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Figure 3 | Breast Cancer Research

Figure 3

From: Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer

Figure 3

iNOS knockdown impairs tumorigenicity and EMT by a dual impact on HIF1α and ER stress/TGFβ/AFT4/ATF3 crosstalk. Proliferation (A), migration (B), and mammosphere-forming efficiency (MSFE) (C) in MDA-MB-231 cells transiently transfected with two different NOS2-directed siRNAs (siRNA1 and siRNA2) compared with scrambled control. Western blot analysis of NOS isoforms (iNOS, eNOS, and nNOS) and EMT transcription factors in MDA-MB-231 and SUM159 cell lines treated with (D) 1400 W and (E) siRNA-mediated NOS2 knockdown. (F) Selective iNOS inhibition reduced hypoxia (HIF1α) and ER stress markers (IRE1α and ATF4). (G) Phospho-Smad2/3, Smad2/3, and mature TGFβ protein levels in MDA-MB-231 and SUM159 cells. (H) Recombinant TGFβ1 (10 ng/mL for 7 days) activates the PERK/eIF2α/ATF4/ATF3 axis in MCF10A. (I) Effects on the PERK/eIF2α/ATF4/ATF3 axis by co-treatment of recombinant TGFβ1 (10 ng/mL, 7 days) and 1400 W (4 mM) for 24 hours in MCF10A cells. iNOS, ATF4, ATF3, and mature TGFβ protein levels in siRNA-mediated NOS2 knockdown (siRNA2) MCF10A cells for 96 hours. (J) Selective iNOS inhibition is postulated to impair EMT and tumor cell migration by an impact on HIF1α, ER stress (IRE1α/XBP1), and the crosstalk between ATF4, ATF3, and TGFβ. Results were normalized to scrambled. Data are presented as mean ± standard error of the mean. ****P <0.0001. 1400 W, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide; ATF3, activating transcription factor 3; ATF4, activating transcription factor 4; EMT, epithelial-mesenchymal transition; ER, endoplasmic reticulum; HIF1α, hypoxia-inducible factor 1α; iNOS, inducible nitric oxide synthase; siRNA, small interfering RNA; TGFβ, transforming growth factor β.

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