Antiproliferative Effects Of Tamoxifen Research Articles

The vitamin D analog EB1089 sensitizes triple-negative breast cancer cells to the antiproliferative effects of antiestrogens

PurposePatients bearing estrogen receptor (ER)α-negative breast cancer tumors confront poor prognosis and are typically unresponsive to hormone therapy. Previous studies have shown that calcitriol, the active vitamin D metabolite, can induce ERα expression in ERα-negative cells. EB1089, a calcitriol analog with reduced calcemic effects, exhibits greater potency than calcitriol in inhibiting cancer cell growth. However, the impact of EB1089 on ERα expression in triple-negative breast cancer (TNBC) cells remains unexplored. This study aims to investigate whether EB1089 could induce functional ERα expression in TNBC cell lines, potentially enabling the antiproliferative effects of antiestrogens. Materials and methodsTNBC cell lines HCC1806 and HCC1937 were treated with EB1089, and ERα expression was analyzed using real-time PCR and Western blots. The transcriptional activity of induced ERα was evaluated through a luciferase reporter assay. The antiproliferative effects of tamoxifen and fulvestrant antiestrogens were assessed using the sulforhodamine B assay in the EB1089-treated cells. ResultsOur findings indicated that EB1089 significantly induced ERα mRNA and protein expression in TNBC cells. Moreover, EB1089-induced ERα exhibited transcriptional activity and effectively restored the inhibitory effects of antiestrogens, thereby suppressing cell proliferation in TNBC cells. ConclusionEB1089 induced the expression of functional ERα in TNBC cells, restoring the antiproliferative effects of antiestrogens. These results highlight the potential of using EB1089 as a promising strategy for re-establishment of the antiproliferative effect of antiestrogens as a possible management for TNBC. This research lays the foundation for potential advancements in TNBC treatment, offering new avenues for targeted and effective interventions.

Agonistic Effects of Deinoxanthin on Tamoxifen Antiproliferative Activity on HER2 Positive Breast Cancer: An In vitro Study on MDA-MB-453

A good understanding of the pathogenesis of breast cancer, in which in 2020 an estimated 684,996 women across the world died, plays a vital role in the development of treatment methods. In recent years, estrogen receptor (ER), progesterone receptor (PR), and Epidermal Growth Factor receptor-2 (HER2) expression levels have been substantial prognostic markers. The combinational therapy approach is the use of two or more active ingredients or methods. The most important benefit of this treatment method is to reduce the development of drug resistance since the probability of the carcinoma being resistant to more than one drug at the same time is low. Tamoxifen, an estrogen receptor competitive and nonsteroidal drug, has been used for nearly 20 years to treat patients with hormone receptor-positive breast cancer. Deinoxanthin is a xanthophyll derivative purified from the cell wall of a radiation-resistant bacterium, Deinococcus radiodurans. This xanthine derivative has been shown in some studies to have proapoptotic and antiproliferative effects on some types of cancer. Within the study's scope, it aimed to increase the effectiveness of Tamoxifen with deinoxanthin. Using the ACTB gene as a reference to investigate the synergistic effect of Tamoxifen and deinoxanthin on the MDA-MB-453 breast cancer cell line; Expression levels of BAX, CASP-3, BCL-2, and HER2 genes were examined by RT-qPCR method. The ELISA method determined the amounts of BAX, CASP-3, BCL-2, and HER2 proteins and compared them with RT-qPCR results. It was determined that the antiproliferative effect of Tamoxifen on the HER2 positive breast cancer cell line was increased with the combination of deinoxanthin.

Abstract P4-02-14: Breast cancer cell/adipocyte crosstalk in obesity hampers the efficacy of tamoxifen

