ERalpha-positive Breast Cancer Research Articles

ZNF213 Facilitates ER Alpha Signaling in Breast Cancer Cells.

BackgroundBreast cancer is the most common women malignancy worldwide, while estrogen receptor alpha positive type accounts for two third of all breast cancers. Although ER alpha positive breast cancer could be effectively controlled by endocrine therapy, more than half of the cases could develop endocrine resistance, making it an important clinical issue in breast cancer treatment. Thus, decoding the detailed mechanism, which controls ER alpha signaling activation and ER alpha protein stability, is of great importance for the improvement of breast cancer therapy. Several zinc finger proteins were shown to mediate the ubiquitination process and modulate protein stability. Thus, we further explore the function of Zinc finger protein 213 on ER alpha protein stability and tamoxifen resistance.MethodsCCK8 and Edu assay was used to measure cell proliferation. RNA sequence was performed by Ingenuity pathway analysis. The ER alpha signaling activities were measured with luciferase assay, real-time quantitative PCR, and western blotting. Protein stability assay and ubiquitin assay were used to determine ER alpha protein degradation and ubiquitination. The immuno-precipitation was utilized to determine ER alpha and ZNF213 interaction. The ubiquitin-based immuno-precipitation assay was sued to detect specific ubiquitination manner on ER alpha.ResultsWe identified ZNF213 as a novel zinc finger protein, which modulated ER alpha protein. ZNF213 expression correlated with poor outcome in endocrine treated patients. ZNF213 depletion inhibited ER alpha signaling and proliferation in breast cancer cells. Further mechanistic studies showed ZNF213 located in cytosol and nuclear, which modulated ER alpha stability via inhibiting ER alpha K48-linked ubiquitination.ConclusionsOur study reveals an interesting post-translational mechanism between ER alpha and ZNF213 in breast cancer. Targeting ZNF213 could be an appealing strategy for ER alpha positive breast cancer.

TRIM3 inhibits P53 signaling in breast cancer cells

BackgroundBeast cancer is the most common women cancer worldwide, while two third of them are ER alpha positive breast cancer. Among the ER alpha positive breast cancer, about 80% are P53 wild type, indicating the potential tumor suppression role in ER alpha positive breast cancer. Since P53 is an important safeguard to inhibit cell malignant transformation, reactivating P53 signaling could a plausible approach to treat breast cancer.MethodsTRIM3 protein levels were measured by western blot, while the P53 classical target genes were measured by real-time PCR. WST1 assay were used to measure cell proliferation, while cleaved caspase-3 was used to detect cell apoptosis. Protein stability and ubiquitin assay were used to detect the P53 protein ubiquitin and stability. The immuno-precipitation assays were used to detect the protein interactions. Immuno-staining was used to detect the protein localization of P53 and TRIM3, while the ubiquitin-based immuno-precipitation assays were used to detect the specific ubiquitination manner of P53.ResultsIn our study, we identified TRIM3 as an endogenous inhibitor for P53 signaling. TRIM3 depletion inhibited breast cancer cell proliferation and promoted apoptosis. In addition, TRIM3 depletion increased P53 protein level in breast cancer cell. Further investigation showed that TRIM3 could associate with P53 and promote P53 K48-linked ubiquitination and degradation.ConclusionOur study identified a novel post-translational modification mechanism between TRIM3 and P53. TRIM3 depletion or blockage could be a promising strategy to rescue P53 signaling and inhibit breast cancer progression.

Abstract 4373: Discovery of ZN-c5, a novel potent and oral selective estrogen receptor degrader

