Long-term Estrogen-deprived MCF-7 Cells Research Articles

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.

Abstract P5-09-02: Cell culture models to study mechanisms for endocrine resistant breast cancer, new treatment options and new biomarkers

Abstract Introduction: Estrogen is the main driver of growth of the majority of breast cancers and many patients benefit from endocrine therapy. However, both innate and acquired resistance is a major problem. In order to study resistance mechanisms and to explore targeted treatment options for endocrine resistant breast cancer, we have developed in vitro cell culture models based on the estrogen receptor (ER) positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D. Methods: Panels of MCF-7 and T47D sublines with acquired resistant to tamoxifen, fulvestrant and aromatase inhibitors were established by selection of colonies surviving long term treatment. Long-term estrogen deprived (LTED) MCF-7/S9 cells were developed by step-wise serum reduction until transfer to serum-free medium. The endocrine resistant sublines were analysed with respect to expression and function of the ER and the HER system, with respect to response to 195 kinase inhibitors and to second-line endocrine therapy. Results: ER expression was reduced in tamoxifen and fulvestrant resistant MCF-7 sublines, unchanged in tamoxifen resistant T47D sublines, whereas fulvestrant resistant T47D sublines were ER negative. In general, ER was functional in the resistant MCF-7 cells. Both tamoxifen and fulvestrant resistant MCF-7 sublines expressed increased level of HER1, the activated form of HER3 and activated ERK1/2, whereas HER4 was severely decreased. In fulvestrant resistant T47D sublines, HER1 and HER4 expression were decreased whereas HER2 expression was highly upregulated. The kinase inhibitor screen disclosed HER receptors as common hits in the tamoxifen and fulvestrant resistant MCF-7 sublines. Regarding T47D, c-Src and Aurora were common hits for tamoxifen and fulvestrant resistant sublines. Tamoxifen resistant MCF-7 and T47D sublines were severely growth inhibited by ER knockdown with either siRNA or fulvestrant, but complete growth arrest required combined treatment with e.g ERK1/2 inhibitor. LTED MCF-7 cells were resistant to aromatase inhibitor treatment and sensitive to both tamoxifen and fulvestrant. Conclusions: In our large panels of MCF-7 and T47D derived endocrine resistant sublines, it was a general finding that ER is the main driver of growth of tamoxifen resistant tumor cells, and also of growth of LTED MCF-7 cells. The ER down modulator fulvestrant inhibited growth of tamoxifen resistant sublines but complete growth arrest required combined treatment with e.g. a kinase inhibitor targeting the activated growth promoting pathways in the resistant cells. In fulvestrant resistant sublines, ER played only a minor role in MCF-7 sublines and no role in T47D sublines. The HER system played a major role in fulvestrant resistant MCF-7 cells, whereas Aurora B and c-Src were new candidate targets for fulvestrant resistant T47D cells. These cell culture models reflect clinical findings and will be available for collaborative studies, e.g. to discover new predictive markers to select targeted therapy for resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-02.

Abstract 786: In-vivo and in-vitro breast cancer profiling to delineate adaptive endocrine resistance: A pilot study

