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Three-dimensional microenvironment confers enhanced sensitivity to doxorubicin by reducing p53-dependent induction of autophagy

Oncogene volume 34, pages 5329–5340 (2015)Cite this article

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Abstract

Preclinical studies of anticancer drugs are typically performed using cancer cell lines maintained in two-dimensional (2D) cultures, ignoring the influences of the extracellular matrix (ECM) and three-dimensional (3D) microenvironment. In this study, we evaluated the microenvironmental control of human breast cancer cells responses to doxorubicin (DOXO) using the 3D laminin-rich ECM (3D lrECM) cell culture model. Under 3D culture conditions, MCF-7 cells displayed drastic morphological alterations, a decrease in proliferation and elevated sensitivity to DOXO. Interestingly, the chemotherapy-mediated activation of autophagy was compromised in the 3D matrix, suggesting an association between the increased cytotoxicity of DOXO and hindered autophagy induction. Indeed, while chloroquine or ATG5 knockdown potentiated DOXO-induced cell death under the 2D culture conditions, the autophagy inducer rapamycin improved the resistance of 3D-cultured cells to this drug. Moreover, in the monolayer-cultured cells, DOXO treatment led to increases in p53 and DRAM-1 expression, which is a p53-dependent activator of autophagy that functions in response to DNA damage. Conversely, p53 and DRAM-1 expression was impaired in 3D-cultured cells. The knockdown of p53 by shRNA blocked DRAM-1 activation, impaired autophagy induction and sensitized only those cells maintained under 2D conditions to DOXO. In addition, 2D-cultured MDA-MB-231 cells (a p53-mutated breast cancer cell line) not only showed increased sensitivity to DOXO compared with MCF-7 cells but also failed to induce DRAM-1 expression or autophagy. Similar to p53 silencing, DRAM-1 knockdown potentiated DOXO cytotoxicity only in 2D-cultured cells. These results suggest that the 3D tissue microenvironment controls tumor cell sensitivity to DOXO treatment by preventing p53-DRAM-autophagy axis activation.

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Acknowledgements

We thank Professor Maria S. Solengas (Department of Dermatology, University of Michigan Comprehensive Cancer Center) for kindly providing the pEGFP-LC3 construct. We are grateful for the excellent technical assistance provided by the Core Facility for Scientific Research-USP (CEFAP-USP) in conducting the confocal fluorescence microscopy assays. This work was supported by FAPESP (Sao Paulo, Brazil), CAPES and CNPq (Brasilia, Brazil).

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  1. Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil

    L R Gomes, A T Vessoni & C F M Menck

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Correspondence to C F M Menck.

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Gomes, L., Vessoni, A. & Menck, C. Three-dimensional microenvironment confers enhanced sensitivity to doxorubicin by reducing p53-dependent induction of autophagy. Oncogene 34, 5329–5340 (2015). https://doi.org/10.1038/onc.2014.461

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