Pleiotropic functions of EAPII/TTRAP/TDP2

Abstract

EAPII (also called TTRAP, TDP2), a protein identified a decade ago, has recently been shown to function as an oncogenic factor. This protein was also proven to be the first 5′-tyrosyl-DNA phosphodiesterase. EAPII has been demonstrated to have promiscuous protein associations, broad responsiveness to various extracellular signals and pleiotropic functions in the development of human diseases including cancer and neurodegenerative disease. Emerging data suggest that EAPII is a multi-functional protein: EAPII repairs enzyme (topoisomerase)-mediated DNA damage by removing phosphotyrosine from DNA adducts; EAPII is involved in multiple signal transduction pathways such as TNF-TNFR, TGF? and MAPK, and EAPII is responsive to immune defense, inflammatory response, virus infection and DNA toxins (chemo or radiation therapy). This review focuses on the current understanding of EAPII biology and its potential relations to many aspects of cancer development, including chromosome instability, tumorigenesis, tumor metastasis and chemoresistance, suggesting it as a potential target for intervention in cancer and other human diseases.

Introduction

EAPII (ETS1-Associated Protein II) was identified as a new protein interacting with ETS1, a transcription factor involved in tumorigenesis and metastasis,^1–3 and this association modulates the transcriptional activity of ETS1.⁴ EAPII was also independently identified and designated as TTRAP (TRAF and TNF receptor-associated protein) through association with the cytoplasmic domain of CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs); this association inhibits NFκB activation.⁵ More recently, EAPII was recognized as the first enzyme that removes topoisomerase-mediated adducts at the 5′-phosphotyrosyl bond, called TDP2 (tyrosyl-DNA phosphodiesterase 2). TDP2 restores 5′-phosphate termini at double strand breaks, preparing them for ligation and overexpression of TDP2 in yeast caused resistance to camptothecin, a topoisomerase I (Top1) inhibitor.⁶ Emerging evidence delineates the physiological and pathological roles of EAPII in many biological processes including embryonic development, neuronal development, and cancer development, progression and chemoresistance. This review summarizes the current understanding of EAPII biology and its biological significance, including gene structure, protein interaction networks, biological functions and related human diseases mainly focusing on cancer. For clarity, we will hereafter use the name EAPII.

EAPII Genomic Organization and Protein Expression

The EAPII gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, mollusca and C. elegans. In humans, the EAPII cytogenetic band is located at 6p22.3–p22.¹. Seven EAPII exons are spread on the reverse strand of 17 kb genomic DNA. A single 2.3 kb transcript of the EAPII gene is ubiquitously expressed in most tissues and cell lines examined, including normal human kidney, lung, liver, thymus, breast, ovary and prostate.^4,5,7 EAPII mRNA encodes a 362-amino acid protein, migrating at an apparent molecular weight of 49 kD. The protein is detectable in most cancer cell lines examined. Interestingly, a variant form of EAPII protein migrating at 43 kD also appears in many cancer cell lines.⁸ Forced expression of exogenous EAPII also produces two bands migrating at 49 kD and 43 kD. Both forms can be specifically recognized by multiple EAPII antibodies and can be knocked down by a variety of short hairpin RNA (shRNA) targeting either the coding sequence or the untranslated region (UTR) of the EAPII transcript.⁸ It is clear that the smaller molecule (43 kD) is a short form of EAPII protein, which exists in mammalian cells. It will be interesting to determine how the short form is produced (cleavage or alternative splicing) and whether it functions differently.

Structure of EAPII protein and potential structural-function correlation.

