Cathy Samayoa

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.

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

Therapeutic Use of Estrogen Receptor β Agonists in Prevention and Treatment of Endocrine Therapy Resistant Breast Cancers: Observations From Preclinical Models

Breast cancer is one of the most common cancers in the world. The majority of breast cancers express estrogen receptor (ER)α, and endocrine therapy is the primary therapeutic approach to treat ER positive breast cancers. However, developing resistance and side effects are common events of these therapeutic strategies. Recent studies have evaluated the role of ERs sub types and demonstrated that ERα is a tumorigenic and ERβ functions as a tumor suppressor. In recent years, preclinical studies focused on the use of natural and synthetic ERβ agonists to treat wide varieties of cancers, including breast cancer. Successful studies conducted so far, have established that ERβ agonists are effective both alone and in combination with chemotherapeutic agents. These data have suggested that use of ERβ agonists in combination with endocrine therapy may be an effective treatment strategy in hormone receptor positive breast cancers. The present review focuses on the tumor suppressive role of ERβ, and the efficacy and mechanisms of several natural and synthetic ERβ agonists against breast cancer.

Abstract 1820: Investigating the role of estrogen receptor β agonists in the prevention and treatment of breast cancer

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Breast Cancer is the primary cause of cancer-associated mortality worldwide. In the United States alone, more than 250,000 women are diagnosed every year. Asian women have the lowest incidence rates. However, their rates increase to those of the US within one generation after migration. Suggesting that diet and lifestyle are major contributing factors in breast cancer risk. Since Asian women consume high amount of soy when compared to other populations, perhaps compounds found in soy may have a protective effect that is lost once they adopt a western diet. Recent studies have identified plant-derived compounds from soy and licorice that activate Estrogen Receptor β. Unlike previous ERβ agonists, these novel compounds are selective for ERβ and have been tested for safety in clinical trials. Current breast cancer treatment strategies focus on Estrogen Receptor α signaling, given that the majority of cases diagnosed are ERα positive. These treatment strategies include endocrine therapies; such as anti-estrogens or aromatase inhibitors. However, in the last decade, the importance of ERβ has emerged. Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer. The objective of this study was to investigate the utility of using ERβ agonists in the prevention and treatment on breast cancer. To investigate the significance of ERβ activation in the prevention of breast cancer, we used an immunocompetent transgenic mouse model of breast cancer. Specifically, HER2/neu overexpressing mice develop premalignant lesions at 4-5 months and tumors starting at month 7. HER2/neu mice were treated with ERβ agonists for 3 months and subsequently mammary glands were analyzed using whole mount and IHC. When compared to controls, ERβ agonist treated mice showed a decrease in branching and ductal hyperplasia, with no change in body weight. These results suggest that ER β agonist treatment is chemopreventive and provided protection against premalignant lesion in the mammary gland. To investigate the utility of ERβ agonists in the treatment of breast cancer, we used in-vitro and in-vivo models systems. Our results demonstrated that treatment with the agonists was able to inhibit the short-term and long-term growth of the breast cancer cell lines; MCF7aro and LTLT which represent post-menopausal breast cancer and letrozole resistant cells, respectively. In addition to growth inhibition, treatment with ERβ agonists also decreased cell migration and invasion. In addition to activating ERβ, we also found that the agonists are also able to increase the expression of ERβ without increasing ERα levels. Our studies also demonstrated that treatment with ERβ agonists modulated key signaling molecules involved in cell death and cell cycle. Our studies suggest that activation of ERβ signaling is a valuable strategy in the treatment and chemoprevention of breast cancer. Citation Format: Cathy Samayoa, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Investigating the role of estrogen receptor β agonists in the prevention and treatment of 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 1820.

