LTED Cells Research Articles

Abstract P2-22-01: Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer

Abstract Late-stage relapse also known as late recurrence breast cancer is a metastatic disease that occurs more than five years after initial adjuvant endocrine therapy treatment. The 5-year relative survival rate is less than 30% and there are limited treatment options for patients with late-stage relapse because of poor performance status. We hypothesize that long non-coding RNA (lncRNA) protein interactions regulate genes to promote late-stage relapse and these interactions can be targeted using RNA therapeutics. To address this, we used RNA-sequencing to identify LINC00355 to be up-regulated in 24 late-stage relapse samples compared to a unique cohort of 72 primary breast cancer samples. Analysis of 480 primary breast cancer samples from The Cancer Genome Atlas (TCGA) also shows decrease expression of LINC00355 compared to late-stage relapse. To better assess LINC00355 cell type expression, we analyzed 26 publicly available primary breast cancer single cell sequencing data samples and detected 0.90% or less of cells expressing LINC00355, which was restricted to cancer epithelial cells. Due to cellular localization playing an important role in lncRNA function, we isolated nuclear and cytoplasmic lysates and orthogonally validated with RNAscope in situ hybridization (RNA-ISH) to show that LINC00355 is located in the nucleus of MCF7 long-term estrogen deprived (MCF7 LTED) cells. LTED cells are deprived of estrogen for longer than three years meant to recapitulate the acquired resistance to aromatase inhibitors. Next, we determined that LINC00355 binds to MENIN protein to regulate p27KIP expression to promote proliferation with UV crosslinking immunoprecipitation and chromatin immunoprecipitation qPCR of LINC00355 overexpression and mutant constructs. Furthermore, RNA-ISH assays validated high nuclear expression of LINC00355 and RNA-ISH protein co-detection assays showed co-localization of LINC00355-MENIN interaction in a panel of late-stage relapse breast cancer patient tissues compared to primary tissues. Lastly to determine clinical significance, we treated MCF7 LTED cells with two locked nucleic acid antisense oligos (ASOs) targeting LINC00355 to observe a significant decrease in proliferation, cell viability, and invasion. Overall, this is the first study to show the importance of lncRNA-protein interactions and therapeutically target lncRNAs with ASOs in late-stage relapse breast cancer. Moving forward, we intend to further understand how LINC00355 contributes to the progression of late-stage relapse with the intent of creating novel cancer diagnostics and therapies. Citation Format: DeAnna Wells, Kyla Gelev, Prasanth Thunuguntla, Reyka Jayasinghe, Li Ding, Jieya Shao, Cynthia Ma, Jessica Silva-Fisher. Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-01.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

Metalloestrogenic effects of cadmium are absent in long-term estrogen-deprived MCF-7 cells: Evidence for the involvement of constitutively activated estrogen receptor α and very low expression of G protein-coupled estrogen receptor 1

Cadmium (Cd) has estrogen-like activities in breast cancer; it acts as a metalloestrogen in humans. Prospective cohort studies of Cd and breast cancer risk suggest a significant relationship between increased Cd intake and cancer incidence, with more pronounced effects for estrogen receptor α (ERα)-positive breast cancers. However, a recent systematic review with the highest level of evidence demonstrated no such relationship in post-menopausal women. Thus, the reported effects of Cd in pre- and post-menopausal ERα-positive breast cancers are inconsistent. MCF-7 human breast cancer cells normally exhibit growth through estradiol-triggered ERα signaling; however, the MCF-7 cells cultured in estrogen-deprived conditions for a long time (∼ 6 months) eventually result in LTED cells that can be used to utilize to study the proliferation of ERα-positive breast cancer cells obtained from post-menopausal women. Our results showed that unlike MCF-7 cells, LTED cells showed estradiol-independent growth because of constitutively activated ERα. Moreover, Cd (∼10 nM) stimulated ERα signaling in MCF-7 cells and ERα-expressing LTED cells, but not in LTED cells; in ERα-expressing LTED cells, this effect was reversed by ICI 182,780 (an ERα antagonist). Furthermore, in comparison with MCF-7 cells, the LTED cells expressed very low levels of G protein-coupled estrogen receptor 1 (GPER1), a membrane ER capable of stimulating the estrogenic activity of Cd. These findings suggest that the estrogenic action of Cd may be suppressed in LTED cells, and potentially in post-menopausal breast cancer.

ISY-24-3 - ESR1 gene alterations related to endocrine resistance in metastatic breast cancer

Estrogen receptor (ER) positive breast cancer can be treated by endocrine therapy; however a certain population of ER-positive patients becomes resistant to endocrine therapies. Expression of the ESR1 gene encoding ER was upregulated in response to estrogen deprivation (LTED cells), which was usually seen in the metastatic breast cancer (MBC) with resistance to aromatase inhibitor. We found that RNAs from ESR1 gene locus were induced both from coding and non-coding regions in LTED cells using both total RNA-sequencing and mRNA-seq analyses. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating non-coding RNAs). Depletion of one of Eleanors, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. The activation of the ESR1 gene locus by the Eleanors is critical for cancer cell adaptation to estrogen deprivation and can be a new therapeutic and diagnostic targets in endocrine therapy resistance. Recent evidences have shown that the key potential mechanisms of the resistance to aromatase inhibitors based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. We initially developed a droplet digital PCR-based method for the sensitive detection of ESR1 mutations, Y537S, Y537N, Y537C, and D538G, which showed ESR1 mutations occurred in 2.5% of primary breast cancer specimens (n = 270) and in 20% of MBC specimens (n = 55). Furthermore, the patients with ESR1 mutations from cell free (plasma) DNA tended to have a poorer response to post-treatment, compared with wild-type patients. Taken together, ESR1 gene alterations including non-coding RNA are profoundly related to the endocrine resistance in metastatic breast cancer and could be a predictor of the usage to combination with the signal transduction inhibitor, such as mTOR inhibitor, PI3K inhibitor.

