TAM-resistant MCF-7 Cells Research Articles

Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance.

Among the several mechanisms accounting for endocrine resistance in breast cancer, autophagy has emerged as an important player. Previous reports have evidenced that tamoxifen (Tam) induces autophagy and activates transcription factor EB (TFEB), which regulates the expression of genes controlling autophagy and lysosomal biogenesis. However, the mechanisms by which this occurs have not been elucidated as yet. This investigation aims at dissecting how TFEB is activated and contributes to Tam resistance in luminal A breast cancer cells. TFEB was overexpressed and prominently nuclear in Tam-resistant MCF7 cells (MCF7-TamR) compared with their parental counterpart, and this was not dependent on alterations of its nucleo-cytoplasmic shuttling. Tam promoted the release of lysosomal Ca2+ through the major transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) and two-pore channels (TPCs), which caused the nuclear translocation and activation of TFEB. Consistently, inhibiting lysosomal calcium release restored the susceptibility of MCF7-TamR cells to Tam. Our findings demonstrate that Tam drives the nuclear relocation and transcriptional activation of TFEB by triggering the release of Ca2+ from the acidic compartment, and they suggest that lysosomal Ca2+ channels may represent new druggable targets to counteract the onset of autophagy-mediated endocrine resistance in luminal A breast cancer cells.

GPER promotes tamoxifen-resistance in ER+ breast cancer cells by reduced Bim proteins through MAPK/Erk-TRIM2 signaling axis.

Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Our previous studies find that GPER and its down-stream signaling play a pivotal role in the development of tamoxifen (TAM) resistance. cDNA array analysis indicated a set of genes associated with cell apoptosis are aberrant in GPER activated and TAM-resistant MCF-7Rcells compared with TAM-sensitive MCF-7cells. Among these genes, Bim (also named BCL2-L11), a member of the BH3-only pro-apoptotic protein family is significantly decreased, and TRIM RING finger protein TRIM2 (a ubiquitin ligase) is highly expressed in MCF-7R. To understand the mechanism of TAM-resistance in GPER activated ER+ breast cancer, the function of TRIM2 and Bim inducing cell apoptosis was studied. By using immunohistochemical and western blot analysis, there is an adverse correlation between TRIM2 and Bim in TAM-resistant breast tumor tissues and MCF-7R cells. Knockdown Bim in TAM-sensitive MCF-7cells or overexpression of Bim in TAM-resistant MCF-7cells significantly changed its sensibility to TAM through altering the levels of cleaved PARP and caspase-3. Activation of GPER and its downstream signaling MAPK/ERK, not PI3K/AKT, led to enhanced TRIM2 protein levels and affected the binding between TRIM2 and Bim which resulted in a reduced Bim in TAM-resistant breast cancer cells. Thus, the present study provides a novel insight to TAM-resistance in ER-positive breast cancer cells.

Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro.

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER+) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER+ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER+ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66-/PR-/HER2-) and ER-α36+/GPER1+ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER+ breast cancer.

Abstract 4421: GPER-mediated activation of the cAMP response element-binding protein (CREB) and subsequent IGFBP-1 expression is altered during the development of tamoxifen resistance

