Tamoxifen-induced Cell Death Research Articles

The DEK oncogene is a target of steroid hormone receptor signaling in breast cancer.

Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated in vitro following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify DEK as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.

The effect of cholesterol depletion in resistant breast cancer cells.

e11031 Background: A leading cause of breast cancer chemotherapeutic treatment failure is resistance of cancerous cells towards current anti-estrogen agents. This project investigated the relationship between inhibition of cholesterol synthesis and its effect on resistant breast cancer cells. Methods: MCF-7 cells were maintained in phenol red free media and pretreated with charcoal stripped media for 24 hours prior to the experiments (ensure all exogenous estrogen was taken out). Cells were plated in 96 well plates at 10,000 to 15,000 cells/well. MCF-7 cells were tested for their sensitivity to BIBB515 and whether the proliferation of cells with estrogen could be blocked with BIBB515. The cells were grown for 6 days, on day 7 the cell proliferation was determined by adding MTT and read at 570nM. Results: Estrogen, tamoxifen and anastrazole induced cell proliferation that was controlled by 10µM BIBB515. Inhibiting the cholesterol pathway in combination with tamoxifen induced cell death in MCF-7 cells (cell proliferation of 0.13 ±0.15) compared to tamoxifen alone (0.57±0.041) p<0.001 by one way ANNOVA and multiple comparison by DUNN test. Anastrozole alone induced statistically significant cell proliferation 0.403 ±0.03 compared to combination treatment with BIBB515 (0.05 ± 0.007 p<0.001 by one way ANNOVA and multiple comparison by DUNN test). BIBB515 treatment could also inhibit the estrogen induced cell proliferation in MCF-7 cells. These results indicate that cholesterol inhibitors can be developed to fill a niche area in breast cancer disease management where clinicians would prolong treatment on first-line anti-hormone therapies of tamoxifen and anastrazole for advanced disease patients. Conclusions: Results show that inhibition of cholesterol sensitizes resistant cells to current anti-estrogen chemotherapeutic agents. This finding is significant because it could potentially translate into reduce rates of breast cancer relapse due to regiment failure. Exploiting this mechanism could alter the current treatment regimens, leading to control of the diseases in advanced stages by either inducing tumors to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

P5-05-04: Bipshenol-A Modulates Estrogen Receptor Alpha and Compromises Tamoxifen-Induced Apoptosis.

Abstract BACKGROUND: Antiestrogens are important for treatment and prevention of breast cancer. Such drugs influence multiple pathways involved in regulation of cell survival — including apoptosis. In previous work, exposure of human, benign, high-risk donor breast epithelial cells (HRBECs) to the xenoestrogen (XE) bisphenol-A (BPA) induced gene expression changes predicting disruption of apoptotic signaling (Dairkee et al, Cancer Research 2008). Therefore, we hypothesized that antiestrogenic, pro-apoptotic effects of tamoxifen are likely modulated under the influence of XEs. Because the environmental XE, BPA is routinely detected in a large percentage of random human blood samples, we employed functional tests to determine whether BPA interferes with tamoxifen-induced apoptosis in XE-exposed HRBECs. METHODS: With IRB approval and written informed consent, HRBECs were obtained by random periareolar fine needle aspiration (RPFNA) from women who were high risk on the basis of personal or family history of breast cancer, breast density, or high-risk histology. Primary HRBEC cultures were treated for one week with a low concentrations of BPA corresponding to levels reported in human body fluids. Estrogen Receptor (ER)-α and -β were measured by Western blot (n=6). Altered response to tamoxifen toxicity was measured by analyzing quantitative changes in the Annexin V-positive apoptotic cell population (n=8). RESULTS: After one-week exposure to BPA, ER-α increased in six of six cases, ER-β decreased in 5 of 6 cases, and we observed a 25 percent reduction in tamoxifen-induced apoptosis. Prior exposure to BPA at the high concentration range detected in the human population reduced tamoxifen efficacy by 50 percent. There was a dose response of evasion of apoptosis to increasing concentrations of BPA (P<0.001, two-sided test for slope fixed effect in a linear mixed effects regression). Prior exposure to another XE, methylparaben (MP), caused a similar dose-dependent decline in tamoxifen-induced apoptosis (p<0.001). CONCLUSION: BPA and MP, in a range of concentrations measured in humans, prevent tamoxifen-induced apoptotic cell death. For BPA this involves upregulation of ER-α and downregulation of ER-β. Because these XEs are widespread in the American population, they may play a role in limiting the prophylactic and therapeutic efficacy of tamoxifen and other hormone-based therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-04.