Bongkrekic Acid as a Warburg Effect Modulator in Long-term Estradiol-deprived MCF-7 Breast Cancer Cells.

An in vitro cell model of long-term estrogen-deprived MCF-7 (LTED) cells has been utilized to analyze the re-growth mechanisms of breast cancers treated with blockers for estrogen receptor α (ERα) signaling. Bongkrekic acid (BKA) is a natural toxin isolated from coconut tempeh contaminated with the bacterium Burkholderia cocovenans. LTED cells, MCF-7 cells and MDA-MB-231 cells were employed in the study. After treatment with BKA (chemically synthesized; purity: >98%), several biochemical analyses were carried out. LTED cells were categorized into an oxidative phenotype. When LTED cells were treated with BKA, lactate dehydrogenase A (LDH-A)/pyruvate dehydrogenase kinase 4 (PDK4) were down-regulated, thereby prompting the aggressive use of glucose via mitochondrial oxidative phosphorylation and induction of cell death responses. These effects of BKA were not observed in the other breast cancer cells analyzed. We suggest the potential of BKA as an experimental tool for the analysis of cancer biology in LTED cells.

Abstract P2-09-02: Epigenetic Regulation of histone variants - a role in hormone therapy resistant breast cancer?

Abstract OBJECTIVE To utilize a genome wide approach to characterize epigenetic changes associated with resistance to estrogen deprivation, thus mimicking resistant to AIs. BACKGROUND Mechanisms of endocrine resistance include the activation of growth factor receptor pathways, overexpression of ER co-activators, and metabolic resistance due to polymorphisms in metabolizing enzymes. Recently, epigenetic mechanisms have been investigated to explain endocrine resistance. The majority of mechanisms for acquired resistance have been described following tamoxifen therapy, but the mechanisms for resistance to aromatase inhibitors (AI) remain less well known. HYPOTHESIS Estrogen deprivation results in altered methylation and altered expression of critical target genes in breast cancer cells, which helps the cell to survive in the absence of estrogen, contributing to acquired AI resistance in breast cancer patients. MATERIALS AND METHODS We utilized derivatives of the ER_ MCF-7 cell line which were isolated after being cultured in estrogen-free media for 9 months (C4-12 cell line) and 18–24 months (LTED, Long term estrogen deprivation cell line). A genome-wide methylation screen was performed using Methyl CpG binding Domain (MBD) pull down assay followed by hybridization into Affymetrix Human Promoter 1.0R Array. Altered DNA methylation was validated by bisulfite genomic sequencing assays while gene expression of candidate genes was studied by qRT-PCR and Western blotting. RESULTS There were a significant amount of differentially methylated genes in C4-12 and LTED when compared to MCF7. Intriguingly, among the genes which were significantly hypomethylated in both endocrine resistant cell lines were a number of histone genes, with the homomorphic variant HIST1H2BE showing the strongest hypomethylation. Canonical histones have both homomorphic and heteromorphic variants, which differ in their timing of expression and similarity to the canonical histone protein's amino acid sequence. Overexpression of various heteromorphic histone variants have been implicated in carcinogenesis, but to date, there have been no studies on homomorphic variants, and specifically H2B variants, in regards to their role in tumorigenesis. We show that hypomethylation of HIST1H2BE was associated with its upregulation in C4-12 and LTED cells. Furthermore, knockdown of of HIST2BE resulted in decreased growth in the endocrine resistant cells, but not in the parental MCF-7 cell lines. CONCLUSION & FUTURE DIRECTIONS The study is novel in two ways; first, this is the first study to show a potential role for histone variants in endocrine resistance. Second, this is one of very few examples where hypomethylation (instead of hypermethylation) results in altered expression of genes critical in tumorigenesis. Current studies include the detailed characterization of the phenotype following over-expression and knockdown of HIST1H2BE, the examination of the timing of expression of this variant in the S phase, and finally we are measuring methylation and expression of HIST2BE in a large collection of tumors from AI-treated breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-09-02.

Abstract 4825: Inhibition of AKT abrogates resistance to endocrine therapy in estrogen receptor positive breast cancer