Abstract P3-04-03: Investigating the therapeutic use of estrogen receptor β agonists in breast cancer

In breast cancer, estrogen receptor status reflects the biology of the tumor and is used to determine the likelihood of responding to endocrine therapy. 70% of all breast cancers express Estrogen Receptor α, which is indicative of estrogen dependence for growth. Current therapies aim to lower estrogen levels, or inhibit estrogen receptor signaling to ultimately prevent recurrence. However, only two-thirds of ERα-positive respond to endocrine therapy. The Estrogen Receptors are ligand-activated transcription factors with similar structures that slightly differ in their DNA binding sites and ligand binding domains. Estrogen Receptor α has been shown to induce the proliferation of mammary gland cells, whereas Estrogen Receptor β exhibits tumor suppressive properties. The objective of this study was to investigate the utility of selective ERβ agonists as a treatment for breast cancer. Using different breast cancer models, we investigated the effects of ERβ agonist treatment on growth, migration, apoptosis, cell cycle distribution and gene expression. In this study, we demonstrate that treatment with ERβ agonist results in inhibition of cell growth and migration. Additionally, treatment also impacts cell cycle distribution and affects key proteins involved in cell cycle regulation. We also demonstrate an increase in ERβ mRNA and protein levels upon treatment. This shift in the ERβ to ERα ratio, represents a viable targeting strategy in the treatment of breast cancer. To determine if our finding translate in-vivo, we employed xenograft and syngeneic mouse models. Our in-vivo studies demonstrated reduced tumor volumes in mice treated with ERβ agonists in combination with conventional therapy, confirming the growth inhibitory activity of ERβ agonists. Selective activation of ERβ, through the use of ERβ agonists has enabled us to exploit its tumor suppressive function and investigate the utility as a treatment for breast cancer. This study suggest that activation of ERβ signaling is a valuable strategy to inhibit breast cancer growth and progression. Citation Format: Samayoa C, Krishnegowda NK, Vadlamudi R, Tekmal RR. Investigating the therapeutic use of estrogen receptor β agonists in breast cancer. [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 P3-04-03.

Abstract P3-04-16: Estrogen receptor β agonists reduce breast cancer tumor growth in syngeneic mouse models

The estrogen receptors (ER) play a significant role in breast cancer, with the majority of breast cancers expressing estrogen receptor alpha (ERα) and depending on its signaling. ERα has proliferative function, however, estrogen receptor beta (ERβ) has anti-proliferative functions. Recently, several selective ERβ agonists have been identified. The objective of this study was to determine the effectiveness of ERβ agonists in inhibiting the growth of distinct breast cancer cells both in-vitro and in-vivo, and to determine the mechanisms involved. The mouse mammary tumor cell lines, D2A1 and MM51, express both ERα and ERβ. Specifically D2A1 cells are dependent on estrogen for growth and have increased expression of aromatase. Additionally, D2A1 cells are very aggressive and highly metastatic; representing a good model of breast cancer progression. MM51 cells are Her2/neu positive and are an adequate model to study the crosstalk between growth factors and ER signaling. Syngeneic mouse tumor models were used to incorporate an intact immune system which plays a role in the tumor’s microenvironment. Our in-vitro studies demonstrate that ERβ agonists significantly inhibit cell growth in a dose dependent manner. Our syngeneic studies show that, in-vivo, ERβ agonists effectively reduce tumor volume and inhibit tumor progression. This study reveals that in addition to acting on ERβ, these agonists reduce the expression of ERα at both the mRNA and protein level, therefore modulating the ratio of ERα to ERβ. Additionally, treatment with ERβ agonists results in increases apoptosis through increased p53 expression, and cell cycle arrest through p27 and cyclin D1. Together, these studies demonstrate the therapeutic potential of ERβ agonists for the treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen K Krishnegowda, Ratna K Vadlamudi, Rajeshwar R Tekmal. Estrogen receptor β agonists reduce breast cancer tumor growth in syngeneic mouse models [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-16.