Abstract Tamoxifen is a commonly prescribed anti-estrogen treatment for breast cancer patients; this selective estrogen receptor modulator functions as an ERα-antagonist in breast tissue and inhibits cell proliferation and survival of breast cancer cells. More recently, the active metabolite of tamoxifen, 4-OHT (Tam), has been shown to activate the G protein-coupled estrogen receptor (GPER). Previous studies from our laboratory identified a novel, GPER-dependent mechanism of Tam action in breast cancer cells that requires the extracellular accumulation of the insulin-like growth factor binding protein-1 (IGFBP-1). The molecular mechanisms of Tam-induced IGFBP-1 production mediated by GPER have not been determined. GPER activation has been shown to stimulate cAMP production and PKA activation in several cell types. We hypothesized the cAMP response element-binding protein (CREB) activation is required for Tam-induced IGFBP-1 production in breast caner cells. To determine if CREB activation may be involved in IGFBP-1 transcription, CREB phosphorylation (S133) status and nuclear accumulation were determined in MCF-7 cells after Tam treatment. The phosphorylation and nuclear accumulation of CREB were observed in Tam-treated MCF-7 cells suggesting that CREB activation may be required for Tam-induced IGFBP-1 transcription. Increased CREB phosphorylation and IGFBP-1 transcription were inhibited by pretreatment with the PKA inhibitor H-89 supporting a role for CREB in Tam-induced IGFBP-1 expression. H-89 pretreatment also significantly decreased the previously reported inhibition of IGF-1-stimulated signaling by Tam treatment in breast cancer cells. We have previously reported that Tam-induced IGFBP-1 expression is dysregulated in Tam-resistant MCF-7 cells (MCF-7TamR). To determine if aberrant CREB signaling results in the observed dysregulation, similar experiments were performed in MCF-7TamR cells. Our results show that CREB phosphorylation and nuclear accumulation are decreased in Tam-resistant cells compared to Tam-sensitive cells. Taken together, these results suggest that activation of GPER results in CREB-dependent IGFBP-1 production and subsequent inhibition of IGF-1-stimulated signaling in breast cancer cells treated with Tam. Furthermore, dysregulation of GPER-mediated CREB activation may be involved in the development of Tam resistance in breast cancer. Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. GPER-mediated activation of the cAMP response element-binding protein (CREB) and subsequent IGFBP-1 expression is altered during the development of tamoxifen resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2015-4421

Abstract P3-04-06: Inhibiting androgen receptor nuclear localization decreases estrogen receptor (ER) activity and tumor growth in ER+ breast cancer

Abstract Background: Androgen receptor (AR) is widely expressed in breast tumors, but the role of AR in estrogen receptor (ER)+ tumors is controversial. In the absence of estradiol (E2), dihydrotestosterone (DHT) increases growth of ER+ breast cancer cells in vitro and in vivo. Anti-androgens such as bicalutamide (Bic) and enzalutamide (ENZ) inhibit this DHT-mediated proliferation. Surprisingly we have found that ENZ, which impairs nuclear entry of liganded AR, also inhibits E2-mediated proliferation of ER+ breast cancer cells, while Bic does not. Hypothesis: We hypothesize that nuclear localization of AR is necessary for maximal E2-mediated proliferation in ER+/AR+ breast cancer cells, and targeting AR with ENZ or other agents that impede AR nuclear entry or cause AR degradation will inhibit growth of ER+/AR+ human breast cancer cell lines and decrease tumor burden in preclinical models. Methods: ER+/AR+ MCF7, BCK4, and ZR-75-1 cells were treated with E2 plus or minus ER and AR antagonists and proliferation was measured by crystal violet staining or Incucyte live cell imaging. Nuclear AR was assessed by immunocytochemistry or nuclear/cytoplasmic fractionation. For in vivo experiments, 1x10^6 luciferase-expressing MCF7 cells were injected into the mammary fat pad of nu/nu mice and tumor growth monitored by caliper and IVIS imaging. Results: ENZ blocked E2-induced proliferation and showed synergistic activity with the ER antagonists 4-hydroxy-Tamoxifen (OH-Tam) and Fulvestrant (Fulv) in vitro. E2-induced expression of ER target genes including PR and SDF1 was also inhibited by ENZ, but not by Bic. Similarly, AR knockdown decreased baseline and E2-induced proliferation of MCF7 cells and E2-induced ER target gene expression. Both DHT and E2 treatment induced nuclear translocation of AR, which was decreased by ENZ. Nuclear translocation of AR in response to E2 occurs only in ER+ cell lines, further supporting a role for nuclear AR in E2-induced ER activity. In vivo, ENZ inhibited E2-induced growth of MCF7 tumors as effectively as Tamoxifen (Tam), and the combination of ENZ plus Tam was more effective than either drug alone. ENZ also inhibited growth of Tam-resistant MCF7 cells in vitro. Conclusions: Our results suggest that nuclear localization of AR plays a previously-unrecognized role in E2-mediated ER activity in ER+/AR+ breast cancer cells. Because of its ability to inhibit nuclear entry of liganded AR, ENZ may serve as an effective therapeutic in ER+/AR+ breast cancers. Importantly, ENZ may be particularly useful in combination with current anti-estrogen therapies (Tam or Fulv) since it affects ER, but via an indirect mechanism acting through AR. ENZ may also be effective in tumors resistant to ER-directed therapy based on in vitro data and published clinical data indicating that many such tumors express more AR protein than ER. Funded by: DOD BCRP Clinical Translational Award BC120183 to JKR, American Cancer Society Postdoctoral Fellowship PF-13-314-01 – CDD to NCD. Citation Format: Nicholas C D'Amato, Britta M Jacobsen, Dawn R Cochrane, Nicole S Spoelstra, Beatrice L Babbs, Anthony Elias, Jennifer K Richer. Inhibiting androgen receptor nuclear localization decreases estrogen receptor (ER) activity and tumor growth in ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-06.