Abstract Estrogen receptor α-positive (ER+) breast cancers eventually adapt to hormone deprivation and acquire resistance to aromatase inhibitors. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Herein we investigated the effects of AKT inhibition on the hormone-independent growth of four ER+ breast cancer cell lines (MCF-7, ZR75-1, MDA-MB-361 and HCC-1428) with acquired resistance to estrogen deprivation (termed long-term estrogen-deprived cells, LTED). Treatment with the ATP-competitive AKT inhibitor AZD5363 inhibited phosphorylation of the AKT/mTOR substrates PRAS40, GSK3β/α, and S6K while inducing hyperphosphorylation of AKT at S473 and T308. AZD5363 suppressed the hormone-independent growth of 3/4 LTED lines. AZD5363 prevented the emergence of hormone-independent MCF-7, ZR75-1, and MDA-361 cells. Further, treatment of ovariectomized athymic mice with AZD5363 suppressed the hormone-independent growth of ER+/PI3K mutant MCF-7 xenografts. Combined treatment with AZD5363 and the ER downregulator fulvestrant more effectively suppressed xenograft growth than either drug alone. AZD5363 also caused dose-dependent inhibition of an ER+ breast cancer explant model unresponsive to fulvestrant or tamoxifen. We next examined whether AKT inhibition results in feedback upregulation of mitogenic signaling pathways. Treatment with AZD5363 resulted in upregulation and phosphorylation of the HER3 receptor tyrosine kinase (RTK), as well as upregulation of insulin receptor (InsR) protein levels and phosphorylation of Src at Y416. A Src family kinase inhibitor suppressed AZD5363-induced upregulation of P-HER3 in MCF-7/LTED cells. Src inhibition or siRNA-mediated knockdown of InsR or IGF-IR significantly enhanced the growth inhibitory effects of AZD5363. Following treatment with AZD5363, phospho-RTK array analysis revealed increased phosphorylation of multiple RTKs in MCF-7/LTED and/or ZR75-1/LTED cells, including InsR, IGF-IR, EGFR, HER2, HER3, HER4, Dtk, and FGFR3. Treatment with AZD5363 also upregulated RTK mRNA levels. Further, treatment of MCF-7/LTED cells with AZD5363 resulted in marked translocation of AKT PH-GFP to the membrane, reflective of increased PIP3 production and, thus, AKT phosphorylation at T308. Pre-treatment with the PI3K inhibitor BKM120 or the IGF-IR/InsR TKI AEW541 prevented AZD5363-induced membrane localization of PH-GFP. These results suggest that 1) AKT signaling is critical for hormone-independent growth of ER+ breast cancer cells in vitro and in vivo; and 2) AKT inhibition in these cells induces feedback upregulation of RTK expression and activity. Thus, inhibitors of AKT merit evaluation as a potential treatment for endocrine-resistant breast cancer. The combination of these agents with inhibitors of upstream RTKs are under evaluation. 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 4825. doi:1538-7445.AM2012-4825

P4-01-01: Preclinical and Clinical Studies of Estrogen Deprivation Support the PDGF/Abl Pathway as a Novel Therapeutic Target for Overcoming Resistance.

Abstract Aim: To determine the relevance of PDGF/Abl signaling pathway as a therapeutic target in endocrine resistance in vitro and in vivo. Rationale: Targeting estrogenic stimulation reduces mortality from ER-positive (ER+) breast cancer (BC) but resistance remains a major clinical problem. To identify the molecular mechanisms associated with resistance to estrogen-deprivation, we previously assessed the temporal changes in gene expression during adaptation to long-term culture of MCF7 human breast cancer cells in the absence of estradiol (E2) (LTED), modeling resistance to an aromatase inhibitor (AI). Platelet-derived growth factor (PDGF)/Abl signaling was the top adaptive pathway at the point of resistance (p=1.15 E-07), but there is limited evidence for it playing a role clinically. Methods: Gene expression data from postmenopausal women with ER+ BC at pre-treatment and at 2-week on-treatment with neoadjuvant anastrozole were interrogated. Immunohistochemical staining using antibodies against PDGFRα, β and Abl was assessed in paired clinical specimen of 45 patients who had received an AI and presented progressive disease. Proliferation assays, immunoblots and ChIP assays were carried out using either the PDGFR/Abl inhibitor nilotinib or siRNA knockdowns in LTED and wild-type MCF7 cells. Results: In vitro: PDGFRβ and Abl expression and phosphorylation were elevated in LTED cells. Cell proliferation was decreased in LTED cells by siRNA knock-down of either PDGFRβ (50% p<0.01), Abl (40% p<0.01) or the combination (70% p<0.01). Inhibition of Abl activity using nilotinib decreased ER protein levels and suppressed ER-mediated transcription by reducing recruitment of AIB1 and CBP to the promoter of E-responsive genes TFF1 and GREB1. Clinical: Data from 81 patients treated with an AI in the neoadjuvant setting, showed increases in PDGFRβ gene expression after two weeks (1.25 fold, p=0.003). Low PDGFRβ at baseline was associated with a better response. Of note, paired pre-AI-treatment and relapse clinical specimens from a cohort of patients treated in the primary or advanced setting revealed increases in tumor protein expression of PDGFRα (1.39 fold, p=0.0065), PDGFRβ (4.32 fold, p=0.006) and Abl (1.8 fold, p=0.001) at the point of relapse. Tumor (T) and stromal (S) expression of PDGFRα as well as PDGFRβ was significantly correlated in pre-treatment and relapse samples (Table 1). High post-treatment tumor and stromal PDGFRβ levels were associated with a short time to treatment failure (TTF) (T: Rs −0.284, p=0.066; S: Rs −0.307, p=0.046). Expression of PDGFRα in relapsing tumor specimens was correlated with Abl expression (Rs 0.342, p=0.027) and Ki67 levels (Rs 0.39, p=0.01). Furthermore, changes in Abl correlated significantly with changes in ER expression (Rs 0.307, p=0.048). Discussion: These in vitro and clinical data support a biological interaction between PDGF/Abl and ER-signaling and suggest that the PDGF signaling pathway warrants clinical evaluation as a therapeutic target in endocrine resistant breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-01.

S3-4: ER Downregulation with Fulvestrant in Combination with pan-PI3K Inhibitor BKM120 Synergizes Against ER+/PI3K-Mutant Breast Cancer Xenografts In Vivo.