Abstract Despite the improvements in the outcomes of breast cancer patients following the implementation of endocrine therapy into daily oncology practice, the development of ‘de novo’ or acquired resistance to these treatments has become a major limitation. To date, several hallmarks of hormonal resistance have been proposed, including ERα loss or mutations, activation of growth factor-mediated signaling pathways, and alterations of key cell cycle checkpoints. More recently, it was proven that a reduced response to these therapeutic regimens may rely not only on tumor-cell intrinsic factors, but it is also dependent on the surrounding microenvironment and on the host characteristics. Indeed, clinical studies have suggested that obesity, in addition to promoting breast cancer development and progression, represents a heavyweight player driving breast cancer endocrine resistance. However, the influence of adipocytes and their secretome on the efficacy of hormone therapy has not properly investigated. In a model of co-culture, we demonstrated that the anti-proliferative effects of Tamoxifen on anchorage-dependent and anchorage-independent growth of MCF-7 breast cancer cells were counteracted by conditioned media (CM) released by fully differentiated 3T3-L1 cells (3T3-L1 A), a widely used 'in vitro' model of white adipocytes. Moreover, pre-incubation of MCF-7 cells with adipocyte-derived CM reduced their sensitivity to the inhibitory effects of Tamoxifen on motility, as evidenced by wound-healing and transmigration assays. Interestingly, the growth and the motility of Tamoxifen-resistant breast cancer cells were dramatically increased in the presence of adipocyte-derived CM, further suggesting a supportive role of adipocyte secretome in the development and progression of endocrine-resistant phenotypes. Quantitative transcriptome profiling of Tamoxifen-resistant cells revealed 917 differentially expressed genes (FDR ≤ 0.05), of which 243 were upregulated and 180 were downregulated in response to adipocyte-CM. Among these major modifications, the most significant down-regulated gene was Thioredoxin-interacting protein (TXNIP, 5-fold), a tumor suppressor participating in the metabolic reprogramming and oxidative stress in various solid cancers. These results well correlated with data obtained in retrospective analyses showing that low TXNIP expression in breast cancer patients was associated with a statistically significant poorer recurrence-free (RFS), overall (OS), post-progression (PPS) and distant metastasis-free (DMFS) survival compared to high TXNIP-expressing patients. This relevant discrimination is also achieved in tamoxifen-treated patients, highlighting a role of TXNIP for identifying patients at high risk of progression. These findings bring insights on adipocytes and mammary cancer cell interactions during Tamoxifen therapy, particularly in overweight/obese people and add evidence to advocating obesity as a parameter to be considered when identifying treatments for patients with ER-positive breast cancer. Citation Format: Amanda Caruso, Luca Gelsomino, Giusi La Camera, Cinzia Giordano, Salvatore Panza, Daniela Bonofiglio, Stefania Catalano, Sebastiano Andò, Ines Barone. Breast cancer cell/adipocyte crosstalk in obesity hampers the efficacy of tamoxifen [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-14.

Erythropoietin Potentiates the Anti-Proliferative Effect of Tamoxifen in Ovarian Adenocarcinoma A2780 Cells

We have recently shown that erythropoietin receptor (EPOR) protects cancer cells from tamoxifen (TAM)-induced cell death in the absence of erythropoietin (EPO). In this study, we analyzed the effect of EPOR silencing and EPO treatment on the response to TAM in human ovarian adenocarcinoma cells A2780. We demonstrated that the EPOR siRNA silencing decreases cell proliferation and sensitizes and/or potentiates the anti-proliferative effect of TAM on A2780 cells. Similarly, the combined effect of EPO and TAM treatment significantly reduced cell proliferation compared to TAM alone. Our in vitro results indicated the need for further investigation of EPO effects on a similar in vivo model.

Abstract P2-06-01: Characterisation of C11orf67, an oncogenic driver in a new subtype of aggressive endocrine receptor positive breast cancer

