Tamoxifen-resistant MCF-7 Cell Line Research Articles

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

Dual in vitro invasion/migration suppressing and tamoxifen response modulating effects of a recombinant anti-ALCAM scFv on breast cancer cells.

It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.

LncRNA CYTOR promotes tamoxifen resistance inbreast cancer cells via sponging miR‑125a‑5p.

Development of resistance to endocrine therapy, such as tamoxifen, remains a tricky clinical problem during the treatment of breast cancer. Accumulating evidence suggested that dysregulation of long noncoding (lnc_RNAs contributes to the development of tamoxifen resistance. In the current study, via screening, cytoskeleton regulator RNA (CYTOR) was identified as the most significantly elevated lncRNA in the established tamoxifen resistant MCF7 cell lines (MCF7/TAM1 and MCF7/TAM2) compared with the parental MCF7 cells (MCF7-P). The CCK-8 assay indicated that silencing of CYTOR increased the sensitivity of MCF7/TAM1 and MCF7/TAM2 to tamoxifen treatment. Using bioinformatic analysis, it was predicted that microRNA (miR)-125a-5p might bind to CYTOR and the expression of miR-125a-5p was negatively correlated with CYTOR in the tumor tissues of breast cancer. In addition, RT-qPCR and dual luciferase assays validated that CYTOR directly repressed miR-125a-5p expression in breast cancer cells. Through regulation of miR-125a-5p, CYTOR elevated serum response factor (SRF) expression and activated Hippo and mitogen associated protein kinase signaling pathways to promote breast cancer cell survival upon tamoxifen treatment. In the collected tumor tissues of breast cancer in the present study, high expression of CYTOR was detected in tissues from patients with no response to tamoxifen compared with those from patients who were not treated with tamoxifen. A positive correlation between CYTOR and SRF mRNA expression was observed in tissues collected from patients with breast cancer. In conclusion, the results of the present study demonstrated a pivotal role of CYTOR in mediating tamoxifen resistance in breast cancer.

CircRNA_0025202 Regulates Tamoxifen Sensitivity and Tumor Progression via Regulating the miR-182-5p/FOXO3a Axis in Breast Cancer.

Tamoxifen is the most commonly used endocrine therapy for patients with hormone receptor (HR)-positive breast cancer. Despite its initial therapeutic efficacy, many patients eventually develop drug resistance, which remains a serious clinical challenge. To investigate roles of circular RNAs (circRNAs) in tamoxifen resistance, a tamoxifen-resistant MCF-7 cell line was established and screened for its circRNA expression profile by RNA sequencing. hsa_circ_0025202, a circRNA that was significantly downregulated, was selected for further investigation. Using a large cohort of clinical specimens, we found that hsa_circ_0025202 exhibited low expression in cancer tissues and was negatively correlated with lymphatic metastasis and histological grade. Gain- and loss-of-function assays indicated that hsa_circ_0025202 could inhibit cell proliferation, colony formation, and migration and increase cell apoptosis and sensitivity to tamoxifen. Bioinformatics and luciferase reporter assays verified that hsa_circ_0025202 could act as a miRNA spongefor miR-182-5p and further regulate the expression and activity of FOXO3a. Functional studies revealed thattumorinhibition and tamoxifen sensitization effects of hsa_circ_0025202 were achieved via the miR-182-5p/FOXO3a axis. Moreover, invivo experiments confirmed that hsa_circ_0025202 could suppress tumor growth and enhance tamoxifen efficacy. Taken together, hsa_circ_0025202 served an anti-oncogenic role in HR-positive breast cancer, and it could be exploited as a novel marker for tamoxifen-resistant breast cancer.

Overexpression miR-24-3p repressed Bim expression to confer tamoxifen resistance in breast cancer.

