Samaya Rajeshwari Krishnan

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.

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.

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.

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

Abstract 622: S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer

Breast cancer is the primary cause of cancer-associated mortality in women worldwide. Estrogen and the Estrogen Receptors (ER) play a significant role in breast cancer, with over two-thirds of breast cancers expressing ERα. Current endocrine therapy, such as aromatase inhibitors, target estrogen biosynthesis and anti-estrogens target ERα. However, therapeutic resistance frequently arises. In addition to the importance of ERα, ERβ has also been shown to play a critical, but opposing role in breast cancer. ERβ has been shown to inhibit the growth of ERα-positive breast cancer cells. The ratio of ERα to ERβ, in addition to the cross talk between ER9s and growth factor signaling, has been implicated in the development of therapeutic resistance. Recently, several plant-derived compounds that exhibit ERβ agonist activity have been identified. S-equol is a potent ERβ agonist and a metabolite from the soy isoflavone daidzein, and has been previously shown to alleviate menopausal symptoms in a clinical trial. Activation of ERβ, or its over expression, shifts the balance of ER9s from the oncogenic action of ERα to the tumor suppressor activity of ERβ, and therefore may be a valuable therapeutic approach in the treatment of breast cancer. In this study we sought out to determine the efficacy of the ERβ agonist, S-equol, in inhibiting the growth and progression of breast tumors using a syngeneic mouse model. We used mouse mammary tumor cells expressing endogenous ERβ, and to determine the contribution of ERβ, cells with knockdown of ERβ were generated using shRNA. ERβ mRNA and protein expression was analyzed using qRT-PCR and western blot respectively. Syngeneic tumors were established and mice were treated with either a vehicle control or S-equol. Treatment with S-equol reduces tumor volume and inhibits the progression of mouse mammary tumor cells in tumors expressing ERβ. Our mechanistic studies show that S-equol reduces the expression of ERα, and increases the expression of p53 and p27 in an ERβ dependent manner. Additionally, S-equol modulates the expression of inflammatory molecules involved in aromatase expression and promotes the differentiation of cancer stem cells. In conclusion, this study suggests that targeting ERβ may be a valuable strategy in treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen K. Krishnegowda, Samaya R. Krishnan, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 622. doi:10.1158/1538-7445.AM2014-622

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 606: Therapeutic efficacy of ERβ agonists on ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer (OC) is the deadliest of all gynecologic cancers in the United States. Despite success with initial chemotherapy, 70% of patients relapse with incurable disease. Development of chemotherapy resistance is the main factor for poor long-term survival in OC. OC stem cells are implicated in tumor initiation and therapy resistance and their elimination is critical for the development of efficient therapeutic strategies. The biological effects of estrogens are mediated through their cognate receptors: estrogen receptor alpha (ERα) and ER beta (ERβ). Emerging evidence suggests that OC cells express ERβ that functions as a tumor suppressor; however, the clinical utility of ERβ agonists in OC remains elusive. Here, we have tested the utility of ERβ to inhibit OC progression by employing ERβ specific agonists. We have used three different ERβ agonists: liquiritigenin (LIQ) isolated from the plant Glycyrrhiza uralensis, S-equol from soy isoflavone daidzein, and the synthetic compound LY500307 (Eli Lilly). Western analysis revealed detectable expression of ERβ in all OC models tested (SKOV3, BG1 and OVCAR3). Using ERβ reporter gene assay and ERβ specific target gene expression, we confirmed functionality of the ERβ pathway in OC cells. All three ERβ ligands showed significant growth inhibition in MTT and colony formation assays, and ERβ agonists promoted apoptosis. Treatment with ERβ agonists significantly upregulated the expression of ERβ via autocrine signaling. Mechanistic studies revealed ERβ mediated non-classical signaling via AP1 and SP1 as playing a critical role in ERβ mediated tumor suppression. Of the three ligands tested, LY500307 showed more potent growth inhibitory activity (IC50: 3μM). ERβ knockdown using shRNA significantly reduced the ERβ agonist mediated growth inhibition. Interestingly, we found that OC stem cells express ERβ and ERβ agonists significantly decrease the sphere formation by OC stem cells. Additional assays revealed that ERβ agonists reduced the stemness of OC stem cells and enhanced their sensitivity to chemotherapy. Accordingly, ERβ agonists significantly reduced the growth of chemotherapy resistant ovarian cancer model cells (cisplatin-resistant ES2, paclitaxel resistant SKOV-3TR cells) and sensitized them to apoptosis. Collectively, our findings show that ERβ agonists have the potential to significantly inhibit ovarian cancer cell growth and reduce stemness; therefore, ERβ agonists represent novel therapeutic agents for the management of ovarian cancer. Citation Format: Gangadhara Reddy Sareddy, Javier E. Chavez, Humberto Salazar, Monica Mann, Jocelyn Hernandez, Samaya Rajeshwari Krishnan, Edward Kost, Rajeshwar Rao Tekmal, Ratna K. Vadlamudi. Therapeutic efficacy of ERβ agonists on ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2014-606

