Cancer Letters

Cancer Letters

Volume 516, 28 September 2021, Pages 84-98
Cancer Letters

Myristoylation-mediated phase separation of EZH2 compartmentalizes STAT3 to promote lung cancer growth

https://doi.org/10.1016/j.canlet.2021.05.035Get rights and content

Highlights

  • EZH2 can be modified by N-myristoylation in lung cancer cells.

  • Myristoylation facilitates EZH2 to undergo liquid-liquid phase separation (LLPS) in cells and in vitro.

  • Myristoylation-mediated LLPS of EZH2 compartmentalizes its non-canonical substrate STAT3 and activates STAT3 signaling.

  • Myristoylation of EZH2 enhances its ability to promote lung cancer cell growth.

Abstract

N-myristoylation is a crucial signaling and pathogenic modification process that confers hydrophobicity to cytosolic proteins. Although different large-scale approaches have been applied, a large proportion of myristoylated proteins remain to be identified. EZH2 is overexpressed in lung cancer cells and exerts oncogenic effects via its intrinsic methyltransferase activity. Using a well-established click chemistry approach, we found that EZH2 can be modified by myristoylation at its N-terminal glycine in lung cancer cells. Hydrophobic interaction is one of the main forces driving or stabilizing liquid-liquid phase separation (LLPS), raising the possibility that myristoylation can modulate LLPS by mediating hydrophobic interactions. Indeed, myristoylation facilitates EZH2 to form phase-separated liquid droplets in lung cancer cells and in vitro. Furthermore, we provide evidence that myristoylation-mediated LLPS of EZH2 compartmentalizes its non-canonical substrate, STAT3, and activates STAT3 signaling, ultimately resulting in accelerated lung cancer cell growth. Thus, targeting EZH2 myristoylation may have significant therapeutic efficacy in the treatment of lung cancer. Altogether, these observations not only extend the list of myristoylated proteins, but also indicate that hydrophobic lipidation may serve as a novel incentive to induce or maintain LLPS.

Introduction

N-myristoylation is a lipid modification process through which a 14-carbon saturated myristic acid is covalently added to the amine group of the N-terminal glycine of different cellular proteins, such as oncoproteins [1]. In addition to the well-known membrane anchoring function, myristoylation mediates protein-protein or protein-lipid interactions, controls protein stability, and regulates transcription [[2], [3], [4], [5]]. In particular, myristoylation drives homo- or hetero-multimerization of modified proteins via hydrophobic interactions [[6], [7], [8]]. The importance of multiple myristoyl-oncoproteins in regulating tumorigenesis is well established [9,10]. The consensus sequence for myristoylation is assumed to be MGXXXSX, in which the G2 residue is essential for myristoylation, while the S6 residue is not a strict requirement [11]. The presence of a glutamine or an asparagine residue at the third position (Q3 or N3) is an alternate feature of myristoylated proteins lacking S6 [12,13]. In addition, a lysine residue at the seventh position (K7) also favors myristoylation [14,15]. Several studies based on large-scale and complementary structural approaches have provided a comprehensive list of myristoylated substrates in different species [11,16,17]. However, a large proportion of predicted myristoylated targets await experimental validation [18]. Therefore, unveiling novel myristoylated proteins is important for understanding their involvement in physiological and pathological processes.

A growing body of evidence points to a pivotal role of protein liquid-liquid phase separation (LLPS) in a wide range of biological processes, including carcinogenesis [[19], [20], [21], [22], [23]]. LLPS is driven by physical interactions between biomolecules. LLPS enables biomolecules to form a highly condensed liquid droplet-like phase, thereby providing an extremely efficient platform for the reaction. Hydrophobic interaction is one of the major factors driving LLPS, because a compound known to disrupt hydrophobic interactions, 1,6-hexanediol, is widely used to inhibit LLPS. Moreover, a number of proteins that form LLPS droplets are more efficient under high salt concentrations and high temperatures, which favor hydrophobic interactions [24,25]. LLPS is regulated through various modifications, such as lysine methylation and acetylation, arginine methylation, and serine and tyrosine phosphorylation [26]. However, little is known about the involvement of long fatty acylation in the formation of LLPS droplets. Based on its hydrophobic property and oligomerization capability, protein myristoylation could be a possible contributor to LLPS.

