Differential Expression of ETS Family Transcription Factors in NCCIT Human Embryonic Carcinoma Cells upon Retinoic Acid-Induced Differentiation

Abstract

E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

The NCCIT human embryonic carcinoma (EC) cell line, derived from a germ cell tumor, exhibits gene expression patterns similar to those of embryonic stem (ES) cells, which have the capacities of unlimited self-renewal and differentiation.^1–3) Differentiation of ES and EC cells can be induced by retinoic acid (RA).^4,5) Upon RA treatment, NCCIT cells lose their pluripotency, undergo morphological changes, flattening and in some cases acquiring the branched and elongated cytoplasmic processes typical of neurons, and exhibit structural modifications of glycosaminoglycan chains produced via the chondroitin sulfate/dermatan sulfate and heparin/heparin sulfate pathways.⁵⁾ Moreover, RA markedly inhibits clonal growth and tumorigenesis in cancer-derived stem cells.^6–8) Therefore, RA-mediated differentiation of EC cells is a useful model for studies of stem cell-derived pluripotency and tumorigenesis.¹⁾

The E26 transformation-specific (ETS) family of transcription factors plays multiple roles in normal and tumorigenic processes such as development, differentiation, homeostasis, proliferation, and apoptosis.^9,10) The canonical family member, the V-ets oncogene, was first identified as a part of the gag-myb-ets fusion gene of the avian transforming retrovirus E26.^11,12) ETS transcription factors are characterized by a highly conserved 85-amino acid DNA-binding domain.¹³⁾ The core recognition sequence of the ETS domain-binding site is a purine-rich sequence [5′-GGA(A/T)-3′] localized in promoter or enhancer regions of target genes.^14,15) ETS family members, of which there are 27 in human and 26 in mouse, are expressed in a variety of tissues and are highly structurally and functionally conserved.¹⁰⁾ The ETS family members can be further classified into 12 subgroups based on the sequences of their ETS DNA-binding domains: ETS, polyomavirus enhancer activator 3 (PEA3), epithelium-specific ETS transcription factor (ESE), ternary complex factor (TCF), E74-like-factor (ELF), translocated ETS leukemia (TEL), ETS-related gene (ERG), ETS repressor factor (ERF), spleen focus forming virus proviral integration oncogene (SPI), ETS-like-gene (ELG), prostate derived ETS transcription factor (PDEF), and ETS-related protein 71 (ER71).¹⁶⁾ It remains unclear how these diverse ETS proteins regulate gene transcription in a specific manner by binding to a common set of target sequences. One possible explanation might be that ETS factors are expressed in a tissue- or cell type-specific manner. Therefore, analysis of the expression of ETS family members in a variety of cell types should facilitate greater understanding of their roles in tumorigenesis. In fact, the expression profiles of ETS family members in cancer cell lines derived from breast and prostate tissues are highly variable and context-dependent.^7–19) In this study, to identify critical ETS transcription factor(s) implicated in the regulatory mechanism of pluripotent and tumorigenic EC cells, we explored the relative mRNA expression levels of ETS factors in NCCIT cells upon RA-mediated differentiation, and then investigated the effects of selected factors on cell proliferation potential. The findings of this study contribute to our understanding of the roles of oncogenic ETS factors in tumorigenic and pluripotent processes of stem cells.

MATERIALS AND METHODS

Cell Culture and Differentiation

As previously described,²⁰⁾ NCCIT cells (American Type Cell Collection; Manassas, VA, U.S.A.) were cultured in Dulbecco’s modified Eagle’s medium (Hyclone Laboratories, Inc., Logan, UT, U.S.A.) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin–streptomycin (Welgene, Daegu, South Korea) in the presence of 5%CO₂ at 37°C. Cells were subcultured every 3 d. To induce differentiation, NCCIT cells were treated with 10 µM RA (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 10 d.

