Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (gamma-glutamylcysteine ligase heavy chain (gamma-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and gamma-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-beta-estradiol or ICI-182,780 did not significantly change gamma-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.     

Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER⁺) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER⁺ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER⁺ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66^?/PR^?/HER2^?) and ER-α36⁺/GPER1⁺ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER⁺ breast cancer.     

Reversal of tamoxifen resistant breast cancer by low dose estrogen therapy

Currently, the standard of care for estrogen receptor (ER)-positive breast cancer is 5 years of tamoxifen (TAM) or an aromatase inhibitor (AI) such as anastrozole. New studies indicate that extending antiestrogen therapy beyond 5 years with sequential regimens will improve disease-free survival. Based on the emerging concept that longer therapies are better, we have developed sequential models of tamoxifen-resistant breast cancer in vivo to mimic the clinical scenario of long-term antiestrogen therapy. The goal of the current study was to investigate the consequences of long-term treatment with tamoxifen on the growth of breast tumors in athymic mice. The results demonstrate that there are distinct phases of resistance to tamoxifen that correlate with time of treatment and expression of HER2/neu mRNA. In the treatment phase, 17β-estradiol (E₂) stimulated growth, while TAM inhibited growth of MCF-7 tumors (MCF-7E₂). The withdrawal of treatment, mimicking the use of an AI, completely prevented growth. In Phase I resistance, the tumors (MCF-7TAMST) were growth-stimulated by either E₂ or TAM, but inhibited by no treatment, fulvestrant, or E₂ + fulvestrant. Phase II-resistant tumors (MCF-7TAMLT) were treated for more than 5 years and growth-stimulated by TAM. However, no treatment, fulvestrant, or E₂ completely inhibited growth. Interestingly, the few tumors (MCF-7TAMLT) that survived in response to E₂ were robustly re-stimulated by E₂ after transplantation into new generations of athymic mice. These E₂-stimulated tumors (MCF-7TAME) were inhibited by TAM in a dose-dependent similar to their parental tumors (MCF-7E₂). In addition, the MCF-7TAME tumors were inhibited by either no treatment or fulvestrant. HER2/neu and HER3 mRNAs were over-expressed in TAM-stimulated MCF-7TAMLT tumors and remained high in E₂-stimulated MCF-7TAME tumors. The data indicate that complete reversal of resistance to TAM can be achieved with the use of low dose E₂ therapy. Also, these data suggest that over-expression of HER2/neu alone is insufficient to predict resistance to TAM. Based on the results, we suggest using an alternating treatment regimen, cycling antiestrogen with estrogen therapy to avoid drug-resistance.     

P-gp 和MDR1 在三种乳腺癌细胞中表达差异研究

目的:比较P-gp和MDR1在人乳腺癌敏感细胞(MCF-7/S)和耐药细胞(MCF-7/ADR、MCF-7/TAM)中的表达差异,初步探讨乳腺癌细胞对阿霉素与对三苯氧胺产生耐药机制的区别。方法:采用免疫细胞化学法、流式细胞术检测P-gp,采用实时荧光定量PCR法检测MDR1在三种乳腺癌细胞中的表达情况。结果:在MCF-7/ADR细胞中P-gp和MDR1均呈高表达,阳性表达率与MCF-7/S细胞比较,有统计学意义(P<0.01)。在MCF-7/TAM细胞中P-gp、MDR1均呈低表达,与MCF-7/S细胞比较,无统计学意义(P>0.05)。结论:P-gp和MDR1的高表达是乳腺癌细胞对阿霉素产生耐药的主要机制,而并非是乳腺癌细胞对三苯氧胺产生耐药的机制。     

Downregulation of Choline Kinase-Alpha Enhances Autophagy in Tamoxifen-Resistant Breast Cancer Cells

