Post-Translational Modifications of EMT Transcriptional Factors in Cancer Metastasis

Open Life Sci. 2016; 11: 237–243

Topical Issue on Cancer Signaling, Metastasis and Targeted Therapy Open Access Review Article

Rui Chang, Peng Zhang*, Jiacong You* Post-translational modifications of EMT transcriptional factors in cancer metastasis

DOI 10.1515/biol-2016-0033 Received April 27, 2016; accepted June 1, 2016 1 Introduction

Abstract: Metastasis is an important reason for death Cancer metastasis is a significant event in the of cancer patients which characterized as the formation development of tumor, which consist of several intricate of secondary cancers at distant sites. Epithelial– steps. It was accepted that the tumor cells may acquire mesenchymal transition (EMT) is a dynamic process the capability to move from the primary site to the distant that appear to facilitate tumor metastasis in various microenvironment. This provides a feasibility for the cancers by switching epithelial cells into mesenchymal establishment of malignant tumor. In the early stage of properties. Although previous investigation suggested a metastasis, the epithelial properties of cells may disappear key role of EMT transcriptional factors in suppression of while their mesenchymal characteristics arise, which lead E-cadherin, the association of these factors with other to epithelial-mesenchymal transition (EMT). EMT was cellular regulators in cancer metastasis need to be fully considered as the primary mechanism that cells acquire the elucidated. Post-translational modifications (PTMs), such metastatic ability [1]. During this morphogenetic process, as acetylation and phosphorylation, have emerged as an repression of E-cadherin, a epithelial-related marker, important mechanism to modulate biological behavior was assumed as the hallmark of EMT. Several signaling of substrate proteins. In this review, we summarized pathways, such as Wnt, Notch and TGF-β, were involved in protein modification and subsequent function changes the promotion of EMT. Moreover, EMT was also regulated of Snail, Twist and ZEB, as well as their influence on by transcriptional factors that inhibit expression level of tumor progression. Acetylation of EMT transcriptional E-cadherin, including Snail, Twist and ZEB1/2. Snail was factors usually cause nuclear localization and/or protein a zinc-finger protein that induce EMT process by binding stabilization thus contribute to E-cadherin repression. to E-box sequences of E-cadherin promoter to suppress Besides, Twist and ZEB were phosphorylated by diverse transcription. The basic helix-loop-helix (bHLH) protein kinases to promote metastasis in many cancers, while Twist is another EMT transcriptional factor that could Snail was negatively regulated by phosphorylation enhance cell motility and activate the EMT process. It was to degradation. Then, the potential of therapy for reported that binding of ZEB proteins to miR-200 promoter metastasis by targeting PTMs-involved regulation of EMT lead to transcriptional repression, while Smad-mediated transcriptional factors were discussed. transcription was conversely regulated by ZEB1/2 proteins. Post-translational modifications (PTMs) refer to Keywords: EMT, acetylation, phosphorylation, the enzyme-dependent modification of proteins after transcriptional factor synthesis of proteins. Acetylation of histone and nonhistone proteins was catalyzed by histone acetyltransferases (HATs), which play significant roles *Corresponding authors: Peng Zhang, Department of in the modulation of transcriptional activation, sub- Cardiothoracic Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China, E-mail: [email protected] cellular localization, half-life of protein, and DNA/protein Jiacong You, Tianjin Key Laboratory of Lung Cancer Metastasis and binding ability. HATs are a large family that possess Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin intrinsic acetyl-transfer capability for modification of Medical University General Hospital, Tianjin 300052, China the substrate, including p300, CBP, and PCAF. It was Rui Chang, Tianjin Key Laboratory of Lung Cancer Metastasis and acknowledged that not only basal transcription, but Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China also signaling pathway, cell cycle, tumorigenesis and diverse intracellular events were regulated by acetylation.

