Abstract
Impairment of the circadian clock is linked to cancer development. However, whether the circadian clock is modulated by oncogenic receptor tyrosine kinases remains unclear. Here we demonstrated that receptor tyrosine kinase activation promotes CK2-mediated CLOCK S106 phosphorylation and subsequent disassembly of the CLOCK–BMAL1 dimer and suppression of the downstream gene expression in hepatocellular carcinoma (HCC) cells. In addition, CLOCK S106 phosphorylation exposes its nuclear export signal to bind Exportin1 for nuclear exportation. Cytosolic CLOCK acetylates PRPS1/2 K29 and blocks HSC70-mediated and lysosome-dependent PRPS1/2 degradation. Stabilized PRPS1/2 promote de novo nucleotide synthesis and HCC cell proliferation and liver tumour growth. Furthermore, CLOCK S106 phosphorylation and PRPS1/2 K29 acetylation are positively correlated in human HCC specimens and with HCC poor prognosis. These findings delineate a critical mechanism by which oncogenic signalling inhibits canonical CLOCK transcriptional activity and simultaneously confers CLOCK with instrumental moonlighting functions to promote nucleotide synthesis and tumour growth.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Mass spectrometry data have been deposited in ProteomeXchange with the accession code PXD037738. UniProt protein database (EMBL-EBI) was used for protein identification. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
References
Kinouchi, K. & Sassone-Corsi, P. Metabolic rivalry: circadian homeostasis and tumorigenesis. Nat. Rev. Cancer 20, 645–661 (2020).
Bass, J. & Takahashi, J. S. Circadian integration of metabolism and energetics. Science 330, 1349–1354 (2010).
Sahar, S. & Sassone-Corsi, P. Metabolism and cancer: the circadian clock connection. Nat. Rev. Cancer 9, 886–896 (2009).
Reinke, H. & Asher, G. Crosstalk between metabolism and circadian clocks. Nat. Rev. Mol. Cell Biol. 20, 227–241 (2019).
Patke, A., Young, M. W. & Axelrod, S. Molecular mechanisms and physiological importance of circadian rhythms. Nat. Rev. Mol. Cell Biol. 21, 67–84 (2020).
Ye, Y. et al. The genomic landscape and pharmacogenomic interactions of clock genes in cancer chronotherapy. Cell Syst. 6, 314–328 e312 (2018).
Papagiannakopoulos, T. et al. Circadian rhythm disruption promotes lung tumorigenesis. Cell Metab. 24, 324–331 (2016).
Fu, L., Pelicano, H., Liu, J., Huang, P. & Lee, C. The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111, 41–50 (2002).
Lee, S., Donehower, L. A., Herron, A. J., Moore, D. D. & Fu, L. Disrupting circadian homeostasis of sympathetic signaling promotes tumor development in mice. PLoS One 5, e10995 (2010).
Ruan, W., Yuan, X. & Eltzschig, H. K. Circadian rhythm as a therapeutic target. Nat. Rev. Drug Discov. 20, 287–307 (2021).
Dong, Z. et al. Targeting glioblastoma stem cells through disruption of the circadian clock. Cancer Discov. 9, 1556–1573 (2019).
Breuhahn, K. & Schirmacher, P. Reactivation of the insulin-like growth factor-II signaling pathway in human hepatocellular carcinoma. World J. Gastroenterol. 14, 1690–1698 (2008).
Xu, D. et al. The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis. Nature 580, 530–535 (2020).
Ji, H. et al. EGF-induced ERK activation promotes CK2-mediated disassociation of α-Catenin from β-Catenin and transactivation of β-Catenin. Mol. Cell. 36, 547–559 (2009).
Paci, G., Caria, J. & Lemke, E. A. Cargo transport through the nuclear pore complex at a glance. J. Cell Sci. https://doi.org/10.1242/jcs.247874 (2021).
Elfgang, C. et al. Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals. Proc. Natl Acad. Sci. USA 96, 6229–6234 (1999).
Ben-Sahra, I., Howell, J. J., Asara, J. M. & Manning, B. D. Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339, 1323–1328 (2013).
Doi, M., Hirayama, J. & Sassone-Corsi, P. Circadian regulator CLOCK is a histone acetyltransferase. Cell 125, 497–508 (2006).
Hirayama, J. et al. CLOCK-mediated acetylation of BMAL1 controls circadian function. Nature 450, 1086–1090 (2007).
