Reese, Jordan

Significance Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and patients exhibit high rates of recurrence and mortality in part due to lack of treatment options beyond standard-of-care chemotherapy regimens. In the subset of TNBCs that express estrogen receptor beta (ERβ), ligand-mediated activation of ERβ elicits potent anticancer effects. We report here the elucidation of the ERβ cistrome and transcriptome in TNBC and identify a mechanism whereby ERβ induces cystatin gene expression resulting in inhibition of canonical TGFβ signaling and a blockade of metastatic phenotypes. These findings suggest that ERβ-targeted therapies represent a treatment option for the subset of women with ERβ-expressing TNBC.

Abstract Background: Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses. Clinical management of TNBC is limited to surgery, chemotherapy and radiation due to lack of estrogen receptor alpha and HER2 expression. Recently, we have shown that approximately 40% of TNBCs express estrogen receptor beta (ERβ) and have begun to explore the possibility that this receptor could be utilized as a novel therapeutic target for this disease. Methods: To examine the biological functions of ERβ in TNBC, novel ERβ expressing TN cell lines (MDA-MB-231 and Hs578T) were developed. In vitro experiments were employed to determine alterations in the global gene expression profiles, biological pathways, proliferation rates, and cell cycle progression following estrogen or ERβ-specific agonist treatment. Cell line xenografts were also established in athymic ovariectomized nude mice to examine tumoral responses to ERβ targeting agents and to investigate gene and protein expression patterns as well as potential serum biomarkers indicative of therapeutic response. Additionally, using the resources of the Mayo Clinic Breast Cancer Genome Guided Therapy Study (BEAUTY), we have identified, and begun to analyze, ERβ+ and ERβ- patient derived xenografts (PDX) established from women with TNBC. Results: Our studies have revealed that both estrogen and multiple ERβ-specific agonists elicit significant anti-proliferative effects in ERβ+ TNBC cells primarily through a G1/S phase cell cycle arrest. These anti-proliferative effects appear to be mediated by cystatins, a family of small secreted cysteine protease inhibitors which are highly induced following estrogen and ERβ-specific agonist treatment. Conditioned media isolated from estrogen or ERβ-specific agonist treated cells decreased the proliferation rates of multiple non-ERβ expressing cell lines; effects that were completely reversed when cystatins were depleted from the media. In addition, we have shown that activation of ERβ, and the subsequent induction of cystatin gene expression, leads to suppression of canonical TGFβ signaling through multiple mechanisms including suppression of TGFβR2 expression, induction of Smad7 expression and blockade of TGFβ ligand-mediated activation of this pathway both in vitro and in vivo. Finally, ERβ+ TNBC PDXs exhibit significantly decreased tumor growth rates in estrogen-treated mice compared to ERβ- TN breast tumors. Conclusions: Our in vitro and in vivo data show that estrogen and ERβ-specific agonists elicit anti-cancer effects in ERβ+ TNBC. These effects appear to be mediated, in part, by cystatins through their inhibitory effects on canonical TGFB signaling, a pathway known to drive TNBC progression. Importantly, these data lay the foundation for studies aimed at examining the ability to therapeutically target ERβ in TNBC patients. Citation Format: Reese J, Bruinsma E, Subramaniam M, Suman V, Pitel K, Kalari K, Yu J, Wang L, Goetz M, Ingle J, Hawse J. ERβ elicits tumor suppressive effects in triple negative breast cancer through the induction of cystatins and suppression of TGFβ signaling. [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 P5-04-01.

Abstract Background: While the biological functions and clinical importance of ERα are well understood in breast cancer, much less is known about its most closely related family member, ERβ, particularly in the setting of triple negative disease. Additionally, the ability to therapeutically target ERβ in triple negative breast cancer (TNBC) has not been fully explored. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in a cohort of 71 TNBCs. To further define the biological functions of ERβ in TNBC, novel ERβ expressing triple negative cell lines (MDA-MB-231 and Hs578T) were developed and comprehensively characterized at the level of global gene expression profiling, modulation of important biological pathways, cellular proliferation and response to targeted therapies. Results: In TNBCs from 71 patients, nuclear and cytoplasmic ERβ was detected at moderate to high levels in 24% and 32% of cases respectively. This moderate to high expression of both nuclear and cytoplasmic ERβ was associated with higher levels of Ki67. Of the 17 tumors expressing ERβ, 13 (76%) were negative for androgen receptor expression. In the triple negative MDA-MB-231-ERβ and Hs578T-ERβ cell lines, expression of ERβ led to inhibition of proliferation in response to both estrogen and multiple ERβ specific agonists. Microarray analysis and RT-PCR profiling of these cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors, while suppressing the expression of many interleukins. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells inhibited the proliferation rates and blocked TGFβ signaling in non-ERβ expressing TNBC cells, effects that were completely reversed following depletion of cystatins from the conditioned media. Conclusions: ERβ is expressed in a substantial proportion of TNBCs, and most do not express the androgen receptor. In TNBC, where targeted therapies are lacking, our data suggest that estrogen or ERβ specific agonists would be expected to elicit anti-tumor effects when ERβ is expressed. These tumor suppressive effects of ERβ appear to be mediated in part through the actions of cystatins, and their inhibition of TGFβ signaling, suggesting that this family of secreted proteins may represent novel biomarkers for monitoring ERβ specific drug responsiveness and/or patient outcomes. Citation Format: Jordan M Reese, Malayannan Subramaniam, Vera J Suman, Xianglin Wu, Vivian Negron, Anne Gingery, Kevin S Pitel, Sejal S Shah, Heather E Cunliffe, Ann E McCullough, Barbara A Pockaj, Thomas C Spelsberg, Matthew P Goetz, James N Ingle, John R Hawse. Therapeutic targeting of ERβ in triple negative breast cancer [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 P6-03-06.

ABSTRACT Xenorhabdus nematophila engages in a mutualistic association with the nematode Steinernema carpocapsae . The nematode invades and traverses the gut of susceptible insects. X. nematophila is released in the insect blood (hemolymph), where it suppresses host immune responses and functions as a pathogen. X. nematophila produces diverse antimicrobials in laboratory cultures. The natural competitors that X. nematophila encounters in the hemolymph and the role of antimicrobials in interspecies competition in the host are poorly understood. We show that gut microbes translocate into the hemolymph when the nematode penetrates the insect intestine. During natural infection, Staphylococcus saprophyticus was initially present and subsequently disappeared from the hemolymph, while Enterococcus faecalis proliferated. S. saprophyticus was sensitive to X. nematophila antibiotics and was eliminated from the hemolymph when coinjected with X. nematophila . In contrast, E. faecalis was relatively resistant to X. nematophila antibiotics. When injected by itself, E. faecalis persisted (∼10 3 CFU/ml), but when coinjected with X. nematophila , it proliferated to ∼10 9 CFU/ml. Injection of E. faecalis into the insect caused the upregulation of an insect antimicrobial peptide, while the transcript levels were suppressed when E. faecalis was coinjected with X. nematophila . Its relative antibiotic resistance together with suppression of the host immune system by X. nematophila may account for the growth of E. faecalis . At higher injected levels (10 6 CFU/insect), E. faecalis could kill insects, suggesting that it may contribute to virulence in an X. nematophila infection. These findings provide new insights into the competitive events that occur early in infection after S. carpocapsae invades the host hemocoel.