Plasmid Construct

Full length PEA3 and ESE-1 cDNAs (clones purchased from Open Biosystems) were amplified using specific primers [PEA3: 5′-TAA TGG ATC CAT GGA GCG GAG GAT GAA AG-3′ (forward) and 5′-TAA TCT CGA GCT AGT AAG AGT AGC CAC CCTT-3′ (reverse); ESE-1: 5′-AAT GAA TTC ATG GCT GCA ACC TGT GAG ATT-3′ (forward) and 5′-ATT GCG GCC GCT CAG TTC CGA CTC TGG AGA-3′ (reverse)]. The polymerase chain reaction (PCR) products were cloned into the pIRES2-EF1α-EGFP (Clontech, Palo Alto, CA, U.S.A.) vector and verified by sequencing, as previously described.²⁰⁾

For RNA interference, short hairpin RNAs (shRNAs) against PEA3 and ESE-1 were prepared. Briefly, two target sequences for each gene were generated using the Gene Link shRNA Design Guidelines website (http://www.genelink.com/sirna/shrnai.asp) for maximum silencing efficiency, as previously described.²¹⁾ Double-stranded oligonucleotides were generated by annealing the primers specific to PEA3 and ESE-1 (Table 1). The resultant double-stranded oligos were inserted into the pGSH1-GFP shRNA vector (Genlantis, San Diego, CA, U.S.A.) digested with BamHI and NotI. The pGSH1-GFP-luciferase shRNA vector (Genlantis) was used as a control.

Transient Transfection, RNA Preparation, and Quantitative Reverse Transcription (qRT)-PCR

NCCIT cells (3×10⁵) were transfected with pGSH1-GFP-PEA3 shRNA 1/2, ESE-1 shRNA 1/2, FLAG-tagged pIRES2-EF1α-EGFP-PEA3, or FLAG-tagged pIRES2-EF1α-EGFP-ESE-1 using the 25-kDa L-polyethylenimine (PEI) transfection reagent (Polysciences, Warrington, PA, U.S.A.), and the cells were harvested 48 h after transfection. pGSH1-GFP-luciferase shRNA and pIRES2-EF1α-EGFP vectors were used for controls. Total RNAs were isolated from NCCIT cells (naïve or differentiated, transfected or untransfected) using the Trizol reagent (Invitrogen, Carlsbad, CA, U.S.A.) and subjected to reverse transcription for cDNA synthesis using 5 µg total RNA, oligo dT primers (Promega), and Moloney murine leukemia virus reverse transcriptase (Invitrogen), as previously described.²²⁾ Fluorescence-based qRT-PCR was carried out using the SYBR Green reagent (AccuPower® Greenstar™ qPCR PreMix; Bioneer, Daejeon, Korea) and the primers specific for the 27 ETS genes previously reported by He et al.¹⁸⁾; OCT4 served a control for RA-induced differentiation (Table 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified from all samples as a normalization control. Relative quantitation of expression levels was determined by the 2^−ΔΔCt method.²³⁾

Table 1. List of Primer Sequences for Generating PEA3 and ESE-1 shRNA Expression Vectors

Primers	Sequence
PEA3 shRNA1	5′-GATCCGTCACTTCCAGGAGACGTGGAAGCTTGCACGTCTCCTGGAAGTGACTTTTTTGGAAGC-3′ (sense)
	5′-GGCCGCTTCCAAAAAAGTCACTTCCAGGAGACGTGCAAGCTTCCACGTCTCCTGGAAGTGACG-3′ (anti-sense)
PEA3 shRNA2	5′-GATCCGCTCAGGTACCAGACAGTGGAAGCTTGCACTGTCTGGTACCTGAGCTTTTTTGGAAGC-3′ (sense)
	5′-GGCCGCTTCCAAAAAAGCTCAGGTACCAGACAGTGCAAGCTTCCACTGTCTGGTACCTGAGCG-3′ (anti-sense)
ESE-1 shRNA1	5′-GATCCGGATGGCATGGCCTTCCAGGAAGCTTGCTGGAAGGCCATGCCATCCTTTTTTGGAAGC-3′ (sense)
	5′-GGCCGCTTCCAAAAAAGGATGGCATGGCCTTCCAGCAAGCTTCCTGGAAGGCCATGCCATCCG-3′ (anti-sense)
ESE-1 shRNA2	5′-GATCCGACGCAGGTTCTGGACTGGGAAGCTTGCCAGTCCAGAACCTGCGTCTTTTTTGGAAGC-3′ (sense)
	5′-GGCCGCTTCCAAAAAAGACGCAGGTTCTGGACTGGCAAGCTTCCCAGTCCAGAACCTGCGTCG-3′ (anti-sense)

PEA3 and ESE-1 sense and antisense sequences are underlined; the hairpin loop structure containing the HindIII restriction site is italicized.