Abstract ER+ breast cancers typically respond to aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To discover mechanisms of resistance to AIs, we maintained four ER+, hormone-dependent human breast cancer cell lines in hormone-depleted conditions for several months until estrogen-independent populations emerged (termed long-term estrogen-deprived, LTED). A siRNA kinome screen identified 42 kinases that suppressed hormone-independent MCF-7/LTED cell growth ≥33% (p<0.05). Pathway analysis revealed phosphatidylinositol 3-kinase (PI3K) as a central hub which integrates signaling from, or emits signaling to many of these 42 kinases. Further, treatment with the PI3K/mTOR dual inhibitor BEZ235 or the pan-PI3K inhibitor BKM120 suppressed LTED cell growth, and prevented the emergence of hormone-independent cells. Activating mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are present in about 35% of ER+ breast cancers. We investigated whether PIK3CA mutations modify the tumor response to estrogen deprivation with letrozole in a pre-surgical clinical trial (NCT00651976). Twenty-one patients with stage I-II operable ER+/HER2− breast cancer were treated with letrozole for 10–21 days up to 24 h prior to surgery. Core tumor biopsies were obtained before treatment and at the time of surgical resection. Ki67 immunohistochemistry (IHC) was scored in pre- and post-letrozole specimens. The change in natural log of the Ki67 score between pre-and post-treatment samples was stratified by PIK3CA mutant vs. wildtype (WT) status. PIK3CA-mutant tumors exhibited a statistically lower reduction in Ki67 score compared to tumors with WT PIK3CA (p=0.03). These findings suggest that mutational activation of PI3K attenuates the response to estrogen deprivation. In some LTED cell lines, downregulation of ER with fulvestrant or siRNA inhibited estrogen-independent growth, suggesting that the unliganded ER can maintain proliferation of cells adapted to estrogen deprivation. Prior work suggested that PI3K-ER crosstalk promotes resistance to estrogen deprivation. However, inhibition of PI3K/AKT/TORC1 signaling did not alter ERS167 phosphorylation in LTED cells, and ER downregulation with fulvestrant did not inhibit PI3K/AKT, suggesting that PI3K and ER modulate non-overlapping pathways in cells adapted to estrogen deprivation. Treatment with BKM120 synergized with fulvestrant to inhibit the growth of 6/8 ER+ breast cancer cell lines in estrogen-depleted conditions. Treatment with single-agent fulvestrant or BKM120 in athymic mice devoid of estrogen supplementation partially inhibited the growth of ER+/PI3K-mutant MCF-7 xenografts, while the combination induced near-complete tumor regression. IHC analysis showed increased cleaved caspase-3+ cells in BKM120-treated tumors and decreased Ki67+ cells in fulvestrant-treated tumors, implying that the combination induced tumor regression by both inhibition of tumor cell proliferation and induction of apoptosis. These data suggest that upon progression on an aromatase inhibitor, patients with ER+/PI3K-mutant breast cancer would benefit from a combination of an ER downregulator and a PI3K pathway inhibitor. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S3-4.

PD01-03: Src Is a Potential Therapeutic Target in Endocrine Resistant Breast Cancer Exhibiting Low Estrogen Receptor (ER)-Mediated Transactivation.

Abstract Aim: Identification of the mechanisms governing sensitivity to dasatinib in endocrine resistant breast cancer. Background: Despite the effectiveness of endocrine therapies over 40% of women relapse. To identify the molecular mechanisms associated with resistance to estrogen-deprivation on an aromatase inhibitor, we previously assessed changes in gene expression during adaptation to long-term culture of MCF7 human breast cancer (BC) cells in the absence of estradiol (E2) (LTED). Analyses of canonical signaling pathways highlighted FAK as one of the major pathways up regulated at the point of resistance and src as a dominant gene in this pathway. Src phosphorylates a plethora of proteins including ER. Surprisingly dasatinib a pan src inhibitor enhanced proliferation of MCF7 cells in the absence of E2 and had limited anti-proliferative effect in the LTED model. In contrast tamoxifen resistant MCF7 cells (TAMR) were exquisitely sensitive to dasantinib [1]. Methods: Proliferation was assessed using Titre Glo™. ER-mediated transcription was measured with an estrogen-response element linked luciferase reporter construct. Protein expression was determined by immunoblotting. Confocal microscopy was used to determine cellular localization of ER. Results: Dasatinib decreased proliferation of the TAMR (IC50 0.05nM) and resensitized them to tamoxifen but had no effect on the wild-type (wt)-MCF7 or LTED cells. Previously a relationship between BC subtype and sensitivity to dasatinib was reported with basal and post-EMT BC cell lines showing the highest sensitivity, which associated with high caveolin (CAV) expression [2]. In ovarian cancer expression of CAV and urinokinase plasminogen activator (uPA) also predicted sensitivity to dasatinib [3]. TAMR cells had a 2-fold increase in CAV-1 and 7-fold increase in uPA compared to wt cells. Comparison of LTED and TAMR showed that the TAMR whilst continuing to express high levels of ER, had a 10-fold lower level of ER-transactivation compared to wt-MCF7 and did not express PGR. In contrast the LTED showed a 7-fold increase in ER-transactivation compared to wt cells in the absence of E2. Strong evidence suggests ER in the TAMR cells acts via a non-genomic mechanism with ER expressed throughout the cell rather than being restricted to the nucleus. This implies the TAMR have an impeded luminal phenotype in relation to ER-genomic function. Dasatinib reduced the expression of ER in the TAMR and resulted in nuclear shuttling of ER whilst having no effect on the wt. BT474 (ER+/HER2 amplified) showed a similar distribution of ER to the TAMR cells but were far less sensitive to the antiproliferative effect of dasatinib. Of note the TAMR have increased phosphorylation of HER2 but no amplification. Conclusion: These data suggest src has differential effects in these endocrine resistant cell lines. The anti-proliferative effect of dasatinib was more pronounced in the endocrine resistant cell line with low ER genomic activity. Src inhibition warrants further clinical evaluation as a therapeutic target in endocrine resistant breast cancer. 1. S.K. Guest, Ann Oncol 2011, 22 (Supp 2):ii29–ii36. 2. Finn RS, Br Can Res Treat. 2007, 105(3):319–26. 3. Konecny GE, BJC. 2009, 101(10):1699–70. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-03.