Abstract Majority of breast cancers express the estrogen receptor alpha (ERα) and the two major strategies for therapeutic targeting of ER-alpha positive breast cancer are aromatase inhibitors (which deprives estrogen) and inhibition of the estrogen receptor alpha. Although these strategies can be effective, many patients develop resistance which ultimately leads to disease progression which continues to rely on estrogen receptor signaling. One of the resistance mechanisms is the acquisition of activating mutations in the ER gene (ESR1) that allow tumors to proliferate without depending on estrogen. One approach to overcome resistance is to develop high affinity small molecules that degrade the estrogen receptor and effectively shut down ER signaling. Fulvestrant, an ER antagonist and a selective estrogen receptor degrader (SERD) is the only small molecule that is approved for the treatment of ER+/HER2− metastatic breast cancer. However, fulvestrant's limitation include low oral bioavailability and administration via intramuscular injection, which limits its exposure and leads to sub-optimal estrogen receptor degradation. The poor pharmacokinetic properties of fulvestrant has fueled the interest in developing an orally bioavailable small molecule estrogen receptor antagonist and degrader that could potentially benefit breast cancer patients. Here we describe the discovery of Zn-c5 a novel, small molecule with potent antagonism and degradative properties against the estrogen receptor both in vitro and in vivo. ZN-c5 showed a high oral bioavailability across several preclinical species as compared to other SERDs. To test if the high oral bioavailability can be translated to potent efficacy in vivo, we evaluated the anti-tumor activity of ZN-c5 in MCF-7 orthotopic tumor xenograft model. Oral ZN-c5 treatment at 5 mg/kg and 10 mg/kg resulted in 89% and 102% tumor growth inhibition respectively. Combination of ZN-c5 with cell cycle inhibitors such as CDK4/6 inhibitors or PI3K inhibitors results in enhanced antitumor activity. In addition to MCF-7 model, we evaluated the activity of ZN-c5 in ER mutant models including WHIM20, a Y537S ESR1 patient derived xenograft model. Treatment with ZN-c5 at 40 mg/kg induced 64% tumor growth inhibition while fulvestrant at 200 mg/kg (exposures 8-fold higher than that achieved in the clinic) resulted in 13% tumor growth inhibition. These data indicate that ZN-c5 has improved antitumor activity over fulvestrant in human tumor xenograft models. Zn-c5 is currently in clinical trials as a single agent and in combination studies. The PK profile of ZN-c5 in breast cancer patients indicates that Zn-c5 has greater than 5-fold exposure than fulvestrant. We believe that the high exposure of ZN-c5 coupled with its potency and degradative properties could therapeutic benefit estrogen receptor positive breast cancer patients. Citation Format: Ahmed A. Samatar, Jiali Li, Sayee Hegde, Peter Huang, Jianhui Ma, Kevin Bunker, Robert Winkler, Fernando Donate, Masha Sergeeva. Discovery of ZN-c5, a novel potent and oral selective estrogen receptor degrader [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4373.

ETS transcription factor ESE-1/Elf3 is an independent prognostic factor of survival in HR+HER2+ breast cancer patients.

The ETS transcription factor ESE-1 has been shown to be important in HER2+ breast cancer and ESE-1 mRNA expression has been shown to associate with prognostic outcomes in the HER2+ subtype, as well as in ER+, HER2+ luminal B patients. However, the clinical significance of ESE-1 protein expression remains unknown. The purpose of the current exploratory study is to evaluate the prognostic value of ESE-1 protein expression in molecular breast cancer subtypes with special emphasis on hormone receptor positive HER2+(HR+ HER2+) and the HER2 positive (HER2+-only) breast cancer patients. We developed a mouse monoclonal anti-ESE-1 antibody, verified its specificity, epitope, and used immunohistochemical staining to assess ESE-1 expression in an IBC approved archive of 957 breast tumor samples. Using Pearson product correlation, contingency analysis, and long rank P value testing, we analyzed the association of ESE-1 expression with clinicopathological features and survival outcomes in HR+HER2-; HR+HER2+; HR- HER2- (Triple negative) and HR-HER2+ (HER2 subtype) patients. ESE-1, nuclear or cytoplasmic, was not significantly associated with survival outcomes in HR+HER2-, triple-negative, or HER2+-only breast cancer patients. However, high nuclear ESE-1 was associated with poor survival outcomes in hormone receptor positive (ERα+, PR+) HER2+ patients and was an independent prognostic marker for that group. This study provides evidence for prognostic significance of nuclear ESE-1 in ERalpha positive breast cancers patients also positive for HER2 indicating that crosstalk between ERalpha and ESE-1 in HER2+ tumors could be important for prognostic outcomes. Further studies regarding the nature of interaction between ESE-1 and ERalpha in these tumors are warranted.

Abstract ES10-2: Mining noncoding mutations for drivers of development in ER alpha-positive breast cancer

Abstract Large-scale analysis of whole genome sequencing of breast tumors has identified thousands of genetic alterations, such as single-nucleotide variants (SNVs), outside of gene coding sequences. Delineating the functional impact of these noncoding SNVs is challenging, limiting their inclusion in precision genomics medicine pipelines. Pan-cancer attempts to detect evidence for positive selection within individual CREs identified promoters of a limited number of genes such as TERT. By considering the enrichment of noncoding mutations in cistromes as opposed to individual CREs, we identify the enrichment of noncoding SNVs in cistromes of breast cancer driver transcription factors, including FOXA1. This parallels enrichment of breast cancer genetic predispositions over these transcription factor cistromes. Furthermore, somatic SNVs accumulate in the regulatory plexus of some of these driver transcription factors providing evidence for a multi-targeted approach to disrupt their activity in breast cancer. In conclusion, our results support a functional impact of somatic SNVs converging on the cistrome of driver transcription factors in breast cancer. Altogether, we show how to interrogate noncoding SNVs to delineate the oncogenic determinants of breast cancer to be considered for therapeutic intervention. Citation Format: Lupien M. Mining noncoding mutations for drivers of development in ER alpha-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES10-2.

