It is increasingly accepted that steroidal receptor coregulators may also function in the cytoplasmic compartment. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel coregulator that plays a role in both the genomic and extranuclear actions of estrogen receptors (ER) in hormonally responsive tissues. In this study using breast tumor arrays, we found that PELP1 was localized only in the cytoplasm in 58% of the PELP1-positive breast tumors. To help explain the significance of the cytoplasmic localization of PELP1 in human breast tumors, we created a mutant protein that was expressed only in the cytoplasm (PELP1-cyto) and then generated a model system wherein MCF-7 breast cancer cells were engineered to specifically express this mutant. We found that PELP1-cyto cells were hypersensitive to estrogen but resistant to tamoxifen. PELP1-cyto cells, but not parental MCF-7 cells, formed xenograft tumors in nude mice. In addition, PELP1-cyto cells exhibited increased association of PELP1 with Src, enhanced mitogen-activated protein kinase (MAPK) activation, and constitutive activation of AKT. The altered localization of PELP1 was sufficient to trigger the interaction of PELP1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K), leading to PI3K activation. In addition, PELP1 interacted with epidermal growth factor receptors and participated in growth factor-mediated ER transactivation functions. Our results suggest that the altered localization of PELP1 modulates sensitivity to antiestrogens, potentiates tumorigenicity, presumably via the stimulation of extranuclear estrogen responses, such as the activation of MAPK and AKT, and also enhance cross-regulation of ER transactivation activity by growth factors. (Cancer Res 2005; 65(17): 7724-32)

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The estrogen receptor plays an important role in breast cancer progression. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also called modulator of nongenomic activity of estrogen receptor (MNAR), a novel coactivator of estrogen receptor, modulates estrogen receptor transactivation functions. The mechanisms by which PELP1 modulates estrogen receptor genomic functions is not known. Here, using biochemical and scanning confocal microscopic analysis, we have demonstrated nuclear localization and functional implications of PELP1. Subnuclear fractionation showed PELP1 association with chromatin and nuclear matrix fractions. Ligand stimulation promoted recruitment of PELP1 to 17␤-estradiol responsive promoters, its colocalization with acetylated H3, and increased PELP1-associated histone acetyltransferase enzymatic activity. Far Western analysis revealed that PELP1 interacts with histone 1 and 3, with more preference toward histone 1. Using deletion analysis, we have identified the PELP1 COOH-terminal region as the histone 1 binding site. The PELP1 mutant lacking histone 1-binding domain acts as a dominantnegative and blocks estrogen receptor ␣-mediated transcription. Chromatin immunoprecipitation analysis showed a cyclic association and dissociation of PELP1 with the promoter, with recruitment of histone 1 and PELP1 occurring in opposite phases. PELP1 overexpression increased the micrococcal nuclease sensitivity of estrogen response element-containing nucleosomes. Our results provide novel insights about the transcription regulation of PELP1 and suggest that PELP1 participates in chromatin remodeling activity via displacement of histone 1 in cancer cells.

We identified dynein light chain 1 (DLC1) as a physiologic substrate of p21-activated kinase 1 (Pak1). Pak1-DLC1 interaction plays an essential role in cell survival, which depends on Pak1's phosphorylation of DLC1 on Ser88. Pak1 associates with the complex of DLC1 and BimL, a proapoptotic BH3-only protein, and phosphorylates both proteins. Phosphorylation of BimL by Pak1 prevents it from interacting with and inactivation of Bcl-2, an antiapoptotic protein. Overexpression of DLC1 but not DLC1-Ser88Ala mutant promotes cancerous properties of breast cancer cells. DLC1 protein level is elevated in more than 90% of human breast tumors. The regulation of cell survival functions by Pak1-DLC1 interaction represents a novel mechanism by which a signaling kinase might regulate the cancerous phenotypes.

Emerging data suggest that p21-activated kinase 1 (Pak1), a downstream signaling molecule of the small GTPases, growth factors, and lipid signaling, is upregulated or hyperactivated in human breast cancer. Until now, however, no direct causative role had been found for Pak1 in mammary tumor formation. We therefore sought to identify the role that Pak1 plays in mammary gland tumorigenesis. Our results showed that in a transgenic mouse model, overexpression of catalytically active Pak1 leads to the development of malignant mammary tumors and to a variety of other breast lesions, including focal solid nodules, ductal hyperplasia, and mini-intraductal neoplasm and adenoma. We also found that Pak1 hyperactivation increases the stimulation of downstream proliferative signaling effectors MEK1/2 and p38-MAPK in mammary tumor epithelial cells. Moreover, in our study, we detected expression of estrogen receptor-alpha expression and progesterone receptor expression during early stages of the lesions, but their expression was lost during the cells' transition to malignant invasive tumors. Finally, we found that consistent with a role in breast tumor progression, Pak1 expression and its nuclear accumulation was increased progressively during the transition from ductal hyperplasia to ductal carcinoma in situ to adenocarcinoma in widely used multistep polyoma-middle T-antigen transgenic mice. Together, these findings provide the first direct evidence that Pak1 deregulation may be sufficient for the formation of mammary gland tumors.

