#3022 Background: The estrogen receptor (ER) plays a central role in the progression of breast cancer. Current endocrine therapy for ER+ve breast cancer involves modulating ER-pathway using Tamoxifen, and blocking peripheral estrogen (E2) synthesis by Aromatase inhibitors. Despite the positive effects, de novo and/or acquired resistance to endocrine therapies frequently occur. Although mechanisms for hormonal therapy resistance remains elusive, most downstream events in these pathways converge upon modulation of cell cycle regulatory proteins including upregulation of Cyclin E and A, along with activation of Cyclin Dependent Kinase 2 (CDK2). ER signaling complexes are known to recruit various co-regulatory proteins and recent evidences suggest that deregulated expression, localization and activity of ER coregulators also plays vital role in hormonal resistance. In this study, we found that CDK signaling regulates ER coregulator PELP1 function via phosphorylation leading to hormonal resistance.
 Methods: Significance of CDK2 axis in the therapy resistance was tested using breast cancer models cells that acquired resistance to endocrine therapy and by using chemical inhibitors that block CDK2 activity. Immunoprecipitation, and confocal analysis was used to confirm protein-protein interactions. In vitro kinase and ortho-phosphate labeling assays were used to test CDK2 phosphorylation of PELP1. Utilizing Breast cancer model cells that express PELP1 mutants that cannot be phosphorylated by CDK2, we examined the significance of PELP1 phosphorylation in cell cycle progression. Using PELP1siRNA nanoparticles, we tested the effect of PELP1 knockdown in hormone therapy resistance.
 Results: ER coregulator PELP1 interacts with CDK2 upon E2 stimulation. In vitro kinase assays using both purified CDK2/CyclinE and CDK2/CyclinA complexes showed that full length PELP1 is a potential substrate of CDK2. PELP1 exhibited phosphorylation at the time points that corresponds to CDK2 activation in MCF7 cells. PELP1 overexpression increases E2F luciferase activity while PELP1 mutants that lack CDK2 sites failed to enhance the E2F activity. CDK2 mediated phosphorylation of PELP1 is important for PELP1 regulation of E2F and ER target genes. Combination therapies using PELP1 siRNA nano particles or Roscovitine along with tamoxifen or letrozole, sensitized the therapy resistance cells for endocrine therapy.
 Conclusions: We have identified ER coregulator PELP1 as a novel substrate of CDK2. Because CDK2 activity is deregulated in breast tumors and implicated in therapy resistance, our findings suggests that CDK2-PELP1 axis deregulation may contribute therapy resistance. Combinatorial therapeutic strategy using Roscovitine along with PELP1 siRNA nanoparticles will provide new therapeutic opportunity to increase the sensitivity of hormone resistant cells to Tamoxifen and Letrozole therapy. These studies were supported by DOD breast cancer grant BC083207. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3022.
#5036 Background: Estrogen stimulates breast tissue to increase cell divisions (mitosis) and is implicated in breast cancer progression. ER action is complex and requires functional interactions with coregulators. Proline-, glutamic acid-, and leucine-rich protein (PELP)-1, also known as modulator of nongenomic actions of estrogen receptor (MNAR), is a novel nuclear receptor (ER) coregulator with multitude of functions. Emerging evidence suggest that PELP1 expression is deregulated in breast cancer and serves as a scaffolding protein that couples various signaling complexes with estrogen receptor. In this study we found that ER coregulator PELP1 plays a novel role in mitosis.
 Material and Methods: To understand the mechanism by which ER coregulator PELP1 contribute to breast cancer progression, we have utilized small RNA interference methodology and established breast cancer model cells that stably express PELP1-shRNA (MCF7-PELP1shRNA). FACS analysis was used to determine the cell cycle status of the model cells. Using confocal microscopy, immunoprecipitation, in vitro kinase assays, site directed mutagenesis and Western analysis using phospho-antibodies we studied the mechanism and significance of PELP1 signaling in mitotic progression. We also developed PELP1 siRNA nanoparticles and used them as well as CDK1 inhibitors in cell proliferation studies.