Abstract The recent integration of both genomic and transcriptomic datasets have added a further dimension to the landscape of breast cancer (BrCa) subtyping, defining novel functional subgroups with distinctive oncogenic drivers that carry important implications for therapy. This integrative clustering has unveiled a novel subtype of hormone receptor positive (HR+) BrCa associated with high proliferation and very poor survival characterised by copy number amplification and overexpression of a cluster of candidate oncogenic drivers at the 11q13.5-14 locus (1). At the heart of this amplicon we have demonstrated the selective overexpression of C11orf67/AAMDC (Adipogenesis associated Mth938 domain containing) which encodes a hypothetical protein of 122 aa with unknown function. In a pilot tissue microarray of 75 BrCa cases C11orf67 amplification and expression were significantly correlated with hormone receptor positivity. These positive cases also demonstrated high risk features with 75% demonstrating lymph node involvement. In functional elucidation studies knockdown of C11orf67 in the highly expressing T47D cell line lead to decreased cell proliferation, cell migration, anchorage independent cell growth and induction of senescence. T47D xenografts with stable shRNA-induced C11orf67 knockdowns introduced into BALB/c mice showed significantly lower tumour volumes relative to T47D with empty vector. A genome wide analysis of these T47D-C11orf67 shRNA cells compared to T47D-empty vector cells using the Illumina HumanHT-12 platform demonstrated 40 differentially expressed genes. Network analysis revealed a proliferation node, enriched in cell cycle proteins, and a metabolic node comprising several biosynthetic enzymes such as MTHFD1L involved in one-carbon folate metabolism. Supporting this link and pointing to potential utility in chemotherapy selection, induction of ectopic C11orf67 expression in MCF7 cells increased sensitivity to fluorouracil and methotrexate but not to paclitaxel. Investigating potential novel binding partners and effectors, in yeast two hybrid screening C11orf67 was a found to associate strongly with RABGAP1L, a protein involved in controlling GTPase signalling, protein trafficking, and autophagy. Exploring the molecular cues that control C11orf67 expression, our data suggest the locus is regulated by transcription factors associated with high proliferation and metabolic control, notably Myc and NFkB, as well as HRs. E2 lead to a significant down-regulation of C11orf67 in T47D cells, which was reversed by the antiestrogen drug tamoxifen, whereas PG significantly increased C11orf67 levels. In keeping with this MCF7 cells ectopically expressing C11orf67 were resistant to the anti-proliferative effects of tamoxifen compared to the parent cell line. These observations endorse C11orf67 as a novel oncogenic driver with exciting therapeutic potential, which could serve to distinguish the HR+ tumours at high risk of relapse and guide both the selection of current chemotherapeutical and endocrine treatments as well as the design of future precision therapeutics, notably anti-folate/one carbon drugs and novel endocrine agents.

Synergy of leptin/STAT3 with HER2 receptor induces tamoxifen resistance in breast cancer cells through regulation of apoptosis-related genes.

Tamoxifen is a major treatment modality for estrogen receptor positive breast cancer, but the occurrence of resistance remains a problem. Recently, obesity-related leptin has been found to interfere with tamoxifen in breast cancer MCF-7 cells. In the present study we investigated the effect of leptin on three tamoxifen-treated breast cancer cell types (i.e., MDA-MB-231, MCF-7 and MCF-7/HER2). The effect of tamoxifen/leptin treatment was evaluated using a MTT cell viability assay. mRNA expression was assessed by real time PCR and protein expression by Western blotting. WWOX, Survivin and BCL2 gene promoter activities were evaluated by chromatin immunoprecipitation. Cell viability assays revealed that estrogen receptor negative MDA-MB-231 cells were resistant, that estrogen receptor positive MCF-7 cells were sensitive and that MCF-7/HER2 cells were relatively resistant to tamoxifen, while leptin co-administration 'rescued' MCF-7 and, especially, MCF-7/HER2 cells from the anti-proliferative effect of tamoxifen. The cell lines also exhibited a different phosphorylation status of STAT3, a transcription factor that is activated by the obesity related leptin receptor b (Ob-Rb). Most importantly, chromatin immunoprecipitation assays revealed differential STAT3 binding to the anti-apoptotic BCL2 and pro-apoptotic WWOX gene promoters in MCF-7 and MCF-7/HER2 cells, leading to concomitant modifications of its mRNA/protein expression levels, thus providing a selective advantage to HER2 over-expressing MCF-7/HER2 cells after treatment with tamoxifen and tamoxifen plus leptin. Our study provides novel evidence indicating that synergy between the leptin/Ob-Rb/STAT3 signalling pathway and the HER2 receptor protects tamoxifen-treated HER2 over-expressing cells from the inhibitory effect of tamoxifen through differential regulation of apoptosis-related genes.

OP0026 Gene expression profiling identifies FYN as important in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy

Background We aimed to elucidate the molecular mechanisms of tamoxifen resistance in breast cancer. Methods We did gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant cell lines versus the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were functionally linked to cell proliferation, death, and control of gene expression, and include FYN, PRKCA, ITPR1, DPYD, DACH1, LYN, GBP1, and PRLR. We also evaluated the subcellular localisation of FYN in primary breast tumours from two cohorts of endocrine-treated ER+ breast cancer patients, one with advanced disease (n = 47) and the other with early disease (n = 76). Findings Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of tamoxifen-resistant cell lines, while exerting no significant effect on MCF-7/S0.5 cells. Moreover, overexpression of FYN in parental tamoxifen-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to tamoxifen treatment, while knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth-promoting and survival-promoting function of FYN in MCF-7 cells. FYN knockdown in tamoxifen-resistant cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, could overcome the antiproliferative effects of tamoxifen. Evaluation of the subcellular localisation of FYN in breast cancer patients showed that in those with advanced disease, plasma membrane-associated FYN expression strongly correlated with longer progression-free survival (p Interpretation Our results indicate that FYN plays an important part in tamoxifen resistance, and its subcellular localisation in breast tumour cells could be an important novel biomarker of response to endocrine therapy in breast cancer.

