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Three-dimensional microenvironment confers enhanced sensitivity to doxorubicin by reducing p53-dependent induction of
L R Gomes1, A T Vessoni1, C F M Menck1
1Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
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.
Insights
Three-dimensional (3D) cultures reveal that the tumor microenvironment enhances breast cancer cell sensitivity to doxorubicin (DOXO) by inhibiting the p53-DRAM-autophagy pathway, unlike traditional 2D methods.
Area of Science:
- Oncology
- Cell Biology
- Biomedical Engineering
Background:
- Traditional preclinical cancer drug studies use 2D cultures, neglecting the extracellular matrix (ECM) and 3D microenvironment's impact.
- The 3D microenvironment significantly influences cancer cell behavior and drug response, necessitating advanced culture models.
Purpose of the Study:
- To investigate the role of the 3D laminin-rich ECM (3D lrECM) microenvironment in modulating human breast cancer cell responses to doxorubicin (DOXO).
- To elucidate the mechanisms underlying altered drug sensitivity in 3D versus 2D culture conditions, focusing on the p53-DRAM-autophagy axis.
Main Methods:
- Utilized a 3D lrECM cell culture model to compare human breast cancer cell (MCF-7 and MDA-MB-231) responses to DOXO against standard 2D cultures.
- Assessed cell morphology, proliferation, DOXO sensitivity, and activation of the autophagy pathway.
- Investigated the expression of p53 and DRAM-1, a key regulator of DNA damage-induced autophagy, and employed gene silencing techniques (shRNA, knockdown).
Main Results:
- 3D culture conditions induced morphological changes, reduced proliferation, and increased MCF-7 cell sensitivity to DOXO.
- Autophagy activation was compromised in 3D cultures, correlating with increased DOXO cytotoxicity.
- DOXO treatment upregulated p53 and DRAM-1 in 2D cultures, but this was impaired in 3D cultures; p53/DRAM-1 knockdown affected DOXO sensitivity primarily in 2D cells.
- p53-mutated MDA-MB-231 cells showed similar responses, failing to induce DRAM-1 or autophagy in 3D, and were more sensitive to DOXO in 2D.
Conclusions:
- The 3D tissue microenvironment significantly alters breast cancer cell response to doxorubicin.
- The p53-DRAM-autophagy axis plays a critical role in mediating doxorubicin sensitivity, with its inhibition in 3D cultures contributing to altered drug efficacy.
- 3D culture models are essential for accurately predicting anticancer drug responses in a more physiologically relevant context.
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