Elevated Expression of Ets2 or Distinct Portions of Ets2 Can Reverse Ras-mediated Cellular Transformation

Abstract

Ets transcription factors are important downstream targets of oncogenic Ras. The transcriptional activity of several Ets family members is regulated by Ras, and interfering with Ets-dependent transcription by expression of just the Ets2 DNA binding domain can inhibit or reverse Ras-mediated cellular transformation. To better understand the role of Ets proteins in Ras transformation, we have now analyzed the effects of stably expressing a variety of Ets2 constructs in Ras-transformed NIH3T3 (DT) cells. Expression of only the Ets2 transactivation domains, which also inhibits Ras or Neu/ErbB-2-mediated activation of Ets-dependent transcription, strongly inhibited anchorage-independent growth, but did not revert the transformed DT cell morphology. Unexpectedly, high expression of full-length Ets2, a transcriptional activator, broadly reversed the transformed properties of DT cells, including anchorage-independent growth, transformed morphology, and tumorigenicity, but did not impair attached cell growth. Increasing full-length Ets2 transcriptional activity by fusing it to the VP16 transactivation domain enhanced its ability to reverse DT cell transformation. Mutational analysis revealed that the mitogen-activated protein kinase phosphorylation site required for Ras-mediated activation, Ets2(T72), was not essential for Ets2 reversion activity. The distinct reversion activities of the highly expressed Ets2 transactivation domains or full-length Ets2, along with the specific reversion activity by Ets2 constructs that either inhibit or activate Ets-dependent transcription, suggests multiple roles for Ets factors in cellular transformation. These results indicate that several distinct approaches for modulating Ets activity may be useful for intervention in human cancers.

Ets2 and protein kinase Cepsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells

“…pcDNA3-based expression plasmids were used for the expression of Ets1, Ets2, Elf-1 and Ese-1 [20]. pCIN4-based expression plasmids, E2full, E2DBD or E2TAD [39], were introduced into cells to express Flag-Ets2, Flag-Ets2-DBD or Flag-Ets2-TAD respectively. Plasmids pHANE-PKC-α-CAT and pHANE-PKC-ε-CAT for the expression of PKCα-CAT and PKCε-CAT respectively, were kindly provided by Jae-Won Soh (Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, U.S.A.).…”

“…Under PMA conditions, Figure 2A) on WT-Ets2-dependent activation of the PTHrP P3 promoter. Effect of two transdominant-negative versions of the Ets2 protein (DBD and TAD) [39] on basal and PMA-dependent P3 activity were also tested. DBD-Ets2 is a Ets2 protein fragment that contains the C-terminal part of the Ets2 protein from amino acids 332-468, which includes the DBD.…”

“…Next, we tested transdominant-negative versions of the Ets2 proteins, the N-terminal deletion mutant DBD-Ets2, containing amino acids 332-348 and the DBD or the C-terminal deletion mutant TAD-Ets2, harbouring amino acids 1-331 including the transactivating domain (TAD) [39], for their ability to interfere with PTHrP P3 promoter activity in MCF-7 cells. We found that both Ets2 mutants significantly reduced basal as well as PMA-induced transcription from the PTHrP P3 promoter ( Figure 3B).…”

SP100 expression modulates ETS1 transcriptional activity and inhibits cell invasion

“…This PCR product was then digested with the respective enzymes and cloned into BamHI-XhoI sites of the modified pcDNA3 vector (Foos et al, 1998), containing a FLAG sequence (provided by Dr Craig Hauser, Burnham Institute). The Ets2 DN sequence was verified by automated sequence analysis.…”

Altered Ets transcription factor activity in prostate tumor cells inhibits anchorage-independent growth, survival, and invasiveness

“…In addition, stable expression of the DNA binding domain (the ETS domain) from several divergent Ets family members was found to reverse the transformed phenotype of stably Ras-transformed NIH3T3 cells, but not to inhibit attached cell growth (Wasylyk et al, 1994). Surprisingly, expression of full-length Ets2 (an activator of Ets-dependent transcription), or an even more powerful activator (VP16-Ets2) in Ras transformed NIH3T3 cells reversed their transformed phenotype more e ciently than expression of just the Ets2 DNA binding domain (Foos et al, 1998). While these approaches using dominant acting Ets constructs do not reveal which particular Ets factors may be involved in transformation, they are of value in establishing the importance of this large family of transcription factors in this process.…”