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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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In vivoeffects of L-NMMA in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 10 per group) treated with vehicle, L-NMMA, docetaxel, and combination. (B) Illustrative images of Ki67 staining in the vehicle, L-NMMA, docetaxel, and combination groups. Original optical objective: 10×. Counterstain: hematoxylin. (C) Cell proliferation of tumor xenografts with Ki67 immunostaining. (D) Nuclear cleaved caspase-3 staining in the chemo and combo groups. (E) Primary and secondary mammosphere of breast cancer cells isolated from tumor tissue. (F) Tumor-initiating capacity of tumor cells. Results were normalized to vehicle. For proliferation and apoptosis, 1,000 cells were counted from 10 different fields, and the percentage was determined. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. L-NMMA, NG-monomethyl-L-arginine; MSFE, mammosphere-forming efficiency.
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Fig5: In vivoeffects of L-NMMA in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 10 per group) treated with vehicle, L-NMMA, docetaxel, and combination. (B) Illustrative images of Ki67 staining in the vehicle, L-NMMA, docetaxel, and combination groups. Original optical objective: 10×. Counterstain: hematoxylin. (C) Cell proliferation of tumor xenografts with Ki67 immunostaining. (D) Nuclear cleaved caspase-3 staining in the chemo and combo groups. (E) Primary and secondary mammosphere of breast cancer cells isolated from tumor tissue. (F) Tumor-initiating capacity of tumor cells. Results were normalized to vehicle. For proliferation and apoptosis, 1,000 cells were counted from 10 different fields, and the percentage was determined. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. L-NMMA, NG-monomethyl-L-arginine; MSFE, mammosphere-forming efficiency.

Mentions: Similarly, docetaxel and L-NMMA (200 mg/kg, daily) were given to MDA-MB-231 xenograft-bearing mice. We found reduced tumor growth in the L-NMMA group compared with vehicle, but there was no change between the docetaxel and combination groups (Figure 5A). These results were further correlated with lower proliferation rate (Ki67) in the L-NMMA and combination groups (Figure 5B and C). Additionally, higher apoptosis levels in docetaxel-treated xenografts were found; these results might offset the high proliferation rate relative to the combination group (Figure 5D). A decrease in MS formation was seen for both L-NMMA and combination compared with vehicle and chemotherapy alone (Figure 5E). LDA showed that the vehicle-treated and docetaxel-treated groups presented 12/12 and 8/12 tumors, respectively, with a significant decrease in L-NMMA-treated (1/12) and combination-treated (4/12) xenografts at 5 weeks with 5 × 104 cells. After 6 weeks, a decrease in both the L-NMMA and combination groups (3/12 and 5/12, respectively) compared with the vehicle and docetaxel groups (6/12 and 8/12, respectively) was observed (P <0.05) (Figure 5F). Our results in the MDA-MB-231 xenograft model do not show additional benefit over chemotherapy in terms of tumor volume; however, it is clear that the benefits of L-NMMA lie in the ability of this inhibitor to reduce self-renewal and tumor initiation capacities of CSCs.Figure 5


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)

In vivoeffects of L-NMMA in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 10 per group) treated with vehicle, L-NMMA, docetaxel, and combination. (B) Illustrative images of Ki67 staining in the vehicle, L-NMMA, docetaxel, and combination groups. Original optical objective: 10×. Counterstain: hematoxylin. (C) Cell proliferation of tumor xenografts with Ki67 immunostaining. (D) Nuclear cleaved caspase-3 staining in the chemo and combo groups. (E) Primary and secondary mammosphere of breast cancer cells isolated from tumor tissue. (F) Tumor-initiating capacity of tumor cells. Results were normalized to vehicle. For proliferation and apoptosis, 1,000 cells were counted from 10 different fields, and the percentage was determined. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. L-NMMA, NG-monomethyl-L-arginine; MSFE, mammosphere-forming efficiency.
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Fig5: In vivoeffects of L-NMMA in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 10 per group) treated with vehicle, L-NMMA, docetaxel, and combination. (B) Illustrative images of Ki67 staining in the vehicle, L-NMMA, docetaxel, and combination groups. Original optical objective: 10×. Counterstain: hematoxylin. (C) Cell proliferation of tumor xenografts with Ki67 immunostaining. (D) Nuclear cleaved caspase-3 staining in the chemo and combo groups. (E) Primary and secondary mammosphere of breast cancer cells isolated from tumor tissue. (F) Tumor-initiating capacity of tumor cells. Results were normalized to vehicle. For proliferation and apoptosis, 1,000 cells were counted from 10 different fields, and the percentage was determined. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. L-NMMA, NG-monomethyl-L-arginine; MSFE, mammosphere-forming efficiency.
Mentions: Similarly, docetaxel and L-NMMA (200 mg/kg, daily) were given to MDA-MB-231 xenograft-bearing mice. We found reduced tumor growth in the L-NMMA group compared with vehicle, but there was no change between the docetaxel and combination groups (Figure 5A). These results were further correlated with lower proliferation rate (Ki67) in the L-NMMA and combination groups (Figure 5B and C). Additionally, higher apoptosis levels in docetaxel-treated xenografts were found; these results might offset the high proliferation rate relative to the combination group (Figure 5D). A decrease in MS formation was seen for both L-NMMA and combination compared with vehicle and chemotherapy alone (Figure 5E). LDA showed that the vehicle-treated and docetaxel-treated groups presented 12/12 and 8/12 tumors, respectively, with a significant decrease in L-NMMA-treated (1/12) and combination-treated (4/12) xenografts at 5 weeks with 5 × 104 cells. After 6 weeks, a decrease in both the L-NMMA and combination groups (3/12 and 5/12, respectively) compared with the vehicle and docetaxel groups (6/12 and 8/12, respectively) was observed (P <0.05) (Figure 5F). Our results in the MDA-MB-231 xenograft model do not show additional benefit over chemotherapy in terms of tumor volume; however, it is clear that the benefits of L-NMMA lie in the ability of this inhibitor to reduce self-renewal and tumor initiation capacities of CSCs.Figure 5

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