Post-translational modifications of EMT transcriptional factors in cancer metastasis

Rui Chang; Peng Zhang; Jiacong You

doi:10.1515/biol-2016-0033

Artikel Open Access

Post-translational modifications of EMT transcriptional factors in cancer metastasis

Rui Chang , Peng Zhang und Jiacong You

Veröffentlicht/Copyright: 13. Oktober 2016

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Abstract

Metastasis is an important reason for death of cancer patients which characterized as the formation of secondary cancers at distant sites. Epithelial– mesenchymal transition (EMT) is a dynamic process that appear to facilitate tumor metastasis in various cancers by switching epithelial cells into mesenchymal properties. Although previous investigation suggested a key role of EMT transcriptional factors in suppression of E-cadherin, the association of these factors with other cellular regulators in cancer metastasis need to be fully elucidated. Post-translational modifications (PTMs), such as acetylation and phosphorylation, have emerged as an important mechanism to modulate biological behavior of substrate proteins. In this review, we summarized protein modification and subsequent function changes of Snail, Twist and ZEB, as well as their influence on tumor progression. Acetylation of EMT transcriptional factors usually cause nuclear localization and/or protein stabilization thus contribute to E-cadherin repression. Besides, Twist and ZEB were phosphorylated by diverse kinases to promote metastasis in many cancers, while Snail was negatively regulated by phosphorylation to degradation. Then, the potential of therapy for metastasis by targeting PTMs-involved regulation of EMT transcriptional factors were discussed.

Keywords: EMT; acetylation; phosphorylation; transcriptional factor

1 Introduction

Cancer metastasis is a significant event in the development of tumor, which consist of several intricate steps. It was accepted that the tumor cells may acquire the capability to move from the primary site to the distant microenvironment. This provides a feasibility for the establishment of malignant tumor. In the early stage of metastasis, the epithelial properties of cells may disappear while their mesenchymal characteristics arise, which lead to epithelial-mesenchymal transition (EMT). EMT was considered as the primary mechanism that cells acquire the metastatic ability [1]. During this morphogenetic process, repression of E-cadherin, a epithelial-related marker, was assumed as the hallmark of EMT. Several signaling pathways, such as Wnt, Notch and TGF-β, were involved in the promotion of EMT. Moreover, EMT was also regulated by transcriptional factors that inhibit expression level of E-cadherin, including Snail, Twist and ZEB1/2. Snail was a zinc-finger protein that induce EMT process by binding to E-box sequences of E-cadherin promoter to suppress transcription. The basic helix-loop-helix (bHLH) protein Twist is another EMT transcriptional factor that could enhance cell motility and activate the EMT process. It was reported that binding of ZEB proteins to miR-200 promoter lead to transcriptional repression, while Smad-mediated transcription was conversely regulated by ZEB1/2 proteins.

Post-translational modifications (PTMs) refer to the enzyme-dependent modification of proteins after synthesis of proteins. Acetylation of histone and nonhistone proteins was catalyzed by histone acetyltransferases (HATs), which play significant roles in the modulation of transcriptional activation, sub-cellular localization, half-life of protein, and DNA/protein binding ability. HATs are a large family that possess intrinsic acetyl-transfer capability for modification of the substrate, including p300, CBP, and PCAF. It was acknowledged that not only basal transcription, but also signaling pathway, cell cycle, tumorigenesis and diverse intracellular events were regulated by acetylation. Besides, phosphorylation represent another common and best characterized modification that engage in a variety of cellular programs. The reversible protein phosphorylation were manipulated by kinases and phosphatases to catalyze phosphorylation and dephosphorylation, respectively. During this modification, the function and activity of substrate proteins maybe altered by addition or removal of phosphoryl group. Dynamic protein phosphorylation modulates a wide range of molecular events that participated in diverse cellular processes such as proliferation, apoptosis and cancer development.