Protein sequence alignment of EAPII indicates that EAPII contains a conserved domain retaining all six motifs that are hallmarks of an endonuclease/exonuclease/phosphatase super-family (Exo_endo_phos, PF03372) (Fig. 1).⁹ This large family of proteins includes magnesium-dependent endonucleases and phosphatases involved in intracellular signaling. Within the Exo_endo_phos family, EAPII is most similar to human HAP1/REF-1/APE1, which functions as an endonuclease involved in recognizing and cleaving apurinic/apyrimidinic (AP) sites during DNA repair.¹⁰ HAP1/REF-1/APE1 is also involved in the modulation of transcriptional activation through regulating redox signaling.^11,12 Although EAPII shares low overall primary sequence identity with the Exo_endo_phos family members (∼12%), the functionally and structurally critical residues are absolutely conserved: E152, G261, D262, N264, D350 and H351 are critical for enzymatic activity and Q151 and S349 are necessary for orienting and/or stabilizing catalytic residues.^13–16 Based on these conserved motifs, EAPII was predicted to be a phosphodiesterase.⁹ Consistent with this prediction, EAPII was discovered as the first enzyme that is capable of removing topoisomerase II-mediated adducts at the 5′-phosphotyrosyl bond.¹⁷ In addition, EAPII protein contains an ubiquitin-associated protein-like domain (UBA-like domain) at the N-terminus (Fig. 1). The UBA-like domain is a sequence motif present in multiple enzyme classes of the ubiquitination pathway,¹⁸ and this domain provides a binding surface for EAPII to ubiquitin or its relatives, suggesting that EAPII may potentially be involved in the ubiquitin-proteasome pathway. Determination of whether these domains are essential or sufficient to the roles of EAPII is important for exploring the mechanisms underlying its functions.

Figure 1.

Schematic representation of EAPII protein structure. The UBA domain is indicated in red and the Exo_endo_phos domain in solid blue. Six exo_endo_phos motifs are indicated by the amino acid sequence and the starting and ending number and the functional critical residues are highlighted in red. The potential function of the motifs are: Asn hydrogen bonds of TWN to catalytic Asp of the GDXN motif; Glu of LQE coordinates Mg2⁺ or Mn2⁺; GDXN catalytic Asp and Asn hydrogen bonds to scissile phosphate in substrate; and the His of SDH paired with catalytic Asp forms a hydrogen bond to scissile phosphate. The phosphorylation sites (T88 and T92) are also indicated. All amino acid residues are indicated by a one-letter symbol.

Biochemical Functions

Signal transduction and the network of EAPII-associated proteins.

Originally, EAPII was identified as a protein that interacts with multiple components of signaling transduction pathways, including signaling adapters such as CD40, TNFR-75 and TRAFs⁵ and signaling effectors such as ETS1, ETS2, etc.⁴ So far, more than 20 proteins have been identified as being associated with EAPII (Table 1). Two major biological functions are implicated by the network of EAPII-associated proteins: transcriptional regulation and signal transduction.

Table 1.

EAPII-associated proteins and related biological functions

Associated Protein	Interacting Regions in EAPII	Experiment Approaches	Related Functions	Ref.
ETS1	36–362 (N-terminal and central regions)	Yeast two-hybrid, IP, co-localization	Transcription modulator	4
ETS2
FLI1
TNFRSF8/CD30	(230–245 aa)	Yeast two-hybrid	TNFα signaling	5
TNFRSF5/CD40
TNFRSF1B/TNF-R75
TRAF2
TRAF3
TRAF5
TRAF6
SMAD3		Yeast two-hybrid, IP, co-localization	TGFβ signaling	19
ACVR1B/ALK4
Hanta virus NP	20–362	Yeast two-hybrid	Virus response	42
SUMO1
UBE2I
ΦC31 integrase	60–180	Yeast two-hybrid, IP	Virus response	24
HIV-1 integrase	N-terminus	Yeast two-hybrid, IP, co-localization	Virus response	43
DJ-1	C-terminus	Yeast two-hybrid, IP, co-localization	Parkinson disease	33
PML	n/a	Yeast two-hybrid, co-localization	NBs function	44
DAXX
Sp100

