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A splicing switch from ketohexokinase-C to ketohexokinase-A drives hepatocellular carcinoma formation

A Corrigendum to this article was published on 27 May 2016

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Abstract

Dietary fructose is primarily metabolized in the liver. Here we demonstrate that, compared with normal hepatocytes, hepatocellular carcinoma (HCC) cells markedly reduce the rate of fructose metabolism and the level of reactive oxygen species, as a result of a c-Myc-dependent and heterogeneous nuclear ribonucleoprotein (hnRNP) H1- and H2-mediated switch from expression of the high-activity fructokinase (KHK)-C to the low-activity KHK-A isoform. Importantly, KHK-A acts as a protein kinase, phosphorylating and activating phosphoribosyl pyrophosphate synthetase 1 (PRPS1) to promote pentose phosphate pathway-dependent de novo nucleic acid synthesis and HCC formation. Furthermore, c-Myc, hnRNPH1/2 and KHK-A expression levels and PRPS1 Thr225 phosphorylation levels correlate with each other in HCC specimens and are associated with poor prognosis for HCC. These findings reveal a pivotal mechanism underlying the distinct fructose metabolism between HCC cells and normal hepatocytes and highlight the instrumental role of KHK-A protein kinase activity in promoting de novo nucleic acid synthesis and HCC development.

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Figure 1: hnRNPH1/2 expression switches KHK-C expression to KHK-A expression in HCC cells.
Figure 2: c-Myc-enhanced hnRNPH1/2 expression switches from KHK-C expression to KHK-A expression in HCC cells.
Figure 3: KHK-A phosphorylates PRPS1 at Thr225.
Figure 4: KHK-A-mediated PRPS1 phosphorylation activates PRPS1.
Figure 5: KHK-A promotes de novo nucleic acid synthesis and reduces ROS production.
Figure 6: KHK-A-dependent phosphorylation of PRPS1 promotes hepatocellular tumorigenesis and is associated with the pathogenesis of HCC.

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  • 27 April 2016

    In the original version of this Article, the affiliations for Gilbert Cote, Hongxia Wang and Liwei Wang were incorrect. In addition, in the Acknowledgements section, 'National Science Foundation of China' should have read 'National Natural Science Foundation of China'. These errors have been corrected in all online versions.

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Acknowledgements

We thank B.-F. Pan for technical support and D. Norwood for critical reading of this manuscript. This work was supported by National Cancer Institute grants 2R01 CA109035 (Z.L.) and 1R0 CA169603 (Z.L.), National Institute of Neurological Disorders and Stroke grant 1R01 NS089754 (Z.L.), MD Anderson Support Grant CA016672, the James S. McDonnell Foundation 21st Century Science Initiative in Brain Cancer Research Award 220020318 (Z.L.), 2P50 CA127001 (Brain Cancer SPORE), a Sister Institution Network Fund from MD Anderson (Z.L.; C.-N.Q.), NIH High-End Instrumentation program grant 1S10OD012304-01 (D.H.H.), CPRIT Core Facility Grant RP130397 (D.H.H.), the Odyssey Fellowship from MD Anderson (X.L.), and research grants 81272340 (C.-N.Q.) and 81472386 (C.-N.Q.) from the National Natural Science Foundation of China. Z.L. is a Ruby E. Rutherford Distinguished Professor.

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This study was conceived by Z.L. and X.L.; Z.L. and X.L. designed the study; X.L., X.Q., L.-X.P., Y.J., D.H.H., Y.Z., Y.X., J.-H.L. and G.C. performed experiments; L.W., H.W. and C.-N.Q. provided reagents and technical assistance; Z.L. and X.L. wrote the paper with comments from all authors.

Corresponding author

Correspondence to Zhimin Lu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 Pathways of fructose metabolism, glycolysis, tricarboxylic acid (TCA) cycle-derived fatty acids, and de novo synthesis of nucleotides and nucleic acids from the pentose phosphate pathway (PPP).

Supplementary Figure 5 hnRNPH1/2 expression switches KHK-C expression to KHK-A expression in HCC cells.