Endocrine therapy resistance represents a major challenge to the successful treatment of patients with breast cancer. The development of tamoxifen resistance commonly occurrs during the treatment of patients with breast cancer whereas its underlying mechanisms remain elusive. Here, we found that miR-24-3p regulated tamoxifen sensitivity in breast cancer cells. Forced overexpression of miR-24-3p augmented tamoxifen-induced cell viability inhibition in breast cancer cells, while knockdown of miR-24-3p partially attenuated the cytotoxicity effect of tamoxifen. Moreover, we discovered Bim as a target gene of miR-24-3p in breast cancer cells by RNA immunoprecipitation, quantitative reverse transcription polymerase chain reaction, Western blot, and dual luciferase reporter assay. In our established tamoxifen resistant MCF7 cell line (MCF7/TAM), there was a significant elevation of miR-24-3p and decrease of BIM expression compared with parental MCF7 cells. In addition, the inhibition of miR-24-3p could reverse the tamoxifen resistance of MCF7/TAM cells by the induction of cell apoptosis. Silencing of Bim expression blocked miR-24-3p inhibitor-induced elevation of tamoxifen sensitivity of MCF7/TAM cells. Using tumor tissues from patients with breast cancer, we also found that the expression of miR-24-3p was negatively correlated with Bim mRNA expression. Collectively, our study highlighted the pivotal role of miR-24-3p overexpression in mediating the development of tamoxifen resistance in breast cancer and suggested miR-24-3p might be a predictor or target for patients with breast cancer.

Regulation of tamoxifen resistance in estrogen receptor positive MCF-7 breast cancer cells by microRNA-10b targeting histone deacetylase 4

Objective To investigate the effect of microRNA-10b (miR-10b) targeting histone deacetylase 4 (HDAC4) on the resistance of estrogen receptor positive (ER+ ) MCF-7 breast cancer cells to tamoxifen. Methods ER+ and tamoxifen resistant MCF-7 cell line MCF-7-TR was established, and miR-10b expression level in MCF-7 cells and MCF-7-TR cells was detected by reverse transcription real-time quantitative polymerase chain reaction (Real-time PCR). Methyl thiazol tetrazolium (MTT) and trypan blue were used to detect the proliferation and activity of MCF-7 cells and MCF-7-TR cells transfected with pre-miR-10b, miR-10b inhibitor and siHDAC4 at different tamoxifen concentrations (0, 5, 10, 15, 20, 30 μmol/L), respectively. The expression of HDAC4 protein was detected by Western blotting. Results Taken expression level and invasive ability of miR-10b in MCF-7 breast cancer cells as the standard, the miR-10b expression level and invasion ability of MCF-7-TR cell breast cancer cells were 7.2±1.1 and 5.3±1.3 respectively. With the increase of the concentration of tamoxifen, decreased the rate of cell proliferation in MCF-7 cells, which is most obvious, followed by MCF-7-TR+ miR-10b inhibitor + siHDAC4 and MCF-7+ pre-miR-10b+ HDAC4 cells, and the proliferation of MCF-7-TR+ siHDAC4 cells and MCF-7-TR cells decreased the rate of the lowest. The migration ability of MCF-7 cells and MCF-7-TR+ miR-10b cells of the inhibitor decreased most, MCF-7+ pre-miR-10b+ and tamoxifen (10 μmol/L) MCF-7-TR+ cells and tamoxifen (10 μmol/L) had no significant difference in the ability of cell migration (t=1.116, P=0.272), but was significantly higher than that of MCF-7 cells and MCF-7-TR+ miR-10b cells (t=0.124, 10.769, 14.569, 11.577, P=0.000). The expression level of HDAC4 protein in MCF-7+ pre-miR-10b cells was lower than that in MCF-7 cells, MCF-7-TR cells, HDAC4 protein expression level, MCF-7-TR+ miR-10b inhibitor cells, MCF-7+ siHDAC4 cells HDAC4 protein expression level was lower than MCF-7 cells. Conclusion The miR-10b-HDAC4 signaling pathway may be a molecular mechanism of tamoxifen resistance in breast cancer. Key words: Breast cancer; Estrogen receptor positive; Tamoxifen resistance; MicroRNA-10b; Histone deacetylase 4

Antrodia cinnamomea extract inhibits the proliferation of tamoxifen-resistant breast cancer cells through apoptosis and skp2/microRNAs pathway

