Upregulation of the Catalytic Telomerase Subunit by the Transcription Factor ER81 and Oncogenic HER2/Neu, Ras, or Raf

Abstract

One hallmark of tumor formation is the transcriptional upregulation of human telomerase reverse transcriptase, hTERT, and the resultant induction of telomerase activity. However, little is presently understood about how hTERT is differentially activated in tumor cells versus normal somatic cells. Specifically, it is unclear if oncoproteins can directly elicit hTERT expression. To this end, we now show that three oncoproteins, HER2/Neu, Ras, and Raf, stimulate hTERT promoter activity via the ETS transcription factor ER81 and ERK mitogen-activated protein (MAP) kinases. Mutating ER81 binding sites in the hTERT promoter or suppression of ERK MAP kinase-dependent phosphorylation of ER81 rendered the hTERT promoter unresponsive to HER2/Neu. Further, expression of dominant-negative ER81 or inhibition of HER2/Neu significantly attenuated telomerase activity in HER2/Neu-overexpressing SKBR3 breast cancer cells. Moreover, HER2/Neu, Ras, and Raf collaborated with ER81 to enhance endogenous hTERT gene transcription and telomerase activity in hTERT-negative, nonimmortalized BJ foreskin fibroblasts. Accordingly, hTERT expression was increased in HER2/Neu-positive breast tumors and breast tumor cell lines relative to their HER2/Neu-negative counterparts. Collectively, our data elucidated a mechanism whereby three prominent oncoproteins, HER2/Neu, Ras, and Raf, may facilitate tumor formation by inducing hTERT expression in nonimmortalized cells via the transcription factor ER81.

FIG. 1.

(A) hTERT mRNA levels correlate with HER2/Neu expression. Relative hTERT mRNA levels in HER2/Neu-positive and -negative human breast tumor specimens were determined by semiquantitative RT-PCR. The highest hTERT-to-GAPDH signal ratio (r) detected was set to 1, and all other values were normalized accordingly. (B) Semiquantitative TRAP assay comparing telomerase activity in HER2/Neu-overexpressing breast cancer cell lines (UACC893, SKBR3, and T47D) versus breast cancer cell lines not overexpressing HER2/Neu (HBL100, HS578T, MDA-MB-435, and MCF7). The internal control band for PCR amplification is indicated. (C) Activation of endogenous hTERT transcription by HER2/Neu-V664E and ER81, but not Ets1 or Ets2, in transfected BJ foreskin fibroblasts. Expression of hTERT and, as a control, GAPDH was detected on agarose gels after RT-PCR. (D) Corresponding TRAP assay. (E) Western blot analysis revealing protein levels of ER81, Ets1, and Ets2 in transfected BJ cells.

FIG. 2.

(A) The hTERT promoter is synergistically activated by HER2/Neu and ER81. Full-length (−3337/+438) or truncated hTERT promoter luciferase constructs or the parental vector pGL2-Basic were cotransfected with HER2/Neu-V664E and ER81 into 293T cells as indicated. Activation of luciferase activity by HER2/Neu and ER81 is depicted. (B) HER2/Neu and ER81 synergize to enhance telomerase activity in 293T cells. Protein levels for HER2/Neu and ER81 are depicted in the upper panel, and the corresponding TRAP assay is shown in the lower panel. (C) Response of the −11/+431 hTERT promoter to HER2/Neu-V664E and ER81 in MDA-MB-231 and OVCAR3 cells. Inserts show anti-ER81 Western blots.

FIG. 3.

(A) Scheme of the hTERT promoter from −200 to +431. The five intragenic ETS core sites of the promoter are designated as numbered boxes 1 through 5. Two upstream ETS sites are marked as numbers 6 and 7. The transcription start site (59) is marked by a black arrow, and the start methionine (ATG) is indicated. (B) Electrophoretic mobility shift assays with ³²P-labeled oligonucleotides corresponding to ETS site 3 or 5 and full-length ER81 expressed in 293T cells in the presence and absence of HER2/Neu-V664E. Where indicated, a 20-fold excess of the respective nonlabeled wild-type or mutated (ETS core, GGAA→CCAA) oligonucleotide was added. (C) Analogous to the above results, binding of purified ER81_249-477 to ETS sites 3 and 5. (D) Similar to the above results, DNA binding of wild-type ER81 and the 6xA mutant that were expressed in 293T cells in the presence of HER2/Neu-V664E. Protein levels are shown in the bottom panel.