Lin, R. et al. CLOCK acetylates ASS1 to drive circadian rhythm of ureagenesis. Mol. Cell 68, 198–209 e196 (2017).
Liu, R. et al. Choline kinase alpha 2 acts as a protein kinase to promote lipolysis of lipid droplets. Mol. Cell 81, 2722–2735 e2729 (2021).
Cuervo, A. M. & Wong, E. Chaperone-mediated autophagy: roles in disease and aging. Cell Res 24, 92–104 (2014).
Kaushik, S. & Cuervo, A. M. The coming of age of chaperone-mediated autophagy. Nat. Rev. Mol. Cell Biol. 19, 365–381 (2018).
Wong, K. M., King, G. G. & Harris, W. P. The treatment landscape of advanced hepatocellular carcinoma. Curr. Oncol. Rep. 24, 917–927 (2022).
Chaudhari, A., Gupta, R., Patel, S., Velingkaar, N. & Kondratov, R. Cryptochromes regulate IGF-1 production and signaling through control of JAK2-dependent STAT5B phosphorylation. Mol. Biol. Cell 28, 834–842 (2017).
Crosby, P. et al. Insulin/IGF-1 drives period synthesis to entrain circadian rhythms with feeding time. Cell 177, 896–909 e820 (2019).
Zhang, J., Lv, H., Ji, M., Wang, Z. & Wu, W. Low circadian clock genes expression in cancers: a meta-analysis of its association with clinicopathological features and prognosis. PLoS ONE 15, e0233508 (2020).
Li, X. et al. Nucleus-translocated ACSS2 promotes gene transcription for lysosomal biogenesis and autophagy. Mol. Cell 66, 684–697 e689 (2017).
Lee, J. H. et al. EGFR-phosphorylated platelet isoform of phosphofructokinase 1 promotes PI3K activation. Mol. Cell 70, 197–210 e197 (2018).
Lee, J. H. et al. Stabilization of phosphofructokinase 1 platelet isoform by AKT promotes tumorigenesis. Nat. Commun. 8, 949 (2017).
Li, X. et al. A splicing switch from ketohexokinase-C to ketohexokinase-A drives hepatocellular carcinoma formation. Nat. Cell Biol. 18, 561–571 (2016).
Qian, X. et al. Conversion of PRPS hexamer to monomer by AMPK-mediated phosphorylation inhibits nucleotide synthesis in response to energy stress. Cancer Discov. 8, 94–107 (2018).
Xu, D. Q. et al. PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity. EMBO J. 35, 496–514 (2016).
Qian, X. et al. KDM3A senses oxygen availability to regulate PGC-1α-mediated mitochondrial biogenesis. Mol. Cell 76, 885–895 e887 (2019).
Qian, X. et al. PTEN suppresses glycolysis by dephosphorylating and inhibiting autophosphorylated PGK1. Mol. Cell 76, 516–527 e517 (2019).
Xu, D. et al. The protein kinase activity of fructokinase A specifies the antioxidant responses of tumor cells by phosphorylating p62. Sci. Adv. 5, eaav4570 (2019).
Lin, S. Y. et al. GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 336, 477–481 (2012).
Du, L. et al. β-Catenin induces transcriptional expression of PD-L1 to promote glioblastoma immune evasion. J. Exp. Med. https://doi.org/10.1084/jem.20191115 (2020).
Xu, D. et al. PAQR3 modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus. Nat. Commun. 6, 8100 (2015).
Yu, R., Craik, D. J. & Kaas, Q. Blockade of neuronal α7-nAChR by α-conotoxin ImI explained by computational scanning and energy calculations. PLoS Comput. Biol. 7, e1002011 (2011).
Yu, R. et al. Molecular determinants conferring the stoichiometric-dependent activity of α-conotoxins at the human α9α10 nicotinic acetylcholine receptor subtype. J. Med. Chem. 61, 4628–4634 (2018).
Yang, W. et al. Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature 480, 118–122 (2011).
Acknowledgements
This study was supported by grants from the Ministry of Science and Technology of the People’s Republic of China (2020YFA0803300, Z.L.; 2021YFA0805600, D.X.), the National Natural Science Foundation of China (92157113 and 82072630, D.X.; 82173114, Z.W.; 82072903 and 82272872, T.L.; 82002811, M. Yan; 82188102 and 82030074, Z.L.), the Zhejiang Natural Science Foundation Key Project (LD22H160002, D.X.; LD21H160003, Z.L.), Zhejiang Natural Science Foundation Discovery Project (LQ22H160023, Z.W.), and the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2019R01001, Z.L.). Z.L. is the Kuancheng Wang Distinguished Chair. The authors received no specific funding for this work. We express our great gratitude to Dr. Hong Wang and his team from the Hangzhou Cosmos Wisdom Mass Spectrometry Center of Zhejiang University Medical School for their technical support in sample analysis utilizing an integrated nanoLC–ESI–MS/MS and data processing platform.