Regulation of Estrogen Signaling and Breast Cancer Proliferation by an Ubiquitin Ligase TRIM56

Breast cancer ranks No.1 in women cancer worldwide, while 60-70% are estrogen receptor alpha positive. The estrogen selective modulators, such as tamoxifen, become the effective drugs for controlling ER alpha breast cancer progression. However, tamoxifen resistance will develop during long-time treatment and cancer progression. Thus, further understanding of ER alpha signaling becomes necessary for the improvement of breast cancer therapy. Here, we identify TRIM56 as a novel regulatory factor in ER alpha signaling. TRIM56 expression is positive correlated with ER alpha and PR in breast cancer samples and is related to poor prognosis in endocrine therapy patients. TRIM56 depletion significantly decreases ER alpha signaling activity and ER alpha positive breast cancer proliferation in vitro and in vivo. TRIM56 associates with AF1 domain of ER alpha via its WD40 domain in the cytoplasm. TRIM56 prolongs ER alpha protein stability, possibly through targeting ER alpha K63 linked ubiquitination. In conclusion, our study reveals an interesting post-translational mechanism between TRIM56 and ER alpha in breast cancer progression. Targeting TRIM56 could be a promising approach for ER alpha positive breast cancer. Funding Statement: The project was supported by the National Science Foundation for Young Scientists of China (No. 8170110153, Ting Zhuang), the Joint Fund of the National Natural Science Foundation of China (No.U1604190, Jian Zhu; U1704169, Xiumin Li), the Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No.15IRTSTHN025, Hui Wang), the National High Technology Research and Development Program of China (2012AA02A201-1), the Foundation of Henan Educational Committee (No.16A310014 and No.17A310025, Jian Zhu and Ting Zhuang), and the Program for Ph.D. starting research funding from Xinxiang Medical University (Ting Zhuang and Jian Zhu). This study is funded by graduate innovative practice base for clinical medicine of Xinxiang Medical University, Yashijie medical laboratory institute，School of laboratory medicine, Xinxiang Medical University Declaration of Interests: The authors claim no conflict of interest. Ethics Approval Statement: This study was reviewed and approved by the Ethical Board at Xinxiang Medical University. This usage of clinical samples was reviewed and approved by the Ethical Board at the Qilu Hospital of Shandong University with written informed consents from all the patients.

SMURF1 facilitates estrogen receptor \u0251 signaling in breast cancer cells

BackgroundEstrogen receptor alpha (ER alpha) is expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression. ER alpha positive breast cancer can be well controlled by ER alpha modulators, such as tamoxifen. However, tamoxifen resistance is commonly observed by altered ER alpha signaling. Thus, further understanding of the molecular mechanisms, which regulates ER alpha signaling, is important to improve breast cancer therapy.MethodsSMURF1 and ER alpha protein expression levels were measured by western blot, while the ER alpha target genes were measured by real-time PCR. WST-1 assay was used to measure cell viability; the xeno-graft tumor model were used for in vivo study. RNA sequencing was analyzed by Ingenuity Pathway Analysis. Identification of ER alpha signaling was accomplished with luciferase assays, real-time RT-PCR and Western blotting. Protein stability assay and ubiquitin assay was used to detect ER alpha protein degradation. Immuno-precipitation based assays were used to detect the interaction domain between ER alpha and SMURF1. The ubiquitin-based Immuno-precipitation based assays were used to detect the specific ubiquitination manner happened on ER alpha.ResultsHere, we identify the E3 ligase SMURF1 facilitates ER alpha signaling. We show that depletion SMURF1 decreases ER alpha positive cell proliferation in vitro and in vivo. SMURF1 depletion based RNA-sequence data shows SMURF1 is necessary for ER alpha target gene expression in the transcriptomic scale. Immunoprecipitation indicates that SMURF1 associates with the N-terminal of ER alpha in the cytoplasm via its HECT domain. SMURF1 increases ER alpha stability, possibly by inhibiting K48-specific poly-ubiquitination process on ER alpha protein. Interestingly, SMURF1 expression could be induced via estradiol treatment.ConclusionsOur study reveals a novel positive feedback between SMURF1 and ER alpha signaling in supporting breast cancer growth. Targeting SMURF1 could be one promising strategy for ER alpha positive breast cancer treatment.