Table 2. List of Primer Sequences Specific to the Genes Encoding 27 ETS Transcription Factors and OCT4 Used for qRT-PCR

Subfamily		Gene	Alternative names		Forward primer		Reverse primer		Expected size (bp)
ETS		ETS1			5′-CAGGAGATGGCTGGGAATTC-3′		5′-TTACAGCTGCAACTGACTTAG-3′		389
		ETS2			5′-GAGACGGATGGGAGTTTAAG-3′		5′-CGTGGTTTGGGATGCAATAAG-3′		405
ESE		ESE-1	ELF3		5′-CACTGATGGCAAGCTCTTC-3′		5′-GGAGCGCAGGAACTTGAAG-3′		261
		ESE-2	ELF5		5′-CAAGACTGTCACAGTCATAG-3′		5′-GTCAACCCGCTCCAAAATTC-3′		260
		ESE-3	EHF		5′-GCGTCTTCAGGTTCTTGAAATC-3′		5′-GTATTGGCAGCTTCAGTTTTC-3′		211
TEL		TEL1	ETV6		5′-CTCCCACCATTGAACTGTTG-3′		5′-CCATGATGTGGTTCATGTAAG-3′		440
		TEL2	ETV7		5′-GAGGAGTCCCTCAACTTATG-3′		5′-CTTGTCCTGGACCATCTTTC-3′		386
ERF		PE-1	ETV3		5′-AGGAGACCGGACCGAAGAC-3′		5′-TTGACCGAATGTTGATGAATG-3′		446
		ERF	PE-2		5′-CACAAGACCAAGGGGAAAC-3′		5′-TCTGACGTGCCATCACTAC-3′		360
ER71		ER71	ETV2		5′-GCACGGACTGTACCATTTC-3′		5′-GGCTCAGCTTCTCGTAATTC-3′		365
PEA3		ER81	ETV1		5′-TTCAGCTCTGGCAGTTTTTG-3′		5′-AGGCCATGCTCTCATCAAAG-3′		361
		PEA3	ETV4		5′-CTCGCTCCGATACTATTATG-3′		5′-CTCATCCAAGTGGGACAAAG-3′		192
		ERM	ETV5		5′-ACTGGAAGGCAAAGTCAAAC-3′		5′-CAGACAAATTTGTAGACGTATC-3′		314
ELF		ELF1			5′-GCTCTTCCGGACTGTTCATG-3′		5′-CCACACTGACGGTTCCATTG-3′		425
		ELF2	NERF		5′-ACTGCATCTGTGTCAGCAAC-3′		5′-CAGGCTGCATGGTGATTTTG-3′		243
		MEF	ELF4		5′-CGTTCACAATGGCATCATAAC-3′		5′-CTGCCTTTGCCATCCTTTG-3′		452
ELG		GABP-α			5′-CTGGAGTCATCTGGAACTTC-3′		5′-CAATTCCATGGAGCTGCATC-3′		583
PDEF		PDEF	PSE		5′-CTGGGGCGATTCACTACTG-3′		5′-TGACCTTGGGCTCTGGAAG-3′		456
TCF		ELK1			5′-TCGCTGCCTCCTAGCATTC-3′		5′-GCATGAGTCTTTCAGTTGAAC-3′		278
		NET	ELK3		5′-CCTGCCATGACTCCGATTC-3′		5′-AACTGGAACAGCGTGCTTG-3′		336
		Sap1-α	ELK4		5′-ATTGAGGGTGACTGTGAAAG-3′		5′-TTCCAGGGAAGGCAGTTTTG-3′		410
SPI		PU-1	SPI-1		5′-AGATGCACGTCCTCGATAC-3′		5′-TCCAACAGGAACTGGTACAG-3′		239
		SPI-B			5′-CATACCCCACGGAGAACTTC-3′		5′-GTACAGGCGCAGCTTCTTG-3′		205
		SPI-C			5′-AGAGGAGCCTGTCTATAATTG-3′		5′-ATGCCATCTCCGGATTATAC-3′		205
ERG		FLI-1	SIC-1		5′-GAATTCTGGCCTCAACAAAAG-3′		5′-CATCGGGGTCCGTCATTTTG-3′		244
		ERG			5′-AAGTAGCCGCCTTGCAAATC-3′		5′-GTGATAGGAGCCCATGTAC-3′		369
		FEV	PET-1		5′-ATCCAGCTGTGGCAGTTTC-3′		5′-GGCCATGAGGTTGAGTTTG-3′		407
		OCT4	POU5F1		5′-CCCCTGGTGCCGTGAA-3′		5′-GCAAATTGCTCGAGTTCTTTCTG-3′		97