Transcriptional regulation. The first major group of EAPII partners are transcription factors including Ets family members [ETS1 (v-ets erythroblastosis virus E26 oncogene homolog 1), ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2), FLI1 (Friend leukemia virus integration 1)],⁴ and smad3.¹⁹ Ets family members control important biological processes, including cellular proliferation, differentiation, lymphocyte development, angiogenesis and tumor metastasis.^1,20 Smad3 is a receptorregulated transcription factor that transduces extracellular signals activated by TGFβ, a multifunctional cytokine involved in the regulation of survival/or apoptosis, proliferation, differentiation and epithelial to mesenchymal transition (EMT) of epithelial cells.^21–23 Through direct protein associations, it has been demonstrated that EAPII inhibits the transactivation by ETS1 ⁴ and SMAD3 ¹⁹ in reporter assays. EAPII and SMAD3 cooperation results in the de-repression of E-cadherin through inhibiting the expression of Snail1a in the embryonic development of zebrafish.¹⁹ EAPII itself is concluded to have no intrinsic transcriptional activity because a Gal4 DNA binding domain-fused EAPII (GAL4-DBD-EAPII) does not transactivate the Gal4 reporter (our unpublished observation). Therefore, although the molecular mechanism is still elusive, EAPII can either positively or negatively modulate the transcriptional activity of other transcription factors. In addition, EAPII may modulate the transcriptional activity of NFκB through regulating the upstream events of the signaling cascade, since no direct associations were identified. Similar repressive effects of EAPII on AP-1 transactivation were also demonstrated.^4,5,24 Another possible mechanism of inhibition is through interfering with the transcriptional activity of ETS1, since ETS1 widely participates in combinatorial transcriptional controls through interaction with AP-1 or NFκB^25–27 (Fig. 2).

Figure 2.

Model of EAPII -mediated transcription modulation: (1) EAPII represses NFκB activity through regulating upstream events of the TRAF-NFκB cascade; (2) EAPII represses transactivation by ETS1 on the MMP1 promoter; and (3) EAPII represses transactivation by Smad3 on Snail1, which is a transcriptional repressor of E-cadherin, subsequently leading to de-repression of E-cadherin transcription. Green lines and arrows denote transactivation and black lines indicate transcriptional repression.

Signal transduction. A large cluster of receptor or adaptor proteins is associated with EAPII. These proteins include TGFβ receptor ACVR1B/ALK4 (activin A type 1B receptor), TNF superfamily receptor and adaptors: TNFRSF8/CD30 (tumor necrosis factor receptor superfamily, member 8), TNFRSF5/CD40 (CD40 molecule, TNF receptor superfamily member 5), TNFRSF1B/TNF-R75 (tumor necrosis factor receptor superfamily, member 1B), and TRAFs (TNF receptor-associated factors: TRAF2, TRAF3, TRAF5 and TRAF6).⁵ The region of the CD40 cytoplasmic domain required for EAPII association (230–245 aa) overlaps with the TRAFs recognition motif. CD40 ligand treatment significantly increases the EAPII-CD40 receptor association and overexpression of EAPII represses receptor-initiated NFκB transactivation but has no effect on that induced by p65 or IKKα, suggesting that EAPII acts on the upstream components of the signaling cascade.⁵ TRAFs participate in diverse signaling pathways, which involve cellular activation, differentiation, cell survival and immune responses,^28,29 and TRAFs play critical roles in the cross-talk among these signaling pathways.^30,31 EAPII is differentially associated with TRAFs, with greatest affinity for TRAF6,⁵ suggesting a complexity of signaling regulation by EAPII. We recently found that EAPII is involved in the modulation of MAPK-ERK signaling in lung cancer cells. EAPII overexpression significantly activates Raf-1 and ERK1/2 but not the JNK and p38 pathways, subsequently regulating downstream targets including MYC and cyclin D1.⁸ These observations indicate that EAPII contributes to lung cancer development through deregulation of EGFR signaling, which has been implicated in the development and progression of NSCLC.³² The phosphorylation of EAPII at threonine 88 or/and 92 (T88 or T92) by TGFβ receptor (ALK4) is essential for the function of EAPII, as demonstrated by the inability of EAPII^T88A/T92A to rescue the defects caused by EAPII knockout during zebrafish gastrulation.¹⁹ This finding suggests that EAPII is likely one of the direct downstream targets of TGFβ receptor (ALK4) in zebrafish. In neuroblastoma cells, interestingly, EAPII represses JNK activity in the presence of wild-type DJ-1. However, mutant DJ-1, which strongly binds to EAPII, induces cell death via the JNK and p38 MAPK pathways,³³ suggesting that EAPII can act as a switch to turn on/off the specific signaling. It is no doubt that these protein associations provide the foundation for the EAPII-related signaling network (Fig. 3).