BackgroundBreast cancer is the most common cancer in women and affects 1.38 million women worldwide per year. Antiestrogens such as tamoxifen, a selective estrogen receptor (ER) modulator, are widely used in clinics to treat ER-positive breast tumors. However, remissions of breast cancer are often followed by resistance to tamoxifen and disease relapse. Despite the increasing understanding of the resistance mechanisms, effective regimens for treating tamoxifen-resistant breast cancer are limited. Antrodia cinnamomea is a traditional medicinal mushroom native only to Taiwan. In this study, we aimed to examine in vitro effect of antrodia cinnamomea in the tamoxifen-resistant cancer.MethodsAntrodia cinnamomea was studied for its biological activity against proliferation of tamoxifen-resistant breast cancer by XTT assay. Next, the underlying mechanism was studied by flow cytometry, qPCR and Western’s blotting assay.ResultsOur results revealed that the ethanol extract of antrodia cinnamomea (AC) can inhibit the growth of breast cancer cells, including MCF-7 cell and tamoxifen-resistant MCF-7 cell lines. Combination treatment with AC and 10− 6 M tamoxifen have the better inhibitory effect on the proliferation of tamoxifen-resistant MCF-7 cells than only AC did. AC can induce apoptosis in these breast cancer cells. Moreover, it can suppress the mRNA expression of skp2 (S-phase kinase-associated protein 2) by increasing the expressions of miR-21-5p, miR-26-5p, and miR-30-5p in MCF-7 and tamoxifen-resistant MCF-7 cells.ConclusionsThese results suggest that the ethanol extract of antrodia cinnamomea could be a novel anticancer agent in the armamentarium of tamoxifen-resistant breast cancer management. Moreover, we hope to identify additional pure compounds that could serve as promising anti-breast cancer candidates for further clinical trials.

Abstract P4-07-10: Epithelial mesenchymal transition associated with high miR-221 and integrin β6 leads to poor prognosis in hormone receptor positive HER2 negative breast cancers

Abstract Background: MicroRNA mediated molecular alterations are involved in the initiation and progression of cancer. Altered expression of multiple microRNAs is associated with endocrine resistance in hormone receptor positive HER2 negative (HR+/HER2-ve) cancer. The role of miR-221 in inducing epithelial to mesenchymal transition (EMT) is well documented especially in cell line model systems. However, the detailed mechanism of specific microRNAs in intrinsic and acquired resistance to endocrine therapy needs to be worked out. In addition, more needs to be done in the documentation of these mechanisms in human breast cancer specimens with complete clinical documentation and long-term follow-up. In this study, we have evaluated the clinical significance of miR-221 and its mechanistic role in EMT using human specimens and cell line models. Materials and Methods: Formalin fixed paraffin embedded tumor from 129 HR+/HER2-ve breast cancer patients with a median follow up of 63 months were used for estimation of miR-221 by quantitative real time PCR. Expression levels of genes which are direct targets of miR-221 and related genes in EMT were analysed from these tumors. Survival between miR-221 high and low groups was compared by Kaplan Meier survival curves and prognostic relevance was estimated by Cox proportional hazard model. Cell line experiments to investigate the role of miR-221 in inducing EMT through integrin β6 are underway in both wild type and tamoxifen resistant MCF-7 cell lines (A gift from Prof Ben Ho Park, Johns Hopkins University School of Medicine). Results: A significant elevated level of miR-221 was observed in small proportion (14%) of HR+/HER2-ve tumors. miR-221 expression had an inverse correlation with both ER protein and ESR1 mRNA levels within HR+/HER2-ve tumors. Tumors with high levels of miR-221 showed significantly higher expression of integrin β6 which is a robust marker of EMT. Patients with high expression of miR-221 had a poorer survival in Kaplan Meier analysis. Results of interrogation of EMT mediated through integrin related pathways involving miR-221 in cell line models will be presented. Discussion: The association between miR-221 and integrin β6 in HR+/HER2-ve breast cancer with endocrine resistance suggests a potential link between an epigenetic regulator and a mediator of tumor-stromal interaction. The other mediators involved in this pathway are being investigated. miR-221 could be potentially used as a marker for identification of a poor prognostic subtype within HR+/HER2-ve breast cancers. Citation Format: Prabhu JS, Kaul R, Korlimarla A, Desai K, Gangadharan C, Rajarajan S, Nair MG, Alexander A, Kaluve R, Manjunath S, Correa M, Prasad MSN, Patil S, Srinath BS, Sridhar TS. Epithelial mesenchymal transition associated with high miR-221 and integrin β6 leads to poor prognosis in hormone receptor positive HER2 negative breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-10.