Abstract P5-09-13: HDAC inhibition re-sensitizes acquired hormone resistant cells to the cytotoxic effect of tamoxifen

Abstract Breast cancer still continues to be a major cause of cancer related deaths in women, second only to lung cancer. Administering anti-estrogens and aromatase inhibitors, both in the adjuvant and metastatic settings, in estrogen receptor (ER) positive disease, is one of the most effective treatment strategies. However, prolonged exposure to these drugs leads to the emergence of resistance in about 40% of those who initially respond. Hence, the emphasis of a multitude of studies is to understand the underlying mechanisms of this acquired resistance and implementing means to overcome it. Recent studies have implicated epigenetic modulation of gene expression in the development of resistance to the anti-estrogen tamoxifen. A clinical trial conducted by our group has demonstrated the efficacy of combining the HDAC inhibitor vorinostat with tamoxifen in patients who had progressed on prior anti-estrogen therapies and showed a total 40% clinical benefit (19% objective response and 21% stable disease for more than 6 months). In an effort to identify patients most likely to benefit from this novel therapy, we sought to elucidate the mechanism behind the clinical efficacy of this combination. To this end, we have generated an in vitro tamoxifen resistant cell line (TAMR) by long-term exposure of MCF7 cells to 4-hydroxy tamoxifen. Significantly reduced anti-proliferative effect of tamoxifen and other anti-estrogens in TAMR compared to the sensitive MCF7 cells demonstrates the establishment of resistance to anti-estrogens in this cell line. In TAMR cells, addition of an HDAC inhibitor reverts resistance to tamoxifen. Although estrogen receptor (ER) expression in TAMR cells appears unaltered, the classical genomic signaling of ER in these resistant cells is suppressed and unresponsive to ligands, as deduced from transcription of ER response genes and luciferase assay of an ERE-luciferase construct. However, the ER remains important, since siRNA mediated depletion of ER inhibits cell growth in TAMR cells. Treatment with the dual EGFR/Her2 kinase inhibitor or an AKT inhibitor significantly inhibits the growth of these cells only when combined with tamoxifen, indicating the importance of crosstalk between pathways. Furthermore, Akt and mTOR protein and activities are down regulated by HDAC inhibition, which are further reduced when combined with tamoxifen. SiRNA mediated depletion of ER leads to reduced Akt and mTOR activities which suggests that ER may act as a driver, possibly through its non-genomic function at the plasma membrane activating members of the growth factor signaling pathways. Studies are ongoing to further characterize the interaction of HDAC and ER inhibition on these signaling pathways and to determine their significance to resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-13.

Abstract 1190: Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy .

Abstract To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer we performed microarray profiling and identified 366 genes with altered expression in 4 tamoxifen-resistant (TamRs) vs. the parental tamoxifen-sensitive MCF-7/S0.5 cell lines. Most of these genes were functionally linked to cell growth, proliferation, death and control of gene expression and included FYN, PRKCα, ITPR1, DPYD, DACH1, GBP1 and PRLR. Increased expression of FYN was observed in TamR cells and treatment with FYN-specific interfering RNA or a specific Src family kinase (SFK) inhibitor restored the anti-proliferative effect of tamoxifen in these cells. TamR cells also expressed increased levels of phosphorylated breast cancer antiestrogen resistance protein 1 (pBCAR1), an important downstream SFK substrate in the MAPK pathway. Evaluation of the association of subcellular localization of FYN in cancer tissue and clinical endpoints was made in 2 cohorts of ER+ breast cancer patients, one with advanced disease (N=47) and the other with early breast cancer (N=76). The result showed that plasma membrane-associated FYN expression in the cancer cells strongly correlated with longer progression-free survival compared to FYN expression not associated with the plasma membrane (p<0.0002) in the advanced disease cohort. Similarly, in the early breast cancer patients, plasma membrane associated FYN expression was associated with longer metastasis-free survival (p<0.003) and overall survival (p<0.0005) independent of tumor size, grade and node status. Our results indicate that FYN plays an important role in tamoxifen resistance, and its subcellular location in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer. Citation Format: Daniel Elias Roro, Henriette Vever, Henrik Ditzel. Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2013-1190

Abstract 4349: Genome-wide kinase and phosphatase siRNA screen identifies clinically relevant targets in IGF1R signaling-mediated antiestrogen resistance in breast cancer.