Here, we investigated the role of P21-activated kinase 1 (Pak1) signaling in the function of estrogen receptor-A (ER-A) as assessed by serine 305 (S305) activation and transactivation activity of ER. We found that Pak1 overexpression interfered with the antiestrogenic action of tamoxifen upon the ER transactivation function in hormone-sensitive cells. In addition, tamoxifen stimulation led to up-regulation of ER target genes in breast cancer cells with increased Pak1 expression. Tamoxifen also increased Pak1-ER interaction in tamoxifenresistant but not in tamoxifen-sensitive cells. Results from the mutational studies discovered a role of ER-S305 phosphorylation in triggering a subsequent phosphorylation of serine 118 (S118), and these effects were further potentiated by tamoxifen treatment. We found that S305 activation-linked ER transactivation function requires a functional S118, and active Pak1 signaling is required for a sustaining S118 phosphorylation of the endogenous ER. All of these events were positively influenced by tamoxifen and thus may contribute toward the loss of antiestrogenic effect of tamoxifen. These findings suggest that Pak1 signaling-dependent activation of ER-S305 leads to an enhanced S118 phosphorylation presumably due to a conformational change, and such structural modifications may participate in the development of tamoxifen resistance.

Here we define a function of metastasis-associated protein 1 (MTA1), a presumed corepressor of estrogen receptor α (ERα), as a transcriptional activator of Breast Cancer Amplified Sequence 3 (BCAS3), a gene amplified and overexpressed in breast cancers. We identified BCAS3 as a MTA1 chromatin target in a functional genomic screen. MTA1 stimulation of BCAS3 transcription required ERα and involved a functional ERE half-site in BCAS3 . Furthermore, we discovered that MTA1 is acetylated on lysine 626, and that this acetylation is necessary for a productive transcriptional recruitment of RNA polymerase II complex to the BCAS3 enhancer sequence. BCAS3 expression was elevated in mammary tumors from MTA1 transgenic mice and 60% of the human breast tumors, and correlated with the coexpression of MTA1 as well as with tumor grade and proliferation of primary breast tumor samples. These findings reveal a previously unrecognized function of MTA1 in stimulating BCAS3 expression and suggest an important role for MTA1-BCAS3 pathway in promoting cancerous phenotypes in breast tumor cells.

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1 (also referred to as MNAR, or modulator of nongenomic activity of estrogen receptor)), a recently identified novel coactivator of estrogen receptors, is widely expressed in a variety of 17␤-estradiol (E 2 )-responsive reproductive tissues and is developmentally regulated in mammary glands. pRb (retinoblastoma protein), a cell cycle switch protein, plays a fundamental role in the proliferation, development, and differentiation of eukaryotic cells. To study the putative function of PELP1, we established stable MCF-7 breast cancer cell lines overexpressing PELP1. PELP1 overexpression hypersensitized breast cancer cells to E 2 signaling, enhanced progression of breast cancer cells to S phase, and led to persistent hyperphosphorylation of pRb in an E 2 -dependent manner. Using phosphorylation site-specific pRb antibodies, we identified Ser-807/Ser-811 of pRb as a potential target site of PELP1. Interestingly, PELP1 was discovered to be physiologically associated with pRb and interacted via its C-terminal pocket domain, and PELP1/pRb interaction could be modulated by antiestrogen agents. Using mutant pRb cells, we demonstrated an essential role for PELP1/pRb interactions in the maximal coactivation functions of PELP1 using cyclin D1 as one of the targets. Taken together, these findings suggest that PELP1, a steroid coactivator, plays a permissive role in E 2 -mediated cell cycle progression, presumably via its regulatory interaction with the pRb pathway.The steroid hormone 17␤-estradiol (E 2 ) 1 plays an important role in controlling the expression of genes involved in a wide variety of biological processes, including development, homeostasis, and breast cancer progression (1, 2). The biological effects of estrogen are mediated by its binding to the structurally and functionally distinct estrogen receptors (ERs) ER␣ and ER␤. ER␣ is the major ER in mammary epithelium (3). Upon binding of E 2 to ER␣, the ligand-activated ER␣ translocates to the nucleus, binds to the 13-bp palindromic estrogen response element (ERE) in the target genes, and stimulates gene transcription, thus promoting cell proliferation (4). In addition to the genomic actions of ER, non-genomic functions are implicated via activation of intracellular signal transduction pathways involved in the regulation of cell proliferation (5-8).ER␣ comprises an N-terminal AF1 (activation function-1) domain, a DNA-binding domain, and a C-terminal ligandbinding region that contains an AF2 domain (9). The AF2 ligand-dependent activation function of ER␣ is located in the ligand-binding domain, whereas AF1 functions in a ligandindependent manner. AF1 and AF2 exhibit cell-type and promoter context specificity (10). Also, the transcription functions of ERs have been shown to be influenced by several coactivators, including SRC1 (steroid receptor coactivator-1), GRIP1, AIB1, CBP (cAMP response element-binding protein-binding protein), p300, PGC1, E6AP, PCAF (p300/CBP associating factor), and SNF2 (11-13). It is ge...