Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1.

    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    Google Scholar 

  2. 2.

    Wang L, Gallo KA, Conrad SE. Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis. Oncotarget. 2013;4:1158–71.

    Article  Google Scholar 

  3. 3.

    Lumachi F, Luisetto G, Basso SMM, Basso U, Brunello A, Camozzi V. Endocrine therapy of breast cancer. Curr Med Chem. 2011;18:513–22.

    CAS  Article  Google Scholar 

  4. 4.

    Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med. 1998;339:1609–18.

    CAS  Article  Google Scholar 

  5. 5.

    Jager NG, Linn SC, Schellens JH, Beijnen JH. Tailored tamoxifen treatment for breast cancer patients: a perspective. Clin Breast Cancer. 2015;15:241–4.

    CAS  Article  Google Scholar 

  6. 6.

    García-Becerra R, Santos N, Díaz L, Camacho J. Mechanisms of resistance to endocrine therapy in breast cancer: focus on signaling pathways, mirnas and genetically based resistance. Int J Mol Sci. 2013;14:108–45.

    Article  Google Scholar 

  7. 7.

    Rodriguez D, Ramkairsingh M, Lin X, Kapoor A, Major P, Tang D. The central contributions of breast cancer stem cells in developing resistance to endocrine therapy in estrogen receptor (ER)-positive breast cancer. Cancers. 2019;11:1028.

    CAS  Article  Google Scholar 

  8. 8.

    Harrison H, Farnie G, Brennan KR, Clarke RB. Breast cancer stem cells: something out of notching? Cancer Res. 2010;70:8973–6.

    CAS  Article  Google Scholar 

  9. 9.

    Acar A, Simões BM, Clarke RB, Brennan K. A role for notch signalling in breast cancer and endocrine resistance. Stem Cells Int. 2016;2016:2498764–6.

    Article  Google Scholar 

  10. 10.

    Aranda E, López-Pedrera C, De La Haba-Rodriguez JR, Rodríguez-Ariza A. Nitric oxide and cancer: the emerging role of S-nitrosylation. Curr Mol Med. 2012;12:50–67.

    CAS  Article  Google Scholar 

  11. 11.

    Peñarando J, Aranda E, Rodríguez-Ariza A. Immunomodulatory roles of nitric oxide in cancer: tumor microenvironment says ‘NO’ to antitumor immune response. Transl Res. 2019;210:99–108.

    Article  Google Scholar 

  12. 12.

    Somasundaram V, Basudhar D, Bharadwaj G, No JH, Ridnour LA, Cheng RYS, et al. Molecular mechanisms of nitric oxide in cancer progression, signal transduction, and metabolism. Antioxid Redox Signal. 2019;30:1124–43.

    CAS  Article  Google Scholar 

  13. 13.

    López-Sánchez LM, Aranda E, Rodríguez-Ariza A. Nitric oxide and tumor metabolic reprogramming. Biochem Pharmacol. 2020;176:113769.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Glynn SA, Boersma BJ, Dorsey TH, Yi M, Yfantis HG, Ridnour LA, et al. Increased NOS2 predicts poor survival in estrogen receptor-negative breast cancer patients. J Clin Invest. 2010;120:3843–54.

    CAS  Article  Google Scholar 

  15. 15.

    Ambs S, Glynn SA. Candidate pathways linking inducible nitric oxide synthase to a basal-like transcription pattern and tumor progression in human breast cancer. Cell Cycle. 2011;10:619–24.

    CAS  Article  Google Scholar 

  16. 16.

    Eyler CE, Wu Q, Yan K, MacSwords JM, Chandler-Militello D, Misuraca KL, et al. Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2. Cell. 2011;146:53–66.

    CAS  Article  Google Scholar 

  17. 17.

    Puglisi MA, Cenciarelli C, Tesori V, Cappellari M, Martini M, Di Francesco AM, et al. High nitric oxide production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the tumour-initiating properties of colon cancer stem cells. J Pathol. 2015;236:479–90.

    CAS  Article  Google Scholar 

  18. 18.

    Peñarando J, López-Sánchez LM, Mena R, Guil-Luna S, Conde F, Hernández V, et al. A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer. BMC Biol. 2018;16:1–14.

    Article  Google Scholar 

  19. 19.

    Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW, Hambardzumyan D, et al. Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells. Cell Stem Cell. 2010;6:141–52.

    CAS  Article  Google Scholar 

  20. 20.

    Cañas A, López-Sánchez LM, Valverde-Estepa A, Hernández V, Fuentes E, Muñoz-Castañeda JR, et al. Maintenance of S-nitrosothiol homeostasis plays an important role in growth suppression of estrogen receptor-positive breast tumors. Breast Cancer Res. 2012;14:R153.