Besides, phosphorylation represent another common and with other factors to enhance expression of E-cadherin best characterized modification that engage in a variety of in breast cancer cells. Moreover, they found that p300 cellular programs. The reversible protein phosphorylation interacted with endogenous E-cadherin promoter to were manipulated by kinases and phosphatases to repress the metastasis potential [4]. catalyze phosphorylation and dephosphorylation, The association between the HAT p300 and EMT has respectively. During this modification, the function and emerged as an important underlying mechanism in cancer activity of substrate proteins maybe altered by addition metastasis and remain to be fully investigated. According or removal of phosphoryl group. Dynamic protein to Hsu et al. [5] and Mu et al. [6], p300 synergized with phosphorylation modulates a wide range of molecular other cellular regulators to induce expression of Snail, events that participated in diverse cellular processes such result in a decrease of E-cadherin and enhancement of as proliferation, apoptosis and cancer development. cell invasiveness. Furthermore, study of RNA helicase The molecular mechanism of EMT and metastasis p68 in colon cancer revealed that the phosphorylation of were investigated in depth in recent years, which p68 lead to disintegration of HDAC1 from Snail1 promoter, contribute to the therapy that targeting EMT and new suggesting a correlation of HDAC1 with activation of drugs development. However, cross-talk between EMT Snail in metastasis [7]. The cooperation of p300-c-Myc transcriptional factors and PTMs that manipulate and DOT1L also facilitates the dissociation of HDAC1 and metastasis and tumor progression has not been thoroughly histone acetylation, thus promote EMT process in human studied. A deeper insights into this research field may pave breast cancer [8]. These data implicated a potential the way for the establishment of a network to explaining mechanism that EMT transcriptional factors such as Snail, the crucial steps in EMT and cancer metastasis. This review trigger EMT and metastasis via an epigenetic switch. focus mainly on the acetylation and phosphorylation of A recent study made by Feng et al. implied that VPA EMT transcriptional factors, the functional consequences (a HDAC inhibitor) could induce EMT in colorectal cancer by these modifications and the influence on the EMT cells. Then they discovered acetylation of Snail by VPA process. Furthermore, the contribution of modifications treatment, which stabilized Snail as well as promote its of EMT transcriptional factors to tumor progression and nuclear localization [9]. In addition, there was an protein- therapy against metastasis by targeting the PTMs pathway protein interaction between CBP/p300 and Snail, and the will be discussed. HAT domain of CBP was involved in these association. To clarify the function of the cross-talk, the author analyzed changes by over-expression of CBP and found 2 Acetylation of EMT transcriptional that Snail was acetylated by CBP at Lys 146 and Lys 187. factors Besides, the nonacetylatable Snail mutant was less stable than the wild-type, which was consistent with previous report. In an effort to assess the functional impact of this 2.1 Acetylation of Snail modification in cancer cells, they observed an inhibition of Snail repressor complex assembly by Snail acetylation, The EMT transcriptional factor Snail exert its metastasis- which lead to an enhancement on tumor metastasis inductive function by down-regulation of E-cadherin. The process [10]. molecular basis of this key step in tumor development has became the hotspot of cancer research. Recently, there were reports suggested that HATs could play roles 2.2 Acetylation of Twist in the regulation of Snail-mediated EMT process. A positive correlation between p300 expression level and As an important member of EMT-activating transcriptional metastasis potential was discovered in hepatocellular factors, Twist was previous showed to exert its oncogenic carcinomas(HCC) by Yokomizo et al. The inhibition of p300 function through multiple pathways. Recently, findings result in up-regulation of E-cadherin, thus they assumed of several groups indicated that both Twist and HATs are that p300 act as a possible modulator of invasion and closely implicated in metastasis and tumor growth. Shiota migration of HCC [2]. Another study in fibroblast-like cells et al. examined regulatory mechanism of cell motility demonstrated that the expression of Snail and p300 were control by Foxo3a and p300 in human bladder cancer. The increased during cardiac EMT, indicating a possibility that repression of p300 was associated with Twist1 up-regulation p300 may be a target to suppress metastasis [3]. However, and E-cadherin reduction by Foxo3a-knockdown in KK47 as observed by Liu et al., p300 act as a positive regulator cell line. These results suggest a putative correlation Post-translational modifications of EMT transcriptional factors in cancer metastasis 239 between p300 and Twist1 in the modulation of urothelial and PCAF may interacted with ZEB1 on miR-200c/141 cancer invasiveness [11]. Epigenetic activation of Twist1 promoter and enhance transcription by acetylation of was confirmed by histone acetyltransferase CBP, which ZEB1. Consequently, the ZEB1-mediated manipulation of cooperated with metadherin to increase Twist1 expression epithelial and mesenchymal properties were depend on in breast cancer [12]. Next, in an attempt to investigate the HAT activity [20]. There was also an interaction between underlying role of Twist1 in progression of human gastric Tip60 and ZEB1, suggesting a role of Tip60 as cofactor of cancer, Qian et al. identified the interaction of p300 with ZEB1 repression [21]. Twist1 by co-immunoprecipitation. Hence, p300 act as a coactivator for Twist-l-mediated regulation of target gene activation [13]. 