Abstract 4854: Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene

Abstract Despite continued administration of antiestrogen therapies, approximately 50% of all estrogen receptor alpha (ERalpha) positive breast cancers will present new metastatic lesions. The acquisition of secondary hormone-resistant metastatic breast cancers represents a significant clinical barrier towards life-long disease free survival for the patient. Somatic mutations to the ERalpha gene (ESR1) Y537S and D538G represent a novel mechanism of acquired antiestrogen resistance because they confer hormone-free transcriptional activity and reduced selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD) potency. Fulvestrant, a SERD, was the only molecule that could completely ablate mutant ERalpha activity. Unfortunately, fulvestrant possesses poor pharmacologic profiles that limit its therapeutic utility. Bazedoxifene (BZA) is a potent mixed SERM/SERD and has improved pharmacokinetics and oral bioavailability compared to fulvestrant. We show that BZA inhibits Y537S and D538G ESR1 somatic mutation transcriptional activity with a greater potency than the SERMs 4-hydroxytamoxifen (TOT) and raloxifene (RAL). Further investigations into the biophysical and structural basis for BZA action suggest that BZA increases the conformational dynamics of helix 12, a key molecular switch that governs ERalpha action resulting in SERD-like properties and improved potency against the somatic mutations compared to TOT and RAL. Citation Format: Sean W. Fanning, Venkat Dharmarajan, Christopher G. Mayne, Weiyi Toy, Kathryn E. Carlson, Teresa A. Martin, Jason Nowak, Jerome Nwachukwu, David J. Hosfield, Emad Tajkhorshid, Sarat Chandarlapaty, Patrick Griffin, Yang Shen, John A. Katzenellenbogen, Geoffrey L. Greene. Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene. [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 4854.

Abstract 5511: Endoxifen inhibits polyamine biosynthetic enzyme activity and up-regulates metabolizing enzymes, spermine oxidase (SMO) and acetyl spermine oxidase (APAO)in breast cancer

Abstract Tamoxifen is the most widely used selective estrogen receptor modulator for anti-estrogen therapy for women with estrogen receptor alpha (ERalpha)-positive breast cancer. Clinical trials recognized that tamoxifen was a pro-drug and got converted to active metabolites in the human body. Earlier studies recognized 4-hydroxytamoxifen as the active metabolite of tamoxifen, although recent studies showed that the concentration of another metabolite, endoxifen was up to 10-fold higher than that of 4-hydorxytamoxifen in patients treated with tamoxifen. These metabolites are formed by the action of cytochrome P450 2D6 (CYP2D6). To elucidate the mechanism of action of endoxifen, we determined its effects on cell growth, activity of polyamine biosynthetic and metabolizing enzymes, and oncogene expression in ERalpha-positive MCF-7 breast cancer cells. Polyamines (putrescine, spermidine and spermine) are ubiquitous cellular components and play important roles in cell proliferation. Estradiol (4 nM) increased the proliferation of MCF-7 cells by 2- to 3-fold by 3 days of treatment. Endoxifen significantly suppressed the effects of estradiol at 100 nM and higher concentrations (P < 0.01; n = 3). Estradiol increased the activity of the polyamine biosynthetic enzymes, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxlase (AdoMetDC), whereas endoxifen suppressed ODC and AdoMetDC activities in a concentration- and time-dependent manner. The change in biosynthetic enzyme activities was reflected in polyamine pools, as estradiol increased and endoxifen suppressed the levels of putrescine and spermidine in MCF-7 cells. There was no significant change in spermine level. Endoxifen had a significant effect on increasing the activities of two polyamine metabolizing enzymes, SMO and APAO. Estradiol suppressed the activities of SMO and APAO by 40% compared to control group at 24 hours of treatment. Endoxifen increased SMO and APAO activities by 2- to 4-fold compared to the activities of these enzymes in estradiol treated group. The expression of c-myc, c-fos and Tiff1 genes was significantly increased by 4 nM estradiol, while the addition of endoxifen suppressed estradiol-mediated increase in gene expression, as measured by quantitative real-time PCR analysis. These results indicate that the polyamine pathway plays an important role in the growth inhibitory and anti-estrogenic effects of endoxifen in breast cancer cells. Upregulation of SMO and APAO might be useful biomarkers for the action of endoxifen. Citation Format: T. J. Thomas, Hui-Chen Hsu, Mervi Hyvonen, Tuomo Keinanen. Endoxifen inhibits polyamine biosynthetic enzyme activity and up-regulates metabolizing enzymes, spermine oxidase (SMO) and acetyl spermine oxidase (APAO)in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5511. doi:10.1158/1538-7445.AM2015-5511

