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Nitric oxide-targeted therapy inhibits stemness and increases the efficacy of tamoxifen in estrogen receptor-positive breast cancer cells


Cancer stem cells (CSCs) are involved in the resistance of estrogen (ER)-positive breast tumors against endocrine therapy. On the other hand, nitric oxide (NO) plays a relevant role in CSC biology, although there are no studies addressing how this important signaling molecule may contribute to resistance to antihormonal therapy in ER+ breast cancer. Therefore, we explored whether targeting NO in ER+ breast cancer cells impacts CSC subpopulation and sensitivity to hormonal therapy with tamoxifen. NO was targeted in ER+ breast cancer cells by specific NO depletion and NOS2 silencing and mammosphere formation capacity, stem cell markers and tamoxifen sensitivity were analyzed. An orthotopic breast tumor model in mice was also performed to analyze the efficacy of NO-targeted therapy plus tamoxifen. Kaplan–Meier curves were made to analyze the association of NOS2 gene expression with survival of ER+ breast cancer patients treated with tamoxifen. Our results show that targeting NO inhibited mamosphere formation, CSC markers expression and increased the antitumoral efficacy of tamoxifen in ER+ breast cancer cells, whereas tamoxifen-resistant cells displayed higher expression levels of NOS2 and Notch-1 compared with parental cells. Notably, NO-targeted therapy plus tamoxifen was more effective than either treatment alone in an orthotopic breast tumor model in immunodeficient mice. Furthermore, low NOS2 expression was significantly associated with a higher metastasis-free survival in ER+ breast cancer patients treated with tamoxifen. In conclusion, our data support that NO-targeted therapy in ER+ breast cancer may contribute to increase the efficacy of antihormonal therapy avoiding the development of resistance to these treatments.

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Fig. 1: NO depletion with c-PTIO abolishes the capacity of breast cancer cells to form mammospheres in vitro.
Fig. 2: NO depletion with C-PTIO inhibits mammosphere formation and decreases the ALDH+ cell subpopulation in SKBR-3 cells.
Fig. 3: NOS2 deficiency inhibits mammosphere formation and the expression of CSCs-specific markers in ER+ breast cancer cells.
Fig. 4: NO depletion or NOS2-silencing increase the antitumoral efficacy of antihormonal treatment with tamoxifen.
Fig. 5: Tamoxifen resistance is associated with increased expression of NOS2 and Notch1 expression in ER+ breast cancer cells.
Fig. 6: NO-targeted therapy with c-PTIO increases the efficacy of tamoxifen in an orthotopic breast tumor model.
Fig. 7: NOS2 expression and metastasis-free survival in patients with ER+ tumors.


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This work was supported by funding from Consejería de Salud, Junta de Andalucía through the project PI-0268-2014 and Instituto de Salud Carlos III through the projects PI13/00553 and PI16/01508 (co-funded by the European Regional Development Fund/ European Social Fund “Investing in your future”). ARA was funded with a researcher contract through the program “Nicolás Monardes” from Junta de Andalucía. We gratefully acknowledge the technical help of Esther Peralbo from the Citometry Unit at the IMIBIC.

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Correspondence to Antonio Rodríguez-Ariza.

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López-Sánchez, L.M., Mena, R., Guil-Luna, S. et al. Nitric oxide-targeted therapy inhibits stemness and increases the efficacy of tamoxifen in estrogen receptor-positive breast cancer cells. Lab Invest 101, 292–303 (2021).

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