Modulation of BAG-1 Expression Alters the Sensitivity of Breast Cancer Cells to Tamoxifen

Background: BAG-1 (bcl-2 associated athanogene) is a multifunctional protein that protects cells from a wide range of apoptotic stimuli including radiation, hypoxia and chemotherapeutic agents. Overexpression of cytoplasmic BAG-1 has been associated with the increased survival and decreased response to treatment with tamoxifen (TAM) in breast cancer. We attempted to assess the expression of BAG-1 in the human breast cancer cells that are resistant to treatment with 4-OH TAM and effect of altered BAG-1 expression on their sensitivity to 4-OH TAM. Methods: BAG-1 expression was examined in the MCF-7 cells that became resistant to 4-OH TAM. The 4-OH TAM-resistant MCF-7 cells were then transfected with the BAG-1 siRNA and the 4-OH TAM-sensitive MCF-7 cells with the plasmids carrying the human BAG-1 isoform-specific expression constructs respectively to investigate the effect of BAG-1 on the TAM-induced apoptosis. Results: Our results showed that the TAM-resistant MCF-7 (TAMR/MCF-7) cells expressed higher level of BAG-1 than that of the MCF-7 cells. Down-regulation of BAG-1 significantly enhanced the sensitivity of the TAMR/MCF-7 cells to TAM treatment. Additionally, we found that BAG-1 p50 was the only isoform that inhibited the TAM-induced apoptosis in the MCF-7 cells, while the other isoforms had little effect. Conclusion: Our study indicated that up and down regulations of the BAG-1 expression were associated with the decreased and increased sensitivity to 4-OH TAM in the estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 respectively, and distinct isoforms of BAG-1 had different anti-apoptotic ability in breast cancer cells treated with the 4-OH TAM.

