Valerie Cortez

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.

Epigenetics of Estrogen Receptor Signaling: Role in Hormonal Cancer Progression and Therapy

Estrogen receptor (ERα) signaling plays a key role in hormonal cancer progression. ERα is a ligand-dependent transcription factor that modulates gene transcription via recruitment to the target gene chromatin. Emerging evidence suggests that ERα signaling has the potential to contribute to epigenetic changes. Estrogen stimulation is shown to induce several histone modifications at the ERα target gene promoters including acetylation, phosphorylation and methylation via dynamic interactions with histone modifying enzymes. Deregulation of enzymes involved in the ERα -mediated epigenetic pathway could play a vital role in ERα driven neoplastic processes. Unlike genetic alterations, epigenetic changes are reversible, and hence offer novel therapeutic opportunities to reverse ERα driven epigenetic changes. In this review, we summarize current knowledge on mechanisms by which ERα signaling potentiates epigenetic changes in cancer cells via histone modifications.

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.

Targeting the PELP1-KDM1 axis as a potential therapeutic strategy for breast cancer.

IntroductionThe estrogen receptor (ER) co-regulator proline glutamic acid and leucine-rich protein 1 (PELP1) is a proto-oncogene that modulates epigenetic changes on ER target gene promoters via interactions with lysine-specific histone demethylase 1 (KDM1). In this study, we assessed the therapeutic potential of targeting the PELP1-KDM1 axis in vivo using liposomal (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DOPC) siRNA to downregulate PELP1 expression and KDM1 inhibitors, pargyline and N-((1S)-3-(3-(trans-2-aminocyclopropyl)phenoxy)-1-(benzylcarbamoyl)propyl)benzamide using preclinical models.MethodsPreclinical xenograft models were used to test the efficacy of drugs in vivo. Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end-labeling immunohistochemical analysis of epigenetic markers was performed on tumor tissues. The in vitro effect of PELP1-KDM axis blockers was tested using proliferation, reporter gene, chromatin immunoprecipitation and real-time RT-PCR assays. The efficacy of the KDM1 targeting drugs alone or in combination with letrozole and tamoxifen was tested using therapy-resistant model cells.ResultsTreatment of ER-positive xenograft-based breast tumors with PELP1-siRNA-DOPC or pargyline reduced tumor volume by 58.6% and 62%, respectively. In a postmenopausal model, in which tumor growth is stimulated solely by local estrogen synthesis, daily pargyline treatment reduced tumor volume by 78%. Immunohistochemical analysis of excised tumors revealed a combined decrease in cellular proliferation, induction of apoptosis and upregulation of inhibitory epigenetic modifications. Pharmacological inhibition of KDM1 in vitro increased inhibitory histone mark dimethylation of histone H3 at lysine 9 (H3K9me2) and decreased histone activation mark acetylation of H3K9 (H3K9Ac) on ER target gene promoters. Combining KDM1 targeting drugs with current endocrine therapies substantially impeded growth and restored sensitivity of therapy-resistant breast cancer cells to treatment.ConclusionOur results suggest inhibition of PELP1-KDM1-mediated histone modifications as a potential therapeutic strategy for blocking breast cancer progression and therapy resistance.

Significance of PELP1 in ER-Negative Breast Cancer Metastasis

Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer. Mol Cancer Res; 10(1); 25–33. ©2011 AACR.

