Molecular Mechanisms Of Tamoxifen Resistance Research Articles

Abstract P3-11-02: CST1 Interaction with RAB1B Modulates Tamoxifen resistance of Breast Cancer by Regulating Autophagy

Abstract Background: Breast cancer is a prominent cause of cancer-related death among women worldwide. Approximately,70% of breast cancers (BC) are estrogen and progestogen receptor-positive (HR+) at diagnosis. Adjuvant endocrine therapy is a pivotal component of treatment for this type of patient. Tamoxifen (TAM), a widely recommended drug in endocrine therapy, is confronted with a high risk of recurrence after 5 years of treatment among breast cancer patients, which shows big clinical demands. Therefore, it is urgent to find new targets for tamoxifen resistance therapy. Recently, our team found that enhanced expression of Cystain SN (CST1) was detected in tissues that had undergone tamoxifen treatment appearing to recurrence and metastasis in ER-positive breast cancer patients. However, the molecular mechanisms of modulating tamoxifen resistance remain unknown. Therefore, our team suspected that there might be a correlation between the abnormal expression of CST1 and tamoxifen resistance. To investigate whether CST1 is involved in tamoxifen resistance and its molecular mechanism, we conducted further research. It is critical to elucidate the molecular mechanisms of tamoxifen resistance and to explore a novel effective therapeutic target. Method: Enhanced expression of CST1 was confirmed by immunohistochemistry in breast cancer tissue samples and by quantitative real-time PCR in Tamoxifen-resistant cancer cell lines MCF-7R/T47DR. To further investigate the molecular function of CST1, we knockdown its expression in MCF-7R/T47DR cells by transfecting with the lentiviral particles of shCST1 and corresponding negative control (Shanghai Genechem Co., Ltd.) and overexpressed CST1 in MCF-7/T47D cells using ove-GST-CST1, ove-HA-RAB1B plasmids. The effects of CST1 on cell viability and proliferation in breast cancer cells were detected by cell counting kit-8 and colony formation assays, respectively. The interaction between CST1 and RAB1B was validated by Co-immunoprecipitation and Western blot. Autophagosomes were found using transmission electron microscopy (TEM) along with Western blotting to detect the transformation of LC3-I to LC3-II protein (i.e., the LC3-II/LC3-I ratio) and P62 protein levels. Results: We found that aberrant expression of Cystatin SN (CST1) was identified in ER-positive breast cancer (BC) cells (Figure 1) and which is crucial in contributing to Tamoxifen resistance (Figure 2). The direct promotion of autophagy by RAPA can faciliate cells to endocrine resistance. And knockdown of CST1 not only suppressed autophagy in Tamoxifen-resistant cells but also attenuated the resistance, which determined the correlation of CST1 with autophagy in BC (Figure 3). Rab1b (Ras-related protein Rab-1B), a membrane protein essential for autophagosome formation, was verified to have interaction with CST1 through Coimmunoprecipitation (CO-IP) experiments (Figure 4). Meanwhile, RAB1B expression was also reduced by CST1 knockdown in the resistant cells. The interaction between CST1 and RAB1B induces autophagy which decreases Tamoxifen’s therapeutic efficacy. Furthermore, rescue experiments suggested that CST1 knockdown–induced sensitization in the efficacy of TAM Therapy and decreased autophagy could be restored via RAB1B overexpression.Conclusions: These results indicate that CST1 interacts with RAB1B to facilitate BC resistant to TAM through regulation of autophagy and CST1 might be a potential therapeutic target to overcome endocrine therapy resistance. Figure 1. Enhanced expression of CST1 in Tamoxifen-resistance breast cancer Figure 1. Enhanced expression of CST1 in Tamoxifen-resistance breast cancer Figure 2. CST1 expression is positively correlated with autophagy in ER-positive breast cancer Figure 2. CST1 expression is positively correlated with autophagy in ER-positive breast cancer Figure 3. CST1 promotes cells resistant to tamoxifen therapy by activating auophagy Figure 3. CST1 promotes cells resistant to tamoxifen therapy by activating auophagy Figure 4. CST1 interacts with RAB1B in cell autophagy Figure 4. CST1 interacts with RAB1B in cell autophagy Citation Format: li liu. CST1 Interaction with RAB1B Modulates Tamoxifen resistance of Breast Cancer by Regulating Autophagy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-02.

