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Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth

Nature Cell Biology volume 18, pages 886–896 (2016)Cite this article

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Abstract

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.

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Figure 1: Hepatocyte-specific overexpression of activated Yap causes hepatomegaly and enhances DMBA-induced liver tumour formation.
Figure 2: Yap alters expression of metabolism-related genes and enhances glul expression.
Figure 3: Yap transcriptionally upregulates GLUL in an evolutionarily conserved fashion.
Figure 4: Yap reprograms nitrogen metabolism by enhancing GLUL-dependent anabolic assimilation of ammonia for de novo nucleotide biosynthesis.
Figure 5: GLUL activity and nucleotide biosynthesis contribute to Yap-induced hepatomegaly and the growth of liver cancer cells.
Figure 6: The mTOR pathway is not deregulated by Yap expression or GLUL inhibition, but it is required for Yap-induced hepatomegaly.
Figure 7: Yap reprograms the relative isotopic enrichment of nutritional nitrogen into nucleotide biosynthesis in a Glul-dependent manner to support liver growth.

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Acknowledgements

This work was supported by an Irwin Arias Postdoctoral Fellowship (A.G.C.) and Liver Scholar Award (A.G.C.) from the American Liver Foundation, an HDDC Pilot Feasibility Grant from the Harvard Digestive Disease Center, P30 DK034854 (A.G.C., D.Y.), NIH NIGMS T32GM007753 (K.L.H.), NIH NCI 5K08CA172288 (K.J.E.), NIH NIDDK R01DK60322 (D.Y.R.S.), NIH NIDDK R01DK090311 (W.G.), NIH K08DK105351 (D.Y.), R01AR064036 (F.D.C.) and R01DK099559 (F.D.C.), and the Packard Foundation (D.Y.R.S.). J.M.A. is partially supported by NIH NCI 5P01CA120964 and 5P30CA006516. G.G.G. is supported by an American-Italian Cancer Foundation postdoctoral research fellowship. K.J.E. was a Robert Black Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-109-10). D.Y. is a Gilead Sciences Scholar in Liver Disease. W.G. is supported by the Claudia Adams Barr Program for Innovative Cancer Research, and is a Pew Scholar in the Biomedical Sciences.

Author information

Author notes

  1. Giorgio G. Galli

    Present address: Present address: Novartis Institutes for BioMedical Research, Disease Area Oncology, 4056 Basel, Switzerland.,

  2. Andrew G. Cox and Katie L. Hwang: These authors contributed equally to this work.

Authors and Affiliations

  1. Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

    Andrew G. Cox, Katie L. Hwang, Sebastian Beltz, Allison Tsomides, Keelin O’Connor, Julia Wucherpfennig, Sahar Nissim, Akihiro Minami, David E. Cohen & Wolfram Goessling

  2. Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, Massachusetts 02115, USA

    Katie L. Hwang

  3. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA

    Kristin K. Brown, Min Yuan, Evan C. Lien & John M. Asara

  4. University of Utah, Salt Lake City, Utah 84112, USA

    Kimberley J. Evason

  5. Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

    Giorgio G. Galli, Dean Yimlamai & Fernando D. Camargo

  6. Weill Cornell Medical College, New York 10065, USA

    Sagar Chhangawala & Yariv Houvras

  7. Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA

    Fernando D. Camargo & Wolfram Goessling

  8. Max Planck Institute, 61231 Bad Nauheim, Germany

    Didier Y. R. Stainier

  9. Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA

    Wolfram Goessling

  10. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    Wolfram Goessling

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  1. Andrew G. Cox
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Contributions

A.G.C. and W.G. conceived the study, reviewed results and wrote the manuscript. K.L.H. contributed to experimental design. A.G.C. and K.L.H. performed the majority of the experiments and data analysis. K.K.B. performed cell culture experiments and immunoblotting. K.J.E. and D.Y.R.S. generated lf:Yap fish and K.J.E. performed pathological analysis of liver tumours. S.B., K.O’C., A.T. and S.N. assisted in zebrafish experiments. M.Y., E.C.L. and J.M.A. developed methods and analysed metabolomics samples. G.G.G. performed ChIP experiments. S.C. and Y.H. analysed RNA-seq data sets. D.Y., A.M., D.E.C., F.D.C., J.M.A., Y.H. and D.Y.R.S. provided overall input. All authors reviewed the manuscript.

Corresponding author

Correspondence to Wolfram Goessling.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Hepatocyte-specific overexpression of activated Yap causes hepatomegaly and accelerates DMBA-induced liver tumor formation.

