Novel sulphamoylated 2‑methoxy estradiol derivatives inhibit breast cancer migration by disrupting microtubule turnover and organization

BACKGROUND : The estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis. In interphase cells, microtubules play an important role in the delivery of proteins to subcellular locations including the focal adhesions. In fact, focal adhesion dynamics and cell migration are in part regulated by microtubules. We hypothesised that novel 2ME2 derivatives can alter cell migration by influencing microtubule dynamics in interphase cells. In this report we describe 2ME2 derivatives that display anti-migratory capabilities in a metastatic breast cancer cell line through their effects on the microtubule network resulting in altered focal adhesion signalling and RhoA activity. METHODS : Cell migration was assayed using wound healing assays. To eliminate mitosis blockage and cell rounding as a confounding factor cell migration was also assessed in interphase blocked cells. Fluorescence confocal microscopy was used to visualise microtubule dynamics and actin cytoskeleton organisation while western blot analysis was performed to analyse focal adhesion signalling and RhoA activation. RESULTS : 2ME2 derivatives, ESE-one and ESE-15-one, inhibited cell migration in cycling cells as expected but equally diminished migration in cells blocked in interphase. While no significant effects were observed on the actin cytoskeleton, focal adhesion kinase activity was increased while RhoA GTPase activity was inhibited after exposure to either compound. Microtubule stability was increased as evidenced by the increased length and number of detyrosinated microtubules while at the same time clear disorganisation of the normal radial microtubule organisation was observed including multiple foci. CONCLUSIONS : ESE-15-one and ESE-one are potent migration inhibitors of metastatic breast cancer cells. This ability is coupled to alterations in focal adhesion signalling but more importantly is associated with severe disorganisation of microtubule dynamics and polarity. Therefore, these compounds may offer potential as anti-metastatic therapies.The National Research Foundation (NRF) of South Africa, the Cancer Association of South Africa (CANSA), the Medical Research Council (MRC) of South Africa, the Struwig Germishuysen Trust and the School of Medicine Research Committee of the University of Pretoria (RESCOM).http://www.cancerci.comgl2020Physiolog

Extracellular Hsp90 and TGFP regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

Tumour metastasis remains the major cause of death in cancer patients and, to date, the mechanism and signalling pathways governing this process are not completely understood. The TGF-ß pathway is the most commonly mutated pathway in cancer, however its role in cancer progression is controversial as it can function as both a promoter and a suppressor of metastasis. Although previous studies have suggested a role for the molecular chaperone Hsp90 in regulating the TGF-ß pathway, the level at which this occurs as well as the consequences in terms of colon cancer metastasis are unknown

Development of a breast cancer spheroid model for use in drug discovery and cancer biology research

The screening of lead compounds during cancer drug discovery still results in many hits that never reach the clinic. This is in part due to the distance between 2D cell culture, which is mostly used for screening assays and the complexity of an in vivo tumour setting. It is thus essential that screening setups are developed that better bridge the gap between in vitro and in vivo studies. Culturing cells in 3D has been shown to provide an architecture and gene expression profile that better resembles that found in tumours. A number of methods have been developed but many are laborious, require specialised equipment, or are prohibitively costly. In Southern Africa, we do not always have access to expensive machinery and reagents. Therefore, in this thesis, we aimed to set up a simple, easy to use, cost-effective 3D spheroid methodology for triple negative breast cancer that could be used in any laboratory with minimal reagents. The non-metastatic triple negative breast cancer cell line, BT-20, was identified as the cell line most amenable to spheroid formation and measurement, compared to the metastatic cell lines MDA-MB-231 and MCF-7. Both these cell lines formed inconsistent, difficult to measure spheroids. A low attachment methodology was settled on to induce cell aggregation and spheroid formation. This proved to be an easy, low-cost, and reproducible method. Using the developed methodology, the efficacy of several novel compounds with anti-proliferative activity in 2D cancer cell line cultures were tested for their ability to affect cell survival in the spheroid model. Our studies show that one non-sulfamoylated 2-methoxyestradiol derivative, EE-15-one, caused a loss of cells in the 2D cell survival assay while it had no effect on BT-20 spheroids. In contrast, two other 2-methoxyestradiol derivatives had similar effects in 2D and 3D. Another novel anticancer compound, STX1972, also had anti-proliferative capabilities in 2D which were lost in cells grown in 3D. As a result, our screening approach on a small number of samples was able to identify 2 of 5 compounds that had no effect on cancer cell growth in 3D. Further analysis of EE-15-one suggests that this compound does not inhibit cell cycle progression like the other derivatives tested but instead inhibits cell adhesion. Our data shows that integrin-based adhesion is replaced by cadherin-dependent cell-cell adhesion in spheroids. Changing the mode of adhesion correlates strongly with the efficacy of this compound suggesting that it is an inhibitor of integrin dependent cell adhesion. Further analysis show that the efficacy of this compound was not dependent on hypoxia, further strengthening the suggestion that it directly acts on cell adhesion. It is commonly accepted that a tumour consists of a heterogeneous mix of subpopulations of cancer cells, each with altered genetic backgrounds. Furthermore, such subpopulations can affect each other changing behaviours of neighbouring cells. To replicate this scenario in vitro, we initiated the development of a co-culture spheroid system of different triple negative breast cancer cell lines to investigate how they behave within a spheroid. Our data shows that when a co-culture spheroid system of BT-20 and MDA-MB-231 cells is generated, the cells with migratory ability were able to migrate away from the spheroid onto rigid surfaces. In contrast, the non-metastatic BT-20 cells remained within the confines of the spheroid. This suggests that indeed different cell populations will continue to behave differently within a 3D cell culture setting. In conclusion, we have developed a robust, cost-effective 3D culture system that has shown great potential for used in high throughput screening of novel anti-proliferative compounds. We have shown that these spheroids use different adhesive strategies than their counterparts kept in 2D. This suggests that these cells also change their cellular behaviour which is essential for better mimicking the in vivo tumour setting.Thesis (PhD)--University of Pretoria, 2020.NRF-DAADMRCCANSAPhysiologyPhDRestricte