Abstract P5-09-14: Plant-derived estrogen receptorb agonists alone or in combination with aromatase inhibitors restore sensitivity in endocrine therapy resistant breast tumors

Estrogen receptors (ER) play an important role in breast cancer. Over two-thirds of all breast cancers express ERa, and current endocrine therapies target its signaling. Antiestrogens (AE) block the binding of estrogen to ERα, while aromatase inhibitors (AI) inhibit local and systemic estrogen production. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, therapeutic resistance frequently arises. There is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Both normal and tumor tissue express ERβ which has anti-proliferative activity and recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (Liq), from the plant Glycyrrhizae uralensis and S-equol, from the soy isoflavone daidzein, are currently in clinical trials for the treatment of vasomotor hot flashes associated with menopause. In this study we sought to determine if ERβ agonists alone or in combination with AI could resensitize Letrozole-resistant breast cancer cells in vivo. Xenografts were established using cells which model post-menopausal breast cancer. Endocrine therapy sensitive (MCF-7aro) and Letrozole resistant (LTLT-Ca) tumors were treated with either ERβ agonists or in combination with AI and progression was measured. In MCF-7aro tumors, ERβ agonist treatment reduced tumor volume and prolonged sensitivity to AI. In the therapy resistant tumors (LTLT-Ca), ERβ agonist treatment blocked tumor growth and restored sensitivity to AI therapy. To determine the molecular mechanism by which ERβ agonists inhibit tumor growth and prolong and restore sensitivity, qRT-PCR and western blot analysis was performed. Our results show changes in the levels of p53, NFkB, cyclin D1, and KLF5 in addition to other genes. To determine the role of ERβ agonists on the activation of these genes specific gene promoter-luciferase reporter constructs were used and the specificity of ERβ-mediated actions was confirmed using a siRNA approach. This study suggests that ERβ acts as a tumor suppressor by suppressing cell growth through the inhibition of cell cycle genes and by inducing apoptosis through the regulation of p53. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-14.

Abstract 1312: The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways.

Estrogen and estrogen receptors (ERα and ERβ) play an important role in breast cancer. Currently, antiestrogen (AE) targeted to ERα and aromatase inhibitors (AI), which block local and systemic estrogen production, are used in the treatment of breast cancer. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, in women with advanced breast cancer, essentially all breast cancers develop resistance to these two classes of agents. Moreover, the side effects associated with currently used AI limit the long-term utility of them as chemopreventative agents. Therefore there is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (LIQ), from the plant Glycyrrhizae uralensis and an active component of MF101, and S-equol, from the soy isoflavone daidzein, are currently being tested to treat vasomotor hot flashes associated with menopause. In addition, S-equol is also being tested as a treatment for BPH in men. However, the exact mechanism of action of these compounds is not clear. In this study we sought to determine the mechanism by which ERβ acts as a tumor suppressor, and whether these ERβ agonists could resensitize Letrozole-resistant breast cancer cells. MCF7aro and LTLT-ca cells were used as models of hormone-responsive and hormone-resistant breast cancer respectively. MTT assay was used to determine the half maximal inhibitory concentrations of the ERβ agonists. Upon treatment, our results show changes in the levels of p53, NFkB, cyclin D1, KLF5 and others using qRT-PCR and western blot analysis. The effects of ERβ agonists on the activation of these genes were confirmed using specific gene promoter-luciferase reporter gene constructs. Specificity of ERβ-mediated actions was confirmed using a siRNA approach in addition to other methods and knockdown efficiency was confirmed through RT-PCR. Interestingly, our results show that the plant-derived ERβ-agonists, LIQ and S-equol, not only affect the growth of breast cancer cells and tumors but treatment increases the expression of p53 and others, and upon ERβ knockdown this effect is ablated. This study suggests that ERβ acts as a tumor suppressor by regulating the expression of p53 and others involved in cell proliferation. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers, as well as provides rationale for new preventive approaches to decrease the incidence of breast cancer. Citation Format: Cathy Samayoa, Anil Kotha, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar Rao Tekmal. The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways. [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 1312. doi:10.1158/1538-7445.AM2013-1312