Estrogen-induced Breast Cancer Cell Proliferation Research Articles

Estrogen-induced miR-196a elevation promotes tumor growth and metastasis via targeting SPRED1 in breast cancer

BackgroundEstrogen plays a critical role in breast cancer (BC) progression through estrogen receptor (ER)-mediated gene regulation. Emerging studies suggest that the malignant progress of BC cells is influenced by the cross talk between microRNAs (miRNAs) and ER-α signaling. However, the mechanism and functional linkage between estrogen and miRNAs remain unclear.MethodsThe expression levels of miR-196a and SPRED1 in BC were tested by qRT-PCR in 46 paired BC and adjacent tissues and by the GEO datasets. The role of miR-196a in estrogen-induced BC development was examined by CCK-8 assay, wound healing assay, Matrigel invasion assay and tumorigenicity assay in nude mice. The binding site of ER-α in miR-196a promoter region was analyzed by ChIP-seq, ChIP assay and luciferase reporter assay. The potential targets of miR-196a in BC cells were explored using the luciferase reporter assay and western blot analysis, and the correlation between miR-196a and SPRED1 was analyzed by Spearman’s correlation analysis in BC specimens and GEO dataset. TCGA BRCA data was used to characterize the ESR1 signatures according to MSigDB gene set.ResultsThe expression levels of miR-196a were higher in ER-positive (ER+) breast tumors compared to ER-negative (ER-) tumor tissue samples. Besides, miR-196a was involved in estrogen-induced BC cell proliferation, migration and invasion. Notably, the up-regulation of miR-196a was mediated by a direct interaction with estrogen receptor α (ER-α) but not estrogen receptor β (ER-β) in its promoter region, and miR-196a expression levels were positively correlated to ER-α signature scores. Furthermore, SPRED1 was a new direct target of miR-196a which participated in miR-196a-promoted BC development and was suppressed by ligand-activated ER-α signal pathway. Finally, forced expression of miR-196a induced tumor growth of MCF7 cells, while inhibition of miR-196a significantly suppressed the tumor progress in vivo.ConclusionsOverall, the identification of estrogen/miR-196a/SPRED1 cascade will shed light on new molecular mechanism of estrogen signaling in BC development and therapy.

Nicotinic Acetylcholine Receptor-Based Blockade: Applications of Molecular Targets for Cancer Therapy

The nicotinic acetylcholine receptor (nAChR) was first characterized in 1970 as a membrane receptor of a neurotransmitter and an ion channel. nAChRs have been shown to be involved in smoking-induced cancer formation in multiple types of human cancer cells. In vitro and in vivo animal studies have shown that homopentameric nAChR inhibitors, such as methyllycaconitine and α-Bgtx, can attenuate nicotine-induced proliferative, angiogenic, and metastatic effects in lung, colon, and bladder cancer cells. Recent publications have shown that α9-nAChR is important for breast cancer formation, and in many in vivo studies, α9-nAChR-specific antagonists (e.g., α-ImI, α-ImI, Vc1.1, RgIA, and It14a) produced an analgesic effect. Vc1.1 functions in a variety of animal pain models and currently has entered phase II clinical trials. For cancer therapy, natural compounds such as garcinol and EGCG have been found to block nicotine- and estrogen-induced breast cancer cell proliferation through inhibition of the α9-nAChR signaling pathway. A detailed investigation of the carcinogenic effects of nAChRs and their specific antagonists would enhance our understanding of their value as targets for clinical translation.

Abstract P4-02-06: Beyond CYP2D6 Genotyping: Personalized Dosing of Tamoxifen (Tam) Based on Low Serum Endoxifen Levels

Abstract Background: The efficacy of Tam varies widely. Tam is biotransformed to endoxifen and 4-OH-Tam by the cytochrome P450 2D6 enzymes. Studies on 2D6 variants as a predictor of Tam's efficacy show conflicting results. Compared to Tam, endoxifen and 4-OH-Tam have a 100-fold greater affinity for the estrogen receptor (ER) and in suppressing estrogen-dependent cell proliferation. However, endoxifen is present at concentrations up to 20-fold higher and is pharmacologically distinct from 4-OH-Tam. Endoxifen's effect on ERα-degredation and transcription is concentration dependent; serum endoxifen levels ≥40nM block ERα-mediated transcriptional activation and inhibit estrogen-induced breast cancer cell proliferation. Methods: Pts taking Tam 20 mg/day, for any reason, were potentially eligible. Serum Tam, Tam metabolites and nineteen 2D6 alleles were analyzed. 2D6 genotyping was performed by PCR-based procedures and metabolites were assessed on Liquid Chromatography/Tandem Mass Spectrometry. Pts were stratified by 2D6 function into 3 groups: extensive metabolizers (EM) who had normal enzyme activity; intermediate metabolizers (IM) who had reduced enzyme activity; and poor metabolizers (PM) who had no enzyme activity. Tam was increased to 30 mg/day if serum endoxifen < 40nMol. Serum Tam and Tam metabolites were measured every 2 weeks for 90 days. Results: 121 patients have enrolled to date. Age, ethnicity, BMI, concomitant medications, and adherence were recorded. 25/117 (21%) pts had low serum endoxifen; all of these pts were increased to Tam 30 mg/day. Of this cohort, 36% were PMs (PM/PM and PM/IM), 32% were IMs (EM/PM and IM/IM) and 32% were EMs (EM/EM and EM/IM). All patients’ serum endoxifen increased to >40nMol by day 30 (Diagram below). Conclusions: 1) Sub-therapeutic serum endoxifen can be found in any 2D6 phenotype. 2) It is possible to achieve therapeutic serum endoxifen/Tam metabolite levels by optimizing the dose of Tam. 3) Subsequently, it is likely that other gene variants are involved in Tam metabolism hence why 2D6 testing may lead to conflicting results. 4). Further investigation is warranted as personalizing the dose of Tam is feasible and may potentially improve the outcome of pts taking Tam regardless of 2D6 genotype. Figure available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-06.

