ETV1 positively regulates transcription of tumor suppressor ARF

Abstract

ETV1 (ETS variant 1) is a transcription factor from the ETS family and an oncogene in several types of human malignancies. Paradoxically, a predicted inactivating mutation in ETV1 was previously found in a clone of HT1080 cells with reduced activity of p53. We report that elevated expression of ETV1 makes p53-null tumor cells hypersensitive to restoration of said tumor suppressor. Furthermore, elevated levels of either wild-type ETV1 or its truncated derivative, dETV1, which mimics the product of an oncogenic rearrangement in certain tumors, results in increased expression of mRNA for p14ARF, a known activator of p53. Accordingly, expression of a luciferase reporter, which is driven by a putative ARF promoter, was elevated by concomitant expression of either ETV1 or dETV1. Our observations point to yet another example of a tumor suppressor gene being activated by a potentially oncogenic signal. A better understanding of the mechanisms that allow a cell to bypass such safeguards is needed in order to predict and prevent the development of an oncogene-tolerant state during cancer evolution.

Keywords: ETV1; oncogenic transformation; p14ARF; p53; tumor suppression.

Figure 1. (A) ETV1 reduces tolerance of Saos2 to re-expression of p53. p53-deficient Saos-2 cells harboring an expression construct for ETV1, anti-sense ETV1 (asETV1) or the corresponding empty vector (pBabePuro) were superinfected with a p53-expressing construct or the corresponding empty vector (pMX12). The cells were selected for the presence of the pMX12-encoded hygromycin resistance marker. The numbers of remaining cells were compared using methylene blue staining and extraction method. Results for each arm of the experiment were normalized for the number of cells in pBabePuro-transduced populations. (B) Tolerance to ETV1 overexpression is enhanced by interference with the function of p53. hTERT-HME1 harboring either dominant-negative p53 fragment (GSE56) or the respective empty vector control (pLXSN) were infected with an ETV1-expressing retrovirus, or the respective empty vector control (pBabePuro). The yield of puromycin-resistant cells was measured using methylene blue staining and extraction method. The results were adjusted for virus titer and are scaled relative to the numbers of cells in pBabePuro-transduced populations.

Figure 2. Effect of ETV1 and dETV1 expression on p14ARF mRNA expression. (A) Schematic representation of ETV1 deletion mutant, dETV1. The dETV1 variant lacks 131 N-terminal amino acids, which results in the loss of a transactivation domain position from amino acids 42 to 73. The DNA-binding ETS domain, as well as another putative transactivation domain spanning amino acids 437–462, remains intact at the C-terminal end. The scheme is not drawn to scale. (B) p14ARF mRNA expression in Saos-2 cells constitutively expressing ETV1 or dETV1 was measured by quantitative RT-PCR, normalized to mean GAPDH transcript levels and reported relative to that in cells harboring the respective empty vector control (pBabePuro). Results are shown as means and standard deviations of three independent replicas.

Figure 3. The effect of ETV1 and dETV1 on ARF promoter activity. (A) The structure of the ARF-luciferase reporter pARF-luc. A fragment of human genome that corresponds to a portion of INK4A exon 1b and the putative ARF promoter was introduced into pGL3Basic plasmid upstream of the firefly luciferase coding region. Translation start sites corresponding to p14ARF and luciferase (show in bold) are maintained in the same open reading. Nucleotide positions are numbered relative to the transcription start site of ARF (“TSS”). The scheme is not drawn to scale. (B) ETV1 increases ARF promoter activity. pARF-luc was co-transfected with a constitutive β-galactosidase reporter pRSV-βgal and either an ETV1-expressing construct or the corresponding empty vector control (pBabePuro). The luciferase activity was normalized for that of β-galactosidase and shown relative to that in vector-transfected cells. (C) ETV1 does not increase the activity of CMV immediate early promoter. CMV-driven luciferase construct pLNCLuc was co-transfected with pRSV-βgal and either an ETV1-expressing construct or the corresponding empty vector control (pBabePuro). (D) ETV1 and dETV1 promote ARF gene expression in Saos-2 cells. p14ARF promoter activity in Saos-2 cells was measured following co-transfection of pARF-luc and pRSV-βgal into Saos-2 cells pre-engineered with the indicated transgenes (see Fig. 2). Luciferase activity was normalized to that of β-galactosidase and is presented relative to the levels in cells expressing empty vector control (pBabePuro). Results are shown as the means and standard deviations of three independent replicas.