Author information
Authors and Affiliations
Contributions
D.X. and Z.L. conceived and designed the study and wrote the manuscript. T.L., Z.W., L.Y., Y.D., H.H., K.W., M. Yan, G.J., Y.S., L.W., L.L., P.Z., B.D., F.S. and Z.Z. performed the experiments; H.J., M. Yang and R.Y. were involved in MD simulation analyses. Y.X., L.X., Q.W. and X.Q. reviewed and edited the manuscript.
Corresponding authors
Ethics declarations
Competing interests
Z.L. owns shares in Signalway Biotechnology (Pearland, TX), which supplied rabbit antibodies that recognize CLOCK pS106 and PRPS1/2 K29ac. Z.L.’s interest in this company had no bearing on its being chosen to supply these reagents. The remaining authors declare no competing interests.
Peer review
Peer review information
Nature Cell Biology thanks Lars Zender, Tsuyoshi Hirota and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 CLOCK S106 phosphorylation disassembles the CLOCK–BMAL1 complex.
(b-e, h, j, k, m, n) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated at least twice independently. Data are the mean ± SD. (a) The luciferase activity of the indicated cells expressing a Per1-driven luciferase reporter was measured after IGF1 treatment (12 h) (n = 6). *P < 0.01; **P < 0.001 by One-way ANOVA post hoc test. (b, c, m, n) The indicated cells were pretreated with or without the indicated inhibitors for 30 min before IGF1 treatment for 30 min (b, c, m) or transfected with the indicated plasmids before IGF1 treatment for 1 h (n). Total cell lysates and cytosolic fractions were prepared. (d) Flag-CK2α immunoprecipitated from Huh7 cells treated with or without IGF1 for 30 min was incubated with or without CIP for 30 min. (e) Purified GST-CK2α was mixed with or without active His-ERK2 for in vitro kinase assay, followed by incubation with purified His-CLOCK. Proteins precipitated by GST pulldown assay were incubated with or without CIP for 30 min. (f) In vitro kinase assays were performed by mixing purified His–CLOCK with or without purified GST-CK2α in the presence of ATP. Mass-spectrometric analysis was performed. (g) Alignment of CLOCK to the consensus CK2α-phosphorylated substrate motif (SXXD/E). (h, i) Huh7 cells expressing Flag-CLOCK were treated with or without IGF1 for 1 h (h). Immunoblotting (h) or IHC analyses of human HCC samples (i) were performed with the indicated antibodies and a CLOCK pS106-blocking peptide. (j-l) The indicated cells expressing the indicated plasmids (j) were treated with or without IGF1 for 1 h (j-l). Total cell lysates and cytosolic and nuclear fractions were prepared (k). The relative CLOCK abundance was quantified (n = 10) (l). *P < 0.05; ***P < 0.0001 by two-tailed Student’s t test.
Extended Data Fig. 2 CLOCK S106 phosphorylation is required for its nuclear exportation.
(e-i) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. All experiments were repeated three times independently with similar results. (a) Alignment of protein sequences spanning CLOCK S106 and the adjacent noncanonical NES from different species. (b, d) MD simulation of CLOCK bound with BMAL1 WT (b) or BMAL1 4Mut (D144A/D231A/D299A/E303A) (d). Evolution of the backbone root-mean-square-deviation (RMSD) of the CLOCK-BMAL1 complex from the initial frame in 250 ns MD simulations. RMSD of the CLOCK-BMAL1 complex with and without CLOCK S106 phosphorylation are shown in red and black, respectively. (c) The conformation of the CLOCK-BMAL1 complex of the last frame extracted from 250 ns MD simulation. CLOCK and BMAL1 structures are shown in green and blue, respectively. The red arrow indicates CLOCK S106. D144, D232, D299, and E303 of BMAL1, which are in a close proximity with CLOCK S106, are shown. (e) Bacterially purified WT GST-CLOCK or GST-CLOCK S106A was incubated with or without His-CK2α in the presence of ATP for an in vitro kinase assay, followed by incubation with the indicated His-BMAL1 proteins. (f) Huh7 cells stably transfected with the indicated plasmids were treated with or without IGF1 for 1 h. Total cell lysates and cytosolic fractions were prepared. (g) Huh7 cells were pretreated with or without TBB for 30 min before treatment with or without IGF1 for 1 h. (h) The indicated cells expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins were harvested. (i) Hep3B cells expressing CLOCK shRNA with reconstituted expression of Flag-rCLOCK proteins were treated with or without IGF1 for 1 h.
