The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique ...in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT-induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high-confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high-confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high-confidence hits have established links to breast cancer. One high-confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.
The emergence of hormone therapy resistance, despite continued expression of the estrogen receptor (ER), is a major challenge to curing breast cancer. Recent clinical studies suggest that epigenetic ...modulation by histone deacetylase (HDAC) inhibitors reverses hormone therapy resistance. However, little is known about epigenetic modulation of the ER during acquired hormone resistance. Our recent phase II study demonstrated that HDAC inhibitors re-sensitize hormone therapy-resistant tumors to the anti-estrogen tamoxifen. In this study, we sought to understand the mechanism behind the efficacy of this combination.
We generated cell lines resistant to tamoxifen, named TAMRM and TAMRT, by continuous exposure of ER-positive MCF7 and T47D cells, respectively to 4-hydroxy tamoxifen for over 12 months. HDAC inhibition, along with pharmacological and genetic manipulation of key survival pathways, including ER and Bcl-2, were used to characterize these resistant models.
The TAMRM cells displayed decreased sensitivity to tamoxifen, fulvestrant and estrogen deprivation. Consistent with previous models, ER expression was retained and the gene harbored no mutations. Compared to parental MCF7 cells, ER expression in TAMRM was elevated, while progesterone receptor (PGR) was lost. Sensitivity of ER to ligands was greatly reduced and classic ER response genes were suppressed. This model conveyed tamoxifen resistance through transcriptional upregulation of Bcl-2 and c-Myc, and downregulation of the cell cycle checkpoint protein p21, manifesting in accelerated growth and reduced cell death. Similar to TAMRM cells, the TAMRT cell line exhibited substantially decreased tamoxifen sensitivity, increased ER and Bcl-2 expression and significantly reduced PGR expression. Treatment with HDAC inhibitors reversed the altered transcriptional events and reestablished the sensitivity of the ER to tamoxifen resulting in substantial Bcl-2 downregulation, growth arrest and apoptosis. Selective inhibition of Bcl-2 mirrored these effects in presence of an HDAC inhibitor.
Our model implicates elevated ER and Bcl-2 as key drivers of anti-estrogen resistance, which can be reversed by epigenetic modulation through HDAC inhibition.
Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying ...molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.
Purpose
The prognostic and predictive values of the MAPK/AKT/ERα phosphorylation axis (pT202/T204MAPK, pT308AKT, pS473AKT, pS118ERα and pS167ERα) in primary tumours were assessed to determine whether ...these markers can differentiate between patient responses for switching adjuvant endocrine therapy after 2–3 years from tamoxifen to exemestane and continued tamoxifen monotherapy in the Intergroup Exemestane Study (IES).
Methods
Of the 4724 patients in IES, 1506 were managed in a subset of centres (
N
= 89) participating in PathIES. These centres recruited 1282 (85%, 1282/1506) women into PathIES of whom 1036 had phospho-marker data. All phospho-markers were analysed by immunohistochemistry staining. Multivariable Cox proportional hazards models of the phospho-markers for disease-free survival (DFS) and overall survival (OS) were adjusted for clinicopathological factors. Treatment effects on the biomarker expression were determined by interaction tests. Benjamini–Hochberg adjustment for multiple testing with a false discovery rate of 10% was applied (
p
BH
).
Results
Phospho-T202/T204MAPK, pS118ERα and pS167ERα were all found to be correlated (
p
BH
= 0.0002). These markers were not associated with either DFS or OS when controlling for the established clinicopathological factors. Interaction terms between the phospho-markers and treatment strategies for either DFS or OS were not statistically significant (
p
BH
> 0.05 for all).
Conclusions
This PathIES study confirmed previously described associations between the phosphorylation site markers of AKT, MAPK and ERα activity in postmenopausal breast cancer patients. No prognostic correlations between the phosphorylation markers and clinical outcome were found, nor were they predictive for clinical outcomes among patients who switched therapy over those treated with tamoxifen alone.
