Abstract 4171: Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts
العنوان: | Abstract 4171: Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts |
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المؤلفون: | Howard Leong-Poi, Krishna K. Singh, Gerald J. Prud'homme, Pratiek N. Matkar |
المصدر: | Cancer Research. 75:4171-4171 |
بيانات النشر: | American Association for Cancer Research (AACR), 2015. |
سنة النشر: | 2015 |
مصطلحات موضوعية: | Cancer Research, Gene knockdown, Mesenchymal stem cell, Transfection, Biology, Cell biology, Endothelial stem cell, medicine.anatomical_structure, Oncology, Biochemistry, Neuropilin 1, medicine, Gene silencing, Fibroblast, Transforming growth factor |
الوصف: | BACKGROUND: Endothelial-mesenchymal transition (EndMT) is an extreme form of endothelial cell plasticity whereby endothelial cells acquire mesenchymal phenotype. While EndMT is a significant component during embryogenesis, maladaptive EndMT is a key contributor to fibrotic pathologies including cancer. With regard to this, EndMT accounts for up to 40% of cancer-associated fibroblasts (CAFs) that play an important role in cancer progression through potentially oncogenic signals like transforming growth factor- β (TGF-β). Recent investigations have demonstrated that the angiogenic co-receptor Neuropilin-1 (NRP1) is aberrantly expressed in cancer and modulates cancer progression through interaction with various ligands, including TGF-β. However, the relationship and driving mechanisms linking NRP1 and EndMT remain unexplored. METHODS: SiRNA-mediated NRP1 gene knockdown studies using Dharmafect-4 transfection reagent and siNRP1 or scramble control (Ambion) were performed on human umbilical vein endothelial cells (HUVECs), at baseline and after TGF-β stimulation. Total RNA was extracted using Trizol® reagent (Invitrogen), and precipitated with isopropanol. Complementary DNA was synthesized using iScript cDNA synthesis kit (Bio-Rad) and real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT and mediators of TGF-β1 signalling were evaluated by RT-PCR, western blotting and immunocytochemistry. RESULTS: RT-PCR and western blots measuring NRP1 mRNA and protein levels in HUVEC lysates confirmed successful silencing. Light and fluorescence microscopy revealed marked morphological changes in TGF-β1 stimulated scramble siRNA-transfected HUVECs. TGF-β1 stimulated HUVECs demonstrated a distinct change from “cobblestone-like endothelial cell morphology” to an enlarged spindle shaped appearance consistent with a “fibroblast like morphology”, accompanied by re-arrangement of the cytoskeleton. We further observed that these characteristic EndMT features were revoked upon NRP1 silencing where cells maintained a conventional cobblestone phenotype. Expression of endothelial cell markers, CD-31 and VE-Cadherin, were significantly higher in NRP1-silenced vs. scramble siRNA-treated HUVECs; additionally, NRP1 silencing was associated with significant reduction in expression of the mesenchymal markers α-smooth muscle actin, Slug and N-Cadherin. Loss of NRP1 was accompanied by decreased expression of TGF-β1 and its receptors as well as significant changes in the key downstream effectors of TGF-β1 signalling. CONCLUSION: Our novel findings demonstrating that loss of NRP1 revokes TGF-β-dependent EndMT-like phenotype switching suggesting that NRP1 may represent a novel therapeutic target to limit the source of EndMT derived CAFs and associated cancer aggression. Citation Format: Pratiek N. Matkar, Krishna Kumar Singh, Gerald Prud'homme, Howard Leong-Poi. Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4171. doi:10.1158/1538-7445.AM2015-4171 |
تدمد: | 1538-7445 0008-5472 |
الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_________::c32bedb80b85916fc988dd6a3de0bba0Test https://doi.org/10.1158/1538-7445.am2015-4171Test |
رقم الانضمام: | edsair.doi...........c32bedb80b85916fc988dd6a3de0bba0 |
قاعدة البيانات: | OpenAIRE |
تدمد: | 15387445 00085472 |
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