(a) Total RNA was extracted from 30 human HCC and paired non-tumor tissue samples. The RT-PCR products were digested by BsmAI and resolved on agarose gels. N, non-tumor tissue; T, tumor tissue. (b) Immunoprecipitation of hnRNPH1/2 (bottom left panel) from Hep3B cells was followed by RT-PCR analysis of hnRNPH1/2-associated RNAs (bottom right panel) with the indicated primers (top panel). (c) Amino acid sequences of hnRNPH1 and hnRNPH2 were aligned. Different amino acids in two proteins are highlighted. (d) Schematic diagram of synthesized pre-mRNAs of AdML and the indicated AdML-KHK exon 3C fused products (left panel). Incubation of the synthesized pre-mRNAs with a Hep3B nuclear extract with or without hnRNPH1/2 depletion was followed by PCR analysis (right panel).

Supplementary Figure 6 KHK-A but not KHK-C phosphorylates PRPS1 at Thr225.

(a) Purified recombinant GST-KHK-A was incubated with immobilized purified recombinant His-PRPS1 or His-PRPS2. An in vitro pulldown assay was performed. Immunoblot analysis was conducted with the indicated antibodies. (b) Schematic diagram of the restriction site of HindIII in PRPS1 cDNA (left panel). Total RNA was extracted from normal hepatocytes and the indicated HCC cells (right panel). (c) The Km of purified GST-KHK-A for fructose with representative plotting of 1/V vs. 1/[fructose] (left panel) or for purified His-PRPS1 with representative plotting of 1/CPM vs. 1/[PRPS1] (right panel) was determined. (d) Hep3 cells were centrifuged and washed with PBS. 100 μl of the cell pellets with removal of PBS were mixed with 900 μl lysate buffer. 10 μl cell lysate was mixed with 10 μl of purified recombinant his-PRPS1 with the indicated concentrations were analyzed by immunoblot assay with an anti-PRPS antibody. (e) Bacterially purified indicated KHK-A and PRPS1 proteins were stained with colloidal coomassie blue. (f) In vitro phosphorylation analysis, SDS-PAGE and autoradiography were performed by mixing purified GST-KHK-A with purified His-PRPS1 or His-PRPS2 in the presence of [γ32P]-ATP. Immunoblot analysis was conducted with the indicated antibodies. (g) Hep3B cells were transiently transfected with a vector expressing the indicated Flag-PRPS proteins. Immunoblot analysis was conducted with the indicated antibodies. (h) GST pull-down analysis was performed by mixing purified immobilized WT GST-KHK-A, GST-KHK-A G257R on glutathione agarose beads with purified His-PRPS1. (i) [γ32P]-ATP was mixed with WT KHK-A or the KHK-A G257R mutant. KHK-A–bound [γ32P]-ATP was measured. The data represent the mean ± SD from n = 3 independent experiments. (j) In vitro phosphorylation assays were performed by mixing purified WT KHK-A or KHK-C with purified PRPS1 in the presence of ATP. Immunoblot analysis was conducted with the indicated antibodies (k) Hep3B cells were cultured in the medium with added fructose with the indicated concentrations. Immunoblot analysis was conducted with the indicated antibodies.

Supplementary Figure 7 Phosphorylated PRPS1 was not regulated by the allosteric regulators.

(a) Activity of bacterially purified WT His-PRPS1 was measured in the presence or absence of phosphate (50 mM) ADP (100 μM). The data represent the mean ± SD from n = 3 independent experiments. (b) The activity of bacterially purified WT His-PRPS1 with or without phosphorylation by GST-KHK-A was measured in the presence or absence of IMP (100 μM), AMP (100 μM), or GMP (100 μM). The data represent the mean ± SD from n = 3 independent experiments. Immunoblot analysis was conducted with the indicated antibodies. (c) The activity of bacterially purified WT His-PRPS1 was measured in the presence or absence of phosphate (50 mM), IMP (100 μM), AMP (100 μM), or GMP (100 μM). The data represent the mean ± SD from n = 3 independent experiments. Immunoblot analysis was conducted with the indicated antibody. (d) Immunoprecipitation analyses with PRPS1 pT225 antibody was performed using Hep3B cell lysates. Immunoblot analysis was conducted with the indicated antibodies. The amount of phosphorylated PRPS1 was quantified. HC represents heavy chain.