Abstract 5578: Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites

Abstract The proliferation of estrogen receptor alpha (ERalpha)-positive breast cancer cells is stimulated by estradiol. Tamoxifen [(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine] is the most commonly used endocrine treatment for ERalpha-positive breast cancer in pre- and post-menopausal women. Tamoxifen is metabolized to 4-hydroxytamoxifen and endoxifen. ICI 182780 (Faslodex or Fulvestrant) is a pure antiestrogen targeted to the ERalpha. We investigated the effects of endoxifen and 4-hydroxytamoxifen on ERalpha-positive MCF-7 breast cancer cells using cell growth assay, oncogene expression and polyamine regulatory pathways. Polyamines are ubiquitous cellular components and are essential for cell growth and differentiation. Treatment of cells for 1-5 days with 4 nM estradiol increased cells number, whereas the addition of endoxifen, 4-hydroxytamoxifen or ICI 182780 in combination with estradiol suppressed cell growth in a concentration- and time-dependent manner. Real time quantitative polymerase chain reaction (RT-qPCR) experiments showed that the addition of 4 nM estradiol to MCF-7 cells increased the expression of c-myc, c-fos and Tiff1 genes significantly, while the addition endoxifen, 4-hydroxytamoxifen, or ICI 182780 suppressed estradiol-mediated increase in gene expression. Polyamine biosynthetic enzymes were also affected by tamoxifen metabolites. Estradiol increased the activities of the polyamine biosynthetic enzyme, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxlase (AdoMetDC), whereas the antiestrogens suppressed ODC and AdoMetDC activities in a concentration- and time-dependent manner. However, these agents had little effect on the activity of the polyamine catabolic enzyme, spermidine/spermine N1-acetyl transferase (SSAT). The change in biosynthetic enzyme activities was reflected in polyamine pools, as decreased putrescine and spermidine levels. There was no significant change in spermine level. These results suggest that the mechanism of action of tamoxifen metabolites involves multiple cell growth regulatory pathways. In addition, endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol on ERalpha-positive breast cancer cells in a manner similar to that of ICI 182780. Note: This abstract was not presented at the meeting. Citation Format: T. Thomas, Huichen Hsu, PingAr Yang, Mervi T. Hyvonen, Tuomo A. Keinanen. Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites. [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 5578. doi:10.1158/1538-7445.AM2014-5578

Tumor suppressor p53 status as a determinant of estrogen receptor beta signaling in breast cancer

Tumor suppressor p53 and estrogen receptor have opposite roles in the onset and progression of breast cancer. The biological actions of estrogens are mediated by genetically distinct estrogen receptors (ER) alpha and beta. In addition to its expression in several ERalpha‐positive breast cancers and normal mammary cells, ERbeta is usually expressed in ERalpha‐negative cancers including triple‐negative breast cancer, but its role in oncogenesis remains unclear. Using glutathione‐S‐transferase (GST)‐pull down and coimunoprecipitation assays, we have demonstrated direct binding of ERbeta to p53 and have delineated the domains required for the interaction. Using the highly sensitive proximity ligation assay (PLA), we show ERbeta‐p53 interaction in situ. Surprisingly, we found that ERbeta has opposite functions depending on the mutational status of p53. A combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real‐time PCR showed that in the cellular context of wild type p53, ER beta is pro‐proliferative, whereas in the context of mutant p53, ER beta is anti‐proliferative. Our finding that ERbeta can elicit opposite functions dependent on the p53 context provides significant insight into the controversy whether ERbeta is pro‐or anti‐tumorigenic with important implications in therapeutic targeting of ERbeta.