Identification of key genes involved in tamoxifen-resistant breast cancer using bioinformatics analysis.

BackgroundThe purpose of the present study was to investigate the molecular mechanisms of tamoxifen resistance in breast cancer and to identify potential targets for antitamoxifen resistance.MethodsDifferentially expressed genes (DEGs) in tamoxifen-resistant and tamoxifen-sensitive breast cancer cells were assessed using the GSE67916 dataset acquired from the Gene Expression Omnibus database. Gene ontology (GO) and pathway enrichment analyses were applied to investigate the functions and pathways of the DEGs. Subsequently, the protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING), and subnetworks were further analyzed by Molecular Complex Detection (MCODE). The PPI network and subnetworks were visualized using Cytoscape software.ResultsIn total, 438 DEGs were identified, of which 300 were upregulated and 138 were downregulated. The DEGs were significantly enriched in the protein binding, cellular response to estradiol stimulus, and immune response GO terms while the most significant pathways included the mitogen-activated protein kinase (MAPK) signaling pathway in cancer. The PPI network of DEGs was constructed with 288 nodes and 629 edges, and 2 subnetworks were screened out from the entire network.ConclusionsA number of significant hub DEGs were identified based on their degree of connectivity in the PPI network, , included MAPK1 (node degree 36), ESR1 (node degree 27), SMARCA4 (node degree 27), RANBP2 (node degree 25), and PRKCA (node degree 21). These critical hub genes were found to be related to tamoxifen resistance in breast cancer. The results of this study further the understanding of tamoxifen resistance at the molecular level and identify potential therapeutic targets for tamoxifen-resistant breast cancer.

Tamoxifen resistance mechanisms in breast cancer treatment

Therapies targeting estrogen receptor (ER) are being widely used to treat ER+ breast cancer patients. Despite early detection and improved survival outcomes, tamoxifen resistance, either intrinsic or acquired- is a major obstacle in effective disease management. Current review will summarize different molecular mechanisms of tamoxifen resistance both intrinsic and acquired in breast cancer treatment. This review not only provides basis to understand the nature of tamoxifen drug resistance but also suggests the mechanisms for its control leading to improved therapeutic interventions.

Tamoxifen resistance mechanisms in breast cancer treatment

Therapies targeting estrogen receptor (ER) are being widely used to treat ER+ breast cancer patients. Despite early detection and improved survival outcomes, tamoxifen resistance, either intrinsic or acquired- is a major obstacle in effective disease management. Current review will summarize different molecular mechanisms of tamoxifen resistance both intrinsic and acquired in breast cancer treatment. This review not only provides basis to understand the nature of tamoxifen drug resistance but also suggests the mechanisms for its control leading to improved therapeutic interventions.

Long non‑coding RNA UCA1 confers tamoxifen resistance in breast cancer endocrinotherapy through regulation of the EZH2/p21 axis and the PI3K/AKT signaling pathway.