Extended Data Fig. 3 CLOCK acetylates PRPS1/2 K29.
(d, j, m) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated at least twice independently. (a) Huh7 cells with or without expression of Flag–CLOCK were treated with or without IGF1 for 1 h. The immunoprecipitated Flag–CLOCK was eluted with Flag peptide and stained with Coomassie Brilliant Blue after SDS-PAGE. Mass spectrometry-identified protein peptide hits are shown. (b) Purified GST–CLOCK was incubated with or without His-CK2α and TBB for an in vitro kinase assay, followed by incubation with or without the indicated His-PRPS1/2 proteins in the presence of acetyl-CoA. Mass-spectrometric analysis was performed. (c) Alignment of protein sequences spanning PRPS1/2 K29 from different species. (d, e) Huh7 cells expressing Flag-PRPS1 were treated with or without IGF1 for 1 h (d). Immunoblotting analyses (d) or IHC analyses of human HCC samples (e) were performed with the indicated antibodies and a PRPS1/2 K29 acetylation-blocking peptide. (f-j) Genomic DNA was extracted from two individual clones of the indicated cells with knock-in expression of PRPS1/2 K29R. PCR products amplified from the indicated DNA fragments were shown (f, g) and sequenced (h, i). The red line indicates the sgRNA-targeting sequence. The black line indicates the PAM. Blue arrows indicate mutated nucleotides. A mutated amino acid and its WT counterpart are indicated by the solid red box (h, i). These cells were transfected with or without constitutively active IGF1R-CA (j). (k) Purified GST–CLOCK were incubated with or without His-CK2α for an in vitro kinase assay, followed by incubation with or without the indicated His-PRPS1/2 proteins in the presence of acetyl-CoA. (l, m) Huh7 (l, m) and Hep3B (l) cells expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins were harvested or (l) treated with or without IGF1 for 1 h (m).
Extended Data Fig. 4 Cytosolic CLOCK promotes PRPS1/2 stabilization.
(a, d-h, j, k, l, o) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated three times independently. (a) The indicated cells were stably transfected with active IGF1R-CA. (b, c) Huh7 cells stimulated with IGF1 for the indicated time (b) or expressing IGF1R-CA (c) were harvested. The mRNA expression levels of the PRPS1/2 genes were measured using qPCR (n = 6). Data are the mean ± SD. N.S., not significant by One-way ANOVA post hoc test (b) or two-tailed Student’s t test (c). (d-g) The indicated cells expressing CLOCK shRNA and active IGF1R-CA with reconstituted expression of the indicated CLOCK protein were treated with CHX for the indicated time (d, f). The quantification of PRPS1/2 levels is shown. Data are the mean ± SD, n = 6, **P < 0.001; ***P < 0.0001 by One-way ANOVA post hoc test (e, g). (h) The indicated cells were treated with DMSO, MG132 (10 μM), PS341 (10 μM), CQ (25 μM) or bafilomycin A1 (BFA) (10 nM) for 8 h. (i) Alignment of protein sequences spanning PRPS1/2 K29 and the adjacent HSC70 recognized motif from different species. (j) LAMP2A shRNA was expressed in the indicated cells. (k-n) Huh7 cells expressing PRPS1/2 shRNA with reconstituted expression of the indicated PRPS1/2 proteins were harvested for immunoblotting (k, l) or qPCR (n = 6) to measure Prps1/2 mRNA levels (m, n). Data are the mean ± SD. N.S., not significant by One-way ANOVA post hoc test. (o) Endogenous CLOCK-depleted Huh7 cells with reconstituted expression of the indicated CLOCK protein were transfected with HA-PRPS1/2 and treated with or without IGF1 for 1 h. Immunoprecipitation using limited amount of HA antibody was performed.
Extended Data Fig. 5 CLOCK-mediated PRPS1/2 stabilization promotes de novo nucleotide synthesis.