Abstract S3-8: RNAi Screening Identifies the Insulin/Insulin-Like Growth Factor-I Receptor Pathway as a Mechanism of Escape from Hormone Dependence in Estrogen Receptor-Positive Breast Cancer

Abstract Estrogen receptor-positive (ER+) breast cancers are treated with strategies aimed at inducing estrogen deprivation such as aromatase inhibitors (AIs). However, many ER+ tumors adapt to hormone deprivation and acquire resistance to AIs. The purpose of this study was to identify kinases essential for the growth of endocrine-resistant ER+ breast cancer cells. To model acquired resistance to AIs, we maintained four ER+, estrogen-dependent human breast cancer cell lines (MCF-7, MDA-361, HCC-1428, and ZR75- 1) under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). We then performed an RNAi screen targeting 779 protein kinases to identify molecules required for growth of MCF-7/LTED cells in hormonefree conditions. Individual knockdown of 42 kinases significantly inhibited MCF-7/LTED cell growth ≥33% (p≥0.05) in at least 3/4 independent experiments. Of these 42 hits, knockdown of the insulin receptor (InsR) inhibited MCF-7/LTED growth (n=4 experiments) compared to control siRNA. Knockdown of InsR expression using independent siRNA oligonucleotides significantly inhibited growth of MCF-7/LTED, ZR75-1/LTED, and HCC-1428/LTED cells. The InsR can heterodimerize with the highly homologous insulin-like growth factor-I receptor (IGF-IR) to potently activate the phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Immunoblot analysis revealed that siRNA knockdown of InsR or IGF-IR caused a compensatory upregulation of IGF-IR and InsR expression, respectively. Knockdown of InsR or IGF-IR inhibited growth of 3/4 LTED lines, but knockdown of both InsR and IGF-IR was required for maximal inhibition of PI3K/AKT. Inhibition of these receptors by treatment with the InsR/IGF-IR tyrosine kinase inhibitor (TKI) OSI-906 decreased PI3K signaling and disrupted the basal association between p85 and the IGF-IR/InsR effectors IRS-1 and IRS-2 in MCF-7/LTED cells. Further, OSI-906 inhibited the hormone-independent growth of all four LTED lines and prevented the emergence of hormone-independent ER+ parental cells following estrogen deprivation. Treatment with the TKI or the neutralizing IGF-IR monoclonal antibody MK-0646 induced feedback upregulation and phosphorylation of the ErbB3/HER3 receptor tyrosine kinase (RTK). Induction of P-HER3 was blocked by the EGFR/HER2 TKI lapatinib. Combined treatment of lapatinib and OSI-906 prevented the emergence of hormone-independent (HER2+/ER+) MDA-361 cells more effectively than either drug alone. Finally, treatment of ovariectomized nude female mice with OSI-906 suppressed the growth of established MCF-7 xenografts. In addition, treatment with OSI-906 decreased PI3K signaling as measured by immunoblot analysis of tumor extracts using P-AKT antibodies. These results suggest that 1) InsR/IGF-IR signaling is required for PI3K/AKT signaling and hormone-independent growth of ER+ breast cancer cells; 2) these cells compensate for the inhibition of InsR/IGF-IR by upregulating alternative RTK pathways such as HER3; 3) combined targeting of InsR/IGF-IR and ER signaling may be effective for the treatment of endocrine-resistant breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S3-8.

Anticancer effect of (E)-2-hydroxy-3′,4,5′-trimethoxystilbene on breast cancer cells by mitochondrial depolarization

TMS (2,3',4,5'-tetramethoxystilbene), a stilbene analog derived from rhapontigenin, was previously demonstrated to induce apoptosis in hormone-resistant breast cancer cells. Therefore, this study investigated the anticancer effect of a new stilbene analog, HTMS ((E)-2-hydroxy-3',4,5'-trimethoxystilbene), and its mechanism in various breast cancer cell lines. The effect of HTMS on cell proliferation of MDA-MB-231, MCF-7, and LTED cells was evaluated using MTT assays. Cell apoptosis was detected by FITC-annexin V staining and flow cytometry analysis, changes in mitochondrial potential were determined by fluorescence microscopy using TMRE staining, and the expression of cleaved PARP and release of cytochrome c were assessed by Western blot analysis. HTMS significantly decreased the cell viability of various types of breast cancer cells in a dose- and time-dependent manner, characterized by G2/M arrest of the cell cycle and the induction of apoptosis. In particular, HTMS disturbed the mitochondrial membrane potential, causing a release of cytochrome c during apoptosis. Furthermore, HTMS was superior to TMS in inhibiting cancer cell growth in a pilot comparison study. HTMS is an effective apoptotic agent for breast cancer cells, making it a candidate therapeutic agent for the treatment of breast cancer.

Upregulation of AIB1, aromatase and ERα provides long-term estrogen-deprived human breast cancer cells with a mechanistic growth advantage for survival.