Abstract Antiestrogen resistance of breast cancer has been linked to increased growth factor receptor signaling such as invoked by the insulin-like growth factor 1 (IGF-1) receptor (IGF1R). We have previously shown that increased IGF1R signaling by ectopic IGF1R overexpression in MCF7 cells (MCF7-IGF1R) directly drives an estrogen receptor (ER)-independent cell proliferation in MCF7 cells that leads to antiestrogen resistance. To identify components of the downstream IGF1R signaling pathways that mediate antiestrogen resistance, we have performed a human genome-wide kinase and phosphatase siRNA screen under tamoxifen resistance condition (1 μM 4-OH-tamoxifen, 1 nM estradiol, and 100 ng/ml IGF-1) in MCF7-IGF1R cells. We identified 174 kinases and phosphatases that increased or decreased tamoxifen resistance. Of these, 14 significantly correlated with the time to disease progression of metastatic breast cancer patients treated with first-line tamoxifen monotherapy, including ADRA1B, BMPR1B, CDK10, CDK5, EIF2AK1 and MAP2K5. Low expression of these six genes in patients correlated with short time to disease progression and silencing of these genes in MCF7-IGF1R cells further increased proliferation under tamoxifen resistance condition. Low expression of the other eight genes in patients correlated with longer time the time to disease progression and silencing of these genes in MCF7-IGF1R cells decreased proliferation under tamoxifen resistance condition Therefore, these latter genes may represent novel drug targets that may be useful to restore tamoxifen sensitivity in breast cancer. Knockdown of two of these genes suppressed IGF1R-mediated effects and restored the anti-proliferative effect of tamoxifen in a dose dependent manner, suggesting synthetic lethality between the knockdowns and tamoxifen. Further study of one of these genes revealed that stable knockdown inhibits proliferation and three-dimensional growth under tamoxifen resistance condition, accompanied with an increase in G1/G0 arrest and mild induction of apoptosis. These effects are independent of PI3K/AKT and MAPK/ERK signaling pathways, and suggest that an as yet unknown pathway plays a decisive role in tamoxifen resistance induced by IGF1R signaling. Citation Format: John H. Meerman, Yinghui Zhang, Maurice P. Jansen, Ramakrishnaiah Siddapa, Sreenivasa Ramaiahgari, Leo Price, Erik Danen, Els M. Berns, John Foekens, Bob van de Water. Genome-wide kinase and phosphatase siRNA screen identifies clinically relevant targets in IGF1R signaling-mediated antiestrogen resistance in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4349. doi:10.1158/1538-7445.AM2013-4349

FOXM1 is a transcriptional target of ERα and has a critical role in breast cancer endocrine sensitivity and resistance

Previous data have shown that FOXM1 expression is elevated in breast cancer tissues and is strongly correlated with the expression pattern of ERα in breast cancer cells. The expression of ERα is a good prognostic factor in breast cancer, as about two-thirds of these ERα-positive patients respond to treatment with antiestrogens. However, approximately one-half of the patients that initially respond to hormonal therapy develop resistance. Since FOXM1 is critical for the progression of the cell cycle, we investigated the regulation of FOXM1 by ERα and its role in endocrine sensitivity and resistance in breast cancer cells. We firstly observed by quantitative RT-PCR a strong and significant positive correlation between ERα and FOXM1 mRNA expression in breast cancer patient samples. We showed that FOXM1 protein and mRNA expression was regulated by ER ligands. We also demonstrated that ectopic conditional expression of ERα, in the presence of estrogens, leads to induction of FOXM1 expression in ER-negative U2OS cells. Using reporter gene assays, we demonstrated that ERα activates FOXM1 transcription through an estrogen-response element site. The direct binding of ERα to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation analysis. Importantly, silencing of FOXM1 by RNA interference abolishes the estrogen-mediated MCF-7 cell proliferation. Conversely, ectopic expression of a constitutively active FOXM1 can abrogate the cell cycle arrest mediated by the antiestrogen tamoxifen. Taken together, the results clearly demonstrate FOXM1 as a key mediator of the mitogenic functions of ERα and estrogen in breast cancer cells. Our findings that antiestrogens repress FOXM1 expression in endocrine-sensitive but not endocrine-resistant breast carcinoma cell lines and that ectopic expression of an active FOXM1 can abrogate the anti-proliferative effects of tamoxifen also suggest that deregulation of FOXM1 may also contribute to antiestrogen insensitivity.