Figure 3.

Model of EAPII-mediated signal transduction: (1) EAPII negatively modulates TNFα signaling; (2) EAPII facilitates or represses the JNK-mediated apoptosis pathway, depending on the genotype of DJ-1 protein; (3) EAPII activates MAPK-ERK signaling; and (4) EAPII negatively modulates Nodal signaling through Smad3 association.

Tyrosyl-DNA phosphodiesterase (TDP) activity and DNA repair.

Using an elegant experimental design, Ledesma et al. discovered that EAPII possesses enzymatic activity that removes topoisomerase protein from 5′-tyrosyl-DNA adducts in mammalian cells.¹⁷ Topoisomerases are nuclear enzymes needed for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix.³⁴ The enzyme creates breaks in DNA and thereby allows the DNA strands to unknot and untangle. In order to carry out its critical physiological functions, topoisomerase generates transient topoisomerase-DNA cleavage complexes, so-called cleavable complexes, in DNA. Oxidation, ionizing radiation or chemotherapeutic agents can stabilize the complex and prevent the enzyme from resealing the DNA break it creates, resulting in enzyme-mediated DNA damage.³⁵ Tyrosyl-DNA phosphodiesterase 1 (TDP1) was identified to specifically remove the phosphodiester bond from topoisomerase I-3′ tyrosyl-DNA while EAPII (also called TDP2) removes the phosphodiester bond from topoisomerase II-5′ tyrosyl-DNA and generates free 5′-phosphate ready for ligation, providing an error-free DNA repair (Fig. 4). The structure of EAPII shares no common functional domain with TDP1, which is a member of phospholipase D superfamily.³⁶ The Exo_endo_phos domain of EAPII suggests it has nuclease activity (removes the phosphodiester bond between nucleotides). Indeed, cfEAPII, an EAPII homolog from C. farreri, was recently identified and was shown to have prominent endonuclease activity by cleaving the genomic DNA from C. farreri, but not from E. coli or Bacillus subtilis.³⁷ Repairing protein-DNA complexes can also be achieved via nonspecific nucleolytic pathways which include the double-strand break repair (DSBR) pathway mediated by Ku70/Ku80 and/or Rad1/Rad10³⁸ and the single-strand break repair (SSBR) pathway mediated by the XRCC-1 complex.³⁹ In addition, Rad32 (Mre11) nuclease or Ctp1 (CtIP) can remove covalently bound topoisomerase I and II from DNA, and this topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs in yeast.⁴⁰ The removal of protein-DNA adducts is essential for maintaining cell survival, and it has been shown that EAPII provides the major 5′-TDP activity, at least, in chicken DT40 cells.⁴¹ However, genetic depletion of EAPII did not change the proliferation rate of these cells.⁴¹ By contrast, EAPII knockdown in human lung cancer cells resulted in apoptotic cell death.⁸ Therefore, how physiologically prominent EAPII-mediated-DNA repair activity is in cellular homeostasis in mammalian cells and how this activity modulates cell behavior during cancer development are critical questions for the study of EAPII. In addition, EAPII was shown to associate with exogenous pathogens that attack human DNA, including hantavirus (HTNV) nucleocapsid proteins (NPs),⁴² ΦC31 integrases²⁴ and HIV-1 integrases.⁴³ EAPII association with ΦC31 integrase inhibits the efficiency of phage C31 integrase-mediated site-specific recombination, but the association with lentivirus HIV-1 integrase results in an enhanced viral integration, suggesting that EAPII is indeed involved in the processes of these pathogenic attacks. The potential models could be the following: (1) the TDP activity of EAPII helps to resolve the topological catenanes produced by the integrase during the recombination; (2) EAPII associates with PML, Daxx, Sp100 and SUMO1, which are colocalized in the promyelocytic leukemia protein nuclear bodies (PML-NBs).⁴⁴ PML-NBs are matrix-associated domains that have been hypothesized to control key pathways for growth suppression, apoptosis and host anti-viral defense through regulating posttranslational modifications of partner proteins like sumoylation, ubiquitination, phosphorylation or acetylation.^45–50 The role of EAPII in the pathogenesis of these infections is warranted for further study.

