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Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model

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Abstract

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible for tumor recurrences.

Key messages

• iNOS expression is involved in bladder tumor development, growth, and angiogenesis.

• iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells.

• iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

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Data availability

Datasets are available from the corresponding author on reasonable request.

Abbreviations

BC:

Bladder cancer

NMI:

No muscle invasive

LG:

Low grade

HG:

High grade

MI:

Muscle invasive

TUR:

Transurethral resection

CSC:

Cancer stem cell

NO:

Nitric oxide

iNOS:

Inducible nitric oxide synthase

L-NAME:

Nω-Nitro-l-arginine methyl ester

TBM:

Tumor-bearing mice

SFE:

Sphere forming efficiency

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Acknowledgments

We are grateful to Dr. Carlos Spector, Maria Pilar Jerabek, Martina Rodriguez Olivera, and Lourdes Otamendi from UCES for their support. The authors thank Dr. Lionel Berthoux for his advice and technical assistance on shRNA iNOS knockdown. We are grateful also to the Emerging Leaders in The Americas Program (ELAP) scholarships of Canada. Also, we thank the animal facility from IOAHR and UQTR for their technical support. We thank Lic. Inés Kletzky for her critical English revision.

Funding

This study was supported by funding from IOAHR, Universidad de Buenos Aires, CONICET (PIP9671/14), UBACyT 20720150100001BA, Agencia de promoción Cientifica y Tecnologica - PICT 2017-0776, Cancer Institute of Canadian Institutes of Health Research (CIHR, number 392334), Cancer Research Society (CRS, number 22471), Fundación hermanos Agustín y Enrique Rocca, Escuela Técnica ORT, and Universidad de Ciencias Empresariales y Sociales (UCES).

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Author notes

  1. Denise Belgorosky and Julie Girouard contributed equally to this work.

Authors and Affiliations

  1. Research Area, Instituto de Oncología Ángel H. Roffo (IOAHR), Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

    Denise Belgorosky, Yanina Veronica Langle & Ana María Eiján

  2. Medical Biology Department, Groupe de Recherche en Signalisation Cellulaire (GRSC), Université du Quebec a Trois-Rivières, Trois-Rivières, QC, Canada

    Julie Girouard, Jovane Hamelin-Morrissete & Carlos Reyes-Moreno

  3. Pathology Department, IOAHR, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

    Lina Marino

  4. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

    Eduardo Imanol Agüero & Ana María Eiján

  5. Urology Department, IOAHR, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

    Héctor Malagrino

  6. Área Investigaciones, Instituto de Oncología “Ángel H. Roffo”, Av. San Martín 5481, CP1417DTB, Buenos Aires, Argentina

    Ana María Eiján

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  1. Denise Belgorosky
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  2. Julie Girouard
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  3. Yanina Veronica Langle
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  9. Ana María Eiján
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Contributions

D. B. has performed the experimental design and carried out the experimental work, the interpretation of the results, drafting of the manuscript, and the discussion of the intellectual content. J. G. and J. H-M. had provided experimental ideas and had participated in silencing iNOS and in vivo imaging experiments. Y. V. L. has also provided experimental ideas, performed some of the qPCR experiments, and helped with the interpretation of the results; and also helped with in vivo assays and contributed in revising the draft critically for important intellectual content. L. M. contributed in ICH experiments. E. I. A. has participated with some qPCR experiments and contributed in revising the draft critically. H. M. provided human samples. Reyes-Moreno has contributed in conceptualization, methodology, resources, review and editing, supervision, and funding acquisition. A. M. E. has contributed in experimental design, funding acquisition, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Ana María Eiján.

Ethics declarations

Studies using patient samples were approved by the Institutional Ethics Committee from IOAHR according to the Declaration of Helsinki (CEI: 6/6/2013), with the corresponding informed consents from each patient. Mice were obtained from the animal facility of the IOAHR or Charles River Laboratories (Montréal, Canada) and handled in accordance with the ARRIVE guidelines. Protocols were approved by the Institutional Review Board of IOAHR (2012/02) and the animal care and use committee of the UQTR, Canada (2015-CRM-3).

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The authors declare that they have no conflict of interest.

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Belgorosky, D., Girouard, J., Langle, Y.V. et al. Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model. J Mol Med 98, 1615–1627 (2020). https://doi.org/10.1007/s00109-020-01973-0

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