(g-l) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated at least twice independently. (a-g) Data are the mean ± SD, n = 6, *P < 0.01; **P < 0.001; ***P < 0.0001; N.S., not significant by One-way ANOVA post hoc test. (a-f) The indicated cells expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins (a, b, e) or the indicated clones with knock-in expression of PRPS1/2 K29R (c, d, f) were transfected with or without active IGF1R-CA, followed by labeling with D-[6-14C] glucose for 30 min. The amounts of 14C-RNA (a, c) and 14C-DNA (b, d) were measured. The 13C-labeled PRPP, IMP, AMP, GMP, UMP, and CMP were measured by LC/MS-MS (e, f). (g) Purified GST–CLOCK was incubated with or without His-CK2α in the presence of ATP for an in vitro kinase assay, followed by incubation with or without the indicated His-PRPS1/2 proteins in the presence of acetyl-CoA. The PRPS1/2 activity was measured. (h, i) Huh7 cells expressing CLOCK shRNA with reconstituted expression of the indicated Flag-rCLOCK were serum-starved for 12 h in the presence of CQ and then treated with or without EGF (h) or FGF1 (i) for 1 h. Cytosolic and whole cell lysates were harvested. (j) Huh7 cells were stimulated with EGF (100 ng/ml) or FGF1 (25 ng/ml) for the indicated time. (k) Huh7 cells stably transfected with constitutively active EGFR-vIII or FGFR1-CA were harvested. (l) Huh7 cells expressing CLOCK shRNA and constitutively active EGFR-vIII or FGFR1-CA with reconstituted expression of the indicated Flag-rCLOCK were treated with CHX for the indicated time. The quantification of PRPS1/2 protein levels is shown. Data are the mean ± SD, **P < 0.001 by One-way ANOVA post hoc test.
Extended Data Fig. 6 Overexpressed CK2α in HCC cells is critical for CLOCK-enhanced PRPS1/2 stability.
(a, b, d-l) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated three times independently. (c, d, g, i) Data are the mean ± SD. n = 6, *P < 0.01; **P < 0.001; ***P < 0.0001 by One-way ANOVA post hoc test. (a, b) The indicated cells expressing CLOCK shRNA with reconstituted expression of the indicated shRNA-resistant CLOCK were constructed (a) and serum-starved for 12 h in the presence of CQ before treatment with or without IGF1 for 1 h (b). Cytosolic and whole cell lysates were harvested. (c) The indicated cells with reconstituted expression of the indicated CLOCK proteins were treated with or without IGF1 for 12 h, followed by labeling with D-[6-14C] glucose for 30 min. The amounts of 14C-RNA and 14C-DNA were measured. (d) The indicated cells expressing CLOCK shRNA and IGF1R-CA with reconstituted expression of Flag-rCLOCK were treated with CHX for the indicated time. The quantification of PRPS1/2 levels is shown. (e, f) The indicated cells were treated with or without IGF1 for 1 h. Whole cell lysates were harvested. (g) L02 cells transfected with the indicated plasmids were treated with CHX for the indicated time in the presence or absence of IGF1. The quantification of PRPS1/2 levels is shown. (h) Huh7 cells transfected with the indicated shRNA were treated with or without IGF1 for 1 h. (i) Huh7 cells transfected with the indicated shRNA were treated with CHX for the indicated time in the presence or absence of IGF1. The quantification of PRPS1/2 levels is shown.
Extended Data Fig. 7 Both nuclear transcriptional activity and cytosolic function of CLOCK contribute to HCC cell proliferation.
(a, d, e, i, j, l, m) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated at least twice independently. (a-c, e-g, h, j, k, n) Data are the mean ± SD. *P < 0.01; **P < 0.001; ***P < 0.0001 by One-way ANOVA post hoc test. (a-c) The indicated cells stably transfected with the indicated plasmids and shRNA were constructed (a) and transiently expressed a Per1-driven luciferase reporter before IGF1 treatment for 12 h. The luciferase activity is shown (n = 6). The mRNA levels of CLOCK-downstream target genes were measured using qPCR (n = 6) (c). Cytosolic and whole cell lysates from Huh7 cells were harvested (d). (e) Huh7 cells transfected with the indicated plasmids and shRNA were treated with CHX. PRPS1/2 protein was quantified (n = 6). (f, g) The indicated cells transfected with the indicated plasmids and shRNA were labeled with D-[6-14C] glucose. The amounts of 14C-RNA (f) and 14C-DNA (g) were measured (n = 6). (h) The indicated cells transfected with the indicated plasmids and shRNA were plated in complete culture medium and counted (n = 6). (i) Huh7 cells were pretreated with or without different dose of Sorafenib for 30 min before IGF1 treatment for 1 h. (j, k) The indicated cells expressing CLOCK shRNA and active IGF1R-CA with reconstituted expression of Flag-rCLOCK WT or S106D were treated with CHX (j) or without CHX (k) in the presence or absence of Sorafenib for the indicted time. PRPS1/2 protein quantification (j) and cell counting (k) were performed (n = 6). (l-n) The indicated cells with reconstituted expression of Flag-rCLOCK WT or S106D were harvested (l) or treated with CHX (m) or without CHX (n) for the indicated time. PRPS1/2 protein quantification (m) and cell counting (n) were performed (n = 6).
