Binoj C. Nair

PELP1 is a reader of histone H3 methylation that facilitates oestrogen receptor‐α target gene activation by regulating lysine demethylase 1 specificity

Histone methylation has a key role in oestrogen receptor (ERα)‐mediated transactivation of genes. Proline glutamic acid and leucine‐rich protein 1 (PELP1) is a new proto‐oncogene that functions as an ERα co‐regulator. In this study, we identified histone lysine demethylase, KDM1, as a new PELP1‐interacting protein. These proteins, PELP1 and KDM1, were both recruited to ERα target genes, and PELP1 depletion affected the dimethyl histone modifications at ERα target genes. Dimethyl‐modified histones H3K4 and H3K9 are recognized by PELP1, and PELP1 alters the substrate specificity of KDM1 from H3K4 to H3K9. Effective demethylation of dimethyl H3K9 by KDM1 requires a KDM1–ERα–PELP1 functional complex. These results suggest that PELP1 is a reader of H3 methylation marks and has a crucial role in modulating the histone code at the ERα target genes.

Extranuclear Functions of ER Impact Invasive Migration and Metastasis by Breast Cancer Cells

The molecular basis of breast cancer progression to metastasis and the role of estrogen receptor (ER) signaling in this process remain poorly understood. Emerging evidence suggests that ER participates in extranuclear signaling in addition to genomic functions. Recent studies identified proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) as one of the components of ER signalosome in the cytoplasm. PELP1 expression is deregulated in metastatic breast tumors. We examined the mechanism and significance of ER-PELP1-mediated extranuclear signals in the cytoskeletal remodeling and metastasis. Using estrogen dendrimer conjugate (EDC) that uniquely activate ER extranuclear signaling and by using model cells that stably express PELP1 short hairpin RNA (shRNA), we show that PELP1 is required for optimal activation of ER extranuclear actions. Using a yeast two-hybrid screen, we identified integrin-linked kinase 1 (ILK1) as a novel PELP1-binding protein. Activation of extranuclear signaling by EDC uniquely enhanced E2-mediated ruffles and filopodia-like structures. Using dominant-negative and dominant-active reagents, we found that estrogen-mediated extranuclear signaling promotes cytoskeleton reorganization through the ER-Src-PELP1-phosphoinositide 3-kinase-ILK1 pathway. Using in vitro Boyden chamber assays and in vivo xenograft assays, we found that ER extranuclear actions contribute to cell migration. Collectively, our results suggest that ER extranuclear actions play a role in cell motility/metastasis, establishing for the first time that endogenous PELP1 serves as a critical component of ER extranuclear actions leading to cell motility/invasion and that the ER-Src-PELP1-ILK1 pathway represents a novel therapeutic target for preventing the emergence of ER-positive metastasis.

Significance of ER–Src axis in hormonal therapy resistance

The estrogen receptor (ER) is implicated in the progression of breast cancer. Despite positive effects of hormonal therapy, initial or acquired resistance to endocrine therapies frequently occurs. Recent studies suggested ERα-coregulator PELP1 and growth factor receptor ErbB2/HER2 play an essential role in hormonal therapy responsiveness. Src axis couples ERα with HER2 and PELP1, thus representing a new pathway for targeted therapy resistance. To establish the significance of ER–Src axis in PELP1 and HER2 mediated therapy resistance, we have generated model cells that stably express Src-shRNA under conditions of PELP1, HER2 deregulation. Depletion of Src using shRNA substantially reduced E2 mediated activation of Src and MAPK activation in resistant model cells. Pharmacological inhibition of Src using dasatinib, an orally available inhibitor substantially inhibited the growth of therapy resistant MCF7–PELP1, MCF7–HER2, and MCF7–Tam model cells in proliferation assays. In post-menopausal xenograft based studies, treatment with dasatinib significantly inhibited the growth of therapy resistant cells. IHC analysis revealed that the tumors were ERα positive, and dasatinib treated tumors exhibited alterations in Src and MAPK signaling pathways. Combinatorial therapy of tamoxifen with dasatinib showed better therapeutic effect compared to single agent therapy on the growth of therapy resistant PELP1 driven tumors. The results from our study showed that ER–Src axis play an important role in promoting hormonal resistance by proto-oncogenes such as HER2, PELP1, and blocking this axis prevents the development of hormonal independence in vivo. Since PELP1, HER2, and Src kinase are commonly deregulated in breast cancers, combination therapies using both endocrine agents and dasatinib may have better therapeutic effect by delaying the development of hormonal resistance.

Therapeutic significance of estrogen receptor β agonists in gliomas.