    Article  Google Scholar 

  21. 21.

    Győrffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010;123:725–31.

    Article  Google Scholar 

  22. 22.

    Ishimura N, Bronk SF, Gores GJ. Inducible nitric oxide synthase up-regulates Notch-1 in mouse cholangiocytes: implications for carcinogenesis. Gastroenterology. 2005;128:1354–68.

    CAS  Article  Google Scholar 

  23. 23.

    Marcato P, Dean CA, Giacomantonio CA, Lee PW. Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle. 2011;10:1378–84.

    CAS  Article  Google Scholar 

  24. 24.

    Kim SY, Kang JW, Song X, Kim BK, Yoo YD, Kwon YT, et al. Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells. Cellular Signal. 2013;25:961–9.

    CAS  Article  Google Scholar 

  25. 25.

    Al-Hussaini H, Subramanyam D, Reedijk M, Sridhar SS. Notch signaling pathway as a therapeutic target in breast cancer. Mol Cancer Ther. 2011;10:9–15.

    CAS  Article  Google Scholar 

  26. 26.

    Rizzo P, Miao H, D’Souza G, Osipo C, Song LL, Yun J, et al. Cross-talk between notch and the estrogen receptor in breast cancer suggests novel therapeutic approaches. Cancer Res. 2008;68:5226–35.

    CAS  Article  Google Scholar 

  27. 27.

    Mocellin S, Bronte V, Nitti D. Nitric oxide, a double edged sword in cancer biology: searching for therapeutic opportunities. Med Res Rev. 2007;27:317–52.

    CAS  Article  Google Scholar 

  28. 28.

    Dontu G, Wicha MS. Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia. 2005;10:75–86.

    Article  Google Scholar 

  29. 29.

    Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 2009;69:1302.

    CAS  Article  Google Scholar 

  30. 30.

    Liu CG, Lu Y, Wang BB, Zhang YJ, Zhang RS, Lu Y, et al. Clinical implications of stem cell gene Oct-4 expression in breast cancer. Ann Surg. 2011;253:1165–71.

    Article  Google Scholar 

  31. 31.

    Han ML, Wang F, Gu YT, Pei XH, Ge X, Guo GC, et al. MicroR-760 suppresses cancer stem cell subpopulation and breast cancer cell proliferation and metastasis: By down-regulating NANOG. Biomed Pharmacother. 2016;80:304–10.

    CAS  Article  Google Scholar 

  32. 32.

    Tsang JY, Huang YH, Luo MH, Ni YB, Chan SK, Lui PC, et al. Cancer stem cell markers are associated with adverse biomarker profiles and molecular subtypes of breast cancer. Breast Cancer Res Treat. 2012;136:407–17.

    CAS  Article  Google Scholar 

  33. 33.

    Ricardo S, Vieira AF, Gerhard R, Leitão D, Pinto R, Cameselle-Teijeiro JF, et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol. 2011;64:937–46.

    Article  Google Scholar 

  34. 34.

    Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008;100:672–9.

    CAS  Article  Google Scholar 

  35. 35.

    Vermeulen L, de Sousa e Melo F, Richel DJ, Medema JP. The developing cancer stem-cell model: clinical challenges and opportunities. Lancet Oncol. 2012;13:e83–9.

    Article  Google Scholar 

  36. 36.

    Schiff R, Massarweh S, Shou J, Osborne CK. Breast cancer endocrine resistance: how growth factor signaling and estrogen receptor coregulators modulate response. Clin Cancer Res. 2003;9:447S–54S.

    CAS  PubMed  Google Scholar 

  37. 37.

    Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, et al. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy. Biochim Biophys Acta. 2010;1806:258–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Kotta-Loizou I, Vasilopoulos SN, Coutts RH, Theocharis S. Current evidence and future perspectives on HuR and breast cancer development, prognosis, and treatment. Neoplasia. 2016;18:674–88.

    CAS  Article  Google Scholar 

  39. 39.

    Hiscox S, Baruha B, Smith C, Bellerby R, Goddard L, Jordan N, et al. Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan. BMC Cancer. 2012;12:458.

    CAS  Article  Google Scholar 

  40. 40.

    Arif K, Hussain I, Rea C, El-Sheemy M. The role of Nanog expression in tamoxifen-resistant breast cancer cells. Onco Targets Ther. 2015;8:1327–34.

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Granados-Principal S, Liu Y, Guevara ML, Blanco E, Choi DS, Qian W, et al. Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer. Breast Cancer Res. 2015;17:25.

    Article  Google Scholar 

Download references


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.

Author information



Corresponding author

Correspondence to Antonio Rodríguez-Ariza.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links