 Results: Down regulation of PELP1 expression resulted in decreased estrogen mediated cell proliferation, delayed mitotic progression and induced accumulation of mitotic cells. Interestingly, PELP1 depleted cells also exhibited multinucleation. Western analysis of various markers of mitotic progression revealed a delay in the kinetics of G2M initiation and progression. Confocal analysis revealed colocalization of CDK1 and PELP1 in G2M. Immunoprecipitation assays demonstrate that endogenous CDK1 form functional complex with PELP1 and Src kinase during mitosis. Using deletion and mutagenesis approach, we have mapped the putative CDK1 phosphorylation sites on PELP1. Down regulation of PELP1 or overexpression of PELP1 mutants (that cannot be phosphorylated by CDK1), reduces the magnitude of Src activation, which is an essential driving force for timely progression of M phase. PELP1 siRNA nanoparticles alone or in combination with CDK1 inhibitors have shown to significantly reduce the proliferation of breast cancer cells and showed increased response in tamoxifen resistant breast cancer cells.
 Discussion: These results suggest that ER coregulator PELP1 play a novel role in G2M progression. Since PELP1 expression is deregulated in breast cancer, PELP1 ability to regulate mitosis could contribute to the progression of cancer by causing genomic instability through the deregulation of mitosis. Taken together our findings suggest that estrogen can promote neoplasia using coregulators by two distinct mechanisms (1) Coregulator induction of target genes and (2) Coregulator mediated actions in mitosis. ER coregulator PELP1 play multiple roles in Estrogen mediated neoplasia, and thus represent a target for novel therapeutic breast cancer strategies by forming the “next generation” of antimitotic drugs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5036.
#601 Back ground: Estradiol (E2) and estrogen receptor (ER) signaling play a key role in development and progression of breast cancer. ER signaling is complex, involves coregulatory proteins and the status of ER coregulators in tumor cells plays an important role in hormonal responsiveness and tumor progression. In addition, ER also participates in non-genomic signaling events in the cytoplasm, however the significance of non-genomic signaling in mammary tumorigenesis remain unknown. PELP1/MNAR is novel ER coregulator that participates in ER genomic and non-genomic actions. PELP1 expression is deregulated in breast tumors and in a subset of tumors PELP1 is predominantly localized in the cytoplasm. Since PELP1 cytoplamsic localization promotes excessive activation of Src and AKT pathways, we hypothesized that PELP1 mediated excessive activation of ER-nongenomic functions may play a role tumorigenesis. To test this, we have generated MMTV-PELP1cyto TG model that uniquely express PELP1 in the cytoplasm of mammary glands that mimics the pathological situation of PELP1 localization seen breast cancer.
 Methods: As a means of targeting the expression of the PELP1 transgene to the mammary gland, we placed the PELP1cyto cDNA under the control of the MMTV promoter. PELP1 transgene integration was verified by PCR and expression levels by Western and IHC in each founder line. Whole-mount preparations and IHC analysis was performed using Tg and age controlled wild type littermates from different developmental stages. Total protein extracts of mammary gland were used for western blot analysis of nongenomic signaling components.
 Results: Preliminary analysis of mammary gland from PELP-cyto mice showed hyperplasia, increased proliferation as analyzed by PCNA staining. Mammary tumors were observed as early as 32 weeks. No spontaneous mammary tumors were found in the wild type cohort. Pathological analysis revealed that these tumor masses represent full blown mammary adenocarcinomas. Mammary tumors showed excessive activation of nongenomic signaling including activation of Src and AKT pathways. A clear induction of aromatase expression was found in PELP1 tumors compared with the wild-type that showed no aromatase expression in the mammary gland.
 Discussion: We have established and characterized a transgenic mouse model that mimics deregulated ER-nongenomic signaling. Our results suggest that PELP1 is a proto-oncogene and demonstrates its in vivo tumorigenic potential. PELP1 driven tumors are ER+ve, express aromatase, thus provide an interesting in vivo model for studying ER-mediated tumorigenesis and to study effect of local E2 on ER-mediated tumorigenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 601.
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