Gliomas are the most common and devastating central nervous system neoplasms. A gender bias exists in their development: females are at lower risk than males, implicating estrogen-mediated protective effects. Estrogen functions are mediated by two estrogen receptor (ER) subtypes: ERα, which functions as tumor promoter, and ERβ, which functions as tumor suppressor. We examined the potential use of ERβ agonists as a novel therapeutic to curb the growth of gliomas. Western analysis of six glioma model cells showed detectable expression of ERβ with little or no ERα. Treatment of glioma cells with ERβ agonists resulted in significant decrease in proliferation. Immunohistochemical analysis of tumor tissues revealed that ERβ expression is downregulated in high-grade gliomas. We found that ERβ agonists promote both expression and tumor-suppressive functions of ERβ in glioma cells. Liquiritigenin, a plant-derived ERβ agonist significantly reduced in vivo tumor growth in a xenograft model. Compared with control mice, animals treated with liquiritigenin had greater than 50% reduction in tumor volume and size. Immunohistochemical analysis of tumors revealed a significant increase in the nuclear ERβ expression with a concomitant decrease in cell proliferation in the liquiritigenin-treated group. Our results suggest that ERβ signaling has a tumor-suppressive function in gliomas. Because ERβ agonists are currently in clinical trials and are well tolerated with fewer side effects, identification of an ERβ agonist as a therapeutic agent can be readily extended to clinical use with current chemotherapies, providing an additional tool for enhancing survival in glioma patients. Mol Cancer Ther; 11(5); 1174–82. ©2012 AACR.

Roscovitine confers tumor suppressive effect on therapy-resistant breast tumor cells

IntroductionCurrent clinical strategies for treating hormonal breast cancer involve the use of anti-estrogens that block estrogen receptor (ER)α functions and aromatase inhibitors that decrease local and systemic estrogen production. Both of these strategies improve outcomes for ERα-positive breast cancer patients, however, development of therapy resistance remains a major clinical problem. Divergent molecular pathways have been described for this resistant phenotype and interestingly, the majority of downstream events in these resistance pathways converge upon the modulation of cell cycle regulatory proteins including aberrant activation of cyclin dependent kinase 2 (CDK2). In this study, we examined whether the CDK inhibitor roscovitine confers a tumor suppressive effect on therapy-resistant breast epithelial cells.MethodsUsing various in vitro and in vivo assays, we tested the effect of roscovitine on three hormonal therapy-resistant model cells: (a) MCF-7-TamR (acquired tamoxifen resistance model); (b) MCF-7-LTLTca (acquired letrozole resistance model); and (c) MCF-7-HER2 that exhibit tamoxifen resistance (ER-growth factor signaling cross talk model).ResultsHormonal therapy-resistant cells exhibited aberrant activation of the CDK2 pathway. Roscovitine at a dose of 20 μM significantly inhibited the cell proliferation rate and foci formation potential of all three therapy-resistant cells. The drug treatment substantially increased the proportion of cells in G2/M cell cycle phase with decreased CDK2 activity and promoted low cyclin D1 levels. Interestingly, roscovitine also preferentially down regulated the ERα isoform and ER-coregulators including AIB1 and PELP1. Results from xenograft studies further showed that roscovitine can attenuate growth of therapy-resistant tumors in vivo.ConclusionsRoscovitine can reduce cell proliferation and survival of hormone therapy-resistant breast cancer cells. Our results support the emerging concept that inhibition of CDK2 activity has the potential to abrogate growth of hormonal therapy-resistant cells.

Cyclin-Dependent Kinase–Mediated Phosphorylation Plays a Critical Role in the Oncogenic Functions of PELP1

Estrogen receptor (ER) signaling plays an important role in breast cancer progression, and ER functions are influenced by coregulatory proteins. PELP1 (proline-, glutamic acid-, and leucine-rich protein 1) is a nuclear receptor coregulator that plays an important role in ER signaling. Its expression is deregulated in hormonal cancers. We identified PELP1 as a novel cyclin-dependent kinase (CDK) substrate. Using site-directed mutagenesis and in vitro kinase assays, we identified Ser(477) and Ser(991) of PELP1 as CDK phosphorylation sites. Using the PELP1 Ser(991) phospho-specific antibody, we show that PELP1 is hyperphosphorylated during cell cycle progression. Model cells stably expressing the PELP1 mutant that lack CDK sites had defects in estradiol (E2)-mediated cell cycle progression and significantly affected PELP1-mediated oncogenic functions in vivo. Mechanistic studies showed that PELP1 modulates transcription factor E2F1 transactivation functions, that PELP1 is recruited to pRb/E2F target genes, and that PELP1 facilitates ER signaling cross talk with cell cycle machinery. We conclude that PELP1 is a novel substrate of interphase CDKs and that its phosphorylation is important for the proper function of PELP1 in modulating hormone-driven cell cycle progression and also for optimal E2F transactivation function. Because the expression of both PELP1 and CDKs is deregulated in breast tumors, CDK-PELP1 interactions will have implications in breast cancer progression.