MCAM/CD146 promotes tamoxifen resistance in breast cancer cells through induction of epithelial–mesenchymal transition, decreased ERα expression and AKT activation

Tamoxifen resistance presents a prominent clinical challenge in endocrine therapy for hormone sensitive breast cancer. However, the underlying mechanisms that contribute to tamoxifen resistance are not fully understood. In this study, we established a tamoxifen resistant MCF-7 cell line (MCF-7-Tam-R) by continuously incubating MCF-7 cells with 4-OH-tamoxifen. We found that melanoma cell adhesion molecule (MCAM/CD146), a unique epithelial-to-mesenchymal transition (EMT) inducer, was significantly up-regulated at both mRNA and protein levels in MCF-7-Tam-R cells compared to parental MCF-7 cells. Mechanistic research demonstrated that MCAM promotes tamoxifen resistance by transcriptionally suppressing ERα expression and activating the AKT pathway, followed by induction of EMT. Elevated MCAM expression was inversely correlated with recurrence-free and distant metastasis-free survival in a cohort of 4142 patients with breast cancer derived from a public database, particularly in the subgroup only treated with tamoxifen. These results demonstrate a novel function of MCAM in conferring tamoxifen resistance in breast cancer. Targeting MCAM might be a promising therapeutic strategy to overcome tamoxifen resistance in breast cancer patients.

A novel chemical, STF-083010, reverses tamoxifen-related drug resistance in breast cancer by inhibiting IRE1/XBP1.

Recent studies show that the unfolded protein response (UPR) within the endoplasmic reticulum is correlated with breast cancer drug resistance. In particular, human X-box binding protein-1(XBP1), a transcription factor which participates in UPR stress signaling, is reported to correlate with poor clinical responsiveness to tamoxifen. In this study, we develop a tamoxifen-resistant MCF-7 cell line by treating the cell line with low concentration of tamoxifen, and we find that XBP1 is indeed up-regulated at both the mRNA and protein levels compared to normal MCF-7 cells. STF-083010, a novel inhibitor which specifically blocks the XBP1 splicing, reestablishes tamoxifen sensitivity to resistant MCF-7 cells. Moreover, co-treatment with STF-083010 and tamoxifen can significantly delay breast cancer progression in a xenograft mammary tumor model. We next investigate the expression of XBP1s in over 170 breast cancer patients' samples and the results demonstrate that XBP1s expression level is highly correlated with overall survival in the ER+ subgroup, but not in the ER- subgroup, suggesting a potential therapeutic application of XBP1 inhibitors in ER+breast cancer treatment.

The effect of Shugan-Yishen formula on HER2-ERK/MAPK-ERα Pathway of TAM-resistant breast cancer cell lines

Objective To investigate the effect of Shugan-Yishen formula on Tamoxifen-resistant MCF-7 cell line（LCC9） through HER2-ERK/MAPK-ERα pathway. Methods Four serums were prepared with the method of serum pharmacology：the serum of Shugan-Yishen formula, Shugan-Yishen formula plus Tamoxifen（TAM）, blank and TAM alone. After LCC-9 cells were affected in the above-mentioed serums in vitro, the inhibition rate and the level of mRNA as well as the protein expression of HER2, ERα, ERK/MAPK, p-ERK/MAPK were detected. Results MTT showed that TAM alone group、Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had higher inhibition rates at 48th hour, the inhibition rates was（6.61±0.129）%, （47.43±2.24）%, （54.19±3.364）%, compared to the blank group, Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had a stronger inhibition on LCC9 at 48th hour （P〈0.05）, and the combination group was better than Shugan-Yishen formula alone（P〈0.05）, there was no difference between TAM group compared to the blank group（P〉0.05）. RT-PCR result showed that compared to the blank group, the expression levels of ESR1 mRNA of group of Shugan Yishen formula plus TAM＆amp;nbsp;werehigher by and large at 48th hour （P〈0.05）, the expression of ESR1 mRNA was（2.282±0.185）;and the levels of ERBB2, MAPK3 mRNAwere lower（P〈0.05）, which levels were（0.71±0.095）, （0.336±0.058）. Western blotting result showed that Shugan-Yishen Formula plus TAM group could markedly decrease the expression of HER2, p-ERK/MAPK of LCC9 at 48th hour （P〈0.05）, which the expressions were（0.708± 0.072）, （0.228±0.034）, and the expression of ER-αelevated（P〈0.05）. There was no significant difference on ERK/MAPK（P〉0.05）. Conclusions ①The combination of Shugan-Yishen formula with tamoxifen can inhibit the LCC9 cells. The inhibition effect reaches the peak at 48th hour. ②The mechanism of Shugan-Yishen formula plus TAM combination effects on human breast cancer TAM-resistant cell line LCC9 may improve the sensitivity of ERαby inhibiting the expression of HER2, ERK/MAPK. Key words: Shugan-Yishen Formula; Breast cancer; HER2-ERK/MAPK-ERαpathway

Differential response to α-oxoaldehydes in tamoxifen resistant MCF-7 breast cancer cells.