The purpose of the present study was to develop Tamoxifen loaded β-cyclodextrin nanosponges for oral drug delivery. The three types of Tamoxifen loaded β-cyclodextrin nanosponges were synthesized by ...varying the molar ratios of β-cyclodextrin to carbonyldiimidazole as a crosslinker viz. 1:2, 1:4 and 1:8. The Tamoxifen nanosponge complex (TNC) with particle size of 400-600 nm was obtained by freeze drying method. Differential scanning calorimetry, Fourier transformed infra-red spectroscopy and X-ray powder diffraction studies confirmed the complexation of Tamoxifen with cyclodextrin nanosponge. AUC and Cmax of TNC formulation (1236.4 ± 16.12 µg·mL−1 h, 421.156 ± 0.91 µg/mL) after gastric intubation were 1.44 fold and 1.38 fold higher than plain drug (856.079 ± 15.18 µg·mL−1 h, 298.532 ± 1.15 µg/mL). Cytotoxic studies on MCF-7 cells showed that TNC formulation was more cytotoxic than plain Tamoxifen after 24 and 48 h of incubation.
In view of future pharmacokinetic studies, a highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed for the simultaneous ...quantification of tamoxifen and three of its main phase I metabolites in human lithium heparinized plasma. The analytical method has been thoroughly validated in agreement with FDA recommendations. Plasma samples of 200
μl were purified by liquid–liquid extraction with 1
ml
n-hexane/isopropanol, after deproteination through addition of 50
μl acetone and 50
μl deuterated internal standards in acetonitrile. Tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and endoxifen were chromatographically separated on an Acquity UPLC
® BEH C18 1.7
μm 2.1
mm
×
100
mm column eluted at a flow-rate of 0.300
ml/min on a gradient of 0.2
mM ammonium formate and acetonitrile, both acidified with 0.1% formic acid. The overall run time of the method was 10
min, with elution times of 2.9, 3.0, 4.1 and 4.2
min for endoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen and tamoxifen, respectively. Tamoxifen and its metabolites were quantified by triple-quadrupole mass spectrometry in the positive ion electrospray ionization mode. The multiple reaction monitoring transitions were set at 372
>
72 (
m/
z) for tamoxifen, 358
>
58 (
m/
z) for N-desmethyl-tamoxifen, 388
>
72 (
m/
z) for 4-hydroxy-tamoxifen and 374
>
58 (
m/
z) for endoxifen. The analytical method was highly sensitive with the lower limit of quantification validated at 5.00
nM for tamoxifen and N-desmethyl-tamoxifen and 0.500
nM for 4-hydroxy-tamoxifen and endoxifen, which is equivalent to 1.86, 1.78, 0.194 and 0.187
ng/ml for tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and endoxifen, respectively. The method was also precise and accurate, with within-run and between-run precisions within 12.0% and accuracy ranging from 89.5 to 105.3%. The method has been applied to samples from a clinical study and cross-validated with a validated LC–MS/MS method in serum.
Recently, we have shown that the new G-protein-coupled estrogen receptor GPR30 plays an important role in the development of tamoxifen resistance in vitro. This study was undertaken to evaluate the ...correlation between GPR30 and tamoxifen resistance in breast cancer patients. GPR30 protein expression was evaluated by immunohistochemical analysis in 323 patients with primary operable breast cancer. The association between GPR30 expression and tamoxifen resistance was confirmed in a second cohort of 103 patients treated only with tamoxifen. Additionally, we evaluated GPR30 expression in 33 primary tumors and in recurrent tumors from the same patients. GPR30 expression was detected in 56.7% of the breast cancer specimens investigated and it correlated with overexpression of HER-2 (
P
= 0.021), EGFR (
P
= 0.024) and lymph node status (
P
= 0.047). In a first cohort, survival analysis showed that GPR30 was negatively correlated with relapse-free survival (RFS) only in patients treated with tamoxifen (tamoxifen with or without chemotherapy). GPR30 expression was associated with shorter RFS (HR = 1.768; 95% CI, 1.156–2.703;
P
= 0.009). In a subset of patients treated only with tamoxifen, multivariate analysis revealed that GPR30 expression is an independent unfavorable factor for RFS (HR = 4.440; 95% CI, 1.408–13.997;
P
= 0.011). In contrast, GPR30 tended to be a favorable factor regarding RFS in patients who did not receive tamoxifen. In 33 paired biopsies obtained before and after adjuvant therapy, GPR30 expression significantly increased only under tamoxifen treatment (
P
= 0.001). GPR30 expression in breast cancer independently predicts a poor RFS in patients treated with tamoxifen.
