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.

Growth and Differentiation Factor 3 Is Transcriptionally Regulated by OCT4 in Human Embryonic Carcinoma Cells

“…38) We also previously reported that OCT4 and GDF3 are highly expressed in pluripotent human EC NCCIT cells and that this expression is reduced upon RA treatment in a time-dependent manner. 30,36,37,39,40) However, there is no report concerning the interaction between OCT4 and GDF3 at the transcriptional level. Therefore, to investigate their transcriptional relationship in pluripotent germ cell-derived EC cells, we first examined the expression of GDF3 and OCT4 in NCCIT cells during RA-mediated differentiation.…”

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

“…FLI1 is a member of the large ETS transcription factor family 43 and has been associated with an increased proliferation, differentiation and evasion from apoptosis in human cancer cells. 44 , 45 Aberrant FLI1 activation induces dysregulated cell division and malignant transformation, 46 , 47 and in the NIH3T3 murine cell line with induced overexpression of FLI1, drug-induced apoptosis was inhibited. 47 In endothelial cells, ETS phosphorylation via the RAS/MAPK pathway is required for CD13 induction, 48 suggesting that there may be a link between the ETS transcription factor family and CD13.…”

GCNF regulates OCT4 expression through its interactions with nuclear receptor binding elements in NCCIT cells

“…4,5,32,[47][48][49] In our previous studies, treatment of NCCIT cells with RA was found to repress the expression of OCT4 and induces differentiation. 33,34,37,38 Therefore, this study demonstrates that GCNF expression transiently increased to the highest at day 2 and then dropped down afterward throughout the period of RA treatment for 10 days, whereas OCT4 expression gradually decreased upon RA treatment in NCCIT cells (Figure 1). We further confirmed that OCT4 expression is reduced by GCNF overexpression and induced by GCNF shRNA (Figure 2), suggesting that the level of OCT4 expression is dependent on GCNF expression.…”

“…We also used the pGL3-ti minimal promoter (an adenovirus major late promoter TATA box and mouse terminal deoxynucleotidyl transferase gene initiator sequence, 35 reporter construct containing the OCT4 promoter conserved region 2 (CR2-ti-Luc) and mutants (CR2-ti-Luc [second binding site mutant, M6], CR2-ti-Luc [third binding site mutant, M7], CR2-ti-Luc [second & third binding site mutant, M8]), which have been described previously. 33,34,[36][37][38] Full-length human GCNF complementary cDNA (cDNA) was obtained from the PCR amplification of NCCIT cells and inserted into Flag-tagged pcDNA3.1+ vector. Target sequence for human GCNF RNA interference was obtained from the previous report.…”

“…Chromatin immunoprecipitation (ChIP) assay was performed as described previously 37,38 with a minor modification using the chromatin DNA purified from NCCIT cells differentiated by RA treatment for 2 days. Lysate was precleared using protein G-agarose beads (Santa Cruz Biotechnology) and incubated with mouse monoclonal antibody against GCNF (Santa Cruz Biotechnology) or normal mouse IgG antibody (Santa Cruz Biotechnology).…”