Expression data from Tamoxifen resistant Breast Cancer Cell lines
ABSTRACT: Tamoxifen Resistant (TR) gene profile from Breast cancer cell lines T47D and ZR75-1 with their oestrogen-deprieved conterparts were analysed for gene associated with TR. We used Microarray Affymetrix HU133plus 2.0 chips for gene expression of TR cell lines, normalised them against GEO data available for normal T47D (GSM70667) and ZR75-1 (GSM70668). We grew parental breast cancer cell lines in tamoxifen containing media (0.1 microM) for 6 months and labelled them tamoxifen resistant (TR). Oestrogen-Deprieved cells were grown in charcoal-stripped media for 6 months then tamoxifen (0.1 microM) was added to the media and cells maintained a further 6 months and termed Oestrogen deprieved-tamoxifen resistant (ODTR) .
Project description:Tamoxifen Resistant (TR) gene profile from Breast cancer cell lines T47D and ZR75-1 with their oestrogen-deprieved conterparts were analysed for gene associated with TR. We used Microarray Affymetrix HU133plus 2.0 chips for gene expression of TR cell lines, normalised them against GEO data available for normal T47D (GSM70667) and ZR75-1 (GSM70668). Overall design: We grew parental breast cancer cell lines in tamoxifen containing media (0.1 microM) for 6 months and labelled them tamoxifen resistant (TR). Oestrogen-Deprieved cells were grown in charcoal-stripped media for 6 months then tamoxifen (0.1 microM) was added to the media and cells maintained a further 6 months and termed Oestrogen deprieved-tamoxifen resistant (ODTR) .
Project description:INTRODUCTION: The tumour microenvironment is hypoglycaemic, hypoxic and acidotic. This activates a stress signalling pathway: the unfolded protein response (UPR). The UPR is cytoprotective if the stressor is mild, but may initiate apoptosis if severe.Activation of the UPR in breast carcinoma is induced by microenvironmental stress such as glucose and oxygen deprivation, but may also be linked to oestrogen stimulation. It may be clinically significant as it may alter chemosensitivity to doxorubicin. METHODS: 395 human breast adenocarcinomas were immunohistochemically stained for UPR activation markers (glucose-regulated protein (GRP-78 and XBP-1). A model of UPR activation in vitro by glucose deprivation of T47D breast cancer cells was developed to determine how the UPR affects cellular sensitivity to doxorubicin and 5-fluorouracil. Cytotoxicity was assessed using a colorimetric cytotoxicity assay (MTT). The effect of oestrogen stimulation and tamoxifen exposure on UPR activation by T47D cells was determined by western blotting measurement of the key UPR protein, GRP-78. RESULTS: Expression of GRP78 and XBP-1 was demonstrated in 76% and 90% of the breast cancers, respectively, and correlated with oestrogen receptor positivity (P=0.045 and 0.017, respectively). In vitro UPR activation induced resistance to both doxorubicin and 5-flurouracil, (P<0.05). Oestrogen stimulation induced GRP78 and XBP1 over-expression on western blotting. Tamoxifen did not block this response and may induce UPR activation in its own right. CONCLUSIONS: The UPR is activated in the majority of breast cancers and confers resistance to chemotherapy. In vitro oestrogen stimulates UPR induction. UPR activation may contribute to breast cancer chemoresistance and interact with oestrogen response elements.
Project description:Tamoxifen is a first-line drug for hormone therapy (HT) in oestrogen receptor-positive breast cancer patients. However, 20% to 30% of those patients are resistant to tamoxifen treatment. Cancer stem cells (CSCs) have been implicated as one of the mechanisms responsible for tamoxifen resistance. Our previous study indicated that decreased expression of the CRB3 gene confers stem cell characteristics to breast cancer cells. In the current investigation, we found that most of the breast cancer patient tissues resistant to tamoxifen were negative for CRB3 protein and positive for ?-catenin protein, in contrast to their matched primary tumours by immunohistochemical analysis. Furthermore, expression of CRB3 mRNA and protein was low, while expression of ?-catenin mRNA and protein was high in tamoxifen resistance cells (LCC2 and T47D TamR) contrast to their corresponding cell lines MCF7 and T47D. Similarly, CRB3 overexpression markedly restored the tamoxifen sensitivity of TamR cells by the MTT viability assay. Finally, we found that CRB3 suppressed the stemness of TamR cells by inhibiting ?-catenin signalling, which may in turn lead to a decrease in the breast cancer cell population. Furthermore, these findings indicate that CRB3 is an important regulator for breast cancer stemness, which is associated with tamoxifen resistance.