Use of Antidepressants in Patients with Breast Cancer Taking Tamoxifen

Tamoxifen, a selective estrogen modulator has been used for more than three decades to treat all stages of estrogen receptor (ER)-positive breast cancer and to prevent the disease. Tamoxifen is a pro-drug that requires metabolic activation to 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) to elicit its pharmacological activity. Endoxifen has identical properties and potency with 4hydroxytamoxifen, but is present in concentrations up to 10-fold higher than 4-hydroxytamoxifen. The cytochrome P450 2D6 (CYP2D6) enzyme plays a key role in converting tamoxifen into its active metabolites with significantly greater affinity for the ER and greater ability to inhibit cell proliferation. Genetic variants in the CYP2D6 gene may result in CYP2D6 enzymes with reduced or null activity, thereby decreasing the anti-cancer effect. In addition to genetic inactivation of CYP2D6, inhibitors of CYP2D6, including some antidepressants to treat hot flashes or depression in patients with breast cancer, may also alter enzyme activity and negatively affect the outcomes of patients receiving adjuvant tamoxifen. This article reviews and discusses the following issues: tamoxifen metabolism, antiproliferative effects of tamoxifen and its metabolites, CYP2D6 genetic polymorphisms, treatment for hot flashes and depression in breast cancer, and the pharmacological interactions between tamoxifen and antidepressants via CYP2D6. Although routine CYP2D6 testing is not recommended yet, coadministration of potent or intermediate CYP2D6 inhibitors in women taking tamoxifen should be avoided.

Serum S100B as a Prognostic Indicator in Patients with Breast Cancer.

Abstract BackgroundThe nuclear receptor co-activator SRC-1 has recently been implicated as an important mediator of breast cancer metastasis in vitro. These findings are in keeping with previous reports from our group reporting significantly reduced disease free survival in patients who express SRC-1 when compared to those who do not. We identified the developmental protein HOXc11 as interacting with SRC-1 in tamoxifen resistant breast cancer cells. Dysregulation of homeobox genes has been reported in a wide variety of both solid organ and haematological malignancies. The putative target gene of HOXc11, S100B is a secreted protein, known to be of prognostic significance in malignant melanoma. The aim of this work was to examine the relationship between HOXc11, SRC-1 and S100B and their relevance to disease progression in breast cancer.Materials and MethodsAll chromatin immunoprecipitation (ChIP) and proliferation assays were performed in tamoxifen resistant breast cancer cells derived from MCF7s. Immunohistochemistry was performed for S100B, HOXc11 and SRC-1 on a tissue microarray of 560 patients with invasive breast cancer with a median follow up of 6.5 years. S100B was assessed using an ELISA based technique (Diasorin, USA) on serum samples of a prospective cohort of patients with breast cancer prior to any treatment.ResultsChIP assays confirmed recruitment of both HOXc11 and SRC-1 to the promoter region of S100B after treatment with tamoxifen at both 45 minutes and 2 hours when compared to control. Treatment of tamoxifen resistant cells with HOXc11 siRNA resensitised the cells to the anti-proliferative effects of tamoxifen. Immunohistochemistry revealed S100B to be a strong predictor of reduced disease free survival in patients receiving adjuvant endocrine treatment (Hazard ratio 5.82; p<0.0001). Tumour expression of S100B was associated with expression of both HOXc11 and SRC-1 providing translational evidence for a relationship between these proteins. Elevated serum levels (>0.15ug/l) of S100B were found in 10.5% of patients with breast cancer prior to treatment. Elevated S100B correlated with HER2 positivity (Fisher's exact: p=0.005) but not with any of the other standard clinico-pathological factors examined. From a cohort of 80 patients with known medium to long term follow up, elevated serum S100B showed a strong positive correlation with disease recurrence (p=0.017, odds ratio 9.38).DiscussionOur investigations into the prognostic significance of S100B in breast cancer have shown considerable promise. Given it's accessibility as a serum protein, quantifiable by ELISA, it offers potential as a marker for disease recurrence in patients with breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4052.

