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

Granados-Principal S, Liu Y, Guevara ML, Blanco E, Choi DS, Qian W, Patel T, Rodriguez AA, Cusimano J, Weiss HL, Zhao H, Landis MD, Dave B, Gross SS, Chang JC - Breast Cancer Res. (2015)

Bottom Line: We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome.High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis.Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.

Methods: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.

Results: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.

Conclusions: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

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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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Fig3: 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.

Mentions: High concentrations of 1400 W (1, 2, and 4 mM) significantly decreased proliferation in both cell lines (Figure 2A). Similar results were observed for L-NAME (Additional file 1: Figure S1A). L-NMMA at the highest concentration (4 mM) showed anti-proliferative activity in both cell lines (Figure 2B). Resistance to treatment and metastasis may arise from a subpopulation of CSCs within a heterogeneous primary cancer [22,23]. iNOS inhibition decreased primary MSs in both cell lines (Figure 2C; Additional file 1: Figure S1B; Additional file 2: Figure S2A; and Additional file 3: Figure S3A). We found reduced secondary MS in both cell lines for all the inhibitors tested (Figure 2D; Additional file 1: Figure S1C; Additional file 2: Figures S2B; and Additional file 3: Figure S3B). We show enhanced iNOS expression in invasive TNBC (Figure 1A); we then investigated the role of iNOS in cell migration. Selective iNOS inhibition with 1400 W caused a marked dose-dependent decrease in migration in both cell lines (Figure 2E and Additional file 1: Figure S1D). L-NMMA-treated cells showed reduction in migration capacity (Figure 2F and Additional file 1: Figure S1E). Similar results were found for L-NAME (Additional file 4: Figure S4A). These results were further confirmed in siRNA-mediated iNOS (NOS2) knockdown MDA-MB-231 (Figure 3A-C) and SUM159 cells (Additional file 5: Figure S5A, B, and C). Collectively, the results indicate that basal levels of iNOS have a major role in CSC self-renewal and migrating properties of TNBC cell lines and a less pronounced effect on proliferation.Figure 2


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

Granados-Principal S, Liu Y, Guevara ML, Blanco E, Choi DS, Qian W, Patel T, Rodriguez AA, Cusimano J, Weiss HL, Zhao H, Landis MD, Dave B, Gross SS, Chang JC - Breast Cancer Res. (2015)

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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Related In: Results  -  Collection

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Fig3: 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.
Mentions: High concentrations of 1400 W (1, 2, and 4 mM) significantly decreased proliferation in both cell lines (Figure 2A). Similar results were observed for L-NAME (Additional file 1: Figure S1A). L-NMMA at the highest concentration (4 mM) showed anti-proliferative activity in both cell lines (Figure 2B). Resistance to treatment and metastasis may arise from a subpopulation of CSCs within a heterogeneous primary cancer [22,23]. iNOS inhibition decreased primary MSs in both cell lines (Figure 2C; Additional file 1: Figure S1B; Additional file 2: Figure S2A; and Additional file 3: Figure S3A). We found reduced secondary MS in both cell lines for all the inhibitors tested (Figure 2D; Additional file 1: Figure S1C; Additional file 2: Figures S2B; and Additional file 3: Figure S3B). We show enhanced iNOS expression in invasive TNBC (Figure 1A); we then investigated the role of iNOS in cell migration. Selective iNOS inhibition with 1400 W caused a marked dose-dependent decrease in migration in both cell lines (Figure 2E and Additional file 1: Figure S1D). L-NMMA-treated cells showed reduction in migration capacity (Figure 2F and Additional file 1: Figure S1E). Similar results were found for L-NAME (Additional file 4: Figure S4A). These results were further confirmed in siRNA-mediated iNOS (NOS2) knockdown MDA-MB-231 (Figure 3A-C) and SUM159 cells (Additional file 5: Figure S5A, B, and C). Collectively, the results indicate that basal levels of iNOS have a major role in CSC self-renewal and migrating properties of TNBC cell lines and a less pronounced effect on proliferation.Figure 2

Bottom Line: We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome.High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis.Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.

Methods: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.

Results: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.

Conclusions: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Show MeSH
Related in: MedlinePlus