The molecular mechanism of EMT and metastasis were investigated in depth in recent years, which contribute to the therapy that targeting EMT and new drugs development. However, cross-talk between EMT transcriptional factors and PTMs that manipulate metastasis and tumor progression has not been thoroughly studied. A deeper insights into this research field may pave the way for the establishment of a network to explaining the crucial steps in EMT and cancer metastasis. This review focus mainly on the acetylation and phosphorylation of EMT transcriptional factors, the functional consequences by these modifications and the influence on the EMT process. Furthermore, the contribution of modifications of EMT transcriptional factors to tumor progression and therapy against metastasis by targeting the PTMs pathway will be discussed.

2 Acetylation of EMT transcriptional factors

2.1 Acetylation of Snail

The EMT transcriptional factor Snail exert its metastasis-inductive function by down-regulation of E-cadherin. The 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 in the regulation of Snail-mediated EMT process. A positive correlation between p300 expression level and metastasis potential was discovered in hepatocellular carcinomas(HCC) by Yokomizo et al. The inhibition of p300 result in up-regulation of E-cadherin, thus they assumed that p300 act as a possible modulator of invasion and migration of HCC [2]. Another study in fibroblast-like cells demonstrated that the expression of Snail and p300 were increased during cardiac EMT, indicating a possibility that p300 may be a target to suppress metastasis [3]. However, as observed by Liu et al., p300 act as a positive regulator with other factors to enhance expression of E-cadherin in breast cancer cells. Moreover, they found that p300 interacted with endogenous E-cadherin promoter to repress the metastasis potential [4].

The association between the HAT p300 and EMT has emerged as an important underlying mechanism in cancer metastasis and remain to be fully investigated. According to Hsu et al. [5] and Mu et al. [6], p300 synergized with other cellular regulators to induce expression of Snail, result in a decrease of E-cadherin and enhancement of cell invasiveness. Furthermore, study of RNA helicase p68 in colon cancer revealed that the phosphorylation of p68 lead to disintegration of HDAC1 from Snail1 promoter, suggesting a correlation of HDAC1 with activation of Snail in metastasis [7]. The cooperation of p300-c-Myc and DOT1L also facilitates the dissociation of HDAC1 and histone acetylation, thus promote EMT process in human breast cancer [8]. These data implicated a potential mechanism that EMT transcriptional factors such as Snail, trigger EMT and metastasis via an epigenetic switch.

A recent study made by Feng et al. implied that VPA (a HDAC inhibitor) could induce EMT in colorectal cancer cells. Then they discovered acetylation of Snail by VPA treatment, which stabilized Snail as well as promote its nuclear localization [9]. In addition, there was an protein-protein interaction between CBP/p300 and Snail, and the 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 that Snail was acetylated by CBP at Lys 146 and Lys 187. 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 modification in cancer cells, they observed an inhibition of Snail repressor complex assembly by Snail acetylation, which lead to an enhancement on tumor metastasis process [10].

2.2 Acetylation of Twist

As an important member of EMT-activating transcriptional factors, Twist was previous showed to exert its oncogenic function through multiple pathways. Recently, findings of several groups indicated that both Twist and HATs are closely implicated in metastasis and tumor growth. Shiota et al. examined regulatory mechanism of cell motility control by Foxo3a and p300 in human bladder cancer. The repression of p300 was associated with Twist1 up-regulation and E-cadherin reduction by Foxo3a-knockdown in KK47 cell line. These results suggest a putative correlation between p300 and Twist1 in the modulation of urothelial cancer invasiveness [11]. Epigenetic activation of Twist1 was confirmed by histone acetyltransferase CBP, which cooperated with metadherin to increase Twist1 expression in breast cancer [12]. Next, in an attempt to investigate the underlying role of Twist1 in progression of human gastric cancer, Qian et al. identified the interaction of p300 with Twist1 by co-immunoprecipitation. Hence, p300 act as a coactivator for Twist-l-mediated regulation of target gene activation [13].