Choline kinase-α (Chk-α) and autophagy have gained much attention, as they relate to the drug-resistance of breast cancer. Here, we explored the potential connection between Chk-α and autophagy in the mechanisms driving to tamoxifen (TAM) resistance, in estrogen receptor positive (ER+) breast cancer cells (BCCs). Human BCC lines (MCF-7 and TAM-resistant MCF-7 (MCF-7/TAM) cells) were used. Chk-α expression and activity was suppressed by the transduction of shRNA (shChk-α) with lentivirus and treatment with CK37, a Chk-α inhibitor. MCF-7/TAM cells had higher Chk-α expression and phosphocholine levels than MCF-7 cells. A specific downregulation of Chk-α by the transduction of shChk-α exhibited a significant decrease in phosphocholine levels in MCF-7 and MCF-7/TAM cells. The autophagy-related protein, cleaved microtubule-associated protein light chain 3 (LC3) and autophagosome-like structures were significantly increased in shChk-α-transduced or CK37-treated MCF-7 and MCF-7/TAM cells. The downregulation of Chk-α attenuated the phosphorylation of AKT, ERK1/2, and mTOR in both MCF-7 and MCF-7/TAM cells. In MCF-7 cells, the downregulation of Chk-α resulted in an induction of autophagy, a decreased proliferation ability and an activation of caspase-3. In MCF-7/TAM cells, despite a significant decrease in proliferation ability and an increase in the percentage of cells in the G0/G1 phase of the cell cycle, the downregulation of Chk-α did not induced caspase-dependent cell death and further enhanced autophagy and G0/G1 phase arrest. An autophagy inhibitor, methyladenine (3-MA) induced death and attenuated the level of elevated LC3 in MCF-7/TAM cells. Elucidating the interplay between choline metabolism and autophagy will provide unique opportunities to identify new therapeutic targets and develop novel treatment strategies that preferentially target TAM-resistance.     

KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway and predicts good prognosis in breast cancer

Tamoxifen resistance represents a daunting challenge to the successful treatment for breast cancer. Krüppel-like factor 4 has critical roles in the development and progression of breast cancer, but its expression, function and regulation in the efficacy of TAM therapy in breast cancer have yet to be investigated. Here, we examined the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 correlated with increased TAM sensitivity in breast cancer cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERα and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness to TAM in T47D and TAM-resistant MCF-7/TAM cells. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion and migration. Moreover, KLF4 expression was down-regulated in breast cancer tumor tissues and high expression of KLF4 was associated with favorable outcomes. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and p38 were more activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors significantly suppressed cell viability. Knockdown of KLF4 activated ERK and p38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and p38 signaling and resulted in increased sensitivity to TAM. Therefore, our findings suggested that KLF4 contributed to TAM sensitivity in breast cancer via phosphorylation modification of ERK and p38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for the TAM-resistant patients.     

UCP2 inhibition sensitizes breast cancer cells to therapeutic agents by increasing oxidative stress

Modulation of oxidative stress in cancer cells plays an important role in the study of the resistance to anticancer therapies. Uncoupling protein 2 (UCP2) may play a dual role in cancer, acting as a protective mechanism in normal cells, while its overexpression in cancer cells could confer resistance to chemotherapy and a higher survival through downregulation of ROS production. Thus, our aim was to check whether the inhibition of UCP2 expression and function increases oxidative stress and could render breast cancer cells more sensitive to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied clonogenicity, mitochondrial membrane potential (ΔΨm), cell viability, ROS production, apoptosis, and autophagy in MCF-7 and T47D (only the last four determinations) breast cancer cells treated with CDDP or TAM, in combination or without a UCP2 knockdown (siRNA or genipin). Furthermore, survival curves were performed in order to check the impact of UCP2 expression in breast cancer patients. UCP2 inhibition and cytotoxic treatments produced a decrease in cell viability and clonogenicity, in addition to an increase in ΔΨm, ROS production, apoptosis, and autophagy. It is important to note that CDDP decreased UCP2 protein levels, so that the greatest effects produced by the UCP2 inhibition in combination with a cytotoxic treatment, with regard to treatment alone, were observed in TAM+UCP2siRNA-treated cells. Moreover, this UCP2 inhibition caused autophagic cell death, since apoptosis parameters barely increased after UCP2 knockdown. Finally, survival curves revealed that higher UCP2 expression corresponded with a poorer prognosis. In conclusion, UCP2 could be a therapeutic target in breast cancer, especially in those patients treated with tamoxifen.     