(a) Schematic diagram of the construct used to generate transgenic fish with hepatocyte-specific (fapb10a) expression of yap1S87A, abbreviated to lf:Yap. (b) Liver histology (H&E stain) in transverse sections of WT and lf:Yap larvae at 5 dpf. Scale bars: 50 μm (upper) and 20 μm (lower) for zoomed images. (c) Quantification of differentiated hepatocyte frequency in WT and lf:Yap larvae at 10 dpf, as determined by FACS. n = 5 biologically independent WT and lf:Yap single cell suspensions, each of which was derived from 10 larvae; see Supplementary Table 4. ∗∗P < 0.01, two-sided Student’s t-test, values represent the mean ± s.e.m. (d) Time course of hepatomegaly during early adulthood as determined by quantification of fluorescent liver area. n = 10, 6, 6, 4, 10, 8, 6 and 4 WT 3 wpf, WT 8 wpf, WT 12 wpf, WT 16 wpf, lf:Yap3 wpf, lf:Yap 8 wpf, lf:Yap 12 wpf and lf:Yap 16 wpf zebrafish respectively; see Supplementary Table 4. ∗P < 0.05, two-sided Student’s t-test, values represent the mean ± s.e.m. (e) Histological assessment of transverse sections from liver of WT and lf:Yap adults. H&E staining at low (10x) and high magnification (25x). Periodic-acid Schiff (PAS) stain for hepatic glycogen (pink inclusions). Bile duct morphogenesis determined by 2F11 staining. Cell proliferation as determined by PCNA staining. Scale bars: 200 μm and 50 μm for zoomed images. (f) Tumor heterogeneity in lf:Yap transgenics exposed to DMBA. Tumors include hepatocellular carcinoma (HCC), HCC with sarcomatoid features, HCC with peliosis/spongiosis-hepatis-like change, and cholangiocarcinoma (CCA). Scale bars: 2 mm for dissected images, 200 μm for histology and 50 μm for zoomed images. (g) Table illustrating the incidence of fibrosis, as determined by Sirius Red staining, glycogen, sarcomatoid cytology, peliosis/spongiosis-hepatis-like change and ascites in DMBA-induced liver tumors. n = 4 and 19 WT and lf:Yap liver tumors respectively.

Supplementary Figure 2 Yap alters expression of metabolism-related genes and enhances glul expression.

(a) Gene set enrichment (GSEA) derived from RNAseq of WT and lf:Yap adult livers identifies a conserved Yap target gene signature. (b) Gene ontology (GO) analysis of the top 20 biological processes upregulated by Yap. Biological processes related to metabolism are highlighted in red. (c) RNAseq analysis of the Wnt target genes cyclind1, axin2, cd44 in dissected WT and lf:Yap livers. n = 3 WT and lf:Yap adult livers; see Supplementary Table 4. Values represent the mean ± s.e.m. (d) qPCR validation of Wnt target genes axin2 and cyclind1 in adult zebrafish livers. n = 3 WT and lf:Yap adult livers; see Supplementary Table 4. Values represent the mean ± s.e.m. (e) RNAseq analysis of yap and taz expression in dissected WT and lf:Yap livers. n = 3 WT and lf:Yap adult livers; see Supplementary Table 4. ∗∗P < 0.01, two-sided Student’s t-test, values represent the mean ± s.e.m.

Supplementary Figure 3 Yap transcriptionally upregulates GLUL in an evolutionarily conserved fashion.

(a) ChIP qPCR analysis of transcriptional activity (H3K27ac) at the glula promoter in dissected WT and lf:Yap livers. Shown is the average of 2 biologically independent WT and lf:Yap adult liver chromatin preps, each of which was derived from a pooled sample of 2; see Supplementary Table 4. (b) Luciferase GLUL reporter assay using truncated promoter constructs in Hek293 cells expressing GFP, YAP or YAP1S127A. n = 3 biologically independent replicates; see Supplementary Table 4. Values represent the mean ± s.e.m. (c) ChIP qPCR analysis of YAP and TEAD4 enrichment at the GLUL promoter in Hep3B and HepG2 cells. Shown is the average of 2 biologically independent replicates; see Supplementary Table 4.

Supplementary Figure 4 Yap reprograms nitrogen metabolism by enhancing GLUL-dependent anabolic assimilation of ammonia for de novo nucleotide biosynthesis.