Enhancer of zeste homolog 2 (EZH2) is a lysine methyltransferase that targets a variety of substrates, including H3K27, AR and STAT3 [27,28]. EZH2 plays an oncogenic role in both histone methylation-dependent and independent manners and is an important anticancer drug target. Tazemetostat is a first-in-class EZH2 inhibitor that was recently approved for the treatment of sarcoma [29]. STAT3 is a bona fide substrate of EZH2 in oncogenic signaling. EZH2 interacts with STAT3 and methylates it at K180, enhancing STAT3 Y705 phosphorylation, a hallmark of STAT3 activation, resulting in elevated tumorigenicity of glioblastoma stem-like cells [28]. EZH2-mediated STAT3 K180 methylation has also been implicated in psoriasis, head and neck cancer, and lung cancer [[30], [31], [32]]. In addition, the K49 residue of STAT3 is methylated by EZH2, and this methylation is crucial for the transcriptional activity of STAT3 in response to IL-6 [33]. Interestingly, the interaction between EZH2 and STAT3 was largely enhanced by EZH2 S21 and T345 phosphorylation catalyzed by AKT and CDK4/6, respectively [28,31]. In contrast, AMPK catalyzes EZH2 T311 phosphorylation and negatively regulates methyltransferase activity as well as the oncogenic performance of EZH2 [34]. Besides phosphorylation, EZH2 is also regulated by methylation, acetylation, ubiquitination, and particularly, palmitoylation [[35], [36], [37]]. The presence of a G2 residue in EZH2 enables its myristoylation.

The N-terminus of EZH2 mediates protein-protein interactions and plays an important role in tumorigenesis [38]. The noncanonical H3K27 methylation-independent oncogenic function of EZH2 is likely to be achieved by N-terminal S21 phosphorylation and the subsequent EZH2-STAT3 interaction, as suggested by multiple studies [27,28,30]. Here, we provide evidence that EZH2 can be myristoylated at the N-terminal glycine in lung cancer cells. We show that myristoylation enables EZH2 to form phase-separated droplets in vitro and liquid-like nuclear puncta in lung cancer cells. Moreover, STAT3 is shown to co-localize with EZH2 in these puncta. The interaction between EZH2 and STAT3 is also enhanced by EZH2 myristolyation, leading to elevated STAT3 Y705 phosphorylation and increased STAT3 transcriptional activity. Furthermore, myristoylation of EZH2 promotes in vivo lung cancer cell growth. These findings provide a rationale for targeting EZH2 myristoylation in lung cancer intervention.

Section snippets

Reagents and cell culture

Reagents for click chemistry assay, including alkyne myristic acid, biotin picolyl azide, and Click-&-Go™ protein reaction buffer, were purchased from Click Chemistry Tools (Scottsdale, AZ, US). Streptavidin-horseradish peroxidase (HRP) was obtained from Beyotime (Shanghai, China). Anti-MYC magnetic beads were obtained from Bimake (Shanghai, China) and Anti-FLAG M2 affinity gels were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used for detection of the

EZH2 can be myristoylated at Gly2 in cells

The H3K27 methylation-independent oncogenic function of EZH2 has been documented in multiple cancers, including prostate cancer, glioblastoma, head and neck cancer, and lung cancer [27,28,30,32]. This H3K27 methylation-independent function of EZH2 appears to be specific to the N-terminal S21 phosphorylation, suggesting the potential importance of its N-terminal region. Moreover, the N-terminal sequence of EZH2 is not only evolutionarily conserved (Fig. 1A) but also matches two alternative

Discussion

Protein N-myristoylation is essential for cell signaling transduction by promoting protein-protein and protein-lipid interactions. Although proteins within the nuclear interior are integral parts of the cell signaling machinery, only a few myristoylated proteins have been identified in the nucleus [18]. In the present study, we demonstrated that EZH2, a nucleus-resident protein, can be modified by myristoylation at its G2 residue in lung cancer cells. Although the N-terminal sequence of EZH2

Authors' contributions

MW, YEC, JZ, and YZ conceived and designed the study; JZ, YZ, YX, XL, LZ, LH, and MW performed the experiments and analyzed the data; MW, YEC, LH, JZ, YZ, and YX wrote the paper. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions and by grants 31801058, 81802885, 81820108023, 31771608, 2018YFC1705505, and 2016YFC1302402 from the China Natural Science Foundation, grant BK20180839 from the Natural Science Foundation of Jiangsu Province, China. L.Z. was supported by Hui-Chun Chin and Tsung-Dao Lee Chinese Undergraduate Research Endowment (CURE).

References (72)

  • A. Erijman et al.

    Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering

    J. Struct. Biol.

    (2011)
  • A. Molliex et al.

    Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization

    Cell

    (2015)
  • A. Patel et al.

    A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation

    Cell

    (2015)
  • S. Boeynaems et al.