PELP1 oncogenic functions involve CARM1 regulation

Estrogen receptor alpha (ERα) is implicated in the initiation and progression of breast cancer and its transcription depends on the modulation of epigenetic changes at target gene promoters via coregulators. There is a critical need to understand the molecular mechanism(s) by which deregulation of epigenetic changes occurs during breast cancer progression. The ERα coregulator PELP1 plays an important role in ERα signaling and is a proto-oncogene with aberrant expression in breast cancer. PELP1 interacts with histones and may be a reader of chromatin modifications. We profiled PELP1s epigenetic interactome using a histone peptide array. Our results show that PELP1 recognizes histones modified by arginine and lysine dimethylation. PELP1 functionally interacts with the arginine methyltransferase CARM1 and their interaction is enhanced by ERα. PELP1-CARM1 interactions synergistically enhance ERα transactivation. Chromatin immunoprecipitation assays revealed that PELP1 alters histone H3 arginine methylation status at ERα target gene promoters. Pharmacological inhibition or small interfering RNA knockdown of CARM1 substantially reduced PELP1 oncogenic functions. The critical role of PELP1 status in modulating arginine methylation status was also observed through in vivo studies where PELP1 knockdown mediated decreased tumorigenesis correlated with decreased arginine dimethylation. Further, immunohistochemical analysis of human breast tumor tissues revealed co-overexpression of PELP1 and CARM1 in a subset of ERα-positive breast tumors. Our findings show PELP1 is a reader of histone arginine methyl modifications and deregulation promotes tumor proliferation via epigenetic alterations at ERα target promoters. Targeting these epigenetic alterations through inhibition of PELP1 and the arginine methyltransferases could be a promising cancer therapeutic.

PELP1 overexpression in the mouse mammary gland results in the development of hyperplasia and carcinoma

Estrogen receptor (ER) coregulator overexpression promotes carcinogenesis and/or progression of endocrine related-cancers in which steroid hormones are powerful mitogenic agents. Recent studies in our laboratory, as well as others, demonstrated that the estrogen receptor coregulator PELP1 is a proto-oncogene. PELP1 interactions with histone demethylase KDM1 play a critical role in its oncogenic functions and PELP1 is a prognostic indicator of decreased survival in patients with breast cancer. However, the in vivo significance of PELP1 deregulation during initiation and progression of breast cancer remains unknown. We generated an inducible, mammary gland-specific PELP1-expressing transgenic (Tg) mouse (MMTVrtTA-TetOPELP1). We found more proliferation, extensive side branching, and precocious differentiation in PELP1-overexpressing mammary glands than in control glands. Aged MMTVrtTA-TetOPELP1 Tg mice had hyperplasia and preneoplastic changes as early as 12 weeks, and ER-positive mammary tumors occurred at a latency of 14 to 16 months. Mechanistic studies revealed that PELP1 deregulation altered expression of a number of known ER target genes involved in cellular proliferation (cyclin D1, CDKs) and morphogenesis (EGFR, MMPs) and such changes facilitated altered mammary gland morphogenesis and tumor progression. Furthermore, PELP1 was hyper-phosphorylated at its CDK phosphorylation site, suggesting an autocrine loop involving the CDK-cyclin D1-PELP1 axis in promoting mammary tumorigenesis. Treatment of PELP1 Tg mice with a KDM1 inhibitor significantly reduced PELP1-driven hyperbranching, reversed alterations in cyclin D1 expression levels, and reduced CDK-driven PELP1 phosphorylation. These results further support the hypothesis that PELP1 deregulation has the potential to promote breast tumorigenesis in vivo and represent a novel model for future investigation into molecular mechanisms of PELP1-mediated tumorigenesis.

Inhibition of mTOR Signaling Reduces PELP1-Mediated Tumor Growth and Therapy Resistance

Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) is a proto-oncogene that modulates estrogen receptor (ER) signaling. PELP1 expression is upregulated in breast cancer, contributes to therapy resistance, and is a prognostic marker of poor survival. In a subset of breast tumors, PELP1 is predominantly localized in the cytoplasm and PELP1 participates in extranuclear signaling by facilitating ER interactions with Src and phosphoinositide 3-kinase (PI3K). However, the mechanism by which PELP1 extranuclear actions contributes to cancer progression and therapy resistance remains unclear. In this study, we discovered that PELP1 cross-talked with the serine/threonine protein kinase mTOR and modulated mTOR signaling. PELP1 knockdown significantly reduced the activation of mTOR downstream signaling components. Conversely, PELP1 overexpression excessively activated mTOR signaling components. We detected the presence of the mTOR signaling complex proteins in PELP1 immunoprecipitates. mTOR-targeting drugs (rapamycin and AZD8055) significantly reduced proliferation of PELP1-overexpressed breast cancer cells in both in vitro and in vivo xenograft tumor models. MCF7 cells that uniquely retain PELP1 in the cytoplasm showed resistance to hormonal therapy and mTOR inhibitors sensitized PELP1cyto cells to hormonal therapy in xenograft assays. Notably, immunohistochemical studies using xenograft tumors derived from PELP1 overexpression model cells showed increased mTOR signaling and inhibition of mTOR rendered PELP1-driven tumors to be highly sensitive to therapeutic inhibition. Collectively, our data identified the PELP1–mTOR axis as a novel component of PELP1 oncogenic functions and suggest that mTOR inhibitor(s) will be effective chemotherapeutic agents for downregulating PELP1 oncogenic functions. Mol Cancer Ther; 13(6); 1578–88. ©2014 AACR.