Tamoxifen-resistant breast cancer cells possess cancer stem-like cell properties

Background Cancer stem cells (CSCs) are the cause of cancer recurrence because they are resistant to conventional therapy and contribute to cancer growth and metastasis. Endocrinotherapy is the most common breast cancer therapy and acquired tamoxifen (TAM) resistance is the main reason for endocrinotherapy failure during such therapy. Although acquired resistance to endocrine treatment has been extensively studied, the underlying mechanisms are unclear. We hypothesized that breast CSCs played an important role in TAM-induced resistance during breast cancer therapy. Therefore, we investigated the biological characteristics of TAM-resistant (TAM-R) breast cancer cells. Methods Mammosphere formation and tumorigenicity of wild-type (WT) and TAM-R MCF7 cells were tested by a mammosphere assay and mouse tumor xenografts respectively. Stem-cell markers (SOX-2, OCT-4, and CD133) and epithelial-mesenchymal transition (EMT) markers were tested by quantitative real-time (qRT)-PCR. Morphological observation was performed to characterize EMT. Results After induction of TAM resistance, TAM-R MCF7 cells exhibited increased proliferation in the presence of TAM compared to that of WT MCF7 cells (P <0.05), indicating enhanced TAM resistance of TAM-R MCF7 cells compared to that of WT MCF7 cells. TAM-R MCF7 cells showed enhanced mammosphere formation and tumorigenicity in nude mice compared to that of WT MCF7 cells (P <0.01), demonstrating the elevated CSC properties of TAM-R MCF7 cells. Consistently, qRT-PCR revealed that TAM-R MCF7 cells expressed increased mRNA levels of stem cell markers including SOX-2, OCT-4, and CD133, compared to those of WT MCF7 cells (P <0.05). Morphologically, TAM-R MCF7 cells showed a fibroblastic phenotype, but WT MCF7 cells were epithelial-like. After induction of TAM resistance, qRT-PCR indicated that MCF7 cells expressed increased mRNA levels of Snail, vimentin, and N-cadherin and decreased levels of E-cadherin, which are considered as EMT characteristics (P <0.05). Conclusion TAM-R MCF7 cells possess CSC characteristics and may be responsible for TAM resistance during breast cancer therapy.

Estrogen Receptor-α Variant, ER-α36, is Involved in Tamoxifen Resistance and Estrogen Hypersensitivity

Antiestrogens such as tamoxifen (TAM) provided a successful treatment for estrogen receptor (ER)-positive breast cancer for the past four decades. However, most breast tumors are eventually resistant to TAM therapy. The molecular mechanisms underlying TAM resistance have not been well established. Recently, we reported that breast cancer patients with tumors expressing high concentrations of ER-α36, a variant of ER-α, benefited less from TAM therapy than those with low concentrations of ER-α36, suggesting that increased ER-α36 concentration is one of the underlying mechanisms of TAM resistance. Here, we investigated the function and underlying mechanism of ER-α36 in TAM resistance. We found that TAM increased ER-α36 concentrations, and TAM-resistant MCF7 cells expressed high concentrations of ER-α36. In addition, MCF7 cells with forced expression of recombinant ER-α36 and H3396 cells expressing high concentrations of endogenous ER-α36 were resistant to TAM. ER-α36 down-regulation in TAM-resistant cells with the short hairpinRNA method restored TAM sensitivity. We also found that TAM acted as a potent agonist by activating phosphorylation of the AKT kinase in ER-α36-expressing cells. Finally, we found that cells with high concentration of ER-α36 protein were hypersensitive to estrogen, activating ERK phosphorylation at picomolar range. Our results thus demonstrated that elevated ER-α36 concentration is one of the mechanisms by which ER-positive breast cancer cells escape TAM therapy and provided a rational to develop novel therapeutic approaches for TAM-resistant patients by targeting ER-α36.

Let-7 MicroRNAs Induce Tamoxifen Sensitivity by Downregulation of Estrogen Receptor α Signaling in Breast Cancer

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3'UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3'UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.

Pomegranate sensitizes Tamoxifen action in ER-α positive breast cancer cells

It is estimated that one in eight women will be affected with cancer during their lives, which means over 1 million women worldwide will be diagnosed with breast cancer in the year of 2011. Roughly, 70% of breast cancer will be estrogen receptor-alpha (ER-α) positive. The presence of ER-α is associated with better prognosis and is able to determine if tumors will respond to the estrogen-blocking/ER-antagonist drug Tamoxifen (TAM). However, a significant fraction of ER-positive tumors respond with minimal or no response to TAM. It is unclear why some breast cancer cells resist TAM and how to make these cells respond. Early evidence suggests Pomegranate fruit extracts (PFEs) exhibit an anticancer effect against some cancers. The objective of the study was to determine whether PFEs may able to enhance/sensitize the TAM's effect in ER-positive MCF-7 breast cancer cells. To test the hypothesis, we determined the effect of PFEs on sensitive and TAM-resistant-MCF-7 cell viability and cell death in the presence or absence of TAM under estrogenic or non-estrogenic culture environment. The present studies demonstrated that PFEs enhance the TAM action in both sensitive and TAM-resistant MCF-7 cells through the inhibition of cell viability (regular or estrogen-induced) by inducing cell-death machinery. Collectively, the results showed for the first time that pomegranate combined with TAM may represent a novel and a powerful approach to enhance and sensitize TAM action.