FIG. 4.

(A and B) Functional effect of mutating from GGAA to CCAA the indicated ETS core sites (indicated by the prefix Δ) of the −11/+431 hTERT luciferase construct in 293T cells. (C) Similarly, analysis of the larger −200/+431 hTERT promoter fused to luciferase cDNA.

FIG. 5.

(A) Electrophoretic mobility shift assays with ³²P-labeled oligonucleotides corresponding to ETS site 3 or 5 incubated with lysates of 293T cells that were or were not transfected with ER81_334-477 expression plasmid. (B) Effect of dominant-negative ER81_334-477 on HER2/Neu- and ER81-mediated −11/+431 hTERT promoter activation in 293T cells. Protein levels corresponding to wild-type ER81 and ER81_334-477 are depicted in the lower panel. (C) Telomerase activity measured by TRAP assay in HER2/Neu-overexpressing SKBR3 cells transfected with or without ER81_334-477.

FIG. 6.

Induction of the −11/+431 hTERT promoter, the TORU promoter, the human −525/+15 MMP-1 promoter, or the human −711/+39 c-fos promoter by HER2/Neu-V664E in the presence of various ETS proteins in 293T cells.

FIG. 7.

(A) Effect of the HER2/Neu inhibitor AG825 on the ability of HER2/Neu-V664E and ER81 to activate the −11/+431 hTERT promoter in 293T cells. (B) ER81 and HER2/Neu-V664E were coexpressed in 293T cells treated with or without AG825. Protein lysates were prepared and utilized in electrophoretic mobility shift assays with radioactively labeled ETS3 and ETS5 oligonucleotides. (C) Respective Western blot showing comparable expression of ER81. (D) TRAP assay demonstrating the effect of AG825 on telomerase activity in HER2/Neu-overexpressing SKBR3 cells.

FIG. 8.

(A) Determination of MAP kinase pathways involved in HER2/Neu induction of the −11/+431 hTERT promoter. 293T cells were transfected as indicated and treated with the ERK MAP kinase pathway inhibitor U0126, the p38 MAP kinase inhibitor SB202190, or the vehicle DMSO. Due to the adverse effect of DMSO on cell growth, absolute luciferase activities were lower than observed before. (B) Electrophoretic mobility shift assays with ³²P-labeled oligonucleotides corresponding to ETS site 3 or 5 incubated with lysates from 293T cells transfected with both ER81 and HER2/Neu-V664 and treated with or without U0126. (C) Corresponding anti-ER81 Western blot. (D) Phosphorylation dependence of ER81 activation of the −11/+438 hTERT promoter. Previously identified phosphorylation sites in ER81 were mutated, and the ability of ER81 to stimulate the hTERT promoter was assessed in transfected 293T cells. The 4xA mutant of ER81 corresponds to an ER81 protein where all MAP kinase phosphorylation sites (S94, T139, T143, and S146) have been mutated to alanine, whereas the 2xA mutant has alanine at the MAP kinase-activated protein kinase phosphorylation sites (S191 and S216) and the 6xA mutant has alanine at all six aforementioned phosphorylation sites.

FIG. 9.

(A) Constitutively active Raf-1 (BXB) enhances the ability of ER81 to activate the −11/+431 hTERT luciferase reporter in 293T cells. The bottom panel shows an anti-ER81 Western blot confirming that comparable amounts of ER81 were expressed in the presence and absence of BXB. (B) Analogous results to those described above, with oncogenic Ras-G12V. (C) RT-PCR illustrating the collaboration of BXB and Ras-G12V with ER81 to stimulate hTERT transcription in nonimmortalized BJ cells. (D and E) Corresponding TRAP assays for ER81 and either BXB or Ras-G12V, respectively.