Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

Abstract

BackgroundThe ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells.ResultsTo map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112) and another to the DNA binding domain (DBD), (NES2: 275LWEFIRDILI284). Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions.ConclusionsThese data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.

ESE-1 Knockdown Attenuates Growth in Trastuzumab-resistant HER2+ Breast Cancer Cells

“…Protein level was quantified using the Bio Rad DC protein assay and 25–50 ug of protein were loaded on SDS PAGE gels for immunoblotting. Immunoblotting for ESE-1 was performed using anti-ESE-1 monoclonal antibody (mAb405) developed in our lab 12 . Antibodies against IGFR (9750S), pIGF-IR (3021S), ERBB3 (12708S), pERBB3 (4791S), mTOR (2972S),pmTOR (2971S), Src (2109), pSrc(2101), HER2 (4290S), pHER2 (2243S),Akt, MAPK were purchased from cell signaling and were used following manufacturer’s protocol at a dilution of 1:1000.…”

Molecular mechanisms of ETS transcription factor-mediated tumorigenesis

“…ESE-1 is able to initiate the transformed phenotype in ESE-1-negative nontransformed MCF10A and MCF12A mammary epithelial cells (MECs) via a cytoplasmic mechanism whereby a 40-amino acid serine-and aspartic rich domain is necessary and sufficient for this transformation process (Fig. 2A) (Prescott et al, 2004) (Prescott, 2011). This mechanism has been discussed at length in section B.4.…”

“…2 and Table 1). Specifically, ESE-1 initiates transformation of MCF12A and MCF10A human mammary epithelial cells (MECs) via a novel cytoplasmic mechanism (Prescott et al, 2004, Schedin et al, 2004, Prescott et al, 2011). ESE-1 is located on chromosome 1q32.1, in a region that is amplified in 50% of the breast cancers.…”

“…A unique 40 amino acid serine and aspartic rich (SAR) domain is necessary and sufficient for the cytoplasmic-mediated transformation of ESE-1 (Fig. 2A) (Prescott et al, 2004, Schedin et al, 2004, Prescott et al, 2011). Enforced nuclear expression of either full-length ESE-1 or of the SAR domain alone abrogates ESE-1’s ability to initiate transformation.…”

“…Taken together, these data suggest that nuclear-cytoplasmic shuttling of ESE-1 is required for initiation of transformation in nontransformed MECs. A single NES (nuclear export signal) lies in the ESE-1 DBD and is required for ESE-1 mediated initiation of MCF-12A transformation (Prescott et al, 2011). The discovery that ESE-1 functions in the cytoplasm to initiate transformation of mammary epithelial cells throws light into subcellular function of ETS transcription factors and defined a novel NES-mediated transformation initiation mechanism independent of nuclear transcriptional function.…”