(a) Immunohistochemical detection of GLUL in WT, lf:Yap transgenic livers and DMBA-induced lf:Yap liver tumors. Scale bar, 50 μm. (b) Ammonia excretion rates in individual WT and lf:Yap adult fish. n = 16 and 12 WT and lf:Yap adult zebrafish respectively; see Supplementary Table 4. Values represent the mean ± s.e.m. (c) Steady-state abundance of urea in WT and lf:Yap livers as determined by selected reaction monitoring (SRM) analysis. n = 5 WT and lf:Yap adult livers; see Supplementary Table 4. Values represent the mean ± s.e.m. (d) Abundance of 15N-labelled Guanosine (M + 2 fraction) from methanol extracted WT and lf:Yap liver lysates, as determined by LC-MS/MS via SRM. n = 5 biologically independent WT and lf:Yap adult liver lysates; see Supplementary Table 4. ∗P < 0.05, two-sided Student’s t-test, values represent the mean ± s.e.m. (e) Abundance of 15N-labelled Cytosine (M + 2 fraction) from methanol extracted WT and lf:Yap liver lysates, as determined by LC-MS/MS via SRM. n = 5 biologically independent WT and lf:Yap adult liver lysates; see Supplementary Table 4. Values represent the mean ± s.e.m. (f) Percentage of 15N-labelled Glutamine isotopologues in WT and lf:Yap transgenic liver lysates following ammonia assimilation in the presence or absence or MSO. n = 5 biologically independent WT and lf:Yap adult liver lysates; see Supplementary Table 4. Values represent the mean ± s.e.m. (g) Percentage of 15N-labelled Histidine isotopologues in WT and lf:Yap transgenic liver lysates following ammonia assimilation in the presence or absence or MSO. n = 5 biologically independent WT and lf:Yap adult liver lysates; see Supplementary Table 4. Values represent the mean ± s.e.m.

Supplementary Figure 5 GLUL activity and nucleotide biosynthesis contribute to Yap-induced hepatomegaly and the growth of liver cancer cells.

(a) RT-PCR validation of morpholinos targeting splice sites in glula and glulb, resulting in alternative transcripts (indicated by arrow heads). (b) Glul activity in WT and lf:Yap larval extracts derived from larvae exposed to MSO from 3–5 dpf. n = 8, 7, 9 and 9 biologically independent WT, WT + MSO, lf:Yap and lf:Yap + MSO larval lysates respectively, each of which was derived from a pool of 20 larvae; see Supplementary Table 4. ∗∗P < 0.01, two-sided Student’s t-test, values represent the mean ± s.e.m. (c) Proliferation of HepG2 liver cancer cells over 4 days in the presence or absence of glutamine (Q), MSO or VP. n = 3 biologically independent replicates; see Supplementary Table 4. Values represent the mean ± s.e.m.

Supplementary Figure 6 The mTOR pathway is not deregulated by Yap expression or GLUL inhibition, but it is required for Yap-induced hepatomegaly.

(a) Immunohistochemical analysis of phospho-S6 (pS6) levels in liver sections from WT and lf:Yap transgenic fish 24 h after exposure to DMSO, MSO or Rapamycin (RAPA). Scale bars: 50 μm. (b) Analysis of the number of liver cells in H + E stained transverse liver sections from WT and lf:Yap transgenic larvae at 5dpf. n = 16 WT and lf:Yap larvae; see Supplementary Table 4. ∗∗∗P < 0.001, two-sided Student’s t-test, values represent the mean ± s.e.m. (c) Analysis of liver area in H + E stained transverse liver sections from WT and lf:Yap transgenic larvae at 5dpf. n = 16 WT and lf:Yap larvae; see Supplementary Table 4. ∗∗∗P < 0.001, two-sided Student’s t-test, values represent the mean ± s.e.m. (d) Analysis of cellularity (cells/unit2) in H + E stained transverse liver sections from WT and lf:Yap transgenic larvae at 5dpf. n = 16 WT and lf:Yap larvae; see Supplementary Table 4. Values represent the mean ± s.e.m.

Supplementary Figure 7 Yap reprograms the relative isotopic enrichment of nutritional nitrogen into nucleotide biosynthesis in a GLUL-dependent manner to support liver growth.

(a) Relative isotopic enrichment of 15N into deoxyguanosine isotopologues derived from hydrolyzed genomic DNA of liver and 15N-spirulina as determined by LC-MS/MS. One experiment is shown. (b) Histological analysis of cell death (TUNEL) from WT and lf:Yap adults derived from the long-term MSO intervention studies. The positive control showing DAB-stained nuclei is derived from a murine liver section. Scale bar, 50 μm.

Supplementary Figure 8 Unprocessed scans of immunoblots accompanied by size markers. Images were obtained by enhanced chemiluminescence.

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Cox, A., Hwang, K., Brown, K. et al. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nat Cell Biol 18, 886–896 (2016). https://doi.org/10.1038/ncb3389

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