ESR1 hotspot mutations have been identified in 30-40% of patients with ER+ MBC and promote resistance to aromatase inhibitors (AIs). Identification of these mutations has been aided by the use of plasma DNA for their detection, however many such tests only survey for hotspot mutations. In this study, we examined the prevalence, biologic and clinical significance of mutations in ESR1 that lie outside previously described hotspots (E380Q, Y537, D538G). Using next generation sequencing of tumor DNA from over 4000 patients with breast cancer, we have identified numerous somatic alterations in ESR1. Among the somatic alterations were mutations detected in the transcription activation function-1 (AF-1), DNA binding domain, dimerization interface and C-terminus of ER. We characterized the functional significance of these non-canonical mutations alongside hotspot mutations, starting with assays of ER driven transcription and deduced several classes of mutations: (1) mutations that weakly promote ligand-independent activity, (2) mutations that led to ligand-independent activity comparable to estradiol stimulation, and (3) mutations that resulted in impaired transcriptional activity. Class 2 mutations remain localized at amino acids 536-538, while the class 1 mutations are observed in various domains of ESR1, including the DNA binding domain and dimerization interface. Several Class 3 mutations were found in proximal to Helix 12, highlighting the critical role of this region. Clinically, non-canonical mutations were not exclusively observed among patients treated with AI, as there were several mutations from SERM/SERD treated patients. We thus examined the effects of different mutants on their sensitivity to ER antagonists, such as fulvestrant or tamoxifen. The data revealed key differences between the different classes of mutants; with majority of the class 2 mutants exhibiting reduced sensitivity to the antagonists compared to wild type. This also correlated with the relative binding affinities (RBA) of the mutants to fulvestrant and 4-hydroxytamoxifen, in which the RBA of class 2 mutants (Y537S and D538G) were significantly lower than wild type, perhaps accounting for their reduced sensitivities. Nevertheless, it appeared that all mutants could be effectively inhibited either by higher concentrations or more potent ER antagonists, implying a continued ability to distort ER into the antagonist conformation. Interestingly, several Class 1 mutants showed weak agonism in response to specific antagonists raising the possibility of their role in SERM/SERD resistance. Taken together, the data demonstrated that not all ESR1 mutations affect receptor function or respond to antiestrogen therapies similarly. These data also imply the importance of more broad sequencing coverage of ESR1 in the clinic to effectively capture the spectrum of biologically relevant alleles. Citation Format: Toy W, Carlson KE, Martin TA, Razavi P, Berger M, Baselga J, Greene G, Katzenellenbogen J, Chandarlapaty S. Non-canonical, clinical ESR1 mutations promote resistance to antiestrogen therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-11.
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Background: Claudin-20 is a member of the claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Claudin-20 was originally described as being distributed in the TJ of intestinal epithelial cells. Due to the increasing evidence supporting the role of TJ proteins in preventing tumour cell metastatic behaviour, this study sought to evaluate the effect of claudin-20 overexpression in human breast cancer cells. Methods: Breast cancer primary tumours (n= 114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR to obtain survival curves. 42 human cancer cell lines were screened for expression of claudin-20. Invasive and non-invasive breast cancer cell lines, MDA-MB-231 (MDAwt) and MCF-7 (MCF-7wt) respectively, which exhibited negligible expression were chosen for over-expression of the claudin-20 gene (MDAexp & MCF-7exp). The gene, after amplification from normal breast cDNA was cloned into a GFP-Fusion TOPO plasmid before electroporation. Expression of the gene product was confirmed by RT-PCR and fluorescent microscopy. Functional testing was carried out using TER (trans-epithelial resistance), paracellular permeability (PCP), cell invasion, cell adhesion and cell growth assays. In addition, in vivo growth was evaluated using an athymic nu/nu mice model. Results: Q-PCR data showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p=0.022). We therefore decided to investigate the effect of claudin-20 over-expression in human breast cancer cell behaviour. Following transformation of the breast cancer cell lines MDA-MB-231 & MCF-7 with a claudin-20 expression construct, the expression of the gene was demonstrated by the presence of mRNA expression and by the protein signal clearly observable at the cell-cell membrane. Claudin-20 transformed cells showed significantly increased invasion compared to WT cells (MDAexp 0.332±0.02 RU, MDAwt 0.304±0.001; p=0.05: MCF-7exp0 0.07±0.002 RU, MCF-7wt 0.05±0.0001; p=0.0014). Both MDAexp and MCF-7exp cells were significantly less adhesive than WT cells, (number of cells adhering to matrix: MDAexp 0.2±0.02 RU versus MDAwt 0.27±0.004, p=0.007; MCF-7exp 0.043±0.0005 RU versus MCF-7wt 0.052±0.0.005, p=0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Over-expression of Claudin-20 resulted in reduced TER (induced by the motogen HGF at 25 ng/ml: MDAexp change in TER at 2h -74+/−1.5 vs MDAwt at 2h -40+/−4.4, p=0.0007). Interestingly, this was not mirrored by PCP, as over-expression of claudin-20 caused a reduction in permeability (MDAexp change in PCP at 2h 182+/−5 vs MDAwt at 2h 260+/−10). Conclusion: The introduction of claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-07.
Introduction: Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily and has been indicated in the pathophysiology of the nervous system and is important in a number of neurological and psychological conditions. Recently, BDNF has also been shown to play a role in the development and progression of solid tumours myeloma, lung cancer and prostate cancer. It has been recently reported that BDNF is also aberrantly expressed in human breast cancer and that a raised level of BDNF is associated with poor clinical outcome and reduced survival. The present study aimed to investigate the biological role of BDNF in human breast cancer. Materials and methods: A panel of human breast cancer cells were used in the present study. The expression profile of BDNF was evaluated using RT-PCR. We constructed a set of anti-BDNF transgenes which were used to transfect breast cancer cells in order to generate BDNF knockdown cells (MCFΔBDNF). The impact of BDNF knockdown on growth and cellular migration were evaluated using standard growth assays and ECIS (electronic cell impedance sensing) technology. Results: BDNF gene transcripts were successfully detected in the breast cancer cell lines, MCF-7, MDA MB-231 and ZR 75-1. MCF-7 wild type cells were subject to transfection of anti-BDNF transgenes, followed by the establishment of BDNF knockdown sub-lines and empty vector control cells (MCFpef). Loss of BDNF in breast cancer cells resulted in reduction of cell growth rate (growth rate in MCFΔBDNF by day 3 1.60+/−0.11, compared with 2.06+/−0.04 in MCFpef, p=0.006). Using electric cell-substrate impedance sensing, it was found that loss of BDNF in breast cancer cells resulted in a significant increase in the rate of cellular attachment and migration (migration rate over 14 h in ohms: MCFΔBDNF 6310.125+/−595.183 compared with MCFpef 5115.5 +/−422.394, p=0.0003). Conclusion: When BDNF was stably knocked down in MCF-7 cell lines, the growth and proliferation were decreased. The ECIS results also showed that cell migration was decreased in MCF-7 cells stably transfected with ribozymes for BDNF compared with the vector control. It is concluded that BDNF, a neurotrophic growth factor aberrantly expressed in cancers such as breast cancer, has a profound impact on the cellular behaviour of breast cancer cells and that loss of BDNF is associated with a reduction of the aggressiveness of breast cancer. BDNF is therefore a potential therapeutic target in breast cancer and its effect in human breast cancer requires further investigation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-21.
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