3 Phosphorylation of EMT Studies from Hamamori et al. also suggested an transcriptional factors interaction between Twist and p300 or p300/CBP- associated factor (PCAF), the domains involved in their association were HAT and CH3 Domains of p300, 3.1 Phosphorylation of Snail C-Terminal HAT and bromodomains of PCAF, respectively. Interestingly, the HAT activity of p300 and PCAF were In the last few years, a number of reports confirmed inhibited by Twist binding, whereas the N terminus of Snail phosphorylation and its functional consequences Twist maybe responsible for this suppression [14]. An in different carcinomas. In an investigation to determine observation made by a Japanese laboratory indicated the signaling pathway underlying E-cadherin, Bauer et al. that PCAF could acetylate Twist1, which promote Twist1 observed the phenomenon that the kinase GSK-3β interacted nucleus localization and its transcriptional potential. with and phosphorylated Snail. Therefore, Snail was Mutation at Lys 73, Lys 76 and Lys 77 result in a reduction blocked in the cytoplasm and inactivated in oral squamous of acetylation level, indicating that these residues maybe cell carcinoma [22]. Interestingly, Snail function was the acetylated sites in Twist1. Silence of PCAF expression dual modulated by GSK-3β-dependent phosphorylation. in KK47 cells suppresses invasion and tumorigenesis The modification of the first domain promote protein in a Twist1-dependent fashion [15]. Moreover, Twist was degradation, whereas the nuclear localization of Snail acetylated by Tip60 at Lys 73 and Lys 76 in basal-like was programmed by phosphorylation of the second breast cancer(BLBC), which was a prerequisite for Twist- domain [23]. The study from Sekiya and Suzuki indicating BRD4 interaction. This association was required for Wnt5a that GSK3-β has a fundamental role in liver regeneration transcription as well as induction of cancer progression in through its phosphorylation of Snail. The subsequent BLBC [16,17]. degradation of Snail lead to hepatocyte proliferation [24]. One the other hand, small C-terminal domain phosphatase (SCP) interacted with and dephosphorylated Snail at two 2.3 Acetylation of ZEB GSK-3β phosphorylation consensus, which stabilized Snail and increase its nuclear accumulation. Hence, The ZEB1/2 are EMT regulators harboring zinc-finger the activity of E-cadherin promoter was repressed while domains that suppress E-cadherin and promote cancer tumor metastasis was enhanced [25]. The degradation metastasis. In human colon carcinomas, a stronger of Snail through GSK3-β-mediated phosphorylation was correlation between ZEB1 and vitamin D receptor was antagonized by association between LOXL2 and Snail [26]. detected when p300 expression was up-regulated. Besides, Slug/Snail2 was also implied to be regulated by However, correlation between ZEB1 and E-cadherin phosphorylation via GSK-3β. In parallel with functional was not influenced by p300 level [18]. Evidence from consequence of Snail, Kim et al. discovered cytoplasm- co-immunoprecipation assay revealed p300 and PCAF localization and degradation of phosphorylated Slug/ interacted with N-terminal domain of ZEB1. Additionally, Snail2 [27]. Fucntional characterization of Ser 4 and Ser acetylation of ZEB1 by PCAF antagonize the binding of 88 as phosphorylation sites in Snail2 implicated that EMT corepressor CtBP, thus change ZEB1 into a transcriptional induction was depended on phosphorylation of Ser 4 [28]. activator [19]. To better understand how epigenetic These results indicated the involvement of PTMs in Slug/ regulation were involved in the ZEB1-modulated EMT Snail2-regulated EMT process. process, Mizuguchi et al. investigate the functional It was recently reported that Snail was phosphorylated mechanism of miR-200 family in human intrahepatic by Ataxia Telangiectasia Mutated (ATM), a kinase that cholangiocarcinoma cells. They discovered that p300 phosphorylates diverse substrates in response to DNA 240 R. Chang, et al. damage. In breast tumor tissues, there was a correlation was ubiquitously phosphorylated in invasive human of ATM-induced phosphorylation of Snail with lymph- breast tumors. Additionally, Twist1 phosphorylation node metastasis. Phosphorylation at Ser 100 stabilize by Akt/protein kinase B(PKB) supports EMT and breast Snail as well as promote invasion and cancer metastasis, cancer metastasis via TGF-β signaling [39]. The half- which is important for cell survival in response to ionizing life of Twist1 was positive regulated by CK2 binding and irradiation [29,30]. The identification of ZEB1 as another phosphorylation on Ser 18 and Ser 20, which enhances ATM substrate for phosphorylation and the mechanism OSC-19 SCCHN cell motility [40]. Significantly, Twist1 behind further confirmed the association between phosphorylation at Thr 125 and Ser 127 within the Thr- radioresistance and EMT process [31]. Gln-Ser (TQS) motif was essential for heterodimerization The findings of Du et al. suggested the protein kinase and Twist1-induced prostate cancer metastasis[41]. D1 (PKD1) as an EMT and metastasis repressor in that The experiments of Bendinelli et al. demonstrated the it regulate subcellular localization of Snail by Ser 11 phosphorylation and increased Twist expression in phosphorylation. PKD1-mediated phosphorylation of nuclear by hepatocyte-growth factor (HGF) in bone- Snail was required for E-cadherin expression in prostate metastatic 1833 cells, suggesting a stimulatory function cancer cells [32]. Furthermore, Ser 11 was involved in of HGF on Twist