Supplementary Figure 8 KHK-A-mediated PRPS1 phosphorylation promotes glucose-derived de novo nucleotide synthesis.

(a) Intracellular IMP levels in lysates of Hep3B cells expressing hnRNPH1/2 shRNA were measured. The data represent the mean ± SD from n = 3 independent experiments. A two-tailed Student’s t test was used. represents P < 0.01 between the cells with or without hnRNPH1/2 depletion. (b) Intracellular IMP levels in lysates of Hep3B cells expressing KHK shRNA with or without reconstituted expression of WT rKHK-A, rKHK-A L83A, or WT rKHK-C were measured. The data represent the mean ± SD from n = 3 independent experiments. A two-tailed Student’s t test was used. represents P < 0.01 between the cells with or without KHK depletion. #represents P < 0.01 between the KHK-depleted cells reconstituted expression of WT rKHK-A and the KHK-depleted cells reconstituted expression of rKHK-A L83A and WT rKHK-C. (c) Intracellular IMP levels in lysates of Hep3B cells expressing PRPS1 shRNA with or without reconstituted expression of WT rPRPS1, rPRPS1 T225A, rPRPS1 A190T were measured. The data represent the mean ± SD from n = 3 independent experiments. A two-tailed Student’s t test was used. represents P < 0.01 between the cells with or without PRPS1 depletion. #represents P < 0.01 between the PRPS1-depleted cells reconstituted expression of WT rPRPS1 and the rPRPS1 T225A. (d,e) Hep3B cells with reconstituted expression of KHK-C or KHK-A were cultured in the presence or absence of fructose (10 mM). The intracellular levels of AT (d) and phosphate (e) were measured. The data represent the mean ± SD from n = 3 independent experiments. A two-tailed Student’s t test was used. represents P < 0.01; NS represents not significant difference between the indicated cells with or without fructose treatment.

Supplementary Figure 9 KHK-A-mediated PRPS1 phosphorylation promotes hepatocellular tumorigenesis and is associated with the pathogenesis of HCC.

(a) The apoptosis of Hep3B and Huh-7 cells with depletion of hnRNP1/2 (left panel), KHK-A (middle panel), and PRPS1 (right panel) and with or without reconstituted expression of the indicated proteins were examined. The data represent the mean ± SD from n = 3 independent experiments. (b) The proliferation rates of Hep3B cells with the depletion of KHK-A and reconstituted expression of the indicated proteins were examined in the presence or absence of added fructose (10 mM) and NAC (2 mM). The data represent the mean ± SD from n = 3 independent experiments. P < 0.05. (c) Lysates of tumors derived from Huh-7 cells with KHK or PRPS1 depletion and with or without reconstituted WT rKHK-A, rKHK-A L83A, WT rKHK-C, WT rPRPS1, or rPRPS1 T225A expression were prepared. Immunoblot analyses with the indicated antibodies were conducted (d) The antibody specificities were validated. Immunohistochemical analysis of the tissues from the tumors derived from Huh-7 cells (left and right panel) or Huh-7 cells with KHK depletion and reconstituted expression of WT rKHK-C (middle panel) with the indicated antibodies was performed in the presence or absence of blocking peptides specific for KHK Exon 3A- or Exon 3C-coded regions or phosphorylated PRPS1 T225. (e) IHC staining of n = 90 human HCC samples with anti-phospho-PRPS1 T225, anti-KHK-A, anti-hnRNPH1/2, and anti-c-Myc antibodies was performed. Chi-square analysis was performed depending on the staining score for each antibody (high staining score 4.1-8.0; low staining score 0-4).

Supplementary Table 1 Multivariate analysis of c-Myc, hnRNPH1/2, KHK-A, and PRPS1 pT225 expression in HCC samples and patient information (Cox regression model).
Supplementary Table 2 The antibody information.
Supplementary Table 3 The PCR primers and shRNA information.

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Li, X., Qian, X., Peng, LX. et al. A splicing switch from ketohexokinase-C to ketohexokinase-A drives hepatocellular carcinoma formation. Nat Cell Biol 18, 561–571 (2016). https://doi.org/10.1038/ncb3338

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