Figure 4.

Model of error-free DNA repair function of EAPII: Transit DNA break (cleavable complex) is re-ligated and DNA topological entanglement is resolved (A→B); Stabilizing the transit cleavable complex by TopoII poison results in enzyme-mediated double strand breaks (A→C); TDP2 removes TopoII from protein-DNA adducts and leaves a free phosphate at 5′ of DNA, which is ready for ligation (C→D) and double strand DNA break is repaired (D→B). The solid yellow ovals represent topoisomerase II , and “∼” indicates ester bond. Phosphodiester bond links nucleotide of DNA.

Biological Significance of EAPII and Relevance for Human Diseases

Embryogenesis.

EAPII expression was found in multiple human fetal organs including the lung, brain and thymus.⁴ In mice, EAPII expression can be identified as early as E7 of embryonic development, and the level decreases along the course of fetal development.⁵ In situ hybridization of EAPII showed that EAPII mRNA appears ubiquitously at E12.5. This widespread expression is reduced at E15.5, but the signals from kidney, small intestine, testis, liver and lung remain strong; in particular, the highest expression levels are in the thymus and discrete brain regions,⁵ suggesting an important role of EAPII in embryonic development. In support of these observations, mouse blastocysts with homozygous deficiency of EAPII (EAPII^-/-) can be identified, but EAPII^-/- embryos die before 7.5 d post coitum (dpc) (our unpublished observation), demonstrating that EAPII is a critical gene, at least, for mouse embryogenesis. Consistent with this observation, it was also demonstrated that in zebrafish development, EAPII modulates Nodal signaling, which is critical for mesoderm formation, positioning and left-right asymmetric development of the heart and viscera.⁵¹ EAPII controls gastrulation movements through regulating expression of Snail1a and E-cadherin,¹⁹ both of which are epithelial-mesenchymal transition (EMT) markers involved in both developmental and cancer-related EMT.⁵² DNA repair activity of EAPII might be important for the maintenance of cellular homeostasis of embryos, and the involvement of EAPII in the regulation of cell migration makes this gene more critical for embryonic development.

Cancer.

Modulation of apoptotic induction. Evasion of apoptosis is one of the hallmarks of cancer.⁵³ EAPII may protect cells from apoptosis or promote apoptosis in a cell-specific manner, and controversial observations have been obtained from different models. EAPII protects neuroblastoma cells from apoptosis induced by MG132, a proteasome inhibitor.³³ EAPII knockdown results in apoptosis in H460, H522 and H1975 lung cancer cells.⁸ However, EAPII plays a pro-apoptotic role in some other cell lines. EAPII promotes apoptosis of HL-60 cells induced by hydroquinone, a cytotoxic agent.⁵⁴ The mutant of DJ-1, an oncogene in cooperation with Ras,⁵⁵ promotes apoptosis through JNK and p38 MAPK pathways in an EAPII-dependent manner.³³ Furthermore, EAPII is upregulated in FOXO3a-induced apoptosis, in which FOXO3a turns TNF receptor signaling to a pro-apoptotic JNK-dependent pathway.⁵⁶ The inconsistency of findings regarding the apoptosis regulation of EAPII may result from the complexity of the EAPII interacting-protein network and the EAPII-associated signaling pathways such as TNF and TGFβ, which involve either pro- and/or anti-apoptosis events,^57–59 depending on the integrity of the cascade of specific pathways and the status of the protein(s) in the signaling cascade.^60–62