intracellular localization and Twist the interaction between Snail and Fbxo11, followed by regulation [42]. Twist2 function was also determined to ubiquitylation and degradation of Snail upon Fbxo11 be manipulated by phosphorylation [43]. On the contrary, E3 ligase activity. Therefore, Fbxo11 appeared to inhibit phosphorylation of Twist by KappaB kinase β (IKKβ) EMT and tumorigenesis by recognizing and destabilizing inhibitor result in nuclear export and destabilization phosphorylated Snail [33]. Phosphorylation at Ser 11, Ser 82, of this EMT transcriptional factor [44]. These reports Ser 92, Ser 104 and Ser 107 were confirmed by MacPherson indicated that the gain of function and tumor metastasis- et al., which demonstrating a multiply phosphorylation driving property of Twist could be dual-regulated by status of Snail1. Snail1 was phosphorylated at S92 by phosphorylation. casein kinase-2(CK2) and at S11 by protein kinase A(PKA), Further, study on Twist1 phosphorylation by respectively. In accordance with previous studies, Vichalkovski et al. suggested Ser 42 and Ser 123 as phospho- phosphorylation of Ser 11 was a vital requisite for Snail- sites by PKB. This modification was required for inhibiting mediated EMT and tumor metastasis [34]. CK1 synergized p53 as well as promoting cell survival after DNA damage- with GSK3-β to phosphorylate Snail, result in ubiquitin- induced stress [45]. Besides, phosphorylation of basic- mediated degradation of Snail. Knockdown of CK1 induce helix I motif by Akt was critical for Twist to cooperated cell migration remarkably, suggesting the participation with Hand1 in the regulation of cardiac remodeling [46]. of CK1 in Snail-regulated EMT process [35]. Yand et al. Therefore, Twist phosphorylation may be involved in the reported that Snail was a kinase substrate of p21-activated cellular programming and development other than cancer kinases 1(Pak1), which phosphorylate Snail at Ser 246. metastasis. Pak1 phosphorylation contribute to translocation of Snail to the nucleus and consequent transcriptional activation, indicating the key role of Pak1 signaling in EMT and breast 4 Conclusion cancer progression [36]. Subcellular localization and stabilization of Snail was programmed by protein kinase Cancer metastasis is the key stage in the tumor Lats2-mediated phosphorylation at Thr 203. Moreover, progression that involves multiple steps with a complex Lats2 promote EMT in a Snail1-dependent manner and of various regulators. EMT transcriptional factors were enhance metastasis potential in breast cancer cells [37]. widely known to suppress the epithelial characteristics and induce metastasis process in several malignant tumors. These transcriptional factors work in cooperation 3.2 Phosphorylation of Twist with other cellular regulators in repression of E-cadherin transcription. For instance, a number of evidence There was evidence of Twist1 phosphorylation demonstrated that diverse signaling pathways converge that enchance EMT and invasion in breast cancer to the induction of EMT transcriptional factors and cells. Mitogen-activated protein kinases (MAPKs)- induce metastasis phenotype. Nonetheless, few studies dependent phosphorylation at Ser 68 prevent Twist1 have focused on the post-translational modifications from ubiquitination and subsequent degradation [38]. and subsequent function change of these transcriptional In a following study, Xue’s group revealed that Twist1 factors in tumor progress. Of note, it will be necessary to Post-translational modifications of EMT transcriptional factors in cancer metastasis 241 study the pivotal role of acetylation, phosphorylation or indicated that multiple sites were phosphorylated by other modifications in regulating the behavior of EMT protein kinases. A majority of serine phosphorylation in transcriptional factors for cell migration and invasion. Snail lead to cytoplasm translocation and degradation. Currently, growing data implied that PTMs-mediated Interestingly, phosphorylation positively regulate Twist signaling of EMT transcriptional factors are crucial in and ZEB function to prolong their half-life or promote development and disease, especially cancer metastasis. nuclear import (Table 2). Considering previous findings, Therefore, we highlighted here the mechanism and we can speculate that phosphorylation of residues biological signiﬁcance of PTMs, which associated with in different motif fulfill diverse functions to EMT modulation of EMT transcriptional factors and cancer transcriptional factors, which may due to their structure progression. The members of HATs family acetylated difference. Besides, ubiquitination of EMT transcriptional histones at promoters of EMT transcriptional factors to factors usually result in ubiquitin-dependent degradation, increase their transcription. In addition, these factors a pathway that was crucial for metastasis repression themselves could be substrates for acetylation to affect [33,44,47]. Overall, the molecular mechanism that Snail, EMT process. Lysines acetylation play a critical role in Twist and ZEB could be dual-regulated by PTMs broaden stabilization and subcellular localization of Snail and our knowledge about function of EMT transcriptional Twist, while destroy the CtBP binding with ZEB1 (Table 1). factors during cancer metastasis. These modifications may have profound effects on cell Taken together, with improved understanding invasiveness and migration when target genes (e.g. of the regulatory mechanism behind PTMs-mediated E-cadherin) are regulated by EMT transcriptional factors EMT process, it would be worth to determine whether acetylation. On the other hand, many investigations manipulation via HATs or kinases pathways help to control