Tamoxifen is the standard adjuvant endocrine therapy for estrogen-receptor positive premenopausal breast cancer patients. However, tamoxifen resistance is frequently observed under therapy. A tamoxifen resistant cell line has been generated from the estrogen receptor positive mamma carcinoma cell line MCF-7 and was analyzed for putative differences in the aldehyde defence system and accumulation of advanced glycation end products (AGE). In comparison to wt MCF-7 cells, these tamoxifen resistant cells were more sensitive to the dicarbonyl compounds glyoxal and methylglyoxal and displayed increased caspase activity, p38-MAPK- and IκBα-phosphorylation. However, mRNA accumulation of the aldehyde- and AGE-defence enzymes glyoxalase-1 and -2 (GLO1, GLO2) as well as fructosamine-3-kinase (FN3K) was not significantly altered. Tamoxifen resistant cells contained less free sulfhydryl-groups (glutathione) suggesting that the increased sensitivity towards the dicarbonyls was due to a higher sensitivity towards reactive oxygen species which are associated with dicarbonyl stress. To further analyse, if these data are of more general importance, key experiments were replicated with tamoxifen resistant MCF-7 cell lines from two independent sources. These cell lines were also more sensitive to aldehydes, especially glyoxal, but were different in their cellular signalling responses to the aldehydes. In conclusion, glyoxalases and other aldehyde defence enzymes might represent a promising target for the therapy of tamoxifen resistant breast cancers.

P5-06-06: The RNA Binding Protein FUS Is a Potential Marker for Breast Cancer Progression and Therapy Response.

Abstract Background The RNA binding protein FUS is a multifunctional protein that has recently been demonstrated to be directed to target genes via non-coding RNA, where it represses transcription via disruption of the transcriptional complex. We have shown that FUS is an important regulator of prostate cancer cell growth; FUS over-expression causes G1 arrest, promotes apoptosis and blocks proliferation in vitro and in vivo, whereas knock-down increases proliferation. This regulation appears to be, at least in part, via direct regulation of cyclin D1. Further, analysis of patient samples demonstrated that FUS levels are inversely correlated with Gleason grade and directly correlated with survival and the presence of bone metastases. We hypothesised that FUS is a predictive and/or prognostic marker for hormone-dependent cancers. Materials and Methods: FUS expression in breast cancer was analysed in silico and using Western blotting and immunohistochemistry on a tumour microarray. FUS levels were knocked down by siRNA in MCF-7 cells and chemosensitivity analysed using caspase 3/7 assays. Results: Analysis of the Finak dataset (2008) demonstrated that FUS expression is significantly lower in breast cancer compared to normal breast. Immunohistochemistry revealed that FUS expression is also inversely correlated with grade. Importantly, FUS expression was also correlated with survival (Kaplan-Meier Plotter); significantly longer relapse free survival was seen in patients whose tumours had high levels of FUS. FUS expression also appears to correlate with therapy response since analysis of the Chang dataset (2003) revealed that FUS levels are significantly lower in patients resistant to Docetaxel and we found FUS levels to be lower in the tamoxifen resistant MCF-7 cell line (MCF-7 TAMR) compared to the parental line. We therefore hypothesised that loss of FUS expression may be driving therapy failure. In support of this, reducing FUS expression in the parental MCF7 line resulted in a significant reduction in docetaxel-induced Caspase 3/7 activity, suggesting that FUS is a regulator of chemosensitivity. Discussion: FUS may be an important regulator of breast cancer progression since its levels are inversely correlated with grade and directly correlated with relapse free survival. Further, lower FUS expression is also correlated with reduced hormone therapy and chemo-sensitivity. Since knock-down of FUS reduced MCF-7 chemosensitivity, we believe that the observed changes in FUS expression are, in part, driving therapy failure and hence propose that FUS is a novel target and biomarker for breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-06.