PI3-kinase/p38 kinase-dependent E2F1 activation is critical for Pin1 induction in tamoxifen-resistant breast cancer cells.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. We have shown that Pin1, a peptidyl prolyl isomerase, is consistently overexpressed in TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) and plays a key role in the enhanced angiogenic potential of TAMR-MCF-7 cells. In the present study, we focused on signaling pathways for Pin1 up-regulation in TAMR-MCF-7 cells. Relative to MCF-7 cells, Pin1 gene transcription and E2 transcription factor1 (E2F1) expression were enhanced in TAMR-MCF-7 cells. E2F1 siRNA significantly reduced both the protein expression and the promoter transcriptional activity of Pin1. Activities of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK) and p38 kinase were all higher in TAMR-MCF-7 cells than in control MCF-7 cells and the enhanced Pin1 and E2F1 expression in TAMR-MCF-7 cells was reversed by inhibition of PI3K or p38 kinase. Moreover, the higher production of vascular endothelial growth factor (VEGF) in TAMR-MCF-7 cells was significantly diminished by suppression of PI3K or p38 kinase. These results suggest that Pin1 overexpression and subsequent VEGF production in TAMR-MCF-7 cells are mediated through PI3-kinase or p38 kinase-dependent E2F1 activation.

Abstract 1745: Pomegranate sensitizes tamoxifen action in breast cancer cells: A novel therapeutic strategy

Abstract It is estimated that in year 2009 over 1 million women worldwide will be diagnosed with breast cancer. Roughly, 70% of breast cancer will be estrogen receptor-alpha (ER-α) positive, which facilitates their growth in the presence of estrogen. The presence of ER-α is able to determine if tumors will respond to the estrogen-blocking/ER-antagonist drug Tamoxifen (TAM). However, a significant fraction (∼40%) of ER-positive tumors is minimally or completely unable to respond to TAM. However, it is unclear why some breast cancer cells resist TAM and how to make these cells respond. Early evidence suggests Pomegranate fruit extracts (PFEs) exhibit an anticancer effect against some cancers. The hypothesis of the study was that PFEs may able to enhance TAM's effect in ER-positive MCF-7 breast cancer cells. To test the hypothesis, we determined the effect of PFEs on sensitive and TAM-resistant-MCF-7 cell viability and cell death (apoptosis) in the presence or absence of TAM under estrogenic or non-estrogenic culture environment. Cell viability was determined by cell counting using Trypan-blue assay and cell cycle distribution. Apoptosis was determined by Annexin-V-apoptotic-detection assay and immuno-Western blotting of apoptosis-related-genes (i.e., BCl-2 and Bax). The present studies demonstrated that PFEs enhance the TAM action in both sensitive and TAM-resistant MCF-7 cells through the inhibition of cell viability (regular or estrogen-induced) by inducing cell-death machinery. Collectively, the results showed for the first time that pomegranate combined with TAM may represent a novel and powerful approach to enhance TAM action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1745. doi:10.1158/1538-7445.AM2011-1745

