Mary Ellen Molloy

Click synthesis of estradiol–cyclodextrin conjugates as cell compartment selective estrogens

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E(2)) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD-E(2) conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer-E(2) conjugates, CD-E(2) was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.

Novel Selective Estrogen Mimics for the Treatment of Tamoxifen-Resistant Breast Cancer

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2. Mol Cancer Ther; 13(11); 2515–26. ©2014 AACR.

Extranuclear ERα is associated with regression of T47D PKCα-overexpressing, tamoxifen-resistant breast cancer

BackgroundPrior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17β-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix.MethodsT47D:A18/PKCα cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1.ResultsWe report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2.ConclusionsExtranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic.

Retraction to: Extranuclear ERα is associated with regression of T47D PKCα-overexpressing, tamoxifen-resistant breast cancer. [Mol Cancer. 12, (2013) (34)] DOI: 10.1186/1476-4598-12-34

Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17β-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix. T47D:A18/PKCα cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1. We report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2. Extranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic.

Abstract C98: Selective estrogen mimics (SEMs) for the treatment of tamoxifen resistant breast cancer

Tamoxifen, an antagonist at estrogen receptor alpha (ERα) in breast tissue, and the prototypical selective estrogen receptor modulator (SERM), is the standard of care for many patients with ER-positive breast cancer. However, continued tamoxifen treatment increases the risk of endometrial cancer and half of ER-positive breast cancer patients do not respond or relapse on treatment with tamoxifen. Tamoxifen-resistant tumors are often ER-positive and endocrine-independent and therefore resistant to aromatase inhibitors. Recent clinical trials have shown the efficacy of estrogen in patients who have undergone exhaustive tamoxifen therapy. Therefore, we propose the development of small molecules that mimic the actions of estradiol in ER-positive breast cancer without the uterotrophic actions of estradiol and tamoxifen: Selective Estrogen Mimics (SEMs). Evidence suggest that a significant subset of ER-positive breast cancer, over-expressing PKCα, and tamoxifen-resistant cancers will respond to SEMs. In order to design and optimize SEMs, tamoxifen-resistant cell lines, MCF-7:5C, T47D: PKCα and T47D: Tam1 were studied. The activation of classical ERα signaling by the estrogen mimics was profiled in MCF-7 cells and cell viability in 2D culture was examined. In an effort to increase throughput and mimic the in vivoenvironment more closely, we assessed the SEMs in a new 3D spheroid model. It was found that in MCF-7:5C cells in 2D and T47D spheroids that SEM induced cell death was mediated by activation of ERα. SEMs caused decreased spheroid viability and growth in all three models of tamoxifen resistance. Extranuclear localization of ER after SEM treatment previously reported in matrigel assays and in vivo xenografts was also observed in spheroids. This emphasizes the capability of the 3D spheroids to replicate in vivo environments, making them a high-throughput translational assay of choice for drug discovery. Three promising SEMs were evaluated in xenograft models of TAM-resistant, PKCα overexpressing breast cancer. While E2 caused regression of these tumors, a significant increase in uterine weight as predicted was observed. More importantly, SEM treated mice had negligible increase in uterine weight underlining the enhanced safety of these molecules. These data suggest that development of SEMs that retain the beneficial properties of estrogen while limiting the side effects is a feasible strategy for the treatment of tamoxifen resistant breast cancer. Citation Format: Hitisha Patel, Rui Xiong, Lauren Gutgesell, Jiong Zhao, Mary Ellen Molloy, Debra Tonetti, Gregory R.J Thatcher. Selective estrogen mimics (SEMs) for the treatment of tamoxifen resistant breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C98.