Project description:Introduction:At present, drug resistance remains a major obstacle for breast cancer (BCa) patients who receive tamoxifen (TAM) chemotherapy. In this study, we aimed to investigate the functional role of long non-coding RNA BLACAT1 in the acquisition of TAM resistance in BCa. Methods:TAM-resistant BCa cells were derived by exposure to 1 ?M of TAM for 6 months. The expression levels of BLACAT1 and miR-503 were detected by RT-qPCR analysis. Chemosensitivity of BCa cells to TAM was measured by MTT assay. Apoptosis of BCa cells was detected by flow cytometric analysis, and the expression levels of apoptosis-related proteins were detected by Western blot analysis. The direct binding relation between BLACAT1 and miR-503 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assay. Results:Our findings showed that BLACAT1 was significantly upregulated in TAM-resistant BCa cells (MCF-7/TR and T47D/TR), and BLACAT1 knockdown markedly reduced the TAM resistance in these cells. Importantly, we observed that BLACAT1 might function as a competing endogenous RNA of miR-503 in MCF-7/TR and T47D/TR cells, thereby increasing the expression of oncogenic Bcl-2 protein. Rescue experiments showed that miR-503 inhibition partly blocked the inhibitory effect of BLACAT1 knockdown on TAM resistance of MCF-7/TR and T47D/TR cells. Conclusion:To conclude, this study revealed that overexpressed BLACAT1 induces TAM resistance in human BCa partly by regulating miR-503/Bcl-2 axis, potentially benefiting BCa treatment in the future.
Project description:INTRODUCTION:Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells. METHODS:In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS:RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS:This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.
Project description:This study addresses the hypothesis that altered expression of oestrogen receptor-beta and/or altered relative expression of coactivators and corepressors of oestrogen receptors are associated with and may be mechanisms of de novo tamoxifen resistance in oestrogen receptor positive breast cancer. All cases were oestrogen receptor +, node negative, primary breast tumours from patients who later had no disease progression (tamoxifen sensitive) or whose disease progressed while on tamoxifen (tamoxifen resistant). Using an antibody to oestrogen receptor-beta that detects multiple forms of this protein (total) but not an antibody that detects only full-length oestrogen receptor-beta 1, it was found that high total oestrogen receptor beta protein expressors were more frequently observed in tamoxifen sensitive tumours than resistant tumours (Fisher's exact test, P=0.046). However, no significant differences in the relative expression of oestrogen receptor beta2, oestrogen receptor beta5 and full-length oestrogen receptor beta1 RNA in the tamoxifen sensitive and resistant groups were found. Also, when the relative expression of two known coactivators, steroid receptor RNA activator and amplified in breast cancer 1 RNA to the known corepressor, repressor of oestrogen receptor activity RNA, was examined, no significant differences between the tamoxifen sensitive and resistant groups were found. Altogether, there is little evidence for altered coregulators expression in breast tumours that are de novo tamoxifen resistant. However, our data provide preliminary evidence that the expression of oestrogen receptor beta protein isoforms may differ in primary tumours of breast cancer patients who prove to have differential sensitivity to tamoxifen therapy.
Project description:Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.
Project description:Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17?-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKC?) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKC?-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKC? and T47D:A18-TAM1 tumor models. T47D:A18/PKC? tumor regression was accompanied by translocation of estrogen receptor (ER) ? to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.
Project description:Specific high-affinity binding sites for non-steroidal anti-oestrogens such as tamoxifen have been identified in many animal and human tissues. The function of these binding sites and the nature of their endogenous ligands are currently unknown. Our laboratory has previously reported that unsaturated fatty acids at micromolar concentrations inhibited [3H]tamoxifen binding to the anti-oestrogen-binding sites in rat liver, raising the possibility that fatty acids might represent endogenous ligands for these sites. These studies have now been extended to examine the mechanism by which fatty acids inhibit [3H]tamoxifen binding to the anti-oestrogen-binding site. Saturation analysis revealed that increasing concentrations of oleic acid progressively decreased the apparent binding affinity of these sites for [3H]tamoxifen without decreasing the total number of binding sites; however, the apparent dissociation constant did not vary linearly with the prevailing oleic acid concentration, suggesting that the inhibition of [3H]tamoxifen binding by fatty acid was not competitive in nature. Kinetic studies of [3H]tamoxifen binding showed that oleic acid did not affect the rate of association, but increased the rate of dissociation of [3H]tamoxifen from the anti-oestrogen-binding site; the latter finding would not be expected if oleic acid acted as a competitive inhibitor. Furthermore, incubation of a rat microsomal fraction with [3H]oleic acid in the absence and presence of excess non-radioactively labelled tamoxifen also failed to demonstrate direct competition between oleic acid and tamoxifen for the same binding site. It is concluded that oleic acid, and presumably other unsaturated fatty acids, do not compete for the anti-oestrogen-binding site and probably reduce its tamoxifen-binding affinity by some other mechanism, such as perturbation of the lipid environment of the binding site. The biological significance of this interaction of unsaturated fatty acids with the anti-oestrogen-binding site remains to be elucidated.