N-Alkoxypyrazoles as biomimetics for the alkoxyphenyl group in tamoxifen

The preparation of a series of novel analogues of the selective antiestrogen tamoxifen is reported. 1Z-alkoxyphenyl group in tamoxifen has been replaced by a N-alkoxypyrazole, while functionalised phenyl groups or heteroaromatics were introduced at the 2Z-position using sequential Suzuki cross coupling of 1,2-(bis)borylpinacol 1-phenylbutene with 4- or 5-iodo-1-N,N-dimethylaminoethyl or propyl-pyrazoles. Approximately 50 tamoxifen analogues were obtained and tested in an estrogen receptor (ERalpha) affinity assay. Several compounds exhibited binding affinities 2-5-fold lower than tamoxifen. Dose-response experiments with six selected compounds were carried out using two different human breast cancer cell lines, MCF-7 and the tamoxifen resistant cell line MCF-/TAM(R)-1. Both cell lines exhibited growth inhibition upon treatment with the tamoxifen analogues. Co-treatment of the cells, with estradiol and the individual compounds, were also performed. The results indicated that the observed growth inhibitory effect was mediated by the ERalpha. Analogues of the potent antiestrogen 4-hydroxytamoxifen (4-OHT) were synthesised where the 1E-4-hydroxyphenyl was replaced by a 1-hydroxypyrazol-4-yl group. However, modest growth inhibition of MCF-7 cells was observed upon treatment with these analogues. In contrast, 1Z-, 2Z-ringclosed tamoxifen analogue (59) was shown to possess antiproliferative effects on MCF-7 and MCF-/TAM(R)-1 cells in lower doses than tamoxifen.     

Epithelial cell adhesion molecule promotes breast cancer resistance protein-mediated multidrug resistance in breast cancer by inducing partial epithelial–mesenchymal transition

Overexpression of breast cancer resistance protein (BCRP) plays a crucial role in the acquired multidrug resistance (MDR) in breast cancer. The elucidation of molecular events that confer BCRP-mediated MDR is of major therapeutic importance in breast cancer. Epithelial cell adhesion molecule (EpCAM) has been implicated in tumor progression and drug resistance in various types of cancers, including breast cancer. However, the role of EpCAM in BCRP-mediated MDR in breast cancer remains unknown. In the present study, we revealed that EpCAM expression was upregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and EpCAM knockdown using siRNA reduced BCRP expression and increased the sensitivity of MCF-7/MX cells to mitoxantrone (MX). The epithelial–mesenchymal transition (EMT) promoted BCRP-mediated MDR in breast cancer cells, and EpCAM knockdown partially suppressed EMT progression in MCF-7/MX cells. In addition, Wnt/β-catenin signaling was activated in MCF-7/MX cells, and the inhibition of this signaling attenuated EpCAM and BCRP expression and partially reversed EMT. Together, this study illustrates that EpCAM upregulation by Wnt/β-catenin signaling induces partial EMT to promote BCRP-mediated MDR resistance in breast cancer cells. EpCAM may be a potential therapeutic target for overcoming BCRP-mediated resistance in human breast cancer.     