To investigate the mechanisms by which breast cancer cells adapt and are able to grow during estrogen deprivation, human estrogen receptor-α (ERα)-positive breast cancer cells stably transfected with the aromatase gene (MCF-7Ca) were cultured in steroid-depleted medium for 6-8months until they started proliferating. Compared with the parental MCF-7Ca cells, long-term estrogen-deprived UMB-1Ca cells exhibited increased aromatase activity (2000%), AIB1 expression (3500%) and ERα expression (100%). When MCF-7Ca cells were isolated from tumors of mice treated for 12months with an aromatase inhibitor, letrozole, ERα was reduced (50%) whereas AIB1 levels were increased (>1000%), suggesting that the mechanism of estrogen deprivation might predetermine the signaling pathway utilized. To a lesser extent long-term estrogen-deprived MCF-7 cells (LTED) displayed an increase in AIB1, ERα and aromatase activity. Consistent with other findings, the growth of the LTED cells was inhibited by estradiol and antiestrogens, whereas the UMB-1Ca cells were slightly stimulated by estradiol and inhibited by antiestrogens and letrozole. In LTED cells treated with estradiol, levels of AIB1 and ERα (95%) were reduced. Interestingly, estradiol treatment caused no change in AIB1 and ERα expression in the UMB-1Ca cells which might explain the differential growth effect of the cells to estradiol. Together, these results demonstrate that estrogen deprivation results in the upregulation of the estrogen signaling pathway at the level of AIB1, ERα and aromatase, which might attenuate ER-mediated transcription representing one mechanism by which tumors adapt to proliferation in a low estrogenic environment.

Abstract 2920: Experimental and clinical studies reveal the PDGF/Abl pathway as a novel therapeutic target in endocrine-resistant breast cancer

Abstract The majority of breast tumours at primary diagnosis are estrogen receptor positive (ER). Estrogen (E) mediates its effects by binding to the ER. E-bound ER associates classically with estrogen response elements (EREs) on target genes controlling proliferation and cell survival. ER's ability to regulate gene transcription is also influenced by the activity of co-regulatory proteins such as AIB1. Therapies targeting the estrogenic stimulation of tumour growth have been a major success in reducing mortality from ER-positive breast cancer. However, resistance remains a major clinical problem. To identify the molecular mechanisms associated with resistance to E-deprivation, we assessed the temporal changes in gene expression during adaptation to long-term culture of MCF7 human breast cancer cells in the absence of E2 (LTED), modelling resistance to an aromatase inhibitor (AI). Analyses of canonical signalling pathways showed that platelet-derived growth factor (PDGF)/Abl was significantly elevated as early as one-week post E-deprivation (p=1.94 E-04). Of note this became the top adaptive pathway at the point of resistance (p=1.15 E-07). Clinical data from 88 patients treated with an AI in the neoadjuvant setting, showed increases in PDGFRβ and PDGF expression after two weeks (1.25 fold, p=0.003 and 1.43 fold, p=4.4E-06). Low PDGFRβ at pre-treatment was associated with a better response. Assessment of the level of protein expression and activation status in a panel of cell lines modelling both endocrine sensitive and resistant breast cancer showed PDGFRβ was elevated in both LTED and tamoxifen resistant MCF7 cells (TAMR) compared to wt-MCF7. Moreover, the level of receptor phosphorylation was increased in the resistant cell lines. PDGFRβ expression was also evident in SKBR3 (ER-/ERBB2+) but not in BT474 (ER+/ERBB2+) cells. Of note both LTED and TAMR cells were sensitive to the inhibitory effect of the PDGFR/Abl tyrosine kinase inhibitor nilotinib, in the presence or absence of E. In contrast wt-MCF7 cells showed limited sensitivity in the presence of E. Both SKBR3 and BT474 cells were insensitive. These data suggested that PDGFRβ was associated with the endocrine resistant phenotype. Assessment using ER/ERE linked reporter assays showed nilotinib suppressed ER-mediated transcription and inhibited expression of two endogenous E-regulated genes TFF1 and PGR. Chromatin immunoprecipitation indicated that nilotinib reduced the recruitment of AIB1 and CBP to the TFF1 promoter. Furthermore we were able to demonstrate that nilotinib in combination with tamoxifen resensitised TAMR cells to the inhibitory effect of tamoxifen. These data for the first time reveal cross talk between ER and PDGF/Abl, strongly suggesting that the PDGF signalling pathway merits clinical evaluation as a therapeutic target in endocrine resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2920.

Resistance to Endocrine Therapy in Estrogen Receptor-Positive (ER+) Breast Cancer Is Dependent upon Phosphatidylinositol-3 Kinase (PI3K) Signaling.