Abstract 2710: Lead based development and evaluation of selective estrogen mimics in tamoxifen resistant breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tamoxifen is the standard of care for many patients with ER+ breast cancer and works by antagonizing the actions of estrogen at ER. However it poses an increased risk of endometrial cancer and thrombotic events, and most significantly, 30-50% women develop resistance to tamoxifen therapy, underlining the need for superior therapeutic options. Paradoxically, prior to tamoxifen therapy, estradiol (E2) and the ER agonist, diethylstilbestrol, had been used in breast cancer therapy, though with serious side effects. Development of Selective Estrogen Mimics (SEMs) that cause regression of tamoxifen-resistant breast cancer, but without the side effects of E2, represents a rational therapeutic strategy. Novel SEMs were developed, which in vitro in 3D cultures and in vivo caused complete regression of tamoxifen-resistant xenografts, with characteristics similar to those of E2. These SEMs did not fuel growth of estrogen-dependent T47D xenografts and did not cause uterine growth. SEM mediation of classical ER-signaling was profiled in MCF7 and MDA-MB231:β41 cells. A tamoxifen-resistant cell line, MCF-7: 5C, was used to assay induction of cell death by both E2 and the SEMs and to probe the mechanism of action. Several SEMs were observed to act as partial agonists at ERα , suggesting ideal characteristics as therapeutics. Structure-activity relationships were explored, suggesting that partial ERα agonist have the capacity to cause regression of tamoxifen-resistant tumors, without adverse effects associated with estrogenic actions in normal gynecological tissues of the breast and uterus, which might contribute to carcinogenesis. The ER-mediated agonist/antagonist activity, regression efficacy, and pharmacokinetic profiles of these SEMs were examined to obtain lead compounds that are effective with minimal estrogenic side effects. In MCF-7:5C cells, SEM-induced cell death was mediated by activation of classical nuclear ERα signaling, via induction of apoptosis. This research paves the way for use of SEMs with partial agonist activity at ER for use in tamoxifen-resistant breast cancer with enhanced safety profiles. Citation Format: Hitisha Patel, Rui Xiong, Jiong Zhao, Mary Ellen Molloy, Debra Tonetti, Gregory R.J Thatcher. Lead based development and evaluation of selective estrogen mimics in tamoxifen resistant 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 2710. doi:10.1158/1538-7445.AM2014-2710

Abstract 619: Selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer

Endocrine-resistant breast cancer, whether de novo or acquired, is a major clinical obstacle. Although recent clinical trials have demonstrated the efficacy of 17β-estradiol (E2) or diethylstilbestrol (DES) following exhaustive use of antiestrogens, E2 treatment is associated with major side effects such as an increased risk of other gynecological cancers, deterring the clinical community from adopting it as a treatment strategy. Our lab has previously shown that Protein kinase C alpha (PKCα) overexpression predicts tamoxifen (TAM) resistance in the clinic (Tonetti DA et al., Br J Cancer. 2003) and may also predict a positive response to an estrogenic therapy (Chisamore MJ et al., Clinical Cancer Research. 2001). Further, the ectopic overexpression of PKCα in T47D breast cancer cells, led to a TAM-resistant, E2-inhibited phenotype in vivo which was accompanied by the translocation of estrogen receptor alpha (ERα) to extranuclear sites (Perez White B et al., Molecular Cancer. 2013). The purpose of this study was to identify novel selective estrogen mimics (SEMs), which could achieve the positive therapeutic effects of E2 treatment in TAM-resistant breast cancers, while minimizing the side effects. In vitro screening identified two SEMs, BTC and TTC-352, which displayed estrogenic activity in breast cancer cell lines. BTC and TTC-352 treatment resulted in significant tumor regression in two xenograft models of TAM-resistant, PKCα-overexpressing breast cancer. Similar to E2, T47D:A18/PKCα tumor regression was accompanied by translocation of ERα to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment, however, did not result in growth of parental, TAM-sensitive xenograft tumors. Endometrial thickening, caused by both E2 and TAM, is directly associated with gynecological carcinogenesis and uterine cancer. Interestingly, SEM treatment did not increase uterine weight in mice suggesting negligible hormonal stimulation in gynecological tissues. Both BTC and TTC-352 resulted in regression of two TAM-resistant breast cancer models, while displaying enhanced safety compared to E2 and TAM. These data suggest that further development of SEMs targeted to TAM-resistant breast cancer is a feasible therapeutic strategy. Citation Format: Mary Ellen Molloy, Bethany Perez White, Huiping Zhao, Bradley T. Michalsen, Hitisha K. Patel, Jiong Zhao, Rui Xiong, Marton I. Siklos, Gregory R.J. Thatcher, Debra A. Tonetti. Selective estrogen mimics for the treatment of tamoxifen-resistant 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 619. doi:10.1158/1538-7445.AM2014-619

