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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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Enhanced nitric oxide synthase 2 (NOS2) expression correlates with poor patient survival in invasive triple-negative breast cancer (TNBC). (A) Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC (P = 3.85 × 10−5). (B) High NOS2 expression correlates with death at 5 years in invasive breast carcinoma (P = 0.037). Kaplan-Meier survival analyses in (C) Van de Vijver (n = 69; P = 0.04) and (D) Curtis (n = 260; P = 0.01) breast databases show that high NOS2 expression correlates with worse overall survival of patients with TNBC. (E) Immunohistochemical analysis of TNBC human samples for inducible nitric oxide synthase (iNOS) protein expression. Weak-to-moderate (3 or 4), moderate-to-strong (5 or 6), and strong (7) were the cutoffs established for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20×). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive control of iNOS staining, respectively (original optical objective: 10×). Counterstain: hematoxylin. (F) Increased iNOS expression is associated with less patient survival when compared with low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n = 83) (P = 0.05). shRNA1, small hairpin RNA 1; TCGA, The Cancer Genome Atlas.
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Fig1: Enhanced nitric oxide synthase 2 (NOS2) expression correlates with poor patient survival in invasive triple-negative breast cancer (TNBC). (A) Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC (P = 3.85 × 10−5). (B) High NOS2 expression correlates with death at 5 years in invasive breast carcinoma (P = 0.037). Kaplan-Meier survival analyses in (C) Van de Vijver (n = 69; P = 0.04) and (D) Curtis (n = 260; P = 0.01) breast databases show that high NOS2 expression correlates with worse overall survival of patients with TNBC. (E) Immunohistochemical analysis of TNBC human samples for inducible nitric oxide synthase (iNOS) protein expression. Weak-to-moderate (3 or 4), moderate-to-strong (5 or 6), and strong (7) were the cutoffs established for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20×). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive control of iNOS staining, respectively (original optical objective: 10×). Counterstain: hematoxylin. (F) Increased iNOS expression is associated with less patient survival when compared with low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n = 83) (P = 0.05). shRNA1, small hairpin RNA 1; TCGA, The Cancer Genome Atlas.

Mentions: iNOS has been described to be mediator of metastasis in different cancer types [20,21]. Elevated iNOS expression has been linked to poor survival in patients with ERα-negative breast cancer [7]. We hypothesized that enhanced iNOS expression in TNBC correlates with poor patient survival and metastases. Oncomine Cancer Microarray database analysis of NOS2 expression in TCGA breast database showed higher NOS2 expression in invasive TNBC (n = 46) versus non-TNBC (n = 250) (fold change 1.425, P = 3.85 × 10−5) (Figure 1A). Patient survival analysis demonstrated a correlation between increased NOS2 expression and worse survival at 5 years in patients with invasive ductal breast carcinoma (n = 79) (fold change 1.275, P = 0.037) (Figure 1B). We further examined whether NOS2 expression correlates with worse survival in two additional databases of TNBC. Analysis of Van de Vijver (n = 69 samples) [17] and Curtis (n = 260 samples) [18] databases confirmed that high NOS2 expression was associated with poor survival in patients with TNBC (Figure 1C and D).Figure 1


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)

Enhanced nitric oxide synthase 2 (NOS2) expression correlates with poor patient survival in invasive triple-negative breast cancer (TNBC). (A) Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC (P = 3.85 × 10−5). (B) High NOS2 expression correlates with death at 5 years in invasive breast carcinoma (P = 0.037). Kaplan-Meier survival analyses in (C) Van de Vijver (n = 69; P = 0.04) and (D) Curtis (n = 260; P = 0.01) breast databases show that high NOS2 expression correlates with worse overall survival of patients with TNBC. (E) Immunohistochemical analysis of TNBC human samples for inducible nitric oxide synthase (iNOS) protein expression. Weak-to-moderate (3 or 4), moderate-to-strong (5 or 6), and strong (7) were the cutoffs established for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20×). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive control of iNOS staining, respectively (original optical objective: 10×). Counterstain: hematoxylin. (F) Increased iNOS expression is associated with less patient survival when compared with low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n = 83) (P = 0.05). shRNA1, small hairpin RNA 1; TCGA, The Cancer Genome Atlas.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Enhanced nitric oxide synthase 2 (NOS2) expression correlates with poor patient survival in invasive triple-negative breast cancer (TNBC). (A) Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC (P = 3.85 × 10−5). (B) High NOS2 expression correlates with death at 5 years in invasive breast carcinoma (P = 0.037). Kaplan-Meier survival analyses in (C) Van de Vijver (n = 69; P = 0.04) and (D) Curtis (n = 260; P = 0.01) breast databases show that high NOS2 expression correlates with worse overall survival of patients with TNBC. (E) Immunohistochemical analysis of TNBC human samples for inducible nitric oxide synthase (iNOS) protein expression. Weak-to-moderate (3 or 4), moderate-to-strong (5 or 6), and strong (7) were the cutoffs established for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20×). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive control of iNOS staining, respectively (original optical objective: 10×). Counterstain: hematoxylin. (F) Increased iNOS expression is associated with less patient survival when compared with low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n = 83) (P = 0.05). shRNA1, small hairpin RNA 1; TCGA, The Cancer Genome Atlas.
Mentions: iNOS has been described to be mediator of metastasis in different cancer types [20,21]. Elevated iNOS expression has been linked to poor survival in patients with ERα-negative breast cancer [7]. We hypothesized that enhanced iNOS expression in TNBC correlates with poor patient survival and metastases. Oncomine Cancer Microarray database analysis of NOS2 expression in TCGA breast database showed higher NOS2 expression in invasive TNBC (n = 46) versus non-TNBC (n = 250) (fold change 1.425, P = 3.85 × 10−5) (Figure 1A). Patient survival analysis demonstrated a correlation between increased NOS2 expression and worse survival at 5 years in patients with invasive ductal breast carcinoma (n = 79) (fold change 1.275, P = 0.037) (Figure 1B). We further examined whether NOS2 expression correlates with worse survival in two additional databases of TNBC. Analysis of Van de Vijver (n = 69 samples) [17] and Curtis (n = 260 samples) [18] databases confirmed that high NOS2 expression was associated with poor survival in patients with TNBC (Figure 1C and D).Figure 1

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