Abstract 2731: PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Estrogen Receptor positive breast cancer is commonly treated with hormonal therapy; however, many patients acquire therapy resistance, a major clinical problem. Recent evidence suggests that the accumulation of chromosomal abnormalities via increased error-prone Alternative-Non-Homologous End Joining (A-NHEJ) DNA repair pathway could contribute to breast cancer therapy resistance. Proline, Glutamic acid, Leucine rich Protein-1 (PELP1), an oncogenic co-regulator for transcription factors, is commonly overexpressed in breast cancer, and its deregulation has been implicated in therapy resistance. Our recent studies identified PELP1 as a novel substrate of the DNA damage response (DDR) kinases. In this study, we discovered that PELP1 regulates the A-NHEJ pathway, and we developed a novel peptide drug that targets PELP1-A-NHEJ axis for the treatment of therapy resistance. Our results suggest that PELP1 is phosphorylated at Ser1033 by DDR kinases and phosphorylated PELP1 co-localizes with the gamma-H2AX foci. Using Homologous Recombination (HR) and NHEJ pathway specific reporter assays, we found that PELP1 knockdown decreases NHEJ, with no apparent effect on the HR pathway. Further, using A-NHEJ specific reporter cell line, we found that PELP1 knockdown decreased the frequency of A-NHEJ, while PELP1 overexpression increased the repair frequency. PELP1s ability to regulate A-NHEJ pathway was further confirmed by sequencing repaired plasmids and by quantitating the degree of end resection. More importantly, metaphase chromosome spreads revealed gross chromosomal abnormalities in PELP1 deregulated breast cancer cells. Mechanistic studies revealed that PELP1 interacts with Mre11, and modulates the degree of end resection at the DNA double strand breaks. Mapping studies identified the C-terminus of PELP1 as the binding site for Mre11. Using yeast based genetic screen composed of 10 million peptides, we identified a unique peptide, PIP3 that binds the C-terminus of PELP1. We developed a stapled peptide of PIP3 (sPIP3), which functioned as a potent cytotoxic agent for breast cancer cells with little activity on normal cells. Further, sPIP3 efficiently inhibited the A-NHEJ pathway and significantly, reduced survival and promoted apoptosis of therapy resistant model cells. Collectively, these findings suggest that PELP1 plays a critical role in the A-NHEJ pathway and sPIP3 represents a novel drug for the treatment of therapy resistant breast cancer. Citation Format: Samaya R. Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi. PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2014-2731

Abstract 674: The lysine demethylase KDM1 is a novel therapeutic target for the treatment of gliomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioma development is a multistep process, involving alterations in genetic and epigenetic mechanisms. Understanding the mechanisms and enzymes that promote epigenetic changes in gliomas are urgently needed to identify novel therapeutic targets. In the present study we explored the significance of histone demethylase KDM1 in glioma progression. In order to know the status of expression of KDM1, we utilized glioma tissue microarrays which consist of different grades of astrocytomas, oligodendrogliomas, ependymomas and normal brain tissues. Immunohistochemical analysis showed that KDM1 expression is overexpressed in the gliomas compared to normal brain, and KDM1 expression levels were positively correlated with histological malignancy. KDM1 expression was also found to be elevated in various established glioma cell lines. To study the functional significance of KDM1 in gliomas, KDM1 expression was silenced using siRNA or its pharmacological inhibition using pargyline or NCL-1. Silencing of KDM1 or inhibition of its enzyme activity significantly reduced the proliferation and colony formation of glioma cells. Mechanistic studies showed that inhibition of KDM1 promoted the acetylation of p53 and up regulation of its target genes p21 and PUMA. Patient-derived primary GBM cells expressed high levels of KDM1 and pharmacological inhibition of KDM1 decreased their proliferation. Further, KDM1 inhibition reduced the expression of stemness markers CD133 and nestin in GBM cells. Mouse xenograft assays revealed that inhibition of KDM1 using pargyline significantly reduced U87 glioma xenograft tumor growth. Inhibition of KDM1 increased levels of H3K4-me2 and H3K9-Ac histone modifications, reduced H3K9-me2 modification and promoted expression of p53 target genes (p21 and PUMA), leading to apoptosis of glioma xenograft tumors. Our results suggest that KDM1 is overexpressed in gliomas and could be a potential therapeutic target for the treatment of gliomas. Citation Format: Gangadhara R. Sareddy, Sandeep Saran, Binoj C. Nair, Samaya R. Krishnan, Vijay K. Gonugunta, Andrew J. Brenner, Ratna K. Vadlamudi. The lysine demethylase KDM1 is a novel therapeutic target for the treatment of gliomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2013-674