Abstract 4216: Protein kinase C ≤ overexpression is associated with loss of membrane-associated E-cadherin and β-catenin in T47D xenograft breast tumors

Expression of protein kinase C ≤ (PKC∈) has been shown to be a clinically relevant indicator of tamoxifen response, recurrence and overall survival in breast cancer (Tonetti, 2003; Assender 2007). To study this association, we developed a model of PKC∈-overexpressing breast cancer by stable transfection of the prkca gene into T47D breast cancer cells (T47D/PKC∈, Tonetti, 2000). We have shown that PKC∈ induces a hormone-independent, tamoxifen-resistant phenotype in vitro and in vivo (Chisamore, 2001). This model is further characterized in vitro by increased migratory and invasive capability, expression of mesenchymal markers and reduced expression of adherens junctions and epithelial proteins (Perez White, AACR 2011). β-Catenin and E-cadherin are components of adherens junctions in normal epithelia and loss of E-cadherin is associated with worse outcome in breast cancer (Prat, 2010). Further, the reduction or cytoplasmic relocalization of β-catenin has been associated with poor prognosis (Lopez-Knowles, 2010; Dolled-Filhart, 2006) though the processes upstream or downstream of these changes are not well characterized in breast cancer. Therefore we hypothesized that xenograft tumors derived from T47D/PKC∈ cells would have loss of membrane-associated β-catenin and E-cadherin. Immunofluorescence staining showed cytoplasmic localization of β-catenin and E-cadherin in T47D/PKC∈ tumors in sharp contrast to membrane association in T47D/neo (vector control) tumors. Western blot showed reduction in the level of E-cadherin in T47D/PKC∈ tumor samples but no change in the level of β-catenin compared to T47D/neo tumors. Based on this, we next wanted evaluate the β-catenin degradation pathway mediated by glycogen synthase kinase-3β (GSK-3β). Cellular extracts show an increase in the levels of the serine 9 inhibitory phosphorylation of GSK-3β in T47D/PKC∈ cells but no increase in total protein compared to T47D/neo cells. Treatment with the proteasomal inhibitor MG132 (10 μM) for 2 h did not increase the levels of β-catenin in vitro in T47D/PKC∈ cells. Current data indicate a correlation between PKC∈ expression and loss of membrane-associated β-catenin and E-cadherin. Further, β-catenin is stabilized in the cytoplasm of T47D/PKC∈ cells possibly allowing for nuclear localization and downstream gene transcription. Taken together with previous data, PKC∈ correlates with markers of poor outcome in addition to hormone-independence and tamoxifen-resistance suggesting that PKC∈ may play a central role in breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4216. doi:1538-7445.AM2012-4216

Abstract 2289: Potential role for Src kinase inhibitors in PKCα-overexpressing breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The purpose of this study was to evaluate the role of Src kinase in estrogen induced growth inhibition in the T47D/PKCα breast cancer cell line. Our lab has previously shown that constitutive overexpression of protein kinase Cα (PKCα) imparts a tamoxifen-resistant, hormone-independent phenotype in the T47D:A18 breast cancer cell line (Chisamore, et al. Clin Cancer Res 2001; 7(10):3156-65). Furthermore 17β-estradiol (E2) inhibits tumor growth in vivo as well as inhibits colony formation when cells are grown in 3D Matrigel (Zhang, et al. Mol Cancer Res 2009; 7(4):498-510). Here we show that the Src kinase inhibitor PP2 (5 μM) significantly inhibited T47D/PKCα colony formation in 3D Matrigel, but was unable to inhibit proliferation in vitro. The combination of PP2 and E2 treatment in 3D Matrigel did not further decrease colony number when compared to either alone. This suggests that these compounds may be acting on the same pathway and that E2 may be inhibiting Src kinase activity. To examine the effect of E2 on Src in T47D/PKCα xenograft tumors we probed the phosphorylation state of Src on Tyr416, known to be necessary for its full kinase activity. Src is basally phosphorylated on Tyr416 in T47D/PKCα cells. Interestingly, E2 treatment in vivo resulted in a decrease in phospho-Src. Erk 1/2, a downstream target of Src, is basally phosphorylated in T47D/PKCα cells. E2 treatment also resulted in a decrease in phospho-Erk1/2, in accordance with decreased Src phosphorylation. A reduction in phosphorylated Src and Erk1/2 was not seen in vitro indicating that the extracellular matrix is necessary for E2 to elicit an inhibitory effect on these proteins. In this study, we report that E2 treatment in vivo inhibited Src phosphorylation in tamoxifen resisitant T47D/PKCα tumors. Additionally pharmacological inhibition of Src inhibited T47D/PKCα colony formation in 3D Matrigel. Taken together, our results suggest that a Src inhibitor may be a potential therapeutic option for patients with PKCα-overexpressing breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2289. doi:10.1158/1538-7445.AM2011-2289