Additional file 1: of Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

Western blot analysis of the intracellular levels of TGF-β1 in SW480 and SW620 cells compared to a histone loading control. Full-length versions of the western blots shown in Fig. 1c. A) SW480 and SW620 whole cell lysates were probed for TGF-β1 using mouse anti-human TGF-β1 (sc-65378, Santa Cruz). Purified recombinant TGF-β1 (Biolegend) in its acid-activated form was included as a positive control (+). B) The membrane was reprobed for histone as a loading control using rabbit anti-human histone (9715 L, Cell Signalling Technologies). (TIF 1216 kb

A novel non-sulphamoylated 2-methoxyestradiol derivative causes detachment of breast cancer cells by rapid disassembly of focal adhesions

Abstract Background 2-Methoxyestradiol (2ME2) is an estradiol metabolite with well documented antiproliferative properties in many cancer cell lines. However, it is rapidly metabolised in vivo which limits its clinical application. Therefore, more stable derivatives with potentially improved clinical features have been designed by our group. Here we describe an estrone-like derivative of 2ME2, namely EE-15-one, that unlike other derivatives which induce cell cycle arrest, induces a rapid loss of cell–substrate adhesion through the inactivation and disassembly of focal adhesions. Methods To assess the effect of 2-ethyl-estra-1,3,5 (10),15-tetraen-3-ol-17-one (EE-15-one) on breast cancer cell lines, cell survival was quantified. The effect of EE-15-one on cell attachment was assessed by measuring cell adhesion and cell rounding via light microscopy. Effects on focal adhesion dynamics and actin cytoskeleton organisation were visualised by immunofluorescence while focal adhesion signalling was assessed by western blot. Cell death was quantified using a lactate dehydrogenase activity (LDH) assay. To investigate specificity towards cell–substrate over cell–cell contact inhibition, EE-15-one effects on 3D cell cultures were assessed. Results Cell survival assays show an almost complete loss of cells within 24 h of EE-15-one exposure in contrast to published sulphamoylated 2ME2 derivatives. Cell loss is linked to rapid detachment and adhesion inhibition. Focal adhesion size and number are rapidly diminished while actin fibres became severed and disappeared within 2 h post exposure. These changes were not due to cell necrosis as LDH activity only slightly increased after 24 h. Cells grown in cell–cell adhesion dependent spheroids did not respond to EE-15-one exposure suggesting that EE-15-one specifically inhibits cell–substrate adhesions but not cell–cell adhesions and does not directly impact the actin cytoskeleton. Conclusion We show that a novel 2ME2 derivative, EE-15-one, induces rapid loss of focal adhesion function leading to cell–substrate detachment through interference with integrin-based cell–substrate adhesions, but not cadherin dependent cell–cell adhesions. Therefore, EE-15-one is the first 2ME2 derivative that has an alternative mode of action to the antimitotic activity of 2ME2. As such EE-15-one shows potential as a lead compound for further development as an inhibitor of cell–substrate adhesion which is essential for metastatic dissemination