Long-term Estrogen-deprived Cells Research Articles

Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer.

Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor-positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17beta-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways.

Abstract 2071: Loss of ER-regulated microRNAs is fundamental to hormone-independent breast cancer cells

Abstract Breast cancer is a disease that affects thousands of women every year where death generally occurs due to metastasis and the development of resistance to chemotherapeutic agents. A large subset of breast cancer is attributed to the dysregulation of estrogen receptor (ER) signaling. Postmenopausal women have decreased systemic estrogen, which presumably leads to a repressed ER signaling program. Treatment of postmenopausal breast cancer patients with aromatase inhibitors, which further decrease the level of estrogen by inhibiting its production from androgens, leads to an improved response when compared to anti-estrogen therapy. Long-term estrogen deprived (LTED) MCF7 breast cancer cells, which were generated by limiting the levels of estrogen in culture models this class of cancer. Previous studies have examined gene expression profiles in LTED cells providing clues to how these cancers are regulated. In our study, we set out to further characterize these cancers by examining microRNA (miRNA) expression in LTED cells. As a means to determine miRNAs that may be essential for the regulation of hormone-independent ER-positive breast cancers, we profiled the expression of 738 miRNAs in MCF7 breast cancer cell lines. We compared the miRNA expression levels of common MCF7 cells to a LTED derivative, MCF7:2A. Of the differentially expressed miRNAs, we discovered two different miRNA clusters that are directly regulated by estrogen in MCF7 cells. We observed induced ER binding near the primary-miRNAs (pri-miRNAs) that contain these clusters and that LTED cells have less overall basal ER interaction at these cis elements. This corresponds to a decreased expression of these pri-miRNAs in the LTED cells versus MCF7. Furthermore, treatment of MCF7s with fulvestrant, an anti-estrogen, reduces pri-miRNA expression. Predicted targets of these miRNAs include IGF1R and HER2, proteins known to be upregulated in LTED cells. These proteins have been shown to be fundamental to the growth and survival of estrogen-independent cell lines. We demonstrate modulation of the expression level of these proteins with the addition of miRNAs to LTED cells. Additionally, we can show endogenous association of the mRNAs encoding HER2 and IGF1R with the Ago1 complex in MCF7 cells and reduced association in the LTED cells. These data provide a mechanism by which growth receptors can be induced to express upon estrogen loss in breast cancer. Preliminary analysis of published datasets show a correlation between pri-miRNA expression and metastatic breast cancer where there is decreased miRNA expression in metastatic tumors. This study identifies both ER-regulated miRNAs as well as a potential postmenopausal breast cancer miRNA signature, which may lead to novel therapy of ER-positive breast cancers in postmenopausal women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2071.

Lapatinib Restores Hormone Sensitivity with Differential Effects on Estrogen Receptor Signaling in Cell Models of Human Epidermal Growth Factor Receptor 2–Negative Breast Cancer with Acquired Endocrine Resistance

Acquired endocrine resistance in estrogen receptor (ER)alpha+/human epidermal growth factor receptor 2-negative (HER2-) breast cancer has been associated with modest adaptive increases in HER2, although exactly how aberrant HER2 signaling affects the ERalpha pathway is poorly understood. We investigated (a) whether the epidermal growth factor receptor/HER2 inhibitor lapatinib could restore endocrine responsiveness in cell models of acquired endocrine resistance with modest increases in HER2, and (b) the nature of ERalpha-HER2 cross-talk in this process. Combination growth studies, ERalpha transcription, immunoblot, and gene expression assays were conducted in two models of acquired resistance to (a) estrogen deprivation (long-term estrogen-deprived cells) and (b) tamoxifen (long-term tamoxifen-treated cells), and in hormone sensitive controls. Changes in ERalpha, PgR, and HER2 were assessed in samples from patients treated with tamoxifen. Both cell models of acquired endocrine resistance showed modest adaptive upregulation in HER2, and lapatinib restored endocrine sensitivity in both. The effect of lapatinib on ERalpha signaling varied markedly depending on the nature of the HER2/ERalpha cross-talk. In long-term estrogen-deprived cells characterized by enhanced ERalpha function, lapatinib suppressed ERalpha genomic activity (as measured by pERSer118, ERalpha transcriptional activity, and PGR gene expression). In contrast, in long-term tamoxifen-treated cells with reduced ERalpha activation, lapatinib reactivated ERalpha genomic function. Twenty percent of tamoxifen-resistant patients relapsed with modest increases in HER2 and either suppressed or enhanced ERalpha/PgR expression. Aberrant GFR signaling can augment or suppress ERalpha function. Regardless, interrupting the HER2/ERalpha cross-talk with lapatinib can restore endocrine sensitivity and should be investigated as a therapeutic strategy in combination with endocrine therapy in ERalpha+/HER2- patients with acquired endocrine resistance.