Cancer development and progression. Although the involvement of EAPII in apoptosis was observed in cultured cells, its biological significance in cancer development remained unknown until recently. Immunohistochemical (IHC) studies showed that EAPII protein is significantly elevated in most non-small cell lung cancer (NSCLC) patients (90%) and that the expression level and localization of EAPII change along the course of lung cancer development: while the nuclear staining of EAPII can be seen in highly proliferative tissue, the majority of lung carcinomas showed cytoplasmic or cytoplasmic and nuclear staining.⁸ This finding suggests that EAPII may contribute to both early and advanced stages of lung cancer development. Indeed, an oncogenic role of EAPII expression was observed in a xenograft model of NSCLC in mice.⁸ This evidence supports our hypothesis that EAPII is an oncogenic factor for lung cancer development. EAPII expression is increased in Barrett's esophagus, a premalignant condition whereby the normal stratified squamous esophageal epithelium undergoes a transdifferentiation program resulting in a simple columnar epithelium reminiscent of the small intestine, and in esophageal adenocarcinoma compared with normal esophagus.^63,64 EAPII is upregulated in cervical cancer and head and neck cancer patients with human papillomavirus (HPV) infection, which is a well-known pathogenic factor for these cancers.⁶⁵ This evidence supports the hypothesis that EAPII contributes to the pathogenesis of cancer. On the other hand, it is noteworthy that EAPII reduction was observed in several types of cancer or precancerous conditions: primary lymphoma cells, primary cutaneous anaplastic large cell lymphoma and primary lymphoma cells of classical Hodgkin lymphoma showed a significant reduction of EAPII expression compared with that in non-neoplastic T- and NK-lymphocytes.⁶⁶ EAPII is downregulated in precancerous adenoma, from which colorectal cancers are believed to predominantly arise, compared with normal mucosa from the same patients.^67–69 Comparison of blood-derived gene-expression profiling indicated that the level of the EAPII transcript was lower in patients with xeroderma pigmentosum (XP)-like syndrome, in which multiple spinocellular carcinomas appear, than in healthy control individuals.⁷⁰ It is known that loss of TDP1, which repairs Top1-mediated DNA damages, may contribute to genomic instability in cancer cells because of the transcription or replication-blocking cleavable complex formation.^71,72 Therefore, it is plausible that DNA repair deficiency due to loss of EAPII disrupts DNA integrity and stability, subsequently resulting in susceptibility of cells to cancer. Altered EAPII expression was also observed in the late stage of cancers: remarkable decreases of EAPII expression were found in a mouse invasive colon cancer cell line (vs. non-invasive cell line) and in human metastatic prostate tumor (vs. the primary prostate tumor),^73,74 consistent with our previous observations that expression of EAPII in prostate cancer cells inhibits cell migration.⁴ Significantly, EAPII may play sophisticated roles in a variety of cancers or cancer developmental stages although the gene arraybased data need to be experimentally validated in the future.

Chemoresistance. Topoisomerase II poisons are widely used in oncology,⁷⁵ and tumor cells may be resistant to these DNA damages due to increased DNA repair.^76–78 Emerging evidence supports that EAPII might be a significant factor involved in chemoresistance. Genetic deletion of EAPII in chicken DT40 cells results in specific and severe sensitivity to Top2 poison etoposide (VP-16), but not to Top1 poison camptothecin (CPT), while EAPII suppresses the sensitivity of yeast cells to CPT.^6,41 In lung cancer A549 cells, which have an elevated EAPII expression, EAPII knockdown results in hypersensitivity to VP-16,^6,41 implying that elevated EAPII levels in cancer cells render them chemoresistant, although a systematic related clinical study has not yet been conducted. Therefore, determining whether altered expression of EAPII could have an impact on the outcome of chemo- or radiation-therapy will have significant implications for cancer therapy.

Neuronal development (reading disability, RD) and Parkinson disease.