Progression of tamoxifen resistance remained as a crucial obstacle to treatment of estrogen receptor positive breast carcinoma patients. Recent studies demonstrated the importance of DNA methylation ...pattern on tamoxifen refractory. This study aimed to investigate the protein expression pattern and clinicopathological significance of DNA methyltransferase 1, 3A and 3B, as leading factors in regulation of DNA methylation process, in breast carcinoma patients with adjuvant tamoxifen therapy. Seventy two Formalin-Fixed Paraffin-Embedded (FFPE) breast tumor tissues of tamoxifen sensitive (TAMS) and tamoxifen resistance (TAM-R) patients were recruited for immunohistochemical experiments. DNMT1, DNMT3a, and DNMT3b expressions were observed in 86, 72.2 and 100% of tamoxifen resistance patients, respectively. Data analysis indicated that DNMTs were overexpressed in TAM-R tumors (P < 0.05). In TAM-S subgroup, DNMT1, DNMT3A and DNMT3B expression was associated with high histologic grade (P = 0.049, P = 0.01 and P = 0.02, respectively). DNMT3B expression was also correlated with lymphatic invasion (P = 0.034). In TAM-R subgroup, DNMT1 expression associated with extracapsular nodal extension (P = 0.019). DNMT3A and DNMT3B expression showed a significant association with high histologic grade (P = 0.001) and DNMT3A expression was also associated with HER-2 status (P = 0.027). Cox proportional hazard model demonstrated that overexpression of DNMT3B remained as an independent and unfavorable prognostic factor for disease free survival (P < 0.001). Taken together, these results suggest that DNMTs could be an effective factor in development of tamoxifen resistance in breast tumors.
Display omitted
•Modifications of DNA methylation pattern have crucial roles in tamoxifen refractory in hormone responsive breast tumors.•This study aimed to investigate protein expression of DNA methyltransferase 1, 3A and 3B in tamoxifen-treated breast tumors.•All three types of DNMTs were overexpressed in tamoxifen resistant tissues in comparison to tamoxifen sensitive ones.•The protein expression of DNMT3A and DNMT3B were positively associated with high histological grade in both studied groups.•Overexpression of DNMT3B remained as an unfavorable prognostic factor for disease free survival.
Endoxifen is the key active metabolite of Tamoxifen, a widely used breast cancer drug. Orally administered Tamoxifen, is extensively metabolized by cytochrome P450 (CYP) enzymes, namely CYP3A4 and ...CYP2D6, into active metabolites, especially Endoxifen. Due to genetic polymorphism of CYP2D6, significant numbers of women metabolize Tamoxifen to varying degree and may not receive the optimal benefit from Tamoxifen treatment. We show that oral administration of Endoxifen achieved the optimally effective systemic levels reliably, which may eliminate variability associated with Tamoxifen metabolism that leads to unpredictability in efficacy. Furthermore, use of Endoxifen may avoid a potential serious drug interaction found between Tamoxifen and commonly used selective serotonin reuptake inhibitors, antidepressants. Endoxifen was active in inhibiting the growth of various breast tumor cell lines in NCI 60-Cell Line Screen. Orally administered Endoxifen is rapidly absorbed and systemically available when tested in female rats. The Endoxifen-treated rats showed 787% higher exposure (AUC₀₋∞) and 1,500% higher concentration (C max) levels of Endoxifen when compared with Tamoxifen. Oral Endoxifen administration once a day for 28 consecutive days at dosages 2, 4, and 8 mg/kg proved safe and resulted in progressive inhibition of the growth of the human mammary tumor xenografts in female mice. This is the first ever in vivo report on Endoxifen as a potentially new therapeutic agent for breast cancer.
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