Genetic variants of kinases suppressors of ras (KSR1) to predict survival in patients with ER-alpha positive metastatic breast cancer.

e11018 Background: In addition to its activating role of MAPK pathway, the protein kinase suppressor of ras 1 (KSR1) has been identified as a novel modulator of estrogen receptor alpha (ERα), as silencing of KSR1 in ERα+ breast cancer (BC) cells lines resulted in dowregulation of ERα transcriptional activity in the presence of oestradiol. Our previously data revealed the existence of single nucleotide polymorphisms (SNPs) in EGFR and ERα pathway genes that may predict clinical outcome in ERα positive metastatic BC (mBC) patients. With respect to those findings we aimed to evaluate the clinical implication of KSR1 SNPs on survival in patients with primary ERα+ mBC treated with tamoxifen. Methods: Tumoral DNA was obtained from 222 patients treated with tamoxifen withERα+ invasive BC who had undergone surgery between 1981 and 2003 (median age 56 [28-90]). Three relevant KSR1 SNPs were selected based on public literature resources and databases, including 2 SNPs localised in the regulating 3’untranslated region (rs224190, rs 1075952) and 1 non-synonymous SNP localised in the coding exon 7 region (rs2293180). All SNP were analysed using DNA sequencing with OS and DFS as primary endpoint. Results: The overall median follow-up was 6.4 years.In univariate analysis the rs2241906 SNP was significantly associated with DFS and OS and patients with the TT genotypedemonstrated shorter DFS (2.1 vs. 7.1 months, p=0.005) and OS ( 2.6 vs. 8.4 months p=0.002) than patients with the x/C genotypes. The associations remained significant in the multivariate analysis adjusting age, lymph node status and HER2 status (HR (95%CI): 4.81 (2.00-11.59) and 5.74 (2.29-14.43), for DFS and OS, respectively). The same significant correlation was retrieved with the SNP rs1075952 in linkage disequilibrium with rs2241906 that was used as a control. No relationship was shown between rs2293180 and survival. Conclusions: Our findings demonstrated that KSR1 polymorphisms could arise as a potential marker to predict survival in patients with mBC treated with tamoxifen. The putative role of KSR1 as a modulator of ERα activity could functionally explain our results yielding insight for further studies.

Abstract 2405: Loss of TBK1 induces an epithelial-mesenchymal transition through upregulation of Slug in the breast cancer cell lines

Abstract Estrogen receptor alpha (ER alpha) is a key regulator of proliferation and differentiation in mammary epithelium through their well-documented effects on transcription and represents a crucial prognostic indicator and therapeutic target in breast cancer. Here, we show that loss of TBK1 in ER alpha positive breast cancer cells causes an epithelial-mesenchymal transition (EMT). We found that TBK1 is downregulated in ER alpha negative breast tumors by analyzing published microarray data sets, suggesting that TBK1 might regulate ER alpha expression. To investigate this hypothesis, we made the ER alpha positive breast cancer cell lines stably expressing TBK1-specific shRNA. Knockdown of TBK1 decreased E-cadhrein expression (epithelial marker) and increased N-cadherin, vimentin, fibronectin expression (mesenchymal marker). Additionally, loss of TBK1 in ER alpha positive breast cancer cell lines leads to the downregulation of ER alpha expression. Interestingly, ER alpha repression by knockdown of TBK1 induced the upregulation of transcriptional repressor Slug, a master regulator of EMT. Because ER alpha expression is suppressed by loss of TBK1, it is suggested that TBK1 plays a role in the inverse correlation between Slug and ER alpha. Our findings indicate that loss of TBK1 expression is associated in between ER alpha expression status and invasive growth of breast cancer. 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 2405. doi:1538-7445.AM2012-2405

Abstract SY36-03: Development of small-molecule inhibitors of autophagy as anticancer therapy

