Abstract The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-β in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44+, CD24−, and CD45−. Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-β), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.
<p>Supplementary Fig. S1: Tissue microarray VN:IGFBP-3 PLA immunoreactivity and patient information; Supplementary Fig. S2: VN peptide array amino acid sequence information; Supplementary Fig. S3: IGFBP-3 peptide array amino acid sequence information; Supplementary Fig. S4: VN and IGFBP-3 immunoreactivity of HMLER-FOXC2 primary tumour xenografts; Supplementary Fig. S5: Single channel images of in situ PLA; Supplementary Fig. S6: IGF-II-induced cell signalling in MCF-7 breast cancer cells; Supplementary Fig. S7: Breast cell migration measured using the xCELLigence assay; Supplementary Fig. S8: Optimisation of ligand binding to VN and its subsequent immunodetection; Supplementary Fig. S9: Identification of ligand binding sites on VN using information from VN peptide arrays; Supplementary Fig. S10: Binding sites on IGFBP-3 for VN using the IGFBP-3 peptide arrays</p>
Emerging treatments for advanced PCa continue to target androgen signalling leading to prolonged androgen deprivation. This induces metabolic dysfunction and hyperinsulinaemia which is specifically associated with more rapid progression to CRPC, but is not clinically addressed. The aims of our research is to understand the mechanisms by which insulin contributes to cancer progression and how these pathways are affected by insulin‐lowering therapies such as metformin. Transcriptomic analyses identified that insulin upregulated cell survival and invasion pathways and modulated metabolic pathways. Insulin upregulated Bcl2 and Bcl‐XL and down‐regulated pro‐apoptotic genes Bim and. Androgen deprivation was associated with a 15% reduction in spare respiratory capacity which was restored with 10 nM DHT, or with 10 nM insulin and correlated to resistance to doxyrubicin‐induced apoptosis, measured via Caspase 3 cleavage. We have observed that metformin counteracts insulin‐driven survival pathways, reduces oxidative capacity in androgen‐deprived PCa cells and sensitises cells to apoptosis. These results provide rationale for the combined therapeutic use of insulin‐lowering therapies with ADT.
<p>Supplementary Figure S1: Optimization of the triple immunostaining on different cell lines with known status of cytokeratins, TF and vimentin expression. Supplementary Figure S2: TF expression and pro-coagulant activity of EMT inducible cell lines. Supplementary Figure S3: Impact of TF blocking antibody on coagulant activity. Supplementary Figure S4: Regulation of TF by EMT transcription factors. Supplementary Figure S5: Induction of TF after de novo expression of Snail in MDA-MB-468. Supplementary Figure S6: Validation of TF shRNA. Supplementary Figure S7: Presence of fibrin fibers around lung colonizing MDA-MB-231 cells as revealed by transmission electron microscopy.</p>
IGF2 is a mitogenic foetal growth factor commonly over-expressed in cancers, including prostate cancer (PC). We recently demonstrated that insulin can activate de novo steroidogenesis in PC cells, a major pathway for reactivation of androgen pathways and PC progression. IGF2 can activate the IGF1 receptor (IGF1R) or insulin receptor (INSR) or hybrids of these two receptors. We therefore hypothesized that IGF2 may contribute to PC progression via de novo steroidogenesis. IGF2 mRNA but not IGF2 receptor mRNA expression was increased in patient samples during progression to castrate-resistant PC as was immunoreactivity to INSR and IGF1R antibodies. Treatment of androgen receptor (AR)-positive PC cell lines LNCaP and 22RV1 with IGF2 for 48 h resulted in increased expression of steroidogenic enzyme mRNA and protein, including steroid acute regulatory protein (StAR), cytochrome p450 family member (CYP)17A1, aldo–keto reductase family member (AKR)1C3 and hydroxysteroid dehydrogenase (HSD)17B3. IGF2 treatment resulted in increased steady state steroid levels and increased de novo steroidogenesis resulting in AR activation as demonstrated by PSA mRNA induction. Inhibition of the IGF1R/INSR signalling axis attenuated the effects of IGF2 on steroid hormone synthesis. We present a potential mechanism for prostatic IGF2 contributing to PC progression by inducing steroidogenesis and that IGF2 signalling and related pathways present attractive targets for PC therapy.
Free to read on publisher website Objectives: Adipokines are adipocyte produced hormones that provide information regarding adipocyte quantity and activity. Within the context of ADMET trial, we assessed the impact of metformin on adipokine levels compared with placebo. We hypothesised that levels of adiponectin, leptin and the ratio of the two may provide important clinical information regarding such as the metabolic health of patients and as a potential predictor of cancer outcome. Methods: Men with newly diagnosed metastatic prostate cancer were consecutively randomized to metformin or placebo 3 months post initiation of ADT (Eligard™ (leuprolide) 45 mg with 4 weeks of bicalutamide). Total and high molecular weight (HMW) adiponectin and leptin levels were detected 6-weekly for 54 weeks. Adipokines were measured by Enzyme linked immunosorbent assay (ELISA) and statistical analysis performed using SPSS. Results: 45 patients were recruited into the clinical trial. At 3 months, there was an increase in HMW adiponectin (67 ± 9 vs. 81 ± 10 ng/mL, p = 0.025), total adiponectin (135 ± 17 vs. 176 ± 22 ng/mL, p < 0.001) and leptin levels (108 ± 16 vs. 161 ± 24 pg/mL, p = 0.003). With metformin, there was statistically significant decline in leptin levels from visit 5 to 7 (12–24 weeks after commencing metformin) in comparison to the placebo group. With both adiponectin types, there was a trend for increase over time with metformin. At recruitment, abnormal leptin/total adiponectin ratio was associated with statistically significant and higher weight/BMI, waist/hip measurements, and levels of triglyceride, insulin, and HOMA. Conclusion: The results suggest adipokines are modifiable factors and metformin is an effective treatment for elevated leptin in patients starting ADT for metastatic prostate cancer. Future studies need to address whether reduction in leptin is associated with improved survival.
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Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.
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Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 1-13. (c)2016 AACR.
<p>Supplementary Fig. S1: Tissue microarray VN:IGFBP-3 PLA immunoreactivity and patient information; Supplementary Fig. S2: VN peptide array amino acid sequence information; Supplementary Fig. S3: IGFBP-3 peptide array amino acid sequence information; Supplementary Fig. S4: VN and IGFBP-3 immunoreactivity of HMLER-FOXC2 primary tumour xenografts; Supplementary Fig. S5: Single channel images of in situ PLA; Supplementary Fig. S6: IGF-II-induced cell signalling in MCF-7 breast cancer cells; Supplementary Fig. S7: Breast cell migration measured using the xCELLigence assay; Supplementary Fig. S8: Optimisation of ligand binding to VN and its subsequent immunodetection; Supplementary Fig. S9: Identification of ligand binding sites on VN using information from VN peptide arrays; Supplementary Fig. S10: Binding sites on IGFBP-3 for VN using the IGFBP-3 peptide arrays</p>
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