Proline, glutamic acid and leucine-rich protein-1 is essential for optimal p53-mediated DNA damage response.

Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogenic protein that functions as a coregulator for a number of nuclear receptors. p53 is an important transcription factor and tumor suppressor that has a critical role in DNA damage response (DDR) including cell cycle arrest, repair or apoptosis. In this study, we found an unexpected role for PELP1 in modulating p53-mediated DDR. PELP1 is phosphorylated at Serine1033 by various DDR kinases like ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related or DNAPKc and this phosphorylation of PELP1 is important for p53 coactivation functions. PELP1-depleted p53 (wild-type) breast cancer cells were less sensitive to various genotoxic agents including etoposide, camptothecin or γ-radiation. PELP1 interacts with p53, functions as p53-coactivator and is required for optimal activation of p53 target genes under genomic stress. Overall, these studies established a new role of PELP1 in DDRs and these findings will have future implications in our understanding of PELP1’s role in cancer progression.

Regulation of rDNA Transcription by Proto-Oncogene PELP1

Background Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive. Methodology/Principal Findings Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDKs activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA. Conclusions/Significance Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions.

Regulation of aromatase induction by nuclear receptor coregulator PELP1

Estradiol (E2), estrogen receptor (ER), ER-coregulators have been implicated in the development and progression of breast cancer. In situ E2 synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms, especially in post-menopausal women. Several recent studies demonstrated activity of aromatase P450 (Cyp19), a key enzyme that plays critical role in E2 synthesis in breast tumors. The mechanism by which tumors enhance aromatase expression is not completely understood. Recent studies from our laboratory suggested that PELP1 (Proline, Glutamic acid, Leucine rich Protein 1), a novel ER-coregulator, functions as a potential proto-oncogene and promotes tumor growth in nude mice models without exogenous E2 supplementation. In this study, we found that PELP1 deregulation contributes to increased expression of aromatase, local E2 synthesis and PELP1 cooperates with growth factor signaling components in the activation of aromatase. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II. Analysis of PELP1 driven mammary tumors in xenograft as well as in transgenic mouse models revealed increased aromatase expression. PELP1-mediated induction of aromatase requires functional Src and PI3K pathways. Chromatin immuno precipitation (ChIP) assays revealed that PELP1 is recruited to the Aro 1.3/II aromatase promoter. HER2 signaling enhances PELP1 recruitment to the aromatase promoter and PELP1 plays a critical role in HER2-mediated induction of aromatase expression. Mechanistic studies revealed that PELP1 interactions with orphan receptor ERRalpha, and histone demethylases play a role in the activation of aromatase promoter. Accordingly, ChIP analysis showed alterations in histone modifications at the aromatase promoter in the model cells that exhibit local E2 synthesis. Immunohistochemical analysis of breast tumor progression tissue arrays suggested that deregulation of aromatase expression occurs in advanced-stage and node-positive tumors, and that cooverexpression of PELP1 and aromatase occur in a sub set of tumors. Collectively, our results suggest that PELP1 regulation of aromatase represent a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.

Novel role of PELP1 in regulating chemotherapy response in mutant p53-expressing triple negative breast cancer cells.

Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, occurs in younger women and is associated with poor prognosis. Gain-of-function mutations in TP53 are a frequent occurrence in TNBC and have been demonstrated to repress apoptosis and up-regulate cell cycle progression. Even though TNBC responds to initial chemotherapy, resistance to chemotherapy develops and is a major clinical problem. Tumor recurrence eventually occurs and most patients die from their disease. An urgent need exists to identify molecular-targeted therapies that can enhance chemotherapy response. In the present study, we report that targeting PELP1, an oncogenic co-regulator molecule, could enhance the chemotherapeutic response of TNBC through the inhibition of cell cycle progression and activation of apoptosis. We demonstrate that PELP1 interacts with MTp53, regulates its recruitment, and alters epigenetic marks at the target gene promoters. PELP1 knockdown reduced MTp53 target gene expression, resulting in decreased cell survival and increased apoptosis upon genotoxic stress. Mechanistic studies revealed that PELP1 depletion contributes to increased stability of E2F1, a transcription factor that regulates both cell cycle and apoptosis in a context-dependent manner. Further, PELP1 regulates E2F1 stability in a KDM1A-dependent manner, and PELP1 phosphorylation at the S1033 residue plays an important role in mediating its oncogenic functions in TNBC cells. Accordingly, depletion of PELP1 increased the expression of E2F1 target genes and reduced TNBC cell survival in response to genotoxic agents. PELP1 phosphorylation was significantly greater in the TNBC tumors than in the other subtypes of breast cancer and in the normal tissues. These findings suggest that PELP1 is an important molecular target in TNBC, and that PELP1-targeted therapies may enhance response to chemotherapies.