Abstract Autophagy is an intracellular catabolic mechanism that sequesters selected cytoplasmic components and organelles into double-membrane vesicles, called autophagosomes; the content of autophagosomes is degraded by lysosomal hydrolases after fusing with lysosomes. Autophagy is an important homeostatic mechanism that regulates intracellular recycling and supports cellular survival under stressful conditions (Moreau et al., 2010). Activation of autophagy confers stress resistance and sustains cell survival under adverse conditions. Consistently, autophagy has been found to play an important role in promoting cancer cell survival in the tumor micro-environment in vivo and contribute to metabolic changes to promote oncogenic transformation (Lock et al., 2010). Furthermore, activation of autophagy has been implicated in mediating resistance to existing anticancer therapy. Multiple anti-cancer treatments, including conventional chemotherapy, radiation, as well as targeted therapies such as histone deacetylase inhibitors and imatinib, have been found to activate autophagy as a pro-survival mechanism in cancer cells (Amaravadi and Thompson, 2007; Amaravadi et al., 2007; Kondo et al., 2005; Schoenlein et al., 2009). However, there is no pharmacological method currently available that can selectively inhibit autophagy. We have developed a series of small molecule inhibitors of autophagy, termed “spautins” for specific and potent autophagy inhibitors (Liu et al., 2011). Spautins target USP10 and USP13, two ubiquitin specific peptidases that regulate the ubiquitination and stability of Vps34 complexes, which are critical for autophagy initiation. Vps34 is the class III PI3 kinase that phosphorylates PI (phosphatidylinositol) to produce PI3P (phosphatidylinositol 3-phosphate). Treatment with spautin effectively reduces the levels of Vps34 complexes and PI3P, a key lipid signaling molecule essential for autopaghy, and consequently, leads to inhibition of autophagy. Since USP10 also mediates the deubiquitination of p53 (Yuan et al., 2010), spautins promote the degradation of p53 (Liu et al., 2011) and mutant p53. Our studies demonstrated that spautins show selective cytotoxicity to a subset of cancer cells while normal cells are relatively resistant to spautins (Liu et al., 2011). We will discuss our new data on the ability of spautin to induce the death of cancer cells in vitro and in vivo. Our study demonstrates a proof-of-principle for developing inhibitors of autophagy by targeting protein deubiquitination. Spautin family of autophagy inhibitors represent promising lead candidates for developing novel anti-cancer drugs. Reference: Amaravadi, R.K., and Thompson, C.B. (2007). The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res 13, 7271-7279. Amaravadi, R.K., Yu, D., Lum, J.J., Bui, T., Christophorou, M.A., Evan, G.I., Thomas-Tikhonenko, A., and Thompson, C.B. (2007). Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 117, 326-336. Hoyer-Hansen, M., and Jaattela, M. (2008). Autophagy: an emerging target for cancer therapy. Autophagy 4, 574-580. Kondo, Y., Kanzawa, T., Sawaya, R., and Kondo, S. (2005). The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5, 726-734. Liu, J., Xia, H.G., Kim, M., Xu, L., Li, Y., Zhang, L., Yu, C., Norberg-Vakifahmetoglu, H., Zhang, T., Furuya, T., et al. (2011). Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell 147, 223-234. Lock, R., Roy, S., Kenific, C.M., Su, J.S., Salas, E., Ronen, S.M., and Debnath, J. (2010). Autophagy Facilitates Glycolysis During Ras Mediated Oncogenic Transformation. Mol Biol Cell 22, 165-178. Moreau, K., Luo, S., and Rubinsztein, D.C. (2010). Cytoprotective roles for autophagy. Curr Opin Cell Biol 22, 206-211. Schoenlein, P.V., Periyasamy-Thandavan, S., Samaddar, J.S., Jackson, W.H., and Barrett, J.T. (2009). Autophagy facilitates the progression of ERalpha-positive breast cancer cells to antiestrogen resistance. Autophagy 5, 400-403. Yuan, J., Luo, K., Zhang, L., Cheville, J.C., and Lou, Z. (2010). USP10 regulates p53 localization and stability by deubiquitinating p53. Cell 140, 384-396. 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 SY36-03. doi:1538-7445.AM2012-SY36-03

Gene Expression Profiling Reveals New Aspects of PIK3CA Mutation in ERalpha-Positive Breast Cancer: Major Implication of the Wnt Signaling Pathway

BackgroundThe PI3K/AKT pathway plays a pivotal role in breast cancer development and maintenance. PIK3CA, encoding the PI3K catalytic subunit, is the oncogene exhibiting a high frequency of gain-of-function mutations leading to PI3K/AKT pathway activation in breast cancer. PIK3CA mutations have been observed in 30% to 40% of ERα-positive breast tumors. However the physiopathological role of PIK3CA mutations in breast tumorigenesis remains largely unclear.Methodology/Principal FindingsTo identify relevant downstream target genes and signaling activated by aberrant PI3K/AKT pathway in breast tumors, we first analyzed gene expression with a pangenomic oligonucleotide microarray in a series of 43 ERα-positive tumors with and without PIK3CA mutations. Genes of interest were then investigated in 249 ERα-positive breast tumors by real-time quantitative RT-PCR. A robust collection of 19 genes was found to be differently expressed in PIK3CA-mutated tumors. PIK3CA mutations were associated with over-expression of several genes involved in the Wnt signaling pathway (WNT5A, TCF7L2, MSX2, TNFRSF11B), regulation of gene transcription (SEC14L2, MSX2, TFAP2B, NRIP3) and metal ion binding (CYP4Z1, CYP4Z2P, SLC40A1, LTF, LIMCH1).Conclusion/SignificanceThis new gene set should help to understand the behavior of PIK3CA-mutated cancers and detailed knowledge of Wnt signaling activation could lead to novel therapeutic strategies.

Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance.