Personalized dosing of tamoxifen (Tam) based on polymorphisms of CYP2D6 (2D6) and/or low serum endoxyfen levels.

e13507 Background: The efficacy of Tam varies widely. Tam is biotransformed to endoxifen and 4-OH-Tam by the cytochrome P450 2D6 enzymes. Compared to Tam, endoxifen and 4-OH-Tam have a 100-fold greater affinity for the estrogen receptor (ER) and in suppressing estrogen-dependent cell proliferation. 2D6 variants are an independent predictor of relapse and death. Serum endoxifen levels ≥40nM block ERα-mediated transcriptional activation and inhibit estrogen-induced breast cancer cell proliferation. Methods: Serum Tam, Tam metabolites, and 19 2D6 alleles were analyzed in pts taking Tam 20 mg/day for 90 days. 2D6 genotyping was performed by PCR-based procedures and metabolites were assessed on Liquid Chromatography/Tandem Mass Spectrometry. Tam was increased to 30 mg/day if serum endoxifen < 40 nMol or 2D6 alleles had no enzymatic function. Results: 110 patients have enrolled to date. Age, ethnicity, BMI, concomitant medications, and adherence were recorded. 17% pts had low serum endoxifen and 2% of pts had no 2D6 allele function, all of these pts were increased to Tam 30 mg/day. All patients' serum endoxifen increased >40 nMol by day 14. Conclusions: Subtherapeutic serum endoxifen can be found in any 2D6 phenotype, although it is more common in pts with decreased or no 2D6 allele function. Subsequently, it is likely that other gene variants are involved in Tam metabolism. It is possible to achieve therapeutic serum endoxifen/Tam metabolite levels by optimizing the dose of Tam. Tam dosages can be individualized. Further investigation is warranted as personalizing the dose of Tam is feasible and may potentially significantly improve the outcome of pts taking Tam. Genotype score Avg(endoxifen)day 0 Avg(endoxifen)day 14 Avg (endoxifen) day 30 Avg (endoxifen) day 42 Avg(endoxifen) day 56 Avg (endoxifen) day 90 0 33* 40 64 43 60 Pending 0.5 39 50 64 66 70 72 1 30 43 46 66 53 61 2 32 56 53 55 59 61 No significant financial relationships to disclose.

The Tamoxifen Metabolite, Endoxifen, Is a Potent Antiestrogen that Targets Estrogen Receptor α for Degradation in Breast Cancer Cells

Tamoxifen has been the most important therapeutic agent for the treatment of estrogen receptor (ER)-positive breast cancer for the past three decades. Tamoxifen is extensively metabolized by cytochrome P450 enzymes, and recent in vivo studies have shown that women with genetically impaired cytochrome P450 2D6 have reduced production of endoxifen and a higher risk of breast cancer recurrence. Despite these observations, the contribution of endoxifen to the overall drug effectiveness of tamoxifen remains uncertain. Here, we provide novel evidence that endoxifen is a potent antiestrogen that functions in part by targeting ERalpha for degradation by the proteasome in breast cancer cells. Additionally, we show that endoxifen blocks ERalpha transcriptional activity and inhibits estrogen-induced breast cancer cell proliferation even in the presence of tamoxifen, N-desmethyl-tamoxifen, and 4-hydroxytamoxifen. All of the effects of endoxifen are concentration dependent and do not occur at concentrations observed in human CYP2D6 poor metabolizers. These results support the theory that endoxifen is the primary metabolite responsible for the overall effectiveness of tamoxifen in the treatment of ER-positive breast cancer.

Interleukin-4 and interleukin-13 inhibit estrogen-induced breast cancer cell proliferation and stimulate GCDFP-15 expression in human breast cancer cells

Human breast carcinomas are frequently infiltrated by inflammatory cells secreting several cytokines which may regulate the activity of both immune cells and neoplastic cells. The present study was designed to examine the potential action of interleukin-4 (IL-4) and interleukin-13 (IL-13) in human breast cancer cells. Exposure of ZR-75-1 breast cancer cells to IL-4 or IL-13 for 10 days decreased the amplitude of the mitogenic action of 17β-estradiol by 75% and 55%, respectively, while these cytokines failed to change basal cell proliferation. These cytokines also exerted a similar action in T-47D cells. Exposure to IL-4 or IL-13 markedly increased gross cystic disease fluid protein-15 (GCDFP-15) release in both ZR-75-1 and T-47D cells. The half-maximal stimulatory effects of IL-4 and IL-13 on GCDFP-15 secretion were exerted at respective values of 16 ± 3 pM and 91 ± 8 pM in T-47D cells incubated for a period of 10 days. The effect of IL-13 was not additive to that elicited by IL-4, whereas the stimulation of GCDFP-15 release by these interleukins were additive to that exerted by maximally effective concentrations of the androgen dihydrogestosterone and the synthetic glucocorticoid dexamethasone. Furthermore, exposure of ZR-75-1 cells to IL-4 and IL-13 increased GCDFP-15 mRNA levels by 5.5- and 6.0-fold, respectively. The present results demonstrate that IL-4 and IL-13 may decrease estrogen-induced breast cancer cell proliferation and induce the expression of a breast cancer marker, thus strongly suggesting that breast cancer cells are targets of both IL-4 and IL-13 action.