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.
Highlights
The ability of these Ets2 expression constructs to reverse Ras-mediated transformation was analyzed in stable clones generated in the doubly transformed (DT) cell line, a v-Ki-Rastransformed derivative of mouse NIH3T3 fibroblasts [28]
For the analysis described below, multiple individual stable DT cell clones generated with each Ets2 expression construct or control plasmid were randomly picked, expanded, and assayed for alterations in the transformed phenotype and for expression of the introduced gene product
It has been previously observed that stable expression of just the DNA binding domain (DBD) of the Ets factors Ets1, Ets2, or PU.1 reverses multiple features of the transformed phenotype in Ras-transformed NIH3T3 cells [23]
Summary
Several lines of evidence suggest that are Ets proteins targets of Ras signaling, but that Ets factors play an important role in mediating cellular transformation. The necessary role of Ets proteins in fibroblast transformation has been revealed by inhibition experiments, where expression of the Ets DNA binding domain alone was found to inhibit oncogenic Ras- or Neu-mediated cellular transformation [11, 22]. Because Ets family proteins bind to similar DNA sequences centered about a conserved GGAA/T core [2], it is likely that high level expression of the ETS domain from Ets inhibits the binding and function of many members of the Ets family. It is possible that the dominant inhibitory mutants do not inhibit transformation by blocking Ets-dependent transcriptional activation, but instead may be overcoming transcription inhibition by Ets family repressors
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