Physical and Functional Interactions between Cas and c-Src Induce Tamoxifen Resistance of Breast Cancer Cells through Pathways Involving Epidermal Growth Factor Receptor and Signal Transducer and Activator of Transcription 5b

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Therapeutic doses of tamoxifen only partially inhibit the non-genomic effects of estrogen: Evidence for additive anti-proliferative effect of tamoxifen with MEK inhibitor

3195 Background: Recent data suggests that non-genomic effects of Estradiol (E2) in estrogen-sensitive carcinoma cell lines result in the activation of the MAP-Kinase (MAPK) pathway (p44/42). MAPK ...

The interaction of the ER with ERBB2 and PI3K results in elevated levels of AKT and p90RSK in tamoxifen-resistant MCF-7 cells

Despite advances in endocrine therapies, the majority of patients receiving tamoxifen will eventually relapse while retaining functional estrogen receptors. We have previously shown, using an MCF-7 cell line (TamR) resistant to the anti-proliferative effects of tamoxifen, that elevated levels of phosphorylated AKT and p90RSK lead to an apparent ligand-independent phosphorylation of ERα ser167. Analysis of the growth factor receptors in these cells indicated elevated levels of both phosphorylated ERBB2 and total ERBB3, which we postulated formed heterodimers and activated the PI3 kinase pathway leading to elevated AKT. However, our recent data suggest that elevated AKT results from an interaction between ERα and ERBB2. This association is knocked out by treatment with the pure anti-estrogen ICI 182,780 and is absent in the WT parental cell line. Similarly, we have demonstrated an association between the p85 subunit of PI3K and ERα in TamR but not in WT cells. Treatment of the cell lines with the specific AKT inhibitor SH6 and the MEK1/2 inhibitor U0126 caused greater decrease in cell proliferation and concomitant ERα-directed transactivation in the TamR cells versus the WT, confirming that these pathways are integral to the TamR phenotype. To establish whether p90RSK or AKT was responsible for the phosphorylation of the estrogen receptor at ser167, TamR and WT cells were treated with SH6, U0126 or a combination of the two. Blocking either pathway individually had little effect on ERα ser167phosphorylation. However, a combination of the two inhibitors resulted in almost complete loss of phosphorylation. These data were confirmed using siRNA technology to suppress MAPK and AKT expression. Taken together these data suggest that, in this setting, the ER functions via a non-genomic mechanism, associating with ERBB2 and PI3K at the cell membrane leading to activation of both p90RSK and AKT. This in turn leads to phosphorylation of ERα ser167, ultimately regulating cell growth via genomic mechanisms. Although several of these complexes have previously been postulated, to our knowledge this is the first demonstration of this phenomenon in a tamoxifen-resistant cell line.

Tamoxifen inhibits cell proliferation via mitogen-activated protein kinase cascades in human ovarian cancer cell lines in a manner not dependent on the expression of estrogen receptor or the sensitivity to cisplatin.

Although tamoxifen (TAM), which is widely used in the treatment of breast cancer, also has a beneficial effect on cisplatin-refractory ovarian cancer, the biological mechanism of this effect has remained obscure. TAM, besides its action as an antiestrogen, also inhibits cell proliferation of estrogen receptor (ER)-negative cells by an unknown mechanism. Therefore, we examined the roles of the MAPK family in the antiproliferative effect of TAM on cisplatin-resistant Caov-3, which expresses ER and cisplatin-sensitive A2780, which does not express ER. The number of viable cells was reduced by TAM dose-dependently. TAM induced the activation of ERK, c-Jun N-terminal protein kinase (JNK), and p38 with different time courses. PD98059 canceled the reduction of the number of viable cells by 1 microM TAM and inhibited the TAM-induced cell-cycle arrest at the G(1) phase and dephosphorylation of the retinoblastoma protein. Either expression of dominant-negative JNK or pretreatment with SB203580 canceled the reduction of the number of viable cells by 5 microM TAM and inhibited the apoptotic nuclear changes and the cleavage of poly (ADP-ribose) polymerase induced by TAM. These results provide evidence that whereas the ERK cascade is involved in the induction of cell-cycle arrest at the G(1) phase by lower concentrations of TAM, the JNK or p38 cascade is involved in the induction of apoptosis by higher concentrations of TAM in both types of cells.