Phosphoinositide-3-Kinase Catalytic Subunit Inhibition Does Not Produce Synthetic Lethality in Estrogen Receptor Positive Breast Cancer Cells with Acquired Resistance to Estrogen Deprivation.

Abstract Background: Several phosphoinositide-3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical development. However, targeting strategies for PI3K catalytic subunits for treatment in estrogen receptor positive (ER+) disease have not been fully developed. We have recently reported that targeted inhibition of the two major PI3K catalytic subunits, p110α and p110β, using RNA interference (RNAi) induces apoptosis in short term estrogen-deprived ER+ breast cancer cells. Similarly, treatment with BEZ235, a clinical grade p110α/mammalian target of rapamycin (mTOR)-selective inhibitor, also promoted apoptosis in estrogen-deprived ER+ breast cancer cells. Importantly, we found that estradiol significantly suppressed death in ER+ cells induced by inhibition of PI3K catalytic subunits through both RNA interference and BEZ235. In contrast estrogen deprivation or PI3K inhibition individually caused little or no increase in apoptosis, indicating that there is a synthetic lethal interaction with estrogen deprivation/PI3K inhibitor combination treatment in ER+ breast cancer. These data suggest that combining estrogen deprivation with PI3K inhibition may improve clinical outcome by eradicating ER+ cells through increased tumor cell kill. We have extended these studies in order to determine the effect of PI3K catalytic subunit inhibition in long-term estrogen deprived (LTED) cells, a model for acquired resistance to endocrine therapy.Methods: LTED sub-lines of the ER+ MCF7 and T47D breast cancer cell lines were derived by depriving cells of estrogen for 6–9 months in phenol red-free medium containing charcoal-stripped serum after which time cells grow readily without supplemental estrogen. The effects of PI3K inhibition using RNAi and treatment with BEZ235 or BGT226, a p110α-selective inhibitor, were compared in short-term estrogen deprived MCF7 and T47D cells and their LTED derivatives by measuring effects on cell signaling, growth and apoptosis. Cell signaling was evaluated through western blotting, cell growth was measured using resazurin reduction and apoptosis was quantified by flow cytometry using the TUNEL assay.Results: PI3K inhibition using p110α RNAi or treatment with BEZ235 inhibited the growth of MCF7 LTED cells in the absence of supplemental estrogen. However, p110α RNAi or treatment with BEZ235 or the clinical grade p110α-selective inhibitor BGT226 failed to significantly induce apoptosis in MCF7 LTED cells. The effects of PI3K inhibition in T47D LTED cells are under investigation and will be presented.Conclusions: These results suggest that that the efficacy of PI3K inhibitors will be blunted after the onset of acquired resistance to estrogen deprivation through treatment with aromatase inhibitors and that a combined ER/PI3K targeting strategy in ER+ breast cancer treatment should be employed in the endocrine therapy sensitive phase of the disease. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3131.

The Effect of the IGF1R Antibody R1507 on ER Positive Breast Cancer Cell Lines Growth and Survival.

Abstract Background: The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF-1 and IGF-2 and is expressed in most breast cancer cells, where receptor activation induces proliferative, cell-survival, and transforming activities. Because crosstalk exists between ER and IGF1R blockade of both pathways could increase antitumor activity. Further, IGF signaling may be enhanced in ER+ cells that have become refractory to long term estrogen deprivation (LTED) explaining resistance to aromatase inhibitor therapy. The effect of the selective IGF1R antibody (R1507) was therefore assessed in a series of breast cancer cell lines in both estrogen-dependent and estrogen-independent growth phases.Methods: The ER positive breast cancer cells lines MCF7, T47D and HCC712 were kept in phenol red-free medium containing 5% charcoal-stripped serum. Several doses of R1507 were used to evaluate effects on cell proliferation, signaling and apoptosis. Western blotting with phospho-specific antibodies was used to observe effects on cell signaling, cell growth was measured using resazurin reduction and apoptosis was quantified by flow cytometry using the TUNEL assay. STED (short-term estrogen deprived) cells were treated with R1507 between 2 and 3 weeks after estrogen depletion and LTED were treated with R1507 after 6+ months of estrogen depletion.Results: R1507 suppressed IGF1R expression and inhibited IGF-1 stimulated IGF1R and AKT phosphorylation in all cell lines. R1507 had anti-proliferative effects in MCF7 cells in both estrogen-dependent and independent growth phases and the magnitude of the effect was similar to fulvestrant. R1507 had a smaller effect on cell proliferation in HCC712 cells and no effects on proliferation were observed in T47D cells. Antibody treatment resulted in significant induction of apoptosis in MCF7 STED but MCF7 LTED cells were resistant to the apoptotic effects of IGF1R blockade.Conclusions: R1507 had an inhibitory effect on IGF1R signaling and an anti-proliferative effect in two of the three cell lines tested. In addition R1507 induced synthetic lethality in combination with acute estrogen deprivation in MCF7 cells, an effect we have recently reported for direct PI3 kinase inhibitors under the same circumstances (1). While the proapoptotic effects of R1507 were lost in the estrogen-independent growth phase of MCF7 cells, the anti-proliferative effect was not, indicating that R1507 might have efficacy in patients with endocrine therapy resistant tumors. In other cell lines the efficacy of R1507 was variable and synthetic lethality was not observed, suggesting that the development of predictive biomarkers for R1507 should be a priority. We are currently addressing the activity of R1507 in a clinical trial with two cohorts, one with acquired resistance to letrozole, where the antibody is added to continued letrozole treatment and a second group of patients with stable disease on letrozole to determine if R1507 can induce tumor regression under these circumstances.1. Crowder et al Cancer Research 69:3955-62, 2009 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3126.

