The following full text is a publisher's version.For additional information about this publication click this link. https://hdl.handle.net/2066/229509Please be advised that this information was generated on 2023-04-03 and may be subject to change.T h e ne w e ngl a nd jou r na l o f m e dicine n engl j med 383;24 nejm.org

BackgroundHormone-refractory prostate cancer remains hindered by inevitable progression of resistance to first-line treatment with docetaxel. Recent studies suggest that phenotypic changes associated with cancer may be transferred from cell-to-cell via microvesicles/exosomes. Here we aimed to investigate phenotypic changes associated with docetaxel-resistance in order to help determine the complexity of this problem and to assess the relevance of secreted exosomes in prostate cancer.Methodology/Principal FindingsDocetaxel-resistant variants of DU145 and 22Rv1 were established and characterised in terms of cross-resistance, morphology, proliferation, motility, invasion, anoikis, colony formation, exosomes secretion their and functional relevance. Preliminary analysis of exosomes from relevant serum specimens was also performed. Acquired docetaxel-resistance conferred cross-resistance to doxorubicin and induced alterations in motility, invasion, proliferation and anchorage-independent growth. Exosomes expelled from DU145 and 22Rv1 docetaxel-resistant variants (DU145RD and 22Rv1RD) conferred docetaxel-resistance to DU145, 22Rv1 and LNCap cells, which may be partly due to exosomal MDR-1/P-gp transfer. Exosomes from prostate cancer patients’ sera induced increased cell proliferation and invasion, compared to exosomes from age-matched controls. Furthermore, exosomes from sera of patients undergoing a course of docetaxel treatment compared to matched exosomes from the same patients prior to commencing docetaxel treatment, when applied to both DU145 and 22Rv1 cells, showed a correlation between cellular response to docetaxel and patients’ response to treatment with docetaxel.Conclusions/SignificanceOur studies indicate the complex and multifaceted nature of docetaxel-resistance in prostate cancer. Furthermore, our in vitro observations and preliminary clinical studies indicate that exosomes may play an important role in prostate cancer, in cell-cell communication, and thus may offer potential as vehicles containing predictive biomarkers and new therapeutic targets.

BackgroundThere is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel.ResultsThe resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel.ConclusionThis study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancers but is responsible for a disproportionate number of deaths. We investigated the relevance, in TNBC, of nano-sized exosomes expelled from cells. Specifically, we compared effects of exosomes derived from the claudin-low TNBC cell line Hs578T and its more invasive Hs578Ts(i) 8 variant, as well as exosomes from TNBC patient sera compared to normal sera. Methods: Exosomes were isolated from conditioned media (CM) of Hs578T and Hs578Ts(i) 8 cells and from sera by filtration and ultracentrifugation. Successful isolation was confirmed by transmission electron microscopy and immunoblotting. Subsequent analysis, of secondary/ recipient cells in response to exosomes, included proliferation; motility/migration; invasion; anoikis assays and endothelial tubule formation assays. Results: Hs578Ts(i) 8 -exosomes versus Hs578T-exosomes significantly increased the proliferation, migration and invasion capacity of all three recipient cell lines evaluated i.e. SKBR3, MDA-MB-231 and HCC1954. Exosomes from Hs578Ts(i) 8 cells also conferred increased invasiveness to parent Hs578T cells. Hs578Ts(i) 8 -exosomes increased sensitivity of SKBR3, MDA-MB-231 and HCC1954 to anoikis when compared to the effects of Hs578T-exosomes reflecting the fact that Hs578Ts(i) 8 cells are themselves innately more sensitive to anoikis. In relation to vasculogenesis and subsequent angiogenesis, Hs578Ts(i) 8 -exosomes versus Hs578T-exosomes stimulated significantly more endothelial tubules formation. Finally, our 0959-8049/$ -see front matter Ó

Exosomes (EVs) have relevance in cell-to-cell communication carrying pro-tumorigenic factors that participate in oncogenesis and drug resistance and are proposed to have potential as self-delivery systems. Advancing on our studies of EVs in triple-negative breast cancer, here we more comprehensively analysed isogenic cell line variants and their EV populations, tissues cell line variants and their EV populations, as well as breast tumour and normal tissues. Profiling 384 miRNAs showed EV miRNA content to be highly representative of their cells of origin. miRNAs most substantially down-regulated in aggressive cells and their EVs originated from 14q32. Analysis of miR-134, the most substantially down-regulated miRNA, supported its clinical relevance in breast tumours compared to matched normal breast tissue. Functional studies indicated that miR-134 controls STAT5B which, in turn, controls Hsp90. miR-134 delivered by direct transfection into Hs578Ts(i)8 cells (in which it was greatly down-regulated) reduced STAT5B, Hsp90, and Bcl-2 levels, reduced cellular proliferation, and enhanced cisplatin-induced apoptosis. Delivery via miR-134-enriched EVs also reduced STAT5B and Hsp90, reduced cellular migration and invasion, and enhanced sensitivity to anti-Hsp90 drugs. While the differing effects achieved by transfection or EV delivery are likely to be, at least partly, due to specific amounts of miR-134 delivered by these routes, these EV-based studies identified miRNA-134 as a potential biomarker and therapeutic for breast cancer.