Abstract ER+ breast cancers typically respond to treatment with aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To model AI resistance, we cultured four ER+, hormone-dependent human breast cancer cell lines under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). LTED cells outgrew parental counterparts under hormone-depleted conditions. While two LTED lines showed increased ER levels and response to estradiol, two LTED lines showed the opposite. Therefore, ER+ breast cancer cells did not consistently overcome hormone deprivation by increasing sensitivity to low estrogen levels.We analyzed protein lysate arrays with 625 antibodies against signaling proteins to discover common mechanisms of hormone-independent growth. All LTED lines showed increased phosphorylation of p70S6K, an mTOR substrate, compared to parental controls. An siRNA library screen targeting 779 kinases revealed that downregulation of kinases linked to the phosphatidylinositol-3 kinase (PI3K) signaling pathway (serum/glucocorticoid-regulated kinase 1; insulin receptor; p110α/PI3K) inhibited the growth of MCF-7/LTED but not parental MCF-7 cells under hormone-depleted conditions. Immunoblot analysis for P-AKT and P-p70S6K showed that PI3K/AKT/mTOR signaling was increased in all LTED lines. Pathway analysis of gene expression microarray data showed significant alteration of genes involved in insulin-like growth factor-I (IGF-I) signaling in 3/4 LTED lines. Receptor tyrosine kinase array analysis revealed increased activation of receptors for IGF-I (IGF-IR) and/or insulin (InsR) in 3/4 LTED lines. These receptors transduce signals to potently activate PI3K. Treatment with the IGF-IR/InsR kinase inhibitor AEW541 decreased P-AKT in 3/4 LTED lines. Treatment with inhibitors of IGF-IR/InsR (AEW541), PI3K/mTOR (BEZ235), or mTOR (RAD001) suppressed the monolayer and anchorage-independent growth of 3/4, 4/4, and 4/4 LTED lines, respectively, and prevented the emergence of hormone-independent cells. Treatment with PI3K and mTOR inhibitors induced apoptosis in 3/4 LTED lines. Finally, we analyzed levels of PI3K pathway markers (P-AKT, P-S6, P-GSK3α/β) in primary tumor lysates from 65 ER+ breast cancer patients using reverse-phase protein arrays. Hierarchical clustering yielded two groups of tumors with high or low PI3K activation. Following adjuvant anastrozole therapy, PI3K-high patients had significantly shorter disease-free survival compared to PI3K-low patients. With a median follow-up of 9.4 months, recurrence rates were 16% and 0%, respectively (p<0.05). These data suggest that upon the acquisition of hormone independence and resistance to endocrine therapy, ER+ breast cancer cells increase their dependence on the PI3K pathway. Thus, resistance to endocrine therapy may be abrogated by combination therapies targeting both the ER and PI3K pathways, which may sometimes be achieved by blocking IGF-IR/InsR signaling upstream of PI3K. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 403.

HMPS (2-hydroxy-4-methoxyphenylstilbene), a stilbene derivative of rhapontigenin, and cell death by mitochondrial apoptotic pathway in breast cancer cells

e22130 Background: Breast cancer with resistance to clinical therapy is a significant threat to live of recurrent breast cancer patients, and chemo-resistant breast cancer is increasing rapidly. During last several decades, natural stilbenoids have been studied on anticancer effects in vitro and in vivo, and resveratrol is the most famous stilbene as a leading compound in the studies of anticancer compounds derived from plants. HMPS (2-hydroxy-4-methoxyphenylstilbene) is an analogue derived from rhapontigenin (3,5,3'-trihydroxy-4'-methoxy-trans-stilbene), which is a stilbene of herbal plant Rheum undulatum. TMS (2,3',4,5'-tetramethoxystilbene), an another stilbene analogue from rhapontigenin, was reported potent anticancer effect on tamoxifen-resistant MCF-7 cells. In this study we investigated inhibitory effect of HMPS on proliferation of breast cancer and a potential for a new therapeutic candidate. Methods: We examined cell viability of MCF-7 and MDA-MB-231 by MTT assay after exposure to various concentrations of HMPS. Apoptotic cell death induced by HMPS was investigated by florescence microscopy, cell cycle analysis and western blotting. Results: Cell viability of breast cancer cells after 24 h exposure to HMPS decreased significantly, and both ER-positive and ER-negative breast cancer cells responded to HMPS. HMPS induced nucleus fragmentation and G2/M arrest followed by sub-G1 accumulation of apoptotic cells in time- and dose-dependent manner. During the process of cell death induced by HMPS, mitochondrial membrane potential was disturbed and caspase-3 and PARP cleavage were observed. Moreover, HMPS decreased cell number of LTED MCF-7 cells(Long term estradiol deprived cell) effectively. Conclusions: Our results demonstrates that proliferation inhibitory effect of HMPS is about 50-fold more potent than those of rhapontigenin and furthermore HMPS also inhibits cell growth of LTED cells which are difficult to treat therapeutic agents. Therefore, HMPS may be a potential therapeutic candidate to treat the recurrent breast cancer by alone or combination with other conventional anticancer agents. No significant financial relationships to disclose.

Lapatinib can activate or supress estrogen receptor (ER) signaling in cell models of endocrine resistant breast cancer.