The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin

Tamoxifen resistance has been largely attributed to genetic alterations in the epithelial tumor cells themselves, such as overexpression of HER-2/Neu. However, in the clinic, only about 15-20% of cases of HER-2/Neu amplification has actually been correlated to the acquisition of endocrine resistance, suggesting that other mechanisms must be involved as well. Using the epithelial LM05-E and the fibroblastic LM05-F cell lines, derived from the estrogen dependent spontaneous M05 mouse mammary tumor, as well as MCF-7 cells, we analyzed whether soluble stromal factors or extracellular matrix components protected against tamoxifen induced cell death. Involvement of signaling pathways was determined by using specific inhibitors and western blot, and phosphorylation of the estrogen receptor alpha by western blot and immunofluorescence. Soluble factors produced by the fibroblastic cells protect the epithelial tumor cells from tamoxifen-induced cell death through a mechanism that involves EGFR and matrix metalloproteinases upstream of PI3K/AKT. Exogenous fibronectin by itself confers endocrine resistance through interaction with β1 integrin and activation of PI3K/AKT and MAPK/ERK 1/2 pathways. The conferred resistance is reversed by blocking β1 integrin. We show also that treatment with both conditioned medium and fibronectin leads to the phosphorylation of the estrogen receptor at serine-118, suggesting stromal factors as modulators of ER activity. Our results show that the tumor microenvironment can modulate tamoxifen resistance, providing an alternative explanation for why patients become refractory to hormone-therapy.

Abstract P4-02-11: Treatment with Fibronectin or Carcinoma Associated Fibroblast Conditioned Media Generate Sustained Tamoxifen Resistance in Breast Cancer Cells

Abstract Background: breast cancer is the most common neoplasm in woman and the second cancer-related cause of death. About 70% of breast cancers are positive for estrogen receptor (ER) and progesterone receptors. Tamoxifen is the main hormonal therapy for ER+ patients. However, one third of the patients that are initially responsive eventually develop resistance to this treatment. We previously showed that treatment with fibronectin or conditioned media from carcinoma associated fibroblasts conferred tamoxifen resistance to otherwise sensitive ER+ breast cancer cells (in particular MCF-7 and LM05-E cells). On the other hand we also found that in the case of conditioned media, the PI3K/AKT pathway was involved downstream of EGFR and ≥1 integrin, whereas for fibronectin both the MAPK/ERK1/2 and the PI3K/AKT pathways were involved downstream of ≥1. The aim of this study was to investigate whether the protective effect conferred by the stromal elements was reversible, or, if on the contrary, it was sustained over time even after they were removed from the culture. In order to do so we pre-treated LM05-E cells with DMEM/F12 containing 1% charcoal stripped serum (CSS) as a control or with the addition of fibronectin (10μg/ml) or conditioned media from the LM05-F carcinoma associated fibroblast cell line. Subsequently cells were trypsinized, washed and allowed to attach ON in 8 well chamber slides in the absence of the stromal factors. They were then treated for 48 hours with estradiol 10-8 M, or estradiol plus 4-OH-tamoxifen 10-6 M in the presence of 1% CSS. Cell death was measured by propidium iodide incorporation. The 48 hour pre-treatment with either conditioned media or fibronectin conferred resistance to tamoxifen induced cell death. This resistance was sustained for at least a week in both cases, as determined by culturing the cells for this period of time in the absence of the stromal factors before testing the effect of 4-OH-tamoxifen. Shorter pretreatments of 6 or 12 hours weren't capable of conferring endocrine resistance. Inhibitors of the MAPK/ERK, PI3K/AKT or EGFR were added to the pre-treatments in order to study involvement of these pathways. Neither addition of the PI3K inhibitor LY294002 (10 μM), the MAPK inhibitor PD98059 (10 μM) or the EGFR inhibitor AG1478 (6,4 μM) were able to reverse the sustained protection induced by either fibronectin or conditioned media. Conclusion: our results suggest that both of these stromal elements are able to induce sustained changes in the breast cancer cells that lead to tamoxifen resistance even after they have been removed from the culture. At least a 48 hour period is required to accomplish this protection and this effect is sustained for at least a week. These results have important clinical implications on the lasting effects that the tumor microenvironment may have on the long term response to endocrine treatment in breast cancer. This work is supported by a grant from the Susan G. Komen for the Cure Foundation to MS (BCTR0600341). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-11.