Western-Blot Analysis

For analysis of endogenous PEA3 and ESE-1 protein expression, total protein was purified from naive or differentiated NCCIT cells, as described previously.²¹⁾ Briefly, NCCIT cells were lysed with 1 mL of radio immunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris (pH 8.0) on ice for 20 min. Protein assays were carried out using the Pro-Measure™ reagent (Intron Biotechnology, Daejoen, Korea). Whole-cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Waukesha, WI, U.S.A.). Membranes were blocked with 1% bovine serum albumin (BSA) solution, and then incubated with anti-PEA3 polyclonal (1 : 2500; US Biological, Swampscott, MA, U.S.A.) or anti-ESE-1 polyclonal (1 : 2500; US Biological) antibody. Membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 5000; Santa Cruz Biotechnology) and developed using an enhanced chemiluminescence detection kit (Thermo Scientific, Rockford, IL, U.S.A.).

Cell-Cycle Analysis

NCCIT cells (3×10⁵) were transfected as described above. Forty-eight hours after transfection, cells were harvested and fixed with 70% ethanol. Fixed cells were pelleted, washed in phosphate-buffered saline (PBS), and resuspended in 50 µg/mL RNase A in PBS. The cells were stained with propidium iodide (50 µg/mL) and analyzed on a FACScan flow cytometer using the CellQuest Pro software (BD Biosciences, San Jose, CA, U.S.A.).

Cell Proliferation Analysis

Proliferation potential was analyzed using the Cell-Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan). NCCIT cells (3×10⁵) were transfected as described above. After 48 h, the cells were re-plated onto 96-well plates and cultured for 3 d; five replicate wells were seeded for each sample. Ten microliters of CCK-8 solution was added into each well and incubated for 1 h. Absorbance was measured at 420 nm to determine the number of live cells in each well.

Statistical Analysis

All data are expressed as means±standard deviation (S.D.) of at least three independent experiments. Data were analyzed by t-test or ANOVA with Duncan’s multiple range procedure for multiple comparisons, using SigmaPlot 10 (Systat Software, Inc., San Jose, CA, U.S.A.). In all experiments, p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

NCCIT human EC cells, which were derived from a germ cell tumor, have both pluripotent and tumorigenic characteristics.¹⁾ RA can induce differentiation and further inhibit clonal growth and tumorigenicity in these cells.^6,7) In this study, we measured the expression patterns of 27 ETS transcription factors in NCCIT cells upon RA-mediated differentiation. Differentiation was induced by treating cells with RA for 10 d, and qRT-PCR was performed using primers specific for genes encoding each of the 27 ETS family transcription factors and the stem cell transcription factor OCT4, as a control ¹⁸⁾ (Table 2). Relative transcript levels were normalized against the level of GAPDH mRNA. Previously, we showed that OCT4 expression is repressed during RA-mediated differentiation in NCCIT cells.^24,25) Indeed, OCT4 expression was significantly downregulated in RA-treated NCCIT cells, confirming that the cells differentiated (Fig. 1B).