Studies from Hamamori et al. also suggested an interaction between Twist and p300 or p300/CBP-associated factor (PCAF), the domains involved in their association were HAT and CH3 Domains of p300, C-Terminal HAT and bromodomains of PCAF, respectively. Interestingly, the HAT activity of p300 and PCAF were inhibited by Twist binding, whereas the N terminus of Twist maybe responsible for this suppression [14]. An observation made by a Japanese laboratory indicated that PCAF could acetylate Twist1, which promote Twist1 nucleus localization and its transcriptional potential. Mutation at Lys 73, Lys 76 and Lys 77 result in a reduction of acetylation level, indicating that these residues maybe the acetylated sites in Twist1. Silence of PCAF expression in KK47 cells suppresses invasion and tumorigenesis in a Twist1-dependent fashion [15]. Moreover, Twist was acetylated by Tip60 at Lys 73 and Lys 76 in basal-like breast cancer(BLBC), which was a prerequisite for Twist-BRD4 interaction. This association was required for Wnt5a transcription as well as induction of cancer progression in BLBC [16,17].

2.3 Acetylation of ZEB

The ZEB1/2 are EMT regulators harboring zinc-finger domains that suppress E-cadherin and promote cancer metastasis. In human colon carcinomas, a stronger correlation between ZEB1 and vitamin D receptor was detected when p300 expression was up-regulated. However, correlation between ZEB1 and E-cadherin was not influenced by p300 level [18]. Evidence from co-immunoprecipation assay revealed p300 and PCAF interacted with N-terminal domain of ZEB1. Additionally, acetylation of ZEB1 by PCAF antagonize the binding of corepressor CtBP, thus change ZEB1 into a transcriptional activator [19]. To better understand how epigenetic regulation were involved in the ZEB1-modulated EMT process, Mizuguchi et al. investigate the functional mechanism of miR-200 family in human intrahepatic cholangiocarcinoma cells. They discovered that p300 and PCAF may interacted with ZEB1 on miR-200c/141 promoter and enhance transcription by acetylation of ZEB1. Consequently, the ZEB1-mediated manipulation of epithelial and mesenchymal properties were depend on HAT activity [20]. There was also an interaction between Tip60 and ZEB1, suggesting a role of Tip60 as cofactor of ZEB1 repression [21].

3 Phosphorylation of EMT transcriptional factors

3.1 Phosphorylation of Snail

In the last few years, a number of reports confirmed Snail phosphorylation and its functional consequences in different carcinomas. In an investigation to determine the signaling pathway underlying E-cadherin, Bauer et al. observed the phenomenon that the kinase GSK-3β interacted with and phosphorylated Snail. Therefore, Snail was blocked in the cytoplasm and inactivated in oral squamous cell carcinoma [22]. Interestingly, Snail function was dual modulated by GSK-3β-dependent phosphorylation. The modification of the first domain promote protein degradation, whereas the nuclear localization of Snail was programmed by phosphorylation of the second domain [23]. The study from Sekiya and Suzuki indicating that GSK3-β has a fundamental role in liver regeneration through its phosphorylation of Snail. The subsequent 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 GSK-3β phosphorylation consensus, which stabilized Snail and increase its nuclear accumulation. Hence, the activity of E-cadherin promoter was repressed while tumor metastasis was enhanced [25]. The degradation of Snail through GSK3-β-mediated phosphorylation was antagonized by association between LOXL2 and Snail [26]. Besides, Slug/Snail2 was also implied to be regulated by phosphorylation viaGSK-3β. In parallel with functional consequence of Snail, Kim et al. discovered cytoplasm-localization and degradation of phosphorylated Slug/ Snail2 [27]. Fucntional characterization of Ser 4 and Ser 88 as phosphorylation sites in Snail2 implicated that EMT induction was depended on phosphorylation of Ser 4 [28]. These results indicated the involvement of PTMs in Slug/ Snail2-regulated EMT process.