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn(2+)) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn(2+) with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl(2) markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn(2+) has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) blunted the increase in Zn(2+) levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn(2+) contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.

IKKϵ Phosphorylation of Estrogen Receptor α Ser-167 and Contribution to Tamoxifen Resistance in Breast Cancer

IKKepsilon has recently been identified as a breast cancer oncogene. Elevated levels of IKKepsilon are associated with cell survival and growth. Here, we show that IKKepsilon interacts with and phosphorylates estrogen receptor alpha (ERalpha) on serine 167 in vitro and in vivo. As a result, IKKepsilon induces ERalpha transactivation activity and enhances ERalpha binding to DNA. Cyclin D1, a major target of ERalpha, is transcriptionally up-regulated by IKKepsilon in a phospho-ERalpha-Ser-167-dependent manner. Further, overexpression of IKKepsilon induces tamoxifen resistance, whereas knockdown of IKKepsilon sensitizes cells to tamoxifen-induced cell death. These data suggest that ERalpha is a bona fide substrate of IKKepsilon and IKKepsilon plays an important role in tamoxifen resistance. Thus, IKKepsilon represents a critical therapeutic target in breast cancer.

MicroRNA-221/222 Confers Tamoxifen Resistance in Breast Cancer by Targeting p27Kip1

We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHT(R))) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHT(R) cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27(Kip1), a known target of miR-221/222, was reduced by 50% in OHT(R) cells and by 28-50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27(Kip1) in the resistant OHT(R) cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker.

Estrogen receptor beta increases the efficacy of antiestrogens by effects on apoptosis and cell cycling in breast cancer cells

Clinical evidence indicates that higher levels of estrogen receptor beta (ERbeta) predicts improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. To better understand the mechanisms in which ERbeta can modulate breast cancer therapies, we introduced ERbeta under an inducible promoter into MCF-7 breast cancer cells. In these cells, induction of ERbeta expression led to a shift in the potency and an increase in the efficacy of tamoxifen to inhibit proliferation. A similar effect on breast cancer cells was observed for two other antiestrogens, raloxifene, and fulvestrant. Induced expression of ERbeta did not enhance the antiproliferative effects of small molecule inhibitors that target the epidermal growth factor receptor, insulin growth factor receptor-1 and histone deacetylase, indicating ERbeta specifically cooperates with antiestrogens. The combination of ERbeta expression, which arrests cells in G2, and tamoxifen, which arrests cells in G1, led to a potent blockade of the cell cycle. ERbeta also increased tamoxifen-induced cell death and cooperated with tamoxifen to induce expression of the pro-apoptotic gene bik. In summary, our data indicates that ERbeta increases the efficacy of antiestrogens by effects on apoptosis and on cell cycling and, together with clinical observations, suggests ERbeta could be a valuable prognostic marker and potential therapeutic target.