Abstract Abstract #3031 Background
 Growth factor (GF) signaling is frequently upregulated in hormone resistant breast cancer (BC) and provides the rationale for using the dual EGFR/HER2 inhibitor, lapatinib (LAP) in an effort to overcome endocrine resistance. Hormone resistant BC can also vary in its degree of continued ER dependence. We previously demonstrated potent synergy between LAP and TAM in cell models of acquired endocrine resistance to tamoxifen (TAMR cells), and to estrogen deprivation (LTED cells)1. This study aimed to characterize the phenotype of these two models with a focus on the ER pathway, and to determine the biological basis for the observed synergy between LAP and TAM.
 Methods
 LTED cells and TAMR cells were both derived from hormone-sensitive MCF7 parental cells as previously described1. The differential effects of LAP, TAM, estrogen deprivation (ED) or the combination were assessed by proliferation assays, ERE reporter assays, quantitative PCR and western blot analyses.
 Results
 In LTEDs: Increased ER expression and ERE transactivation were seen in LTEDs compared to MCF7 cells, as well as continued sensitivity to the ER downregulator, fulvestrant (FUL), indicating that in LTEDs, ER activity is not only enhanced but is also a significant driver of growth. In LTEDs, LAP alone suppressed ER activity as measured by both ERE transactivation and ER phosphorylation at Ser118; the most effective suppression of ER activity was achieved with a combination of LAP+TAM/ED where the effects were additive.
 In TAMRs: In contrast, TAMRs demonstrated decreased ER activity (low basal ERE transactivation and loss of PgR) and ER independent proliferation (FUL had minimal growth inhibitory effects despite ER downregulation). In TAMRs, neither LAP nor TAM had significant antiproliferative effects on their own: LAP alone decreased pMAPK/pAkt but increased basal ERE transactivation and transcription of the ER-responsive gene pS2. TAM alone inhibited ERE activation but increased HER2 gene and protein expression. Inhibiting both HER2 and ER pathways with LAP+TAM resulted in synergistic growth inhibition.
 Conclusions
 In LTED cells with enhanced ER function attributable in part to GF receptor activation, the synergistic antiproliferative effects from LAP and TAM or ED may be largely due to enhanced inhibition of ER signaling. In contrast in TAMR cells with suppressed ER activity, LAP alone appears to enhance ER signaling, while TAM alone inhibits ER activity but increases HER2 expression. In TAMRs, the combination of TAM+LAP is required to interrupt both ER and GF signaling. These results suggest that the basis for synergy between LAP and endocrine therapy in hormone resistant BC may vary depending on the nature of the cross-talk between the ER and GF pathways.
 1SABCS, 2006 Abs 303. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3031.

219 POSTER NVP-BEZ235, a dual pan-PI3K/mTOR kinase inhibitor, is effective in human lung cancer models harboring EGFR mutations

Acquired resistance is a major problem limiting the clinical benefit of endocrine therapy. To investigate the mechanisms involved, two in vitro models were developed from MCF-7 cells. Long-term culture of MCF-7 cells in estrogen deprived medium (LTED) mimics aromatase inhibition in patients. Continued exposure of MCF-7 to tamoxifen represents a model of acquired resistance to antiestrogens (TAM-R). Long-term estrogen deprivation results in sustained activation of the ERK MAP kinase and the PI3 kinase/mTOR pathways. Using a novel Ras inhibitor, farnesylthiosalicylic acid (FTS), to achieve dual inhibition of the pathways, we found that the mTOR pathway plays the primary role in mediation of proliferation of LTED cells. In contrast to the LTED model, there is no sustained activation of ERK MAPK but enhanced responsiveness to rapid stimulation induced by E2 and TAM in TAM-R cells. An increased amount of ERα formed complexes with EGFR and c-Src in TAM-R cells, which apparently resulted from extra-nuclear redistribution of ERα. Blockade of c-Src activity drove ERα back to the nucleus and reduced ERα–EGFR interaction. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that different mechanisms are involved in acquired endocrine resistance and the necessity for individualized treatment of recurrent diseases.

Mechanisms of acquired resistance to endocrine therapy in hormone-dependent breast cancer cells

Acquired resistance is a major problem limiting the clinical benefit of endocrine therapy. To investigate the mechanisms involved, two in vitro models were developed from MCF-7 cells. Long-term culture of MCF-7 cells in estrogen deprived medium (LTED) mimics aromatase inhibition in patients. Continued exposure of MCF-7 to tamoxifen represents a model of acquired resistance to antiestrogens (TAM-R). Long-term estrogen deprivation results in sustained activation of the ERK MAP kinase and the PI3 kinase/mTOR pathways. Using a novel Ras inhibitor, farnesylthiosalicylic acid (FTS), to achieve dual inhibition of the pathways, we found that the mTOR pathway plays the primary role in mediation of proliferation of LTED cells. In contrast to the LTED model, there is no sustained activation of ERK MAPK but enhanced responsiveness to rapid stimulation induced by E 2 and TAM in TAM-R cells. An increased amount of ERα formed complexes with EGFR and c-Src in TAM-R cells, which apparently resulted from extra-nuclear redistribution of ERα. Blockade of c-Src activity drove ERα back to the nucleus and reduced ERα–EGFR interaction. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that different mechanisms are involved in acquired endocrine resistance and the necessity for individualized treatment of recurrent diseases.

The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling

Long-term culture of MCF-7 wild-type (wt) cells in steroid-depleted medium (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of ERalpha and enhanced growth factor signalling. In this study, we aimed to compare the effects of the pure anti-oestrogen ICI 182,780 (ICI) with the competitive anti-oestrogen tamoxifen (TAM) on oestrogen and IGF signalling in these cells. Wt MCF-7 and LTED cells were treated with a log 7 concentration range of E2, TAM or ICI. Effects on cell growth, ERalpha transactivation, expression of ERalpha, ERbeta and components of the IGF pathway were measured with and without insulin. In the presence of insulin, growth of LTED cells was refractory to TAM but inhibited by ICI and E2. In the absence of insulin, LTED cells showed persistent hypersensitivity to E2, and remained inhibited by ICI but were largely unaffected by TAM. ICI but not TAM inhibited ER-mediated gene transcription and treatment with ICI resulted in a dose-dependent reduction in ERalpha levels whilst having no effect on ERbeta expression. IGF-I receptor and insulin receptor substrate 2 levels were increased in LTED versus the Wt MCF-7 cells, and ICI but not TAM reduced their expression in a dose-dependent fashion. Thus IGF signalling as well as ERalpha expression and function are enhanced during LTED. While the resultant cells are resistant to TAM, ICI down-regulates ERalpha, reducing IGF signalling and cell growth. These results support the use of ICI in women with ER-positive breast cancer who have relapsed on an aromatase inhibitor.