Abstract A substantial fraction of estrogen receptor (ER) positive breast cancers fail to respond to endocrine treatment, i.e. tamoxifen and aromatase inhibitors. While tamoxifen failure has been partially attributed to primary resistance, for example CYP2D6 deficiency (Schroth et al JAMA 2009), adaptive resistance during ER blockade/estrogen deprivation plays a prominent role. Little is known about shared molecular patterns driving both resistance types. Long-term estrogen deprived (LTED) breast cancer can be induced to undergo apoptosis by pulse estrogen (Song et al JNCI 2001; Jordan JCO 2008). We hypothesize that it should be possible to identify predictive markers for adaptive endocrine resistance and sensitivity to apoptosis to recognize endocrine refractory tumors. To study a putative interface of primary and acquired resistance we performed gene expression profiling with low-density arrays (Fluidigm BioMark system) in tumors of 12 breast cancer patients treated with tamoxifen. They were selected to model CYP2D6 independent primary resistance: half of the patients had functional CYP2D6 but were tamoxifen resistant due to relapse (median time-to-event 3.8 years). The other half of patients were CYP2D6 deficient but tamoxifen sensitive due to the absence of relapse (median follow-up 13.5 years). Expression profiles have been compared to LTED MCF-7 cells (3 and 6 months estrogen deprived) and MCF-7 cells grown in the presence of estrogen thereby modeling adaptive resistance. Gene targets were selected based on literature i) involved in endocrine resistance (N=62), ii) playing a role in transport and estrogen supply (N=30), and iii) being p53 targets for apoptosis and cell-cycle check points (N=46). We observed both shared and specific expression phenotypes for adaptive and primary resistance. The proliferation genes Cyclin B1 and STK15 were downregulated in LTED MCF-7 cells in line with lowered proliferation rates and, unexpectedly, Cyclin B1 expression was also decreased in tamoxifen insensitive tumors suggesting an involvement of G2/M cell cycle transition. The chemokine receptor CXCR4 was upregulated in both LTED cells and resistant tumors, confirming its prominent role in breast cancer progression. The 25-hydroxyvitamin D3 hydroxylase CYP27B1 was upregulated in LTED, but not in resistant tumors suggesting a different role between the two models. Other potential markers involve differentiation, inflammatory response, cell cycle/apoptosis, and estrogen disposition pathways. Our preliminary data demonstrate the feasibility to discern between short-term adaptive versus primary resistance patterns. Expression profiling of LTED cells following estrogen-induced apoptosis is currently underway. 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 786. doi:1538-7445.AM2012-786

Abstract 939: ErbB-2 inhibits Notch-4 expression and activity in breast cancer independent of ER function

Abstract Amplification, over-expression, and/or hyperactivity of ErbB-2 occur in 50% of breast tumors which include both luminal A/B and the HER2+ subtypes. Metastatic breast tumors that overexpress ErbB-2 are generally resistant to anti-hormonal or anti-HER2-targeted therapy. We have published that either 17beta-estradiol or overexpression of ErbB-2 suppresses Notch activity and this is reversed by inhibitors of estrogen receptor (ER) or ErbB-2. Furthermore, we demonstrated that when breast tumors are treated with a Notch inhibitor (gamma-secretase inhibitor or GSI) in combination with tamoxifen (ER+ cells) or trastuzumab (ErbB-2+ cells), ER+ breast tumor xenografts regress or ErbB-2+ xenografts do not recur. Based on these published findings, we asked in the current study by what mechanism does ErbB-2 inhibit Notch signaling. We used both genetic and pharmacologic approaches to address the hypothesis that ErbB-2 hyperactivity was sufficient to suppress Notch independent of ER function. Our results showed that Notch-4 transcription is specifically and significantly decreased when ErbB-2 or Heregulin-beta1 is stably-overexpressed in MCF-7 cells. Furthermore, the data demonstrated that while 17beta-estradiol inhibits Notch-4 mRNA and protein expression, which was reversed by fulvestrant, the inhibition on Notch-4 by ErbB-2 or by Heregulin-beta1 overexpression was independent of ER activity. In addition, we confirmed that ER status was an independent factor by specifically targeting ErbB-2 using lapatinib in ER negative SKBr3 breast cancer cells and the results showed that Notch-4 mRNA and protein increased to more than 100 fold compared to vehicle treatment. We have recently published that c-Fos is a potential transcriptional repressor of Notch-4 in breast cancer cells and in agreement, we identified that c-Fos levels were significantly elevated in breast cancer cells stably-overexpressing either ErbB-2 or Heregulin-beta1. The biological significance of Notch-4 overexpression was investigated in two relevant resistant models in vitro and in vivo. Resistance to lapatinib was prevented by co-treatment of a GSI plus lapatinib. Resistance to anti-hormonal therapy using long-term estrogen deprived MCF-7 cells was reversed by a GSI treatment. MCF-7/HER2 breast tumor xenografts regressed to undetectable levels with the combination of a GSI plus trastuzumab under conditoions where estrogen was deprived. In conclusion, results from the current study suggest that ErbB-2 overexpression or hyperactivity via Heregulin-beta1 is a potent inhibitor of Notch-4 expression possibly by increasing c-Fos expression. Furthermore, the regulation of Notch-4 expression is independent of ER function. More importantly, anti-ER or anti-EbB-2 targeted therapy de-repress Notch-4 expression and thus sensitize breast cancer cells to a Notch inhibitor such as a GSI to prevent or reverse resistance. 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 939. doi:1538-7445.AM2012-939