Table 1. Acetylation of EMT transcripational factors

EMT-TFs HATs Substrate Function Reference residue(s)

Snail VPA — Increase stabilization and nucleus localization [9] (HDACi) CBP K146,K187 Increase stabilization and association with coactivators [10] Twist PCAF K73,K76,K77 Increase nucleus localization and activate transcription [15] Tip60 K73,K76 Promote protein interaction [16,17] ZEB PCAF K741,K774,K775 Suppress protein interaction,activate transcription [19,20]

Table 2. Phosphorylation of EMT transcripational factors

EMT-TFs Kinases Substrate residue(s) Function Reference

Snail GSK3-β S96, S100 Promote degradation [22,23] S107, S111, S115, S119 Increase nuclear export [22,23] CK1 S104, S107 Promote degradation [35] CK2 S92 Promote degradation [34] PKA S11 Promote degradation [34] PKD1 S11 Increase nuclear export, [32] promote degradation ATM S100 Increase stabilization [29,30] Lats2 T203 Increase stabilization and nucleus localization [37] Pak1 S246 Increase stabilization and nucleus localization [36] Snail2 GSK3-β S92, S196 Promote degradation [27] S100, S104 Increase nuclear export [27] — S4, S88 Increase stabilization [28] Twist MAPKs S68 Increase stabilization [38] PKB S42 activate transcription [39,45] CK2 S18, S20 Increase stabilization [40] HGF — Increase stabilization and nucleus localization [42] Akt T121, S123 — [46] — T125, S127 Dimerization [41] ZEB ATM S585 Increase stabilization [31] 242 R. Chang, et al.

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