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. 2020 Apr 30;43(4):384-396.
doi: 10.14348/molcells.2020.2230.

Biological Functions and Identification of Novel Biomarker Expressed on the Surface of Breast Cancer-Derived Cancer Stem Cells via Proteomic Analysis

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Free PMC article

Biological Functions and Identification of Novel Biomarker Expressed on the Surface of Breast Cancer-Derived Cancer Stem Cells via Proteomic Analysis

Eun-Young Koh et al. Mol Cells. .
Free PMC article

Abstract

Breast cancer is one of the most common life-threatening malignancies and the top cause of cancer deaths in women. Although many conventional therapies exist for its treatment, breast cancer still has many handicaps to overcome. Cancer stem cells (CSCs) are a well-known cause of tumor recurrences due to the ability of CSCs for self-renewal and differentiation into cell subpopulations, similar to stem cells. To fully treat breast cancer, a strategy for the treatment of both cancer cells and CSCs is required. However, current strategies for the eradication of CSCs are non-specific and have low efficacy. Therefore, surface biomarkers to selectively treat CSCs need to be developed. Here, 34 out of 641 surface biomarkers on CSCs were identified by proteomic analysis between the human breast adenocarcinoma cell line MCF-7 and MCF-7-derived CSCs. Among them, carcinoembryonic antigen-related cell adhesion molecules 6 (CEACAM6 or CD66c), a member of the CEA family, was selected as a novel biomarker on the CSC surface. This biomarker was then experimentally validated and evaluated for use as a CSC-specific marker. Its biological effects were assessed by treating breast cancer stem cells (BCSCs) with short hairpin (sh)-RNA under oxidative cellular conditions. This study is the first to evaluate the biological function of CD66c as a novel biomarker on the surface of CSCs. This marker is available as a moiety for use in the development of targeted therapeutic agents against CSCs.

Keywords: CD66c; apoptosis; breast cancer stem cell; surface biomarker.

Conflict of interest statement

CONFLICT OF INTEREST

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. Scheme 1.
Experimental processing for the identification of novel biomarkers on the surface of breast cancer-derived CSCs.
Fig. 2
Fig. 2. Scheme 2.
Manufacturing process of BCSCs derived from MCF-7 cells. MCF-7 was seeded in 6-well ultra-low attachment plates. To expand the CSC populations, the seeded cells were grown on several 6-well plates until mammospheres were formed.
Fig. 3
Fig. 3. Scheme 3.
Isolation of BCSCs formed from mammospheres by magnetic activated cell sorting (MACS).
Fig. 4
Fig. 4. Characterization of isolated BCSCs.
(A) BCSCs in formed mammospheres were observed using an optical microscope on days 7, 14, and 21. The size of the cells increased in a time-dependent manner. (B) Flow cytometric analysis of cells for CD24/CD44+. To identify the characteristics of BCSCs, the cell population expressing CD44+ and CD24 were analyzed by flow cytometry. After 3 weeks, the highest level of CD24/CD44+ CSC marker expression was observed after 14 days. (C) Quantitative data of BCSCs expressing CD24/CD44+ in a time-dependent manner. Data are expressed as the mean ± SEM. ***P < 0.01.
Fig. 5
Fig. 5. Comparative proteome analysis of isolated BCSCs and MCF-7 cells.
(A) Venn diagram of isolated proteins of BCSCs by mass spectrometry (MS). The 31 proteins indicated on the right were upregulated in BCSCs compared to MCF-7 cells. (B) The classification according to molecular function of the 31 proteins represented and (C) GO analysis of in various biological processes in the plasma membrane of BCSCs.
Fig. 6
Fig. 6. The expression of the CD66c gene at the transcriptomic level in cultured BCSCs in a time-dependent manner.
(A) The expression of CD66c in BCSCs was confirmed by RT-PCR using mRNA extracted from BCSCs at the transcriptomic level. (B) Quantitative data of the expressed CD66c gene normalized by GAPDH in BCSCs. Data are expressed as the mean ± SEM. *P < 0.05 or **P < 0.02 versus MCF-7 group.
Fig. 7
Fig. 7. The expression of CD66c at the protein level in cultured BCSCs in a time-dependent manner.
The expression of CD66c protein increased in a time-dependent manner in BSCSs compared to MCF-7 cells, in agreement with the transcriptomic results. These results indicate that the expression of CD66c is related to the maintenance period in CSCs, but not in cancer cells. (A) Confirmation of the expression of CD66c protein by western blotting. (B) Quantitative graph of the western blot. Data are expressed as the mean ± SEM. ***P < 0.01 versus MCF-7 group. (C) Resulting peaks of the CD66c protein levels as a factor of time expressed on the surface of BCSCs in the form of a histogram according to FACS data. (D) Quantitative graph of the shifted peak in the FACS data. Data are expressed as the mean ± SEM. *P < 0.05 and ***P < 0.01 for MCF-7 versus BCSC (7 days) or BCSC (14 days); **P < 0.02 for BCSC (14 days) versus BCSC (21 days). (E) The images of CD66c expressed on the surface of the BCSCs according to cell binding assay. Data are expressed as the mean ± SEM. **P < 0.02 versus MCF-7 group. (F) Identification of the characteristics of CSCs in BCSCs expressing CD66c by immunocytochemistry.
Fig. 8
Fig. 8. Biological effects of CD66c in BCSCs by CD66c gene silencing using shRNA.
To determine the biological function of CD66c in BCSCs, BCSCs were treated with several shRNAs. The knock-down of the CD66c gene by shRNA at the transcriptomic (A) and protein (B) level. (C) Cell death and adhesion in BCSCs treated with shRNA-C against CD66c by crystal violet staining. (D) Cell viability in BCSCs treated with shRNA-C. Scrambled shRNA (SC) was used as a negative control here. ***P < 0.01 for NC group versus shRNA-C