Fig. 1. Differential Gene Expression of 27 ETS Transcription Factors in NCCIT Cells upon Retinoic Acid (RA)-Induced Differentiation

(A) Cells were induced to differentiate by exposure to RA for 10 d, and morphological changes were confirmed by microscopic observation. (B) Quantitative RT-PCR (qRT-PCR) was performed using primers specific for genes encoding of the stem cell transcription factor OCT4. (C) qRT-PCR was performed using primers specific for genes encoding each of the 27 ETS family transcription factors. The same mRNA samples were used for the qRT-PCR analyses in B and C. GAPDH mRNA was used as an internal control for normalization of cDNA content. * p<0.05.

ETS factors play multiple roles in embryogenesis and normal organ development, including processes such as angiogenesis and blood cell lineage differentiation.^9,10,26,27) To facilitate analyses of the expression of ETS transcription factors in NCCIT cells in different states, we divided the ETS factors into 12 subgroups based on the sequences of their ETS DNA-binding domains: PEA3, ESE, ETS, TCF, ELF, TEL, ERG, ERF, SPI, ELG, PDEF, and ER71.¹⁶⁾ The ETS, PEA3, and TCF subgroups were relatively easily detectable in naïve NCCIT cells. After 10 d of RA-induced differentiation, seven genes (ETS2, ESE-1, ETV6, ERF, ELF2, ELK1, and ELK3) were upregulated, and five genes (ER81, PEA3, FLI1, ERM, and ELF1) were downregulated, relative to their levels in naive cells. Two genes (ETV3 and ELK4) remain at similar levels. Nine genes (ETS1, ESE-3, TEL2, ETV2, ELF4, SPI-1, SPI-B, SPI-C, and ERG) were expressed at very low levels, and four genes (ESE-2, GABP-a, PDEF, and FEV) were undetectable in both naïve and differentiated cells (Fig. 1C).

Among the ETS factors, PEA3 and ESE-1 exhibited the largest changes in their gene expression levels upon RA-induced differentiation. Expression of PEA3 decreased more than 30-fold, whereas expression of ESE-1 increased more than 16-fold. Western blots confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated NCCIT cells (Fig. 2). Both of these factors have been implicated in the physiology of cancer and development: polyomavirus enhancer activator 3 (PEA3) plays critical roles in invasion, migration, and proliferation of cancer cells,^28–30) and epithelium-specific ETS transcription factor-1 (ESE-1) mRNA is upregulated at least 6-fold after mouse EC cells differentiate.³¹⁾ Together with the previously published findings, our expression data suggest that the ETS factors PEA3 and ESE-1 play important roles in pluripotent and tumorigenic EC cells.

Fig. 2. Differential Expression of PEA3 and ESE-1 Proteins in NCCIT Cells upon RA-Induced Differentiation

Cells were induced to differentiate by exposure to RA for 10 d, and Western-blot analysis was performed using antibodies specific for PEA3 and ESE-1. β-Actin was used as a loading control.

To understand the biological significance of these genes, we investigated the effect of overexpression and knockdown of PEA3 and ESE-1 on the cell cycle and proliferation in naïve NCCIT cells. First, we confirmed by qRT-PCR that transfection of the overexpression and shRNA 1/2 constructs caused the predicted changes in PEA3 and ESE-1 transcript levels (Fig. 3A). Indeed, PEA3 and ESE-1 transcript levels were significantly increased by overexpression of the wild-type (WT) cDNAs and decreased by shRNA-mediated RNA interference. Next, we assessed the cell proliferation potential of NCCIT cells using the CCK-8 proliferation assay (Fig. 3B). Overexpression of PEA3 WT in NCCIT cells significantly increased cell growth, whereas shRNA-mediated knockdown of PEA3 significantly reduced growth. By contrast, overexpression of ESE-1 decreased proliferation, whereas shRNA-mediated ESE-1 knockdown increased growth significantly (p<0.05).