Tamoxifen is the gold standard for breast cancer endocrinotherapy. However, drug resistance remains a major limiting factor of tamoxifen treatment. Long non‑coding (lnc) RNA serves an important role in drug resistance; however, the molecular mechanisms of tamoxifen resistance in breast cancer endocrinotherapy are largely unclear. lncRNA urothelial cancer associated1 (lncRNA UCA1, UCA1) has been proven to be dysregulated in human breast cancer and promotes cancer progression. In the present study, it was demonstrated that UCA1 was significantly upregulated in breast cancer tissues compared with healthy tissues. Furthermore, the expression level of UCA1 was significantly greater in tamoxifen‑resistant breast cancer cells (LCC2 and LCC9) when compared with those in the tamoxifen‑sensitive breast cancer cells (MCF‑7 and T47D). UCA1 silencing in LLC2 and LLC9 cells increased tamoxifen drug sensitivity by promoting cell apoptosis and arresting the cell cycle at the G2/M phase. Notably, the induced overexpression of UCA1 in MCF‑7 and T47D cells decreased the drug sensitivity of tamoxifen. The molecular mechanism involved in UCA1‑induced tamoxifen‑resistance was also investigated. It was identified that UCA1 was physically associated with the enhancer of zeste homolog2 (EZH2), which suppressed the expression of p21 through histone methylation (H3K27me3) on the p21 promoter. In addition, it was demonstrated that UCA1 expression was paralleled to the phosphorylation of CAMP responsive element binding protein (CREB) and AKT. When LCC2 cells were treated with the phosphoinositide 3‑kinase (PI3K)/protein kinaseB(AKT) signaling pathway inhibitor LY294002, the phosphorylation levels of CREB and AKT were significantly downregulated. Taken together, it was concluded that UCA1 regulates the EZH2/p21 axis and the PI3K/AKT signaling pathway in breast cancer, and may be a potential therapeutic target for solving tamoxifen resistance.

Regulation of tamoxifen resistance in estrogen receptor positive MCF-7 breast cancer cells by microRNA-10b targeting histone deacetylase 4

Objective To investigate the effect of microRNA-10b (miR-10b) targeting histone deacetylase 4 (HDAC4) on the resistance of estrogen receptor positive (ER+ ) MCF-7 breast cancer cells to tamoxifen. Methods ER+ and tamoxifen resistant MCF-7 cell line MCF-7-TR was established, and miR-10b expression level in MCF-7 cells and MCF-7-TR cells was detected by reverse transcription real-time quantitative polymerase chain reaction (Real-time PCR). Methyl thiazol tetrazolium (MTT) and trypan blue were used to detect the proliferation and activity of MCF-7 cells and MCF-7-TR cells transfected with pre-miR-10b, miR-10b inhibitor and siHDAC4 at different tamoxifen concentrations (0, 5, 10, 15, 20, 30 μmol/L), respectively. The expression of HDAC4 protein was detected by Western blotting. Results Taken expression level and invasive ability of miR-10b in MCF-7 breast cancer cells as the standard, the miR-10b expression level and invasion ability of MCF-7-TR cell breast cancer cells were 7.2±1.1 and 5.3±1.3 respectively. With the increase of the concentration of tamoxifen, decreased the rate of cell proliferation in MCF-7 cells, which is most obvious, followed by MCF-7-TR+ miR-10b inhibitor + siHDAC4 and MCF-7+ pre-miR-10b+ HDAC4 cells, and the proliferation of MCF-7-TR+ siHDAC4 cells and MCF-7-TR cells decreased the rate of the lowest. The migration ability of MCF-7 cells and MCF-7-TR+ miR-10b cells of the inhibitor decreased most, MCF-7+ pre-miR-10b+ and tamoxifen (10 μmol/L) MCF-7-TR+ cells and tamoxifen (10 μmol/L) had no significant difference in the ability of cell migration (t=1.116, P=0.272), but was significantly higher than that of MCF-7 cells and MCF-7-TR+ miR-10b cells (t=0.124, 10.769, 14.569, 11.577, P=0.000). The expression level of HDAC4 protein in MCF-7+ pre-miR-10b cells was lower than that in MCF-7 cells, MCF-7-TR cells, HDAC4 protein expression level, MCF-7-TR+ miR-10b inhibitor cells, MCF-7+ siHDAC4 cells HDAC4 protein expression level was lower than MCF-7 cells. Conclusion The miR-10b-HDAC4 signaling pathway may be a molecular mechanism of tamoxifen resistance in breast cancer. Key words: Breast cancer; Estrogen receptor positive; Tamoxifen resistance; MicroRNA-10b; Histone deacetylase 4

Integrative analysis of miRNA and gene expression reveals regulatory networks in tamoxifen-resistant breast cancer.

Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer, however, tamoxifen resistance is frequently observed. To elucidate the underlying molecular mechanisms of tamoxifen resistance, we performed a systematic analysis of miRNA-mediated gene regulation in three clinically-relevant tamoxifen-resistant breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive cell line. Alterations in the expression of 131 miRNAs in tamoxifen-resistant vs. parental cell lines were identified, 22 of which were common to all TamRs using both sequencing and LNA-based quantitative PCR technologies. Although the target genes affected by the altered miRNA in the three TamRs differed, good agreement in terms of affected molecular pathways was observed. Moreover, we found evidence of miRNA-mediated regulation of ESR1, PGR1, FOXM1 and 14-3-3 family genes. Integrating the inferred miRNA-target relationships, we investigated the functional importance of 2 central genes, SNAI2 and FYN, which showed increased expression in TamR cells, while their corresponding regulatory miRNA were downregulated. Using specific chemical inhibitors and siRNA-mediated gene knockdown, we showed that both SNAI2 and FYN significantly affect the growth of TamR cell lines. Finally, we show that a combination of 2 miRNAs (miR-190b and miR-516a-5p) exhibiting altered expression in TamR cell lines were predictive of treatment outcome in a cohort of ER+ breast cancer patients receiving adjuvant tamoxifen mono-therapy. Our results provide new insight into the molecular mechanisms of tamoxifen resistance and may form the basis for future medical intervention for the large number of women with tamoxifen-resistant ER+ breast cancer.

ER-α36, a novel variant of ERα, is involved in the regulation of Tamoxifen-sensitivity of glioblastoma cells

Although accumulating evidence has confirmed that adjuvant Tamoxifen (TAM) treatment is able to sensitize glioblastoma cells to radiotherapy and inhibit their proliferation, TAM is not a suitable treatment for all types of glioblastoma cells; furthermore, long-term TAM usage may lead to TAM resistance. Therefore, understanding the underlying molecular mechanism of TAM resistance is necessary in order to improve TAM clinical therapy and the quality of life of patients suffering from glioblastomas. In this study, the significance of ER-α36 to TAM resistance in glioblastoma cells was examined. First, an analysis of ER-α36 expression in two glioblastoma cell lines U87-MG and U251, showed that ER-α36 was anchored to the cytoplasmic membrane of these cells via Caveolin-1. Subsequent experiments investigating the mechanism of TAM-induced inhibition of U87-MG cell growth showed that TAM exerts its effect by inducing apoptosis via a down-regulation of Survivin expression and an up-regulation of Caspase-3 expression. Furthermore, TAM also arrested the cell cycle at S-phase. However, when U87-MG cells were preconditioned with an ER-α36-specific agonist, IC162, this neutralized TAM-induced inhibition of cell growth. This contrasted with the effect of ER-α36 depletion by RNAi, which enhanced TAM-induced inhibition of cell growth. These findings suggest that resistance to TAM involves ER-α36, which probably acts as a negative regulator of TAM-induced inhibition of glioblastoma cell growth. These findings provide a novel insight into the basis of TAM resistance during glioblastoma therapy and a further study is underway to reveal more about the specific molecular mechanisms associated with ER-α36-mediated TAM resistance.

Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4.

BackgroundFor breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors, treatment with tamoxifen is the gold standard. A significant number of patients, however, develop resistance to tamoxifen, and management of such tamoxifen-resistant patients is a major clinical challenge. With an eye to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed that tamoxifen-resistant cells derived from ER-positive MCF-7 cells (MCF7TR) exhibit an increased expression of microRNA-10b (miR-10b). A role of miR-10b in drug-resistance of breast cancer cells has never been investigated, although its is very well known to influence invasion and metastasis.MethodsTo dileneate a role of miR-10b in tamoxifen-resistance, we over-expressed miR-10b in MCF-7 cells and down-regulated its levels in MCF7TR cells. The mechanistic role of HDAC4 in miR-10b-mediated tamoxifen resistance was studied using HDAC4 cDNA and HDAC4-specific siRNA in appropriate models.ResultsOver-expression of miR-10b in ER-positive MCF-7 and T47D cells led to increased resistance to tamoxifen and an attenuation of tamoxifen-mediated inhibition of migration, whereas down-regulation of miR-10b in MCF7TR cells resulted in increased sensitivity to tamoxifen. Luciferase assays identified HDAC4 as a direct target of miR-10b. In MCF7TR cells, we observed down-regulation of HDAC4 by miR-10b. HDAC4-specific siRNA-mediated inactivation of HDAC4 in MCF-7 cells led to acquisition of tamoxifen resistance, and, moreover, reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen-resistance.ConclusionsWe propose miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance which can potentially be expolited as a novel targeted therapeutic approach for the clinical management of tamoxifen-resistant breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1561-x) contains supplementary material, which is available to authorized users.