Inhibition of angiogenesis by quercetin in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem among breast cancer patients. Previously, we have reported that TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) showed increased angiogenic intensity through Pin1-dependent vascular endothelial growth factor (VEGF) production. Among six flavonoids tested in the current study, VEGF gene transcription in MCF-7 cells with stable Pin1 overexpression was inhibited most effectively by quercetin. Reporter gene assays using minimal reporters containing hypoxia response elements and activator protein-1 (AP-1) elements revealed that the activities of hypoxia inducible factor-1α (HIF-1α) and AP-1, key transcription factors for VEGF gene transcription, were suppressed by quercetin. Western blot analyses confirmed that the increased nuclear levels of c-Jun and HIF-1α in TAMR-MCF-7 cells were blocked by quercetin. Moreover, quercetin inhibited the enhanced VEGF secretion and Pin1 expression in TAMR-MCF-7 cells, which was dependent on its phosphatidyl inositol 3-kinase inhibiting effect. Chick chorioallantoic membrane assays demonstrated that the enhanced angiogenesis intensity of TAMR-MCF-7 cells was also suppressed significantly by quercetin. These results demonstrate that quercetin may have therapeutic potential for the treatment of TAM-resistant breast cancer via Pin1 inhibition.

ABCC2 and Clinical Outcome of Tamoxifen Therapy

TO THE EDITOR: The study by Kiyotani et al is another important contribution to the field of tamoxifen (TAM) pharmacogenomics in favor of CYP2D6 genetic testing for the improvement of TAM response in postmenopausal Japanese patients with breast cancer. In addition, for the first time, the authors identified a significant association between the rs3740065 G allele (intron 29, IVS29 154A G) of the ABC efflux drug transporter ABCC2 (multidrug related protein [MRP2]) and longer recurrence-free survival compared with individuals carrying the A allele in their study population of 282 patients. Because TAM-resistant MCF-7 cells express higher levels of ABCC2 than nonresistant MCF-7 cells, it has been suggested that ABCC2 induction could play a role in TAM resistance. On these grounds, Kiyotani et al speculated that the rs3740065 variant may be associated with increased ABCC2 expression and enhanced transport activity, thereby lowering the exposure of breast cancer cells to endoxifen, the active metabolite of TAM. However, in their study, the G allele was associated with longer recurrence-free survival, which would rather indicate a lower expression of ABCC2 and, hence, reduced transporter activity in breast cancer cells, resulting in higher intracellular endoxifen levels. Recently, a systematic DNA sequencing study that included 236 healthy individuals of Japanese ethnic background showed that a common promoter variant in ABCC2 ( 1774delG) is in strong linkage disequilibrium with rs3740065. Moreover, functional studies revealed that the 1774delG variation decreased ABCC2 promoter activity by 36% and that the promoter carrying the 1774delG allele had a defect in chenodeoxycholic acid–induced induction of promoter activity. Finally, a haplotype containing the 1774delG polymorphism showed a strong association with cholestatic or mixedtype hepatitis. We, therefore, propose that it is the 1774delG variant that can explain the findings of an association between ABCC2 and TAM outcome reported by Kiyotani et al. Unfortunately, the authors did not include the 1774delG variant in their investigation. Therefore, the issue of ABCC2 variants that predict clinical outcome must await more specific studies.

Enhancement of vascular endothelial growth factor–mediated angiogenesis in tamoxifen-resistant breast cancer cells: role of Pin1 overexpression

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. Here, we found that TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) produced higher levels of vascular endothelial growth factor (VEGF) than control MCF-7 cells. Molecular analyses using reporter genes and Western blots supported the involvement of c-Jun/activator protein-1 and hypoxia-inducible factor 1alpha in enhanced VEGF transcription in TAMR-MCF-7 cells. Pin1, a peptidyl prolyl isomerase, was consistently overexpressed in TAMR-MCF-7 cells, and c-Jun/activator protein-1-dependent VEGF transcription in TAMR-MCF-7 cells was almost completely inhibited by Pin1 siRNA and by the Pin1 inhibitor juglone. Chick chorioallantoic membrane assays confirmed that the increased angiogenic intensity of TAMR-MCF-7 cells was significantly suppressed by Pin1 inhibition. These results show that Pin1 overexpression is closely associated with VEGF-mediated angiogenesis and suggest that Pin1 is a potential therapeutic target of excessive angiogenesis in TAM-resistant breast cancer cases.