2-(4-Hydroxyphenyl)-Benzo[b]thiophen-6-ol, an Estrogen-Like Compound, Induces Apoptosis in T47D/PKCα Breast Cancer Cells.

Background: Tamoxifen (TAM) treatment failure is a major obstacle encountered in the clinical setting. Our lab has previously shown that constitutive overexpression of PKCα imparts a TAM resistant/ hormone independent phenotype in the T47D:A18 breast cancer cell line. Furthermore 17β-estradiol (E2) inhibits tumor growth in vivo as well as inhibits colony formation when cells are grown in 3D Matrigel (Zhang, 2009). Before TAM treatment was introduced, breast cancer patients received high-dose E2 and diesthystilbesterol (DES) treatment. DES treated patients may have a survival advantage over TAM treated patients in the long term (Peethambaram,1999). Others have shown that E2 can have inhibitory effects on MCF-7 cells both in vitro and in vivo (Lewis-Wambi, 2009). Taken together, this suggests an estrogenic compound may be efficacious for TAM-resistant breast cancer. The present study was designed to evaluate the effect of an estrogenic compound, 2-(4-hydroxyphenyl)-benzo[b]thiophen-6-ol (BTC), related to the SERMs raloxifene and arzoxifene, on the T47D:A18/PKCα breast cancer cell line.Methods: To determine if BTC can activate estrogen regulated genes we preformed an ERE-luciferase reporter assay 24 hours after BTC treatment. 3D Matrigel colony formation assay was performed to determine if BTC could inhibit colony formation. Colonies were stained and counted on day 10. To determine if apoptosis was occurring, cells were grown in Matrigel and the TUNEL Assay was performed on Day 6.Results: T47D:A18/PKCα cells showed a 31-fold induction in ERE-luciferase reporter activity when treated with BTC (10 -7 M) compared to 7-fold induction when treated with E2. In the T47D:A18/Neo parental cell line, BTC induced a 40-fold induction whereas estradiol induced a 96-fold induction. In the Matrigel colony formation assay T47D:A18/PKCα cells formed significantly fewer colonies when treated with BTC. Conversely, in the T47D:A18/Neo parental cell line BTC enhanced colony formation similar to E2 treatment. Our results indicate that BTC was able to induce significant apoptosis on day 6 (P= 0.038) as determined by the TUNEL Assay. Therefore BTC acts similarly to estradiol by inhibiting T47D:A18/PKCα colony formation and inducing apoptosis when cells are grown in Matrigel.Conclusion: These findings suggest that BTC is a potential lead compound in the treatment of PKCα overexpressing breast cancer. Furthermore, BTC was shown to be a potent inducer of the cytoprotective enzyme NQO1 in the Hepa 1c1c7 cell system as well as a strong activator of antioxidant responsive elements (Yu, 2007) indicating it may have the added benefit of chemoprotective effects which are absent in estradiol treatment. Further understanding of the mechanism in which BTC induces apoptosis in the T47D:A18/PKCα cell line may result in a clinical advantage to estradiol treatment including fewer side effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5140.