Monica Mann

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.

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.

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.

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.

PELP1 oncogenic functions involve alternative splicing via PRMT6

Proline‐, glutamic acid‐, and leucine‐rich protein 1 (PELP1) is a proto‐oncogene that functions as coactivator of the estrogen receptor and is an independent prognostic predictor of shorter survival of breast cancer patients. The dysregulation of PELP1 in breast cancer has been implicated in oncogenesis, metastasis, and therapy resistance. Although several aspects of PELP1 have been studied, a complete list of PELP1 target genes remains unknown, and the molecular mechanisms of PELP1 mediated oncogenesis remain elusive. In this study, we have performed a whole genome analysis to profile the PELP1 transcriptome by RNA‐sequencing and identified 318 genes as PELP1 regulated genes. Pathway analysis revealed that PELP1 modulates several pathways including the molecular mechanisms of cancer, estrogen signaling, and breast cancer progression. Interestingly, RNA‐seq analysis also revealed that PELP1 regulates the expression of several genes involved in alternative splicing. Accordingly, the PELP1 regulated genome includes several uniquely spliced isoforms. Mechanistic studies show that PELP1 binds RNA with a preference to poly‐C, co‐localizes with the splicing factor SC35 at nuclear speckles, and participates in alternative splicing. Further, PELP1 interacts with the arginine methyltransferase PRMT6 and modifies PRMT6 functions. Inhibition of PRMT6 reduced PELP1‐mediated estrogen receptor activation, cellular proliferation, and colony formation. PELP1 and PRMT6 are co‐recruited to estrogen receptor target genes, PELP1 knockdown affects the enrichment of histone H3R2 di‐methylation, and PELP1 and PRMT6 coordinate to regulate the alternative splicing of genes involved in cancer. Collectively, our data suggest that PELP1 oncogenic functions involve alternative splicing leading to the activation of unique pathways that support tumor progression and that the PELP1–PRMT6 axis may be a potential target for breast cancer therapy.

Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers

The majority of human breast cancer is estrogen receptor alpha (ER) positive. While anti-estrogens/aromatase inhibitors are initially effective, resistance to these drugs commonly develops. Therapy-resistant tumors often retain ER signaling, via interaction with critical oncogenic coregulator proteins. To address these mechanisms of resistance, we have developed a novel ER coregulator binding modulator, ERX-11. ERX-11 interacts directly with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. ERX-11 is orally bioavailable, with no overt signs of toxicity and potent activity in both murine xenograft and patient-derived breast tumor explant models. This first-in-class agent, with its novel mechanism of action of disrupting critical protein-protein interactions, overcomes the limitations of current therapies and may be clinically translatable for patients with therapy-sensitive and therapy-resistant breast cancers. DOI: http://dx.doi.org/10.7554/eLife.26857.001

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

Abstract B08: ESR1 coregulator binding site inhibitors (ECBIs) as novel therapeutics to target hormone therapy-resistant breast cancer

Estrogens contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens (AE) or aromatase inhibitors (AI). However, most patients develop resistance to these drugs. Critically, therapy-resistant tumors retain ESR1-signaling. Mechanisms of therapy resistance involve the activation of ESR1 in the absence of ligand or mutations in ESR1 that allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. For patients with therapy-resistant breast cancers, there is a critical unmet need for novel agents to disrupt ESR1 signaling by blocking ESR1 interactions with its coregulators. Methods: Using rational design, we synthesized and evaluated a small organic molecule (ESR1 coregulator binding inhibitor, ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Using in vitro cell proliferation and apoptosis assays, we tested the effect of ECBI on several breast cancer cells and therapy-resistant model cells. Mechanistic studies were conducted using established biochemical assays, reporter gene assays, RTqPCR and RNASeq analysis. Gene differential expression lists were analyzed using Ingenuity Pathway Analysis (IPA). ESR1+ve (MCF7 and ZR75) xenografts were used for preclinical evaluation and toxicity. The efficacy of ECBI was tested using an ex vivo cultures of freshly extirpated prrimary human breast tissues. Results: In estrogen induced proliferation assays using several ESR1+ve model cells, we found that ECBI inhibit growth (IC50=300-500 nM). Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells, directly interacted with MT-ESR1 with high affinity and significantly inhibited MT-ESR1 driven oncogenic activity. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 reporter gene activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive xenograft tumors with ECBI (10 mg/Kg/oral) reduced tumor volume by 67% compared to control. Further, ECBI also significantly reduced the proliferation of coregulator-overexpressed breast cancer cells in xenograft model. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumor cells. Conclusions: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBIs block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tissues ex vivo. Thus development of ECBI represents a quantum leap in therapies to target ESR1 Citation Format: Ratna K. Vadlamudi, Gangadhara Reddy Sareddy, Suryavathi Viswanadhapalli, Tae-Kyung Lee, Shi-Hong Ma, Wan Ru Lee, Monica Mann, Samaya Rajeshwari Krishnan, Vijay Gonugunta, Douglas W. Strand, Rajeshwar Rao Tekmal, JungMo Ahn, Ganesh V. Raj. ESR1 coregulator binding site inhibitors (ECBIs) as novel therapeutics to target hormone therapy-resistant breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B08.