(-)-Epigallocatechin-3-gallate downregulates Pg-P and BCRP in a tamoxifen resistant MCF-7 cell line

We investigated the anticancer effect of EGCG treatment on a breast carcinoma cell line resistant to tamoxifen (MCF-7Tam cells). As there are no reports about the molecular mechanisms implicated in EGCG treatment of tamoxifen resistant breast carcinoma cells, we studied the effects of EGCG treatment on three plasma membrane proteins that are involved in the mechanism of drug-resistance: Multidrug Resistance Protein (MRP1), P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). EGCG treatment (10-100 microg/ml for 24-72 hours) caused cell growth inhibition and dose-dependent apoptosis: after 100 microg/ml EGCG treatment for 24 hours, Bax expression increased and Bcl2 expression decreased (p<0.05). Coherently, Annexin V-FITC apoptosis assay detected a significant increase in labelled cells (p<0.05). EGCG did not affect MRP1: in contrast, 100 microg/ml EGCG administration caused P-gp decrease to 53% of control cells (p<0.001) and this effect was not due to downregulation of P-gp gene expression. EGCG induced P-gp decrease even when MG132, a strong proteasome inhibitor, was given together with EGCG to MCF-7Tam cells. EGCG treatment also inhibited BCRP activity: mRNA transcription and protein level did not change after treatment, but mitoxantrone test demonstrated a strong inhibition of BCRP activity (p<0.001). In conclusion, the present results showed that EGCG could down-regulate the activity of two molecules that play a key role in drug metabolism and transport and that are highly expressed in tamoxifen resistant breast carcinoma cells. The interaction of EGCG and drugs used in the therapy of estrogen sensitive breast carcinoma ought to be subject of studies and the potential use of EGCG in drug-resistant diseases ought to be better considered.

Tamoxifen Resistance in Estrogen Receptor Positive (ER+) Breast Cancer Is Driven by Estrogen Receptor Negative (ER-) Cancer Stem-Like Cells.

Abstract Introduction: Intrinsic and acquired resistance to endocrine therapy significantly reduces survival rates of women with ER+ breast cancer. In the normal breast, multipotent stem cells are ER-. We hypothesised that tumourigenic breast cancer stem-like cells (CSCs) are ER- and therefore function independently of estrogen, representing a novel mechanism of resistance to endocrine therapy.Methods: Three ER+ cell lines, MCF-7, T47D and BT474 were assessed for ER expression by immunocytochemistry (ICC) in monolayer and after CSC enrichment. Putative CSC number was assessed by the non-adherent mammosphere (MS) assay, in the presence and absence of tamoxifen, and by Fluorescence Activated Cell Sorting (FACS) for the markers CD44+/CD24-/low/epithelial specific antigen(ESA)+. The effect of acquired Tamoxifen resistance on the CSC population of two independent Tamoxifen-resistant (TAM-R) MCF-7 variants, from Cardiff (TAM-RCARD) and Copenhagen (TAM-RCOP) was compared to parental, tamoxifen sensitive (TAM-S) controls. In the MS assay both the number and size of colonies were assessed.Results: A small sub-population of ER_ cells were demonstrated in MCF7 (6.1 ± 1.4%), T47D (6.8± 0.5) and BT474 (3.7±1.3) cells by ICC. However, the majority of putative breast CSCs (CD44+/CD24-/low/ESA-) in MCF7 cells were ER_ (73.2 ± 4.6 % p= 0.0007). There was a significant increase in the proportion of ER- cells in TAM-R cell lines (TAM-RCARD 11.6 ± 1.4% p=0.01 and TAM-RCOP 22 ± 4.8% p=0.02 respectively) compared to the parental TAM-S controls. Both TAM-R cell lines demonstrated increased mammosphere forming efficiency (MFE) compared to TAM-S cells (TAM-RCARD 4% vs. 2.5% p=0.03, and TAM-RCOP 8% vs. 3% p=0.007). The putative CSC population (CD44+/CD24-/low/ESA+) was enriched more than 2.5 fold in TAM-R compared to TAM-S parental controls. Compared with controls, the absolute MFE was not affected by Tamoxifen treatment, although mammospheres formed were significantly smaller.Conclusions: The majority of the putative CSC populations in ER+ breast cancer do not express ER. There is an increase in the proportion of putative ER- CSCs in TAM-R MCF7 cell lines. The reduction in MS size suggests that Tamoxifen targets transit amplifying cells but does not reduce the absolute number of CSC in ER+ breast cancers. These findings demonstrate a novel mechanism of endocrine resistance in ER+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5125.

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.