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. 2021 Nov 18;22(22):12438.
doi: 10.3390/ijms222212438.

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells through Autocrine Signaling

Anjugam Paramanantham  1   2 , Eun-Joo Jung  1 , Hye-Jung Kim  3 , Bae-Kwon Jeong  4 , Jin-Myung Jung  5 , Gon-Sup Kim  2 , Hong-Soon Chan  6 , Won-Sup Lee  1
Affiliations

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells through Autocrine Signaling

Anjugam Paramanantham et al. Int J Mol Sci. .

Abstract

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial-mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (β-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.

Keywords: CSCs; EGFR; MDA-MB-231; breast cancer; doxorubicin-resistant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The schedule for the establishment of DRM cells and cell viability of DRM cells with (A) MDA-MB-231 human breast cancer cells were treated with doxorubicin with the increasing concentration ranges from 10 nM to 100 nM. Based on the human plasma concentrations of doxorubicin, the final concentration (100 nM) was designed. The cells were treated and maintained with doxorubicin, and the number of passages is represented as P1, P2… Cells were maintained in doxorubicin-containing media for 38 weeks. (B) The cell viability assay for the three clones (22.5, 50 and, 100 nM DRM cells) that showed morphological changes while establishing doxorubicin-resistant cells. The cells showed resistance to doxorubicin as compared to parental cells. The values expressed as mean ± standard deviation (SD) (n = 5) (* p < 0.05 vs. control; ** p < 0.001 vs. control). DRM: Doxorubicin-resistant MDA-MB-231.
Figure 2
Figure 2
Morphological analysis of DRM cells (A) Morphological analysis performed under a light microscope 48 h after cells were seeded. DRM cells showed an increase in cell size and cell-to-cell communication. The cells changed from spindle-like structures to cobblestone-like giant cells as acquiring resistance to doxorubicin (Magnification, ×200; scale bar, 200 µm) (B) Whole-cell morphology analyzed with Mayer’s staining. DRM cells showed morphological changes with acquiring resistance to higher concentrations of doxorubicin. (Magnification, ×100; scale bar, 100 µm) (C) DAPI staining showing the nuclear morphology of DRM cells. DRM cells showed an increase in nuclear size with advancement in doxorubicin resistance. Results were confirmed by three independent experiments. (Magnification, ×100; scale bar, 100 µm). DRM: Doxorubicin-resistant MDA-MB-231.
Figure 3
Figure 3
Cell proliferation ability. DRM cells showed high proliferation rates compared to parental cells. Parental MDA-MB-231 and DRM cells were seeded, and media was changed every 3 days. After 10 days, cells were stained with Giemsa stain and photographed. Results were confirmed by three independent experiments. DRM: Doxorubicin-resistant MDA-MB-231. (* p < 0.05 vs. control; ** p < 0.001 vs. control). DRM: Doxorubicin-resistant MDA-MB-231.
Figure 4
Figure 4
Invasion effect of DRM cells. The transwell invasion assay showed a significant increase in the invasion of DRM cells with the advancement of resistance to higher concentrations of doxorubicin. The cells were seeded with the seeding density of 5 × 104 cells/well to the upper chamber of the Boyden chamber which is pre-coated with Matrigel and the cells invaded through the Matrigel were stained with DAPI. (A) The stained cells were photographed and (B) counted through ImageJ software and a number of invasive cells were graphed. The values expressed as mean ± standard deviation (SD) (n = 5) (* p < 0.05 vs. control; ** p < 0.001 vs. control). DRM: Doxorubicin-resistant MDA-MB-231.
Figure 5
Figure 5
DRM cells have an increased migration potential. The wound healing assay indicated the area of wound closure in 22.5 nM, 50 nM, 100 nM DRM cells, and the parental cells which were 47%, 22%, 15%, and 58%, respectively. The cells were grown with 100% confluence and a wound was created with the use of a 1 mL tip. (A) The migratory effect of the cells was analyzed with the photograph of the etched area with the mentioned durations. (B) The area of the cell migration was measured with the use of ImageJ software and graphed. The values expressed as mean ± standard deviation (SD) (n = 5) ((* p < 0.05 vs. prental cells;, ** p < 0.001 vs. parental cells). DRM: Doxorubicin-resistant MDA-MB-231.
Figure 6
Figure 6
Doxorubicin-resistant cells have a high adhesion effect to umbilical vein endothelial cells (ECs). The adhesion of DRM cells to Endothelial cells (ECs) was significantly increased with the advancement of resistance to higher concentrations of doxorubicin. The cells were washed, and the resistant cells adhered to ECs were counted. (A) The attached cells were photographed using a light microscope (B) Graphical representation of a number of cells attached. The values expressed as mean ± standard deviation (SD) (n = 5) (* p < 0.05, **p < 0.001 vs. control). DRM: Doxorubicin-resistant MDA-MB-231.
Figure 7
Figure 7
DRM cells showed high cancer stem cells, high proliferation, and high EMT phenotype. Western blot analysis of markers for (A) cancer stem cells, (B) proliferation (C) EMT phenotype related proteins, and (D) Gelatin zymography of MMP proteins. Cells were seeded with a seeding density of 5 × 104 cells and grown for 72 h. The control cells were left untreated. The whole-cell protein lysate was prepared, and 30 µg of proteins were resolved in SDS-polyacrylamide gels. Western blots were confirmed by three independent experiments. DRM: Doxorubicin-resistant MDA-MB-231.
Figure 8
Figure 8
DRM cells showed high EGFR activity. Western blot analysis of EGFR and the downstream signal, Akt and ERK. Cells were seeded with a seeding density of 5 × 104 cells and grown for 72 h. The control cells were left untreated. The whole-cell protein lysate was prepared, and 30 µg of proteins were resolved in SDS-polyacrylamide gels. Western blots were confirmed by three independent experiments. DRM: Doxorubicin-resistant MDA-MB-231.
Figure 9
Figure 9
Autocrine signaling in maintaining DRM cells. The parental cells were grown in resistant cell media showed an almost similar pattern of DRM cells in terms of the expression of CSC and EMT phenotype. Parental- MDA-MB-231 cells were grown in Doxorubicin-resistant cells grown media. (A) Morphological analysis of Parental- MDA-MB-231 cells, Parental- MDA-MB-231 cells grown in Doxorubicin resistant cells grown media. (B) Western blot analysis of autocrine signaling by analyzing the cancer stem cell markers and EMT phenotype. Results were confirmed by three independent experiments. DRM: Doxorubicin-resistant MDA-MB-231.
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