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

To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs 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. Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of TamR 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, whereas knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth- and survival-promoting function of FYN in MCF-7 cells. FYN knockdown in TamR cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, may overcome the anti-proliferative effects of tamoxifen. Evaluation of the subcellular localization of FYN in primary breast tumors from two cohorts of endocrine-treated ER+ breast cancer patients, one with advanced disease (N=47) and the other with early disease (N=76), showed that in the former, plasma membrane-associated FYN expression strongly correlated with longer progression-free survival (P<0.0002). Similarly, in early breast cancer patients, membrane-associated expression of FYN in the primary breast tumor was significantly associated with increased metastasis-free (P<0.04) and overall (P<0.004) survival independent of tumor size, grade or lymph node status. Our results indicate that FYN has an important role in tamoxifen resistance, and its subcellular localization in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer.

Gene expression profiling for identification of FYN in tamoxifen resistance and as predictor of early recurrence in patients treated with endocrine therapy.

580 Background: Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Methods: To elucidate the molecular mechanisms of tamoxifen resistance in br...

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 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&amp;lt;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&amp;lt;0.003) and overall survival (p&amp;lt;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

Tamoxifen Resistance in Breast Cancer

Tamoxifen is a central component of the treatment of estrogen receptor (ER)-positive breast cancer as a partial agonist of ER. It has been clinically used for the last 30 years and is currently available as a chemopreventive agent in women with high risk for breast cancer. The most challenging issue with tamoxifen use is the development of resistance in an initially responsive breast tumor. This review summarizes the roles of ER as the therapeutic target of tamoxifen in cancer treatment, clinical values and issues of tamoxifen use, and molecular mechanisms of tamoxifen resistance. Emerging knowledge on the molecular mechanisms of tamoxifen resistance will provide insight into the design of regimens to overcome tamoxifen resistance and discovery of novel therapeutic agents with a decreased chance of developing resistance as well as establishing more efficient treatment strategies.

Abstract A1: Functional significance of miR-10b in tamoxifen resistance of breast cancer cells

Abstract Tamoxifen is a conventional treatment option for breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors. However, a significant number of these patients eventually develop resistance to tamoxifen during therapy, and thus the management of such tamoxifen-resistant patients remains a clinical challenge. In an effort to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed increased expression of microRNA 10b (miR-10b) in ER-positive MCF-7 cells-derived tamoxifen-resistant cells (MCF7TR). The miR-10b has been implicated in invasion and metastasis but its role, if any, in drug resistance of breast cancer cells has never been investigated. To ascertain the mechanistic role of miR-10b in tamoxifen resistance, we downregulated its levels in MCF7TR cells and in addition we also overexpressed miR-10b by transfecting MCF-7 cells with pre-miR-10b. We found that the downregulation of miR-10b in MCF7TR cells led to increased sensitivity to tamoxifen whereas overexpression of miR-10b in MCF-7 cells led to increased resistance to tamoxifen. During our mechanistic investigation, we found that increased expression of miR-10b downregulated the expression of HDAC4 in MCF7TR cells. Inactivation of HDAC4 using HDAC4-specific siRNA transfection in MCF-7 cells led to the acquisition of tamoxifen resistance. Moreover, further reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen resistance. In summary, we have identified miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance, and thus targeting this nexus could become a novel targeted therapeutic approach for the treatment of tamoxifen-resistant breast cancer.

Downregulation of miR-342 is associated with tamoxifen resistant breast tumors

BackgroundTumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive.ResultsHere we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators.ConclusionsOur findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.

Tamoxifen-stimulated growth of breast cancer due to p21 loss

Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21's cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-alpha, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.