Demethylation of promoter C region of estrogen receptor α gene is correlated with its enhanced expression in estrogen-ablation resistant MCF-7 cells

Long-term estrogen deprivation (LTED) MCF-7 cells showing estrogen-independent growth, express estrogen receptor (ER) α at a much higher level than wild-type MCF-7 cells. Enhanced expression of ERα associated with partial localization of ERα to the plasma membranes in LTED cells is thought to be an important step for acquisition of estrogen-ablation resistance. In this study, we compared the regulation of ERα gene expression between wild type and LTED cells, examining the usage of the promoters A and C as well as their methylation status. We found that transcription from the promoter C was drastically enhanced in LTED cells, compared with that in wild-type cells. Furthermore, the promoter C region was highly unmethylated in LTED cells, but partially methylated in wild-type cells. Our findings imply that demethylation of promoter C region in the ERα gene is in part responsible for the enhanced expression of ERα gene in LTED cells.

Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: Conference Summary Statement

Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: Conference Summary Statement

Activation of the MAPK pathway enhances sensitivity of MCF-7 breast cancer cells to the mitogenic effect of estradiol.

Long-term estrogen deprivation causes human breast cancer cells to develop hypersensitivity to the mitogenic effect of estradiol (E(2)). Our prior studies demonstrated an association between enhanced MAPK activation and hypersensitivity in long-term estrogen-deprived (LTED) MCF-7 cells. Herein, we report that MAPK is constitutively activated in LTED cells and not dependent on serum factors. Additionally, activated MAPK levels fall upon reversion of the hypersensitivity. Importantly, we now provide direct evidence that enhanced MAPK causes hypersensitivity to E(2). We activated MAPK in wild-type MCF-7 cells using TGFalpha, and demonstrated a 2-3 log enhancement of sensitivity to E(2). PD98059 abrogated the TGFalpha-induced effect, indicating that MAPK activation is responsible for E(2) hypersensitivity. To determine the level at which MAPK activation enhanced E(2) sensitivity, we examined the dose-response effects of E(2) on several transcriptional readouts, including ERE-reporter activity and the levels of progesterone receptor and pS2. Wild-type and LTED cells exhibited nearly identical responses to E(2), suggesting that mechanisms downstream of estrogen receptor-mediated transcription are involved in inducing hypersensitivity. In support of this possibility, LTED and TGFalpha-treated wild-type cells were hypersensitive to the effects of E(2) on the key cell cycle regulator, E2F1.

Short- and long-term estrogen deprivation of T47D human breast cancer cells in culture

The effects of short- and long-term estrogen deprivation on T47D human breast cancer cells was studied. Cells were routinely grown in an estrogenized environment in media containing fetal bovine serum with phenol red indicator. Cells were estrogen deprived (grown in media containing dextran-coated charcoal-stripped fetal bovine serum without phenol red) for either 10 days or at least 8 months, and effects on genotype, receptor content, and cell growth responsiveness were studied. Cells grown in an estrogenized environment are hypertetraploid, whereas long-term estrogen-deprived cells have become hyperdiploid. Short-term estrogen-deprived cells exhibit a decreased growth rate and progesterone receptor (PgR) content, while estrogen receptor (ER) content is not significantly altered. ER mRNA levels are significantly decreased in these cells. Incubation of these cells with estradiol (10−10 M) for 6 days causes a 5-fold stimulation in cell growth and this stimulation can be inhibited by the antiestrogens 4-hydroxytamoxifen (4-OHT), ICI 164,384, and RU 39411.Cells cultured under long-term estrogen deprivation exhibited an increased growth rate and were refractory to the effects of estradiol and of 4-OHT on cell growth. These cells were ER negative with low levels of PgR; however, one clone of this line was found to be ER and PgR negative. No mRNA for the ER was detected in this line or its clone. With these cell lines it is possible to study the biological characteristics necessary for the outgrowth of a receptor negative, hormone nonresponsive cell population from a receptor positive, hormone-responsive population grown in a estrogen-free environment.