    Protein phase separation: a new phase in cell biology

    Trends Cell Biol.

    (2018)
  • A.A. Hyman et al.

    Beyond stereospecificity: liquids and mesoscale organization of cytoplasm

    Dev. Cell

    (2011)
  • J.W. Darnowski et al.

    Stat3 cleavage by caspases: impact on full-length Stat3 expression, fragment formation, and transcriptional activity

    J. Biol. Chem.

    (2006)
  • O. Hantschel et al.

    A myristoyl/phosphotyrosine switch regulates c-Abl

    Cell

    (2003)
  • M. Loreti et al.

    The regulatory proteins DSCR6 and Ezh2 oppositely regulate Stat3 transcriptional activity in mesoderm patterning during Xenopus development

    J. Biol. Chem.

    (2020)
  • Z. Feng et al.

    Formation of biological condensates via phase separation: characteristics, analytical methods, and physiological implications

    J. Biol. Chem.

    (2019)
  • M. Jiang et al.

    S-Palmitoylation of junctophilin-2 is critical for its role in tethering the sarcoplasmic reticulum to the plasma membrane

    J. Biol. Chem.

    (2019)
  • A. Sing et al.

    A vertebrate Polycomb response element governs segmentation of the posterior hindbrain

    Cell

    (2009)
  • A.L. Pranada et al.

    Real time analysis of STAT3 nucleocytoplasmic shuttling

    J. Biol. Chem.

    (2004)
  • Y. Wang et al.

    GdX/UBL4A specifically stabilizes the TC45/STAT3 association and promotes dephosphorylation of STAT3 to repress tumorigenesis

    Mol. Cell.

    (2014)
  • C. Huang et al.

    Acetylation within the N- and C-terminal domains of Src regulates distinct roles of STAT3-mediated tumorigenesis

    Canc. Res.

    (2018)
  • R.T. Timms et al.

    A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

    Science

    (2019)
  • X.G. Zhu et al.

    CHP1 regulates compartmentalized glycerolipid synthesis by activating GPAT4

    Mol. Cell.

    (2019)
  • H. Li et al.

    Myristoylation is required for human immunodeficiency virus type 1 gag-gag multimerization in mammalian cells

    J. Virol.

    (2007)
  • M. Dolezal et al.

    Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

    Retrovirology

    (2016)
  • D.S. Spassov et al.

    A dimerization function in the intrinsically disordered N-terminal region of Src

    Cell Rep.

    (2018)
  • S. Kim et al.

    Blocking myristoylation of Src inhibits its kinase activity and suppresses prostate cancer progression

    Canc. Res.

    (2017)
  • B. Castrec et al.

    Structural and genomic decoding of human and plant myristoylomes reveals a definitive recognition pattern

    Nat. Chem. Biol.

    (2018)
  • S. Kumar et al.

    Novel myristoylation of the sperm-specific hexokinase 1 isoform regulates its atypical localization

    Biol. Open

    (2015)
  • G. Bologna et al.

    N-Terminal myristoylation predictions by ensembles of neural networks

    Proteomics

    (2004)
  • S. Yamauchi et al.

    The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system

    FEBS J.

    (2010)
  • E. Thinon et al.

    Global profiling of co- and post-translationally N-myristoylated proteomes in human cells

    Nat. Commun.

    (2014)
  • J.A. Traverso et al.

    High-throughput profiling of N-myristoylation substrate specificity across species including pathogens

    Proteomics

    (2013)
  • Cited by (22)

    • Lysine demethylase KDM1A promotes cell growth via FKBP8–BCL2 axis in hepatocellular carcinoma

      2022, Journal of Biological Chemistry
      Citation Excerpt :

      Pictures were acquired with Epson document scanner. Transfection-co-IP assay was performed as previously reported (43, 47). About 48 h after transfection, cells were collected into IPE150 buffer.

    • Long non-coding RNAs and exosomal lncRNAs: Potential functions in lung cancer progression, drug resistance and tumor microenvironment remodeling

      2022, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      EZH2 is considered as a promising target in lung cancer therapy, as it induces the malignant behavior of lung cancer cells via chromatin remodeling [265]. EZH2 modification at N-terminal glycine by myristoylation leads to STAT3 signaling induction and promoting lung cancer progression [266]. Interestingly, a recent experiment has shown that EZH2 downregulation paves the way for chemoresistance in NSCLC via MET expression and phosphorylation [239].

    View all citing articles on Scopus
    1

    J. Zhang and Y. Zeng are co-first authors of this article.

    View full text