Effects of catechin, diclofenac and celecoxib on the proliferation of MCF-7 and LTED MCF-7 cells

Breast cancer is common among women especially after menopause. Inflammation is now considered as a risk factor of cancer and cyclooxygenase-2 (COX-2) enzymes are highly expressed in many cancers. COX-2 is the inducible enzyme responsible for the synthesis of pro-inflammatory prostaglandins. There are evidences that COX inhibitors especially those which inhibit COX-2 have a role in the management of cancers. Since breast tumors are generally estrogen sensitive, whether or not their response to COX inhibitors is changed when they are under the condition of estrogen deprivation. This in vitro study aimed to investigate the effects of different COX inhibitors, i.e . catechin − selective COX-1 inhibitor, diclofenac − COX-1/COX-2 inhibitor, and celecoxib − selective COX-2 inhibitor, at concentrations of 10 −10–10 −4 M on the proliferation of MCF-7 cells and long-term estrogen-deprived (LTED) MCF-7 cells. The effects of these COX inhibitors were assessed on day 2 and day 6 of incubation using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Morphological features of these cells on day 2 of incubation with COX inhibitors were visualized after Ho33342 staining and apoptosis was confirmed by DNA fragmentation. The results indicated that diclofenac and celecoxib, but not catechin, at high concentrations could effectively inhibit the proliferation of both cell types. Celecoxib was, however, the only drug used in this study that could markedly induce DNA fragmentation in LTED MCF-7 cells at the highest concentration (10 −4 M). This study provides the in vitro evidence to support the beneficial effect of COX-2 inhibitors against breast tumors whether or not at the estrogen-deprived stage.

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.

Adaptive hypersensitivity following long-term estrogen deprivation: involvement of multiple signal pathways.

Long-term estrogen deprivation causes hypersensitivity of MCF-7 cells to the mitogenic effect of estradiol (E2) which is associated with activation of mitogen-activated protein kinase (MAPK). However, several lines of evidence indicate that MAPK activation is not the exclusive mechanism for E2 hypersensitivity and multiple signal pathways might be involved. The current study explores the possible role of the PI3 kinase (PI3K) pathway in development of E2 hypersensitivity. Basal PI3K activity in long-term estrogen deprived MCF-7 cells (LTED) was elevated as evidenced by increased phosphorylation of three downstream effectors, Akt, p70 S6 kinase, and eukaryotic initiation factor-4E binding protein (4E-BP1), which was blocked by the specific inhibitor of PI3K, LY294002. Dual blockade of both MAPK and PI3K completely reversed E2 hypersensitivity of LTED cells. Enhancement in aromatase activity is another phenomenon accompanied with E2 hypersensitivity. In aromatase over-expressing MCF-7 cells, aromatase activity was reduced by inhibitors of MAPK and PI3K suggesting the involvement of protein phosphorylation in the regulation of aromatase activity. Our data suggest that in addition to the MAP kinase pathway, activation of the PI3 kinase pathway is involved in E2 hypersensitivity, which develops during adaptation of MCF-7 cells to the low estrogen environment.