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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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Decrease in tumor initiation and lung metastases in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 5 per group) after daily injection of L-NAME (80 mg/kg, intraperitoneal). (B) Primary and secondary mammosphere of cancer cells isolated from tumor tissue. (C) Luminescence of MDA-MB-231 L/G tumor cells in lungs of vehicle- and L-NAME-treated mice. (D) Tumor-initiating capacity of tumor cells (limiting dilution assay). Results were normalized to vehicle. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001. L-NAME, N5-[imino(nitroamino)methyl]-L-ornithine methyl ester; MSFE, mammosphere-forming efficiency.
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Fig4: Decrease in tumor initiation and lung metastases in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 5 per group) after daily injection of L-NAME (80 mg/kg, intraperitoneal). (B) Primary and secondary mammosphere of cancer cells isolated from tumor tissue. (C) Luminescence of MDA-MB-231 L/G tumor cells in lungs of vehicle- and L-NAME-treated mice. (D) Tumor-initiating capacity of tumor cells (limiting dilution assay). Results were normalized to vehicle. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001. L-NAME, N5-[imino(nitroamino)methyl]-L-ornithine methyl ester; MSFE, mammosphere-forming efficiency.

Mentions: Considering our in vitro data, we next investigated whether L-NAME was able to prevent tumor initiation and metastasis of MDA-MB-231 L/G cells in mice. L-NAME significantly reduced tumor growth (P = 0.001) (Figure 4A) as well as primary MS (Figure 4B). Additionally, tumor-initiating capacity of CSCs was assessed with LDA. All of the animals of the vehicle group developed tumors, but treatment with L-NAME yielded 3/5 tumors at 1.5 weeks with 5 × 105 cells. The same results were observed in the vehicle group at 2.5 weeks with 1 × 105 cells compared with the L-NAME-treated group (0/5 tumors) (P <0.05) (Figure 4D). Ex vivo imaging of lungs in the presence of luciferin showed higher fluorescence in the vehicle group compared with the L-NAME group (Figure 4C). These results suggest that iNOS inhibition with daily L-NAME may prevent metastasis to lungs in a TNBC mouse model.Figure 4


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)

Decrease in tumor initiation and lung metastases in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 5 per group) after daily injection of L-NAME (80 mg/kg, intraperitoneal). (B) Primary and secondary mammosphere of cancer cells isolated from tumor tissue. (C) Luminescence of MDA-MB-231 L/G tumor cells in lungs of vehicle- and L-NAME-treated mice. (D) Tumor-initiating capacity of tumor cells (limiting dilution assay). Results were normalized to vehicle. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001. L-NAME, N5-[imino(nitroamino)methyl]-L-ornithine methyl ester; MSFE, mammosphere-forming efficiency.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384389&req=5

Fig4: Decrease in tumor initiation and lung metastases in MDA-MB-231 xenografts. (A) Tumor volume of MDA-MB-231 breast xenografts (n = 5 per group) after daily injection of L-NAME (80 mg/kg, intraperitoneal). (B) Primary and secondary mammosphere of cancer cells isolated from tumor tissue. (C) Luminescence of MDA-MB-231 L/G tumor cells in lungs of vehicle- and L-NAME-treated mice. (D) Tumor-initiating capacity of tumor cells (limiting dilution assay). Results were normalized to vehicle. Data are presented as mean ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001. L-NAME, N5-[imino(nitroamino)methyl]-L-ornithine methyl ester; MSFE, mammosphere-forming efficiency.
Mentions: Considering our in vitro data, we next investigated whether L-NAME was able to prevent tumor initiation and metastasis of MDA-MB-231 L/G cells in mice. L-NAME significantly reduced tumor growth (P = 0.001) (Figure 4A) as well as primary MS (Figure 4B). Additionally, tumor-initiating capacity of CSCs was assessed with LDA. All of the animals of the vehicle group developed tumors, but treatment with L-NAME yielded 3/5 tumors at 1.5 weeks with 5 × 105 cells. The same results were observed in the vehicle group at 2.5 weeks with 1 × 105 cells compared with the L-NAME-treated group (0/5 tumors) (P <0.05) (Figure 4D). Ex vivo imaging of lungs in the presence of luciferin showed higher fluorescence in the vehicle group compared with the L-NAME group (Figure 4C). These results suggest that iNOS inhibition with daily L-NAME may prevent metastasis to lungs in a TNBC mouse model.Figure 4

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