Inhibition of GSK-3β activity can result in drug and hormonal resistance and alter sensitivity to targeted therapy in MCF-7 breast cancer cells

The PI3K/Akt/mTORC1 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance, and metastasis. One molecule regulated by this pathway is GSK-3β. GSK-3β is phosphorylated by Akt on S9, which leads to its inactivation; however, GSK-3β also can regulate the activity of the PI3K/Akt/mTORC1 pathway by phosphorylating molecules such as PTEN, TSC2, p70S6K, and 4E-BP1. To further elucidate the roles of GSK-3β in chemotherapeutic drug and hormonal resistance of MCF-7 breast cancer cells, we transfected MCF-7 breast cancer cells with wild-type (WT), kinase-dead (KD), and constitutively activated (A9) forms of GSK-3β. MCF-7/GSK-3β(KD) cells were more resistant to doxorubicin and tamoxifen compared with either MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells. In the presence and absence of doxorubicin, the MCF-7/GSK-3β(KD) cells formed more colonies in soft agar compared with MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells. In contrast, MCF-7/GSK-3β(KD) cells displayed an elevated sensitivity to the mTORC1 blocker rapamycin compared with MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells, while no differences between the 3 cell types were observed upon treatment with a MEK inhibitor by itself. However, resistance to doxorubicin and tamoxifen were alleviated in MCF-7/GSK-3β(KD) cells upon co-treatment with an MEK inhibitor, indicating regulation of this resistance by the Raf/MEK/ERK pathway. Treatment of MCF-7 and MCF-7/GSK-3β(WT) cells with doxorubicin eliminated the detection of S9-phosphorylated GSK-3β, while total GSK-3β was still detected. In contrast, S9-phosphorylated GSK-3β was still detected in MCF-7/GSK-3β(KD) and MCF-7/GSK-3β(A9) cells, indicating that one of the effects of doxorubicin on MCF-7 cells was suppression of S9-phosphorylated GSK-3β, which could result in increased GSK-3β activity. Taken together, these results demonstrate that introduction of GSK-3β(KD) into MCF-7 breast cancer cells promotes resistance to doxorubicin and tamoxifen, but sensitizes the cells to mTORC1 blockade by rapamycin. Therefore GSK-3β is a key regulatory molecule in sensitivity of breast cancer cells to chemo-, hormonal, and targeted therapy.     

Inhibition of IGF-IR increases chemosensitivity in human colorectal cancer cells through MRP-2 promoter suppression

The emergence of multidrug resistance (MDR) in cancer cells has made many of the currently available chemotherapeutic agents ineffective. However, the mechanism involved in mediating this effect is not yet fully understood. Here, we found the overexpression of type I insulin-like growth factor receptor (IGF-IR) in established colorectal MDR cells. Specific siRNA of IGF-IR decreases cell proliferation, exert synergistic effect with anticancer drugs. The downstream signaling of IGF-IR, PI3K/AKT pathway, was altered upon IGF-IR silencing. The expression of multidrug-resistance-associated protein 2 (MRP-2) was suppressed due to the nuclear translocation of nuclear factor-like 2 (Nrf2). Then the intracellular drug concentration was increased and the drug-resistant phenotype was reversed. Our findings improve current understanding of the biology of IGF-IR and MDR and have significant therapeutic implications on colorectal MDR cancer.     

S6K1 inhibition enhances tamoxifen-induced cell death in MCF-7 cells through translational inhibition of Mcl-1 and survivin

S6 kinase 1 (S6K1) was suggested to be a marker for endocrine therapy resistance in breast cancer. We examined whether tamoxifen’s effect can be modulated by S6K1 inhibition. S6K1 inhibition by PF4708671, a selective inhibitor of S6K1, acts synergistically with tamoxifen in S6K1-high MCF-7 cells. Similarly, the knockdown of S6K1 with small interfering RNA (siRNA) significantly sensitized MCF-7 cells to tamoxifen. Inhibition of S6K1 by PF4708671 led to a marked decrease in the expression levels of the anti-apoptotic proteins Mcl-1 and survivin, which was not related to mRNA levels. In addition, suppression of Mcl-1 or survivin, using specific siRNA, further enhanced cell sensitivity to tamoxifen. These results showed that inhibition of S6K1 acts synergistically with tamoxifen, via translational modulation of Mcl-1 and survivin. Based on these findings, we propose that targeting S6K1 may be an effective strategy to overcome tamoxifen resistance in breast cancer.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

Tamoxifen induces TGF-β1 activity and apoptosis of human MCF-7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.