Abstract 4171: Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts
Bibliographic record
Abstract
Abstract 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
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".