It was recently reported that Snail was phosphorylated by Ataxia Telangiectasia Mutated (ATM), a kinase that phosphorylates diverse substrates in response to DNA damage. In breast tumor tissues, there was a correlation of ATM-induced phosphorylation of Snail with lymph-node metastasis. Phosphorylation at Ser 100 stabilize Snail as well as promote invasion and cancer metastasis, which is important for cell survival in response to ionizing irradiation [29,30]. The identification of ZEB1 as another ATM substrate for phosphorylation and the mechanism behind further confirmed the association between radioresistance and EMT process [31].

The findings of Du et al.suggested the protein kinase D1 (PKD1) as an EMT and metastasis repressor in that it regulate subcellular localization of Snail by Ser 11 phosphorylation. PKD1-mediated phosphorylation of Snail was required for E-cadherin expression in prostate cancer cells [32]. Furthermore, Ser 11 was involved in the interaction between Snail and Fbxo11, followed by ubiquitylation and degradation of Snail upon Fbxo11 E3 ligase activity. Therefore, Fbxo11 appeared to inhibit EMT and tumorigenesis by recognizing and destabilizing phosphorylated Snail [33]. Phosphorylation at Ser 11, Ser 82, Ser 92, Ser 104 and Ser 107 were confirmed by MacPherson et al., which demonstrating a multiply phosphorylation status of Snail1. Snail1 was phosphorylated at S92 by casein kinase-2(CK2) and at S11 by protein kinase A(PKA), respectively. In accordance with previous studies, phosphorylation of Ser 11 was a vital requisite for Snail-mediated EMT and tumor metastasis [34]. CK1 synergized with GSK3-β to phosphorylate Snail, result in ubiquitin-mediated degradation of Snail. Knockdown of CK1 induce cell migration remarkably, suggesting the participation of CK1 in Snail-regulated EMT process [35]. Yand et al. reported that Snail was a kinase substrate of p21-activated kinases 1(Pak1), which phosphorylate Snail at Ser 246. 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 cancer progression [36]. Subcellular localization and stabilization of Snail was programmed by protein kinase Lats2-mediated phosphorylation at Thr 203. Moreover, Lats2 promote EMT in a Snail1-dependent manner and enhance metastasis potential in breast cancer cells [37].

3.2 Phosphorylation of Twist

There was evidence of Twist1 phosphorylation that enchance EMT and invasion in breast cancer cells. Mitogen-activated protein kinases (MAPKs)-dependent phosphorylation at Ser 68 prevent Twist1 from ubiquitination and subsequent degradation [38]. In a following study, Xue’s group revealed that Twist1 was ubiquitously phosphorylated in invasive human breast tumors. Additionally, Twist1 phosphorylation by Akt/protein kinase B(PKB) supports EMT and breast cancer metastasis via TGF-β signaling [39]. The half-life of Twist1 was positive regulated by CK2 binding and phosphorylation on Ser 18 and Ser 20, which enhances OSC-19 SCCHN cell motility [40]. Significantly, Twist1 phosphorylation at Thr 125 and Ser 127 within the ThrGln-Ser (TQS) motif was essential for heterodimerization and Twist1-induced prostate cancer metastasis[41]. The experiments of Bendinelli et al. demonstrated the phosphorylation and increased Twist expression in nuclear by hepatocyte-growth factor (HGF) in bone-metastatic 1833 cells, suggesting a stimulatory function of HGF on Twist intracellular localization and Twist regulation [42]. Twist2 function was also determined to be manipulated by phosphorylation [43]. On the contrary, phosphorylation of Twist by KappaB kinase β (IKKβ) inhibitor result in nuclear export and destabilization of this EMT transcriptional factor [44]. These reports indicated that the gain of function and tumor metastasis-driving property of Twist could be dual-regulated by phosphorylation.