Abstract 860: ESR1 coregulator binding inhibitor (ECBI): a novel agent for treating hormone therapy-resistant breast cancer

Background: Estrogen contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens (AE) or aromatase inhibitors (AI). However, most patients develop resistance to these drugs. In resistant tumors, activation of ESR1 in the absence of ligand or mutations in ESR1 allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. Here we, developed a novel ESR1 coregulator binding inhibitor (ECBI) that targets persistent ESR1 signaling that commonly occur in therapy resistant breast tumors. Methods: Using rational design, we synthesized and evaluated a small organic molecule (ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Mechanistic studies were conducted using reporter gene assays, RT-qPCR., ChIP, and RNA-Seq analysis. Xenografts and patient derived tumors were used for preclinical evaluation and toxicity. Results: In estrogen induced proliferation assays using several ESR1+ve model cells, ECBI significantly inhibited growth and promoted apoptosis. Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 driven ERE reporter gene activity. Further, ECBI directly interacted with mutant-ESR1 with high affinity and significantly inhibited mutant-ESR1 driven oncogenic activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive and therapy resistant as well as syngeneic xenograft tumors with ECBI (10 mg/kg/day/oral) significantly reduced the tumor volume compared to control. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumors. Conclusions: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBI block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tumor tissues ex vivo. This first-in-class agent with its novel mechanism of action overcomes the limitations of current therapies. Citation Format: Ratna K. Vadlamudi, Gangadhara Reddy Sareddy, Suryavathi Viswanadhapalli, Tae-Kyung Lee, Shi-Hong Ma, Wan Ru Lee, Monica Mann, Samaya Rajeshwari Krishnan, Vijay Gonugunta, Yang Liu, Douglas W. Strand, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ganesh V. Raj. ESR1 coregulator binding inhibitor (ECBI): a novel agent for treating hormone therapy-resistant breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 860.

Abstract P1-06-09: Proto-oncogene PELP1 signaling regulates breast cancer stem cells via G9a/EHMT2

BACKGROUND: Evolving evidence suggests that cancer stem cells (CSCs) evade hormonal therapy and therapy resistance occurs due to regrowth of tumor cells from cancer stem cells that escaped hormonal therapy or remained in the body after tumor resection. Recent studies suggest that estrogen stimulates breast cancer stem-cells and G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene that functions as a critical coregulator of several nuclear receptors and other transcription factors. PELP1 is commonly overexpressed in hormone-related cancers, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered PELP1 interacts with G9a/EHMT2. However, it remains unknown whether PELP1-G9a signaling plays a role in breast cancer stem cell proliferation. The objective of this study is to develop small molecular inhibitors that block G9a/EHMT2 interactions and to test their utility. METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. To test the effect of PELP1 inhibitors on CSCs, we cultured CSCs in SFM in the presence or absence of PELP1 inhibitors for a period of 7-10 days. Cells were analyzed for spheroid formation, morphological changes, immunofluorescence for differentiation markers, protein (Western) and RNA (RT-qPCR) analysis. Expression of differentiation markers K19 and K14 and stem cell markers CD133, CD44, Id1, Nestin, Musashi-1, SOX2, Notch2, and OCT1 was determined. RESULTS: Using mapping studies, we identified a small peptide inhibitor (PIP1) that interferes PELP1 interaction with G9/EHMT2. Utilizing Hit-Ligand interaction site with the PELP1 hot spot residues based on 3D alignment and shape, we have identified 61 potential hits from Ligand-Based screening using a 10,000 Diverse Set. Screening of these 61 potential hits using MTT based cell viability assays identified three small organic molecule inhibitors (peptidomimetics) as leads. All three peptidomimetics (#20, #29, #34) showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in assays using three different breast cancer cell lines. Further, PELP1 targeting peptidomimetic disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen therapy resistant cells. In mechanistic studies, we found that knockdown of PELP1 inhibited stem cell maintenance. In FACS analysis of ZR75, ZR75-PELP1 and ZR75-PELP1KD cells, the percentage of CD44high/CD24low cells correlated with PELP1 status. Accordingly, in mammosphere formation assays, PELP1 targeting peptidomimetic significantly inhibited the formation of mammospheres and the size of the mammospheres was also substantially decreased. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity. CONCLUSIONS: Collectively, our studies have discovered an essential role for PELP1 in breast cancer stem cell maintenance and identified the PELP1- G9a/EHMT2 axis as a potential therapeutic target for reducing stemness. Further, the novel small molecule inhibitors of PELP1 could be used for therapeutic targeting of breast cancer stem cells and therapy resistance. Citation Format: Viswanadhapalli S, Mann M, Sareddy GR, Xaionan L, Vankayalapati H, Brann D, Vadlamudi RK. Proto-oncogene PELP1 signaling regulates breast cancer stem cells via G9a/EHMT2. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-09.