High EAPII expression in the developing mouse brain and fetal human brain indicates the functional role of EAPII in neuronal development. Reading disability (RD) or developmental dyslexia (DD) is a condition in which a sufferer displays difficulty reading resulting primarily from neurological factors.⁷⁹ It was hypothesized that impaired neuronal migration is a cellular neurobiological antecedent to RD.⁸⁰ Genetic linkage studies have identified Chromosome 6p23–21.3, where EAPII is located, as one of the potential loci related to RD.^7,81–87 Within this locus, a 77-kilobase region spans the EAPII gene, portions of KIAA0319 and the upstream region of THEM2.^88–91 Current results have converged on KIAA0319 as one of the likeliest RD susceptibility genes at this locus.^92,93 Although a significant association of a haplotype spanning the EAPII gene with reading ability was observed,⁹¹ data are currently inconclusive as to whether EAPII could be another RD susceptibility gene. However, EAPII has shown to be related to the pathogenesis of Parkinson disease. EAPII associates with DJ-1, mutations in which have been demonstrated to cause autosomal recessive Parkinson disease.⁹⁴ The mutant DJ-1 forms aggresome structures in the cytoplasm through recruiting EAPII, facilitating apoptosis of neurons.³³ Since many neurodegenerative diseases are characterized by the accumulation of misfolded proteins that adversely affect neuronal connectivity and plasticity and trigger cell death signaling pathways,^95–100 the appearance of EAPII in the aggresome suggests that EAPII may contribute to Parkinson disease through a similar mechanism.

Regulation of EAPII

EAPII expression.

Understanding the regulation of EAPII expression will reveal its biological significance and define how EAPII coordinates its diverse functions in physiological or pathological conditions. Although such studies are currently limited, array data from public databases allowed us to sketch the processes controlling EAPII regulation. In Table 2, we summarize the available data, albeit the data need to be further validated. It seems that EAPII regulation is controlled by many aspects: (1) transcriptional modulators such as Myb, HOXA9, DEAF1, MDM2 and histone deacetylases (HDAC8), which may directly or indirectly control the transcription of EAPII gene; (2) inflammatory or immune cytokines including IL-19, IL-24, IL-4, GM-CSF, TREM-1 and TRAIL. All of these factors increase EAPII expression, suggesting that EAPII is not only a component of the TNF-TNFR signaling cascade, as evidenced by EAPIIprotein interacting networks, but also an important responsive gene for immune/inflammatory stimuli. Interestingly, significant downregulation of EAPII expression by bacterial lipopolysaccharide (LPS) was found in mammalian (mouse) cells^101–103 and invertebrate (mollusc) cells.³⁷ LPS is one of the most important inflammatory mediators, which activates a potential immune response through Toll-like receptor 4 (TLR4)-TRAF6-p38/JNK/NFκB pathway.¹⁰⁴ Significantly, the inhibition of EAPII expression is important for the activation of key inflammatory pathways such as NFκB. Therefore, a possible feedback loop mediated by EAPII could be a key switch that coordinates immune defense and inflammatory responses. It is noteworthy that EAPII expression can be downregulated by PCDH24 and SERPINA1. The former is related to cell contact inhibition,¹⁰⁵ and the latter is a serine protease inhibitor which represses protease enzymes such as elastase, plasmin, thrombin and plasminogen activator.¹⁰⁶ The substrates of these enzymes are part of the extracellular matrix, implying another potential link of the tumor microenvironment to the function of EAPII.

Table 2.

Regulation of EAPII expression

Modulator	Function	EAPII changes	System/Models	References
knockdown HDAC8 (Histone deacetylases)	Transcription Modulator	↓	prostate cancer cells	107
FOXO3a	Transcription Modulator	↑	Apoptosis	56
HOXA9 knockdown	Transcription Modulator	↓	human AML cells	108
Myb	Transcription Modulator	↑	human monocytes	109, 110
Knockout DEAF1	Transcription Modulator	↑	knockout mice	111
Knockdown MDM2	Transcription Modulator	↑	cancer cell lines: A549, TOV-21G	112
Chronic antigen stimulation		↑	Regulatory T cell	113
TRAIL	TNF-TNFR signaling	↑	T42 breast cancer cells
IL-19	Inflammatory response			114
IL-24		↑
TRE M-1 activation	inflammatory responses	↑		115
IL-4, GM-CSF	T cell differentiation/inflammatory response	↑	Monocyte to DC differentiation	116
LPS	Inflammatory mediator	↓	ATF3-/- mouse wild-type mouse	101–103
Insulin	Glucose metabolism	↓		117
Knockout SIRT6	glucose metabolism/histone deacetylase	↓	liver of SIR T6 knockout mice	118
Protocadherin LKC (PCDH24)	Contact inhibition	↓	Colon cancer cells	Na, 105
α1antitrypsin (SERPINA1)	Serine protease inhibitor	↓	Transgenic mice	119
Knockdown UPF1	eukaryotic surveillance	↑		120
IFNγ	eukaryotic surveillance	↑, NB-localized		44

Data presented in this table are derived from NextBio (www.nextbio.com), which is based on multiple sources including NCBI GEO and EBI ArrayExpress. Original articles are reviewed and cited in the references if the array data have been published.

