss of KHK when comparing 10-week to age-matched normal. (D) Plasma [2-¹³C]fructose, (E) [2-¹³C]glucose, and (F) [2-¹³C]lactate measured in mice, 5 min after injection of [2-¹³C]fructose. (G) [2-¹³C]fructose and (H) [2-¹³C]fructose-1-phsophate in the mouse liver per tumor 5 min after injection of [2-¹³C]fructose (means ± SD, n = 5 normal, n = 3 4-week, n = 5 8-week, and n = 4 10-week tumor biological replicates). All P values were calculated using a Student’s t test with *P < 0.05, **P < 0.01, and ***P < 0.001.