Correction: Targeting the PELP1-KDM1 axis as a potential therapeutic strategy for breast cancer

Author details 1Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, TX 78229, USA. 2Department of Cell and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229, USA. 3Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 13 Taishogun Nishitakatsukasa-Cho, Kita-ku, Kyoto 403-8334, Japan. 4PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan. 5Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8673, Japan. 6Department of Gynecologic Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 78030, USA. 7Center for RNA Interference and Noncoding RNA, University of Texas MD Anderson Cancer Center, Houston, TX 78030, USA. 8Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX 78030, USA. 9Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, TX 78229, USA. Published: 21 December 2012

Potential Role of Pargyline in Overcoming Adaptive Resistance in Breast Cancer Cells.

Background: Estrogen-induced breast carcinogenesis is shown to be characterized by global changes in histone modifications. LSD1 (KDM1), a histone demethylase enzyme, play a key role in establishing specific histone methyl marks at target gene promoters. Recent evidence suggest that LSD1 is recruited to a significant fraction of estrogen receptor (ER) target genes and is required to demethylate proximal histones to enable productive ER transcription. These emerging findings also suggest that deregulation of LSD1 epigenetic pathway could contribute to hormonal independence and adaptive resistance in breast cancer cells. In this study, we examined the therapeutic efficacy of treating breast tumor cells with Pargyline, an FDA approved drug for blocking LSD1 functions, and evaluated the therapeutic benefit.Methods: To test this hypothesis, we used model cells that acquired resistance to hormonal therapy including (a) MCF7-HER2 that overexpress oncogene neu/HER2, (b) MCF7-Tam that have acquired resistance to Tamoxifen, (c) MCF7-Ca-LTLT cells that have acquired resistance to Letrozole, (d) MCF7 cells that overexpress proto-oncogene PELP1 (MCF7-PELP1). Parental MCF7 cells were used as a control. Cells were treated with LSD1 inhibitor Pargyline either alone or in combination with Letrozole and Dasatinib. Activation of ER genomic functions was studied using luciferase reporter gene assays. Epigenetic modifications at target promoters were analyzed by Chromatin immune precipitation (CHIP) assays using H3 methyl (di and Tri -H3K4, -K9) specific antibodies. Biological significance and hormonal therapy sensitivity was measured by in vitro cell proliferation assays. Xenograft studies were used to validate the drug effect in vivo. Pilot studies were performed for delivery of drug combinations using nanoparticle formulation.Results: Reporter gene assays showed that LSD1 has potential to enhance ER mediated transcription. LSD1 functionally interacts with ER coregulator PELP1 and is recruited to ER target genes. Pargyline substantially inhibited ER transactivation functions. ChIP analysis revealed that aggressively growing breast cancer cells and therapy resistant cells have distinct activating histone methyl modifications at growth regulatory ER target genes. Treatment of breast cancer models with LSD1 inhibitor Pargyline facilitated reversal of these specific modifications and thereby inhibited the growth of breast cancer cells in vitro and in vivo models. Combinatorial therapy using three agents; (a) that block Estrogen receptor genomic actions (Tamoxifen or Letrozole), (b) ER nongenomic actions (Dasatinib) and (c) ER epigenetic modifications (Pargyline) showed the most promising therapeutic effect compared to single agent therapy on the growth of therapy resistant cells.Conclusions: Our results suggest that histone methyl modification plays a role in therapy resistance and validates the therapeutic potential of Pargyline in combinational therapies. Collectively, these results suggest that targeting LSD1 axis with Pargyline in combination with current endocrine therapies will have better therapeutic effect and may inhibit or delay development of hormonal resistance, thus providing major benefits to patients care. This study is funded by Komen grant KG090447. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 409.