Potentiation of antiproliferative effects of tamoxifen and ethanol on mouse hepatoma cells by melatonin: possible involvement of mitogen-activated protein kinase and induction of apoptosis.

Melatonin, the major secretory product of the pineal gland, is in focus of many research areas because of its ability to scavenge free oxygen radicals and thereby protect cells and tissues from radical damage. Some studies suggest melatonin may be a possible therapeutic agent with potential clinical applications against pathological states due to reactive oxygen species. Here, we investigated the effects of melatonin on the mouse hepatoma cell line HEPA 1-6, coincubated with ethanol, and tamoxifen, respectively. Cell proliferation rates were detected by the 3-[4,5 dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. A dose-dependent inhibition of the proliferative activity by melatonin was observed from 640 microM to 3 mM, which was significantly higher (P < 0.01) than with the solvent (ethanol) alone. Concentrations of 320 microM and less had no effect on cell proliferation. This antiproliferative effect might be because of the prolonged activation of mitogen-activated protein kinase which was activated by phosphorylation 15 min after the induction with melatonin. Furthermore, apoptosis was found to be enhanced by melatonin (75% more than with the solvent alone, P < 0.001). Finally, we show that the inhibitory effect of tamoxifen (25 microM) is markedly enhanced by the coincubation with melatonin (1.3 mM) up to 75% (P < 0.001). These data show that the antiproliferative effects of tamoxifen and ethanol, respectively, on mouse hepatoma cell line HEPA 1-6 are enhanced by melatonin. Although at the conditions described here the antiproliferative effects of melatonin occur at supraphysiological concentrations, these data may help to support clinical studies where melatonin is given simultaneously with tamoxifen or other standard chemotherapeutica.

Combination effects of tamoxifen plus 5-fluorouracil on gastric cancer cell lines in vitro

We tested the effect of tamoxifen alone and tamoxifen plus 5-fluorouracil (5-FU) on proliferation of two different types of gastric cancer cell lines using the WST-1 method. A high dose of tamoxifen suppressed the proliferation of KATOIII cells (poorly differentiated adenocarcinoma), but MKN28 cells (well-differentiated adenocarcinoma) were not affected. The combination of the two drugs resulted in a synergistic anti-proliferative activity on KATOIII cells. On the other hand, in the combination therapy, tamoxifen stimulated MKN28 cells to proliferate in a dose-dependent manner. TGF-β1 secretion was not changed in KATOIII cells by tamoxifen plus 5-FU treatment but was down-regulated in MKN28 cells. Both cancer cell lines were judged as intracellular estrogen receptor (ER) negative. These data suggest that the anti-proliferative effects of tamoxifen plus 5-FU on KATOIII cells were not dependent on ER expression or TGF-β1 secretion. On the other hand, their proliferative effects on MKN28 cells might be, in part, caused by the reduced secretion of TGF-β1.

Sequential α-interferon and tamoxifen: in-vitro reversal of MDR-1 mediated multidrug resistance in the MCF-7 cell line

Abstract Chemotherapy drug resistance in breast cancer is mediated in part by the MDR-1 gene product, P-glycoprotein (PGP). Tamoxifen (TMX) is able to modulate the MDR-1 phenotype in vitro. Interferons modulate other biologic effects of TMX and may enhance the effect of TMX on PGP function. We have investigated the effects of combinations of α-interferon (IFN) and TMX on the MDR-1 phenotype and function in MCF-7 cells. IFN and TMX were both growth inhibitory to MCF-7 cells. Preincubation with IFN potentiated the antiproliferative effect of TMX even at low concentrations. Immunocytochemically detected staining for the external domain of PGP was increased by both TMX and IFN and the effect of the two agents appeared to be synergistic. Despite the increased expression of the external domain of PGP, in functional studies, as assessed by inhibition of 3 H vinblastine (VBL) efflux, TMX partially reversed the MDR-1 phenotype in both parental MCF-7 and a multidrug resistant subline MCF-7 mdr . Prolonged exposure to IFN during culture, followed by TMX + IFN resulted in a further and significant decrease of VBL efflux in this cell line as compared to after TMX alone. TMX and IFN both have antiproliferative as well as anti-PGP functional effects in this model. Further studies with these two agents in combination, in the setting of clinical drug resistance, appear to be warranted.