Further, study on Twist1 phosphorylation by Vichalkovski et al. suggested Ser 42 and Ser 123 as phospho-sites by PKB. This modification was required for inhibiting p53 as well as promoting cell survival after DNA damage-induced stress [45]. Besides, phosphorylation of basic-helix I motif by Akt was critical for Twist to cooperated with Hand1 in the regulation of cardiac remodeling [46]. Therefore, Twist phosphorylation may be involved in the cellular programming and development other than cancer metastasis.

4 Conclusion

Cancer metastasis is the key stage in the tumor progression that involves multiple steps with a complex of various regulators. EMT transcriptional factors were widely known to suppress the epithelial characteristics and induce metastasis process in several malignant tumors. These transcriptional factors work in cooperation with other cellular regulators in repression of E-cadherin transcription. For instance, a number of evidence demonstrated that diverse signaling pathways converge to the induction of EMT transcriptional factors and induce metastasis phenotype. Nonetheless, few studies have focused on the post-translational modifications and subsequent function change of these transcriptional factors in tumor progress. Of note, it will be necessary to study the pivotal role of acetylation, phosphorylation or other modifications in regulating the behavior of EMT transcriptional factors for cell migration and invasion.

Currently, growing data implied that PTMs-mediated signaling of EMT transcriptional factors are crucial in development and disease, especially cancer metastasis. Therefore, we highlighted here the mechanism and biological significance of PTMs, which associated with modulation of EMT transcriptional factors and cancer progression. The members of HATs family acetylated histones at promoters of EMT transcriptional factors to increase their transcription. In addition, these factors themselves could be substrates for acetylation to affect EMT process. Lysines acetylation play a critical role in stabilization and subcellular localization of Snail and Twist, while destroy the CtBP binding with ZEB1 (Table1). These modifications may have profound effects on cell invasiveness and migration when target genes (e.g. E-cadherin) are regulated by EMT transcriptional factors acetylation. On the other hand, many investigations indicated that multiple sites were phosphorylated by protein kinases. A majority of serine phosphorylation in Snail lead to cytoplasm translocation and degradation. Interestingly, phosphorylation positively regulate Twist and ZEB function to prolong their half-life or promote nuclear import (Table 2). Considering previous findings, we can speculate that phosphorylation of residues in different motif fulfill diverse functions to EMT transcriptional factors, which may due to their structure difference. Besides, ubiquitination of EMT transcriptional factors usually result in ubiquitin-dependent degradation, a pathway that was crucial for metastasis repression [33,44,47]. Overall, the molecular mechanism that Snail, Twist and ZEB could be dual-regulated by PTMs broaden our knowledge about function of EMT transcriptional factors during cancer metastasis.

Taken together, with improved understanding of the regulatory mechanism behind PTMs-mediated EMT process, it would be worth to determine whether manipulation viaHATs or kinases pathways help to control cancer metastasis. More importantly, the molecular basis of tumor development requires to be comprehensively investigated owing to the opposite effects of PTMs on various cancer cells. Advances of this field not only pave the way for basic research of tumorigenesis, but also facilitate development of therapeutic drugs for metastasis by targeting PTMs. There were reports described application of HDAC or DNMT inhibitor, singly or in combination, as epigenetic drugs to suppress cancer migration and invasion [48-50]. Except for used as prognosis markers of tumor progression, HATs or kinases will be alternative targets against cancer metastasis. Small molecule subject to control acetylation or phosphorylation status of EMT transcriptional factors will be valuable for regulating nuclear localization, stabilization or binding partners of these factors, thus have an influence on E-cadherin expression. Selective inhibition of differential modified residues will further provide potential therapeutic targets to regulate metastasis for personalized cancer therapy.

Table 1

Acetylation of EMT transcripational factors

EMT-TFs	HATs	Substrate residue(s)	Function	Reference
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]

Acknowledgements

This study was partly supported by the grants from TMUGH funding (No. ZYYFY2014002).

Conflicts of Interest: The authors have no conflicts of interest to declare.

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Received: 2016-4-27

Accepted: 2016-6-1

Published Online: 2016-10-13

Published in Print: 2016-1-1

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