Nucleus-cytoplasm translocation.

In agreement with the function of EAPII in signaling transduction, the cytoplasmic translocation of EAPII is observed under certain circumstances. Overexpressed EAPII was found in the nucleus,⁴ but endogenous EAPII is expressed in both the nucleus and the cytoplasm: the elevated nuclear staining of EAPII occurs in the highly proliferative bronchial epithelium, and the cytoplasmic staining of EAPII can be observed in many NSCLC tissues.⁸ Therefore, it is possible that an elevated nuclear expression of EAPII favors enhanced proliferative activity in both normal bronchial epithelium and tumor cells, and the cytoplasmic EAPII may be more closely related to its oncogenic role, suggesting that EAPII plays different roles in the cytoplasm and nucleus. Upon proteasome impairment, EAPII relocalizes to the cytoplasm of neuroblastoma cells and forms aggresome-like structures.³³ Furthermore, accumulation of EGFP-EAPII can be found at sites of laser-induced DNA damage,⁶ and occasionally, EAPII also appears in PML-NBs.⁴⁴ These observations indicate that the compartmentalization of EAPII protein may determine its specific targets and unique function, and the regulation of nucleus-cytoplasm translocation is critical for the modulation of EAPII function. In many cases, EAPII interacts with both receptor and endeffector, but it is unknown whether EAPII alone or together with its partner(s) shuttles around in the cytosol or between the cytoplasm and nucleus. Further study of how EAPII localization is regulated and how its localization is correlated with specific functions of EAPII would be helpful in understanding its pleiotropic role. In addition, the interactions of EAPII with UBE2I (ubiquitin-conjugating enzyme E2I) and SUMO1 (SMT3 suppressor of mif two 3 homolog 1) ⁴² suggested that EAPII is potentially SUMO-modified although there is as yet no experimental evidence for this.

Conclusions and Further Directions

Evidence strongly supports that EAPII is a multi-functional protein that plays important roles in many aspects of cell biology, including topological entanglement of DNA catenanes, cellular responsiveness to microenvironments, cell survival and cell migration. These functions are essential for either embryonic or neuronal development or involve many aspects of cancer development, including chromosome instability, tumorigenesis, tumor metastasis and chemoresistance (Fig. 5). Particularly, EAPIImediated signals, including immune defense, inflammatory response and cellular stress such as hypoxia and DNA toxicity, are key players in the orchestration of the tumor microenvironment. The involvement of EAPII in multiple steps of cancer development and chemoresistance implicates it as a potential target for cancer intervention. In future studies, determination of EAPII-mediated signaling pathway(s) and its mechanism is critical for understanding the role of EAPII in cancer development. Other important open questions in the study of EAPII include: (1) Does EAPII have other types of substrate besides DNA or protein-DNA in mammalian cells, and is this related to EAPII's function in signal transduction? The phosphoester bond can be found in nucleic acid (DNA and RNA), protein-DNA, phosphoprotein, protein phosphoglycosylation, phospholipid and second message cAMP and cGMP. (2) How are the signal transduction, transcription and DNA repair aspects of EAPII functions related? Theoretically, the 5′TDP activity of EAPII may generally enhance transcriptional activity by facilitating the opening of the DNA template at the transcription bubble. However, EAPII appears to have specific transcriptional repression activity depending on its interacting partners. (3) Does EAPII play differential functions in embryonic development and cancer progression? How is EAPII reactivated during tumorigenesis and cancer progression? (4) How does the promiscuous protein association of EAPII transduce signals and then change the fate of cancer cells and how is EAPII activity regulated by the tumor microenvironment?

Figure 5.

EAPII-related functions.