Substance P mediates antiapoptotic responses in human colonocytes by Akt activation

We examined the hypothesis that substance P (SP) and the neurokinin-1 receptor (NK-1R), both in vitro and in vivo, promote mucosal healing during recovery from colitis by stimulating antiapoptotic pathways in human colonic epithelial cells. For the in vitro experiments, human nontransformed NCM460 colonocytes stably transfected with NK-1R (NCM460-NK-1R cells) were exposed to SP, and cell viability assays, TUNEL assays, and Western blot analyses were used to detect apoptotic and antiapoptotic pathways. SP exposure of NCM460-NK-1R colonocytes stimulated phosphorylation of the antiapoptotic molecule Akt and inhibited tamoxifen-induced cell death and apoptosis evaluated by the cell viability assay and poly(ADP-ribose) polymerase cleavage, respectively. SP-induced phosphorylation of Akt and cleavage of poly(ADP-ribose) polymerase were inhibited by blockade of integrin alphaVbeta3, Jak2, and activation of phosphatidylinositol 3-kinase. For the in vivo experiments, C57BL/6 mice, administered 5% dextran sulfate (DSS) dissolved in tap water for 5 days followed by a 5-day recovery period, were treated with the NK-1R antagonist CJ-12,255 or vehicle. Vehicle-treated mice showed increased colonic Akt phosphorylation and apoptosis compared with mice that received no DSS. In contrast, daily i.p. administration of CJ-12,255 for 5 days post-DSS suppressed Akt activation, exacerbated colitis, and enhanced apoptosis, and pharmacologic inhibition of Akt, either alone or together with CJ-12,255, produced a similar effect. Thus, SP, through NK-1R, possesses antiapoptotic effects in the colonic mucosa by activating Akt, which prevents apoptosis and mediates tissue recovery during colitis.

Tamoxifen-induced cell death and expression of neurotrophic factors in cultured C6 glioma cells

The effects of ( Z)-2[ p -(1,2-diphenyl-1-butenyl)phenoxy]-N ,N -dimethylamine citrate (tamoxifen) on cell survival and the expression of neurotrophic factors (NTF) were investigated in rat C6 glioma cells (C6). C6 cells do not express the estrogen receptor. Cytotoxic effect was detected from 24 h after the treatment with 10 microM tamoxifen and increased with time in a dose-dependent manner. C6 cells treated with tamoxifen also displayed various morphological types such as elliptical, round and aggregated form. As the treatment time increased, the proliferation of C6 cells was reduced remarkably and most of them became the round or aggregated form. To examine the relationship of the expression of NTF and the cytotoxicity of tamoxifen, the mRNA level of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and basic fibroblast growth factor (bFGF) was measured after 24 h treatment with tamoxifen by RT-PCR. The expression of mRNA of BDNF or GDNF in C6 cells treated with various concentrations of tamoxifen was comparable to controls. The expression of bFGF mRNA was significantly reduced in C6 cells treated with 10 or 15 microM tamoxifen. The results suggest that tamoxifen exerts cytotoxic effect on estrogen receptor-negative C6 cells through the inhibition of the transcription of bFGF.

Tamoxifen-induced cell death in malignant melanoma cells: possible involvement of the insulin-like growth factor-1 (IGF-1) pathway

Recent data indicate that the estrogen receptor (ER) blocker tamoxifen (TAM) can induce cell death in malignant melanoma cells. However, as shown in the present study and several other studies melanoma cells usually do not express classical ERs. In the present study we investigated whether the cytotoxic effect of TAM on melanoma cells could depend on interference with the expression or function of the insulin-like growth factor-1 receptor (IGF-1R), a plasma membrane receptor important for cell survival in this tumor cell type. Several melanoma cell lines were included in the analysis. Administration of TAM at a concentration of 15 μm or more resulted in cell death of the melanoma cells within 48 h. TAM treatment was correlated to a slight to moderate inhibition of IGF-1 binding to IGF-1R. Since it has been reported that TAM can increase the release of IGF binding proteins (IGFBPs) we then investigated whether this mechanism could underly the decreased IGF-1 binding. However, we could demonstrate that the amount of released IGFBPs were unchanged or decreased in TAM-treated cells. Whereas TAM did not have any strong effect on IGF-1 binding and the expression of IGF-1R at the cell surface, it was was found to efficently block tyrosine phosphorylation of IGF-1R β-subunit. Taken together, our data suggest that TAM-induced cytotoxicity of malignant melanoma cells can be due to inactivation of IGF-1R.