Endocrine therapy is the main therapeutic option for patients with estrogen receptor (ERalpha)-positive breast cancer. Resistance to this treatment is often associated with estrogen-independent activation of ERalpha. In this study, we show that in ERalpha-positive breast cancer cells, activation of the receptor tyrosine kinase RET (REarranged during Transfection) by its ligand GDNF results in increased ERalpha phosphorylation on Ser118 and Ser167 and estrogen-independent activation of ERalpha transcriptional activity. Further, we identify mTOR as a key component in this downstream signaling pathway. In tamoxifen response experiments, RET downregulation resulted in 6.2-fold increase in sensitivity of MCF7 cells to antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In tamoxifen-resistant (TAM(R)-1) MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERalpha-positive tumors and that in primary tumors from patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment, there was a twofold increase in the number of RET-positive tumors. Together these findings identify RET as a potentially important therapeutic target in ERalpha-positive breast cancers and in particular in tamoxifen-resistant tumors.

The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers

Transcriptomic screens in breast cancer cell lines have identified a protein named anterior gradient-2 (AGR2) as a potentially novel oncogene overexpressed in estrogen receptor (ER) positive tumours. As targeting the ER is responsible for major improvements in cure rates and prevention of breast cancers, we have evaluated the pro-oncogenic function of AGR2 in anti-hormone therapeutic responses. We show that AGR2 expression promotes cancer cell survival in clonogenic assays and increases cell proliferation and viability in a range of cancer cell lines. Chromatin immunoprecipitation and reporter assays indicate that AGR2 is transcriptionally activated by estrogen through ERalpha. However, we also found that AGR2 expression is elevated rather than inhibited in response to tamoxifen, thus identifying a novel mechanism to account for an agonistic effect of the drug on a specific pro-oncogenic pathway. Consistent with these data, clinical analysis indicates that AGR2 expression is related to treatment failure in ERalpha-positive breast cancers treated with tamoxifen. In contrast, AGR2 is one of the most highly suppressed genes in cancers of responding patients treated with the anti-hormonal drug letrozole. These data indicate that the AGR2 pathway represents a novel pro-oncogenic pathway for evaluation as anti-cancer drug developments, especially therapies that by-pass the agonist effects of tamoxifen.

Modulation of tumorigenesis and oestrogen receptor-α expression by cell culture conditions in a stem cell-derived breast epithelial cell line

The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ER alpha (oestrogen receptor alpha) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T-antigen transfection and X-ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone-deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and alpha 6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44+/high/CD24-/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial-to-mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX-2 (cyclo-oxygenase-2) through a CD44-mediated mechanism. An oestrogen-responsive cell line with ER alpha and CD44+/CD24-/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ER alpha-positive breast cancers, (2) CD44+/CD24-/low breast tumour stem cells and (3) the metastatic ability of breast cancer.

A ferrocenyl derivative of hydroxytamoxifen elicits an estrogen receptor-independent mechanism of action in breast cancer cell lines

The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERalpha positive (MCF-7, T-47D, ZR-75-1) and 3 ERalpha negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERalpha. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERalpha positive and ERalpha negative cells. In the presence of ERalpha, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERalpha, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERalpha positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERalpha, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.

Adenosine A1 receptor, a target and regulator of estrogen receptorα action, mediates the proliferative effects of estradiol in breast cancer

Estrogen receptor-α (ERα) and its ligand estradiol (E2) play critical roles in breast cancer growth and are key therapeutic targets. Here, we report a novel dual role of the adenosine A1 receptor (Adora1) as an E2/ERα target and a regulator of ERα transcriptional activity. In ERα-positive breast cancer cells, E2 up-regulated Adora1 mRNA and protein levels, an effect that was reversed by the E2 antagonist ICI 182,780. siRNA ablation of Adora1 in ERα-positive cells reduced basal and E2-dependent proliferation, whereas Adora1 over-expression in an ERα-negative cell line induced proliferation. The selective Adora1 antagonist, DPCPX, reduced proliferation, establishing Adora1 as a mediator of E2/ERα-dependent breast cancer growth. Intriguingly, Adora1 ablation decreased both mRNA and protein levels of ERα and, consequently, estrogen responsive element-dependent ERα transcriptional activity. Moreover, Adora1 ablation decreased binding activity of ERα to the promoter of its target gene TFF1 and led to reduced TFF1 promoter activity and mRNA levels, suggesting that Adora1 is required for full transcriptional activity of ERα upon E2 stimulation. Taken together, we demonstrated a short feed-forward loop involving E2, ERα, and Adora1 that favors breast cancer growth. These data suggest that Adora1 may represent an important target for therapeutic intervention in hormone-dependent breast cancer.