Fig. 3. Effects of PEA3 and ESE-1 on Cell Cycle and Proliferation in NCCIT Cells

NCCIT cells were transfected with constructs that induced overexpression or shRNA-mediated knockdown of PEA3 and ESE-1. (A) Levels of PEA3 and ESE-1 mRNA transcripts were measured by qRT-PCR, normalized against the level of GAPDH in the same samples, and further normalized to the levels in control transfectants. pGSH1-GFP-luciferase shRNA and pIRES-EF1α-EGFP vectors were used as controls for the knockdown and overexpression constructs, respectively. * p<0.05, ** p<0.01. (B) Proliferation assays. Absorbance was measured at 420 nm to determine the number of live cells in each well. Values labeled with different letters (a–c) are significantly different from one another (p<0.05). (C) Flow-cytometric analysis was performed to assess the cell-cycle distributions of the indicated samples. Naive cells were used as a control. ^abc p<0.05.

Flow-cytometric analysis of GFP-expressing cells revealed changes in cell-cycle patterns (Fig. 3C). Overexpression of PEA3 WT resulted in a significant decrease in the proportion of cells in G1 phase (from 53.1% to 46.6%), and a modest increase in the S-phase (from 24.3 to 27.8%) and G2/M phase (from 23 to 26.5%) populations, compared with the control cells (p<0.05). On the other hand, shRNA-mediated knockdown of PEA3 resulted in a significant increase in the proportion of cells in G1 phase (from 53.1 to 59.8%), a reduction in S-phase (from 24.3 to 19.3%), and a reduction in G2/M phase (from 23 to 20.4%), relative to control cells (p<0.05). The proportion of cells in G1 phase increased from 53.1% in the control to 60.6% in cells overexpressing ESE-1 WT, and decreased from 53.1% in the control to 47.1% in cells expressing ESE-1 shRNA 1/2. The S-phase population decreased from 24.6% in the control to 19.5% in cells overexpressing ESE-1 WT, and increased from 24.6 to 27.5% in cells expressing ESE-1 shRNA 1/2. The G2/M phase population decreased from 22.3% in the control to 19.8% in cells overexpressing ESE-1 WT, and increased from 22.3 to 25.4% in cells expressing ESE-1 shRNA 1/2. These results suggest that PEA3 promotes proliferation of NCCIT cells by increasing the proportion of cells in S-phase, whereas ESE-1 delays proliferation by inhibiting progression from G1 to S. These results suggest that PEA3 and ESE-1 act as an activator and repressor, respectively, of cell-cycle progression and growth of NCCIT cells.

The expression profiles of ETS family members in various cancer cell lines derived from breast and prostate tissues are highly variable and context-dependent.^7–19) In this study, we measured the expression patterns of 27 human ETS factors in NCCIT cells upon RA-induced differentiation. The results suggested that PEA3 and ESE-1 play key roles in these pluripotent and tumorigenic EC cells. PEA3 (also called ETV4 and E1AF) belongs to the PEA3 subfamily of ETS transcription factors; in addition to its ETS DNA-binding domain, it also contains functional acidic domains at both the N- and C-termini. PEA3 is expressed in multiple organs during embryonic and adult development.^32–34) During normal development, PEA3 plays a role in transcriptional control during motor neuron and mammary gland development.^35,36) Deregulation of PEA3 is associated with a variety of cancers including colon, breast, ovarian, prostate, and esophageal cancer.^37–39) ESE-1 (also called ELF3, ESX, jen, and ERT) is expressed in various cancer-derived cell lines, as well as in epithelial cell-rich tissues, and exerts multiple roles in pathophysiological processes.^40–43) Null mutation of Elf3, the mouse homolog of human ESE-1, increases the risk of embryonic death in utero, suggesting that ESE-1 plays an important role in embryonic development.⁴⁴⁾ Currently, in contrast, little is known about the functional significance of PEA3 and ESE-1 in differentiation, pluripotency, and tumor progression in germ cell-derived cancer cells. The identification of critical ETS transcription factors in this study will contribute to our understanding of the complicated roles of ETS factors in processes related to tumorigenesis and pluripotency in these cells.

Acknowledgment

This work was supported by the Basic Research Program (NRF-2013R1A1A2A10009965) and the Next-Generation BioGreen21 Program (No. PJ009620) funded by the Rural Development Administration, Republic of Korea.

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