Extended Data Fig. 8 CLOCK-mediated PRPS1/2 stabilization promotes HCC cell proliferation and liver tumor growth.
(a, b) Huh7 cells (1 × 106) expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins (a) expressing WT PRPS1/2 or PRPS1/2 K29R knock-in mutants (b) were subcutaneously injected into athymic nude mice (n = 6 per group). The mice were euthanized and examined for tumor growth 28 days after injection. Tumor volumes were calculated, and the tumors were weighed. Data are the mean ± SD, n = 7, *P < 0.01; **P < 0.001 by One-way ANOVA post hoc test. (c, d) IHC analyses of the indicated tumor samples were performed with an anti-Ki67 antibody. Ki67-positive cells were quantified. Data are the mean ± SD, n = 10, ***P < 0.0001 by One-way ANOVA post hoc test. (e, f) TUNEL analyses of the indicated tumor samples were performed (upper). Apoptotic cells were stained brown and quantified in n = 10 microscopic fields (lower). Data are the mean ± SD, ***P < 0.0001 by One-way ANOVA post hoc test. (g) Huh7 cells (1 × 106) expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins were intrahepatically injected into athymic nude mice (n = 6 per group). The mice were euthanized and examined for tumor growth 22 days after injection. The arrows indicate tumors (left). The tumor volumes were measured (right). Data are the mean ± SD, n = 6, *P < 0.01 by two-tailed Student’s t test. (h) Huh7 cells (1 × 106) expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins were subcutaneously injected into athymic nude mice (n = 7 per group). The resulting tumors were resected 22 days after injection (left). The growth of xenografted tumors in the mice was measured (middle) and the tumors were weighed (right). Data are the mean ± SD, **P < 0.001 by two-tailed Student’s t test.
Supplementary information
Source data
Source Data Fig. 1
Unprocessed WBs.
Source Data Fig. 2
Unprocessed WBs.
Source Data Fig. 3
Unprocessed WBs.
Source Data Fig. 4
Unprocessed WBs.
Source Data Extended Data Fig. 1
Unprocessed WB.
Source Data Extended Data Fig. 2
Unprocessed WBs.
Source Data Extended Data Fig. 3
Unprocessed WB.
Source Data Extended Data Fig. 4
Unprocessed WBs.
Source Data Extended Data Fig. 5
Unprocessed WB.
Source Data Extended Data Fig. 6
Unprocessed WBs.
Source Data Extended Data Fig. 7
Unprocessed WBs.
Source Data Table Fig. 1
Statistical source data.
Source Data Table Fig. 2
Statistical source data.
Source Data Table Fig. 4
Statistical source data.
Source Data Table Fig. 5
Statistical source data.
Source Data Table Fig. 6
Statistical source data.
Source Data Table Fig. 7
Statistical source data.
Source Data Extended Data Fig. 1
Statistical source data for Extended Data Fig. 1.
Source Data Extended Data Fig. 4
Statistical source data for Extended Data Fig. 4.
Source Data Extended Data Fig. 5
Statistical source data for Extended Data Fig. 5.
Source Data Extended Data Fig. 6
Statistical source data for Extended Data Fig. 6.
Source Data Extended Data Fig. 7
Statistical source data for Extended Data Fig. 7.
Source Data Extended Data Fig. 8
Statistical source data for Extended Data Fig. 8.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Liu, T., Wang, Z., Ye, L. et al. Nucleus-exported CLOCK acetylates PRPS to promote de novo nucleotide synthesis and liver tumour growth. Nat Cell Biol 25, 273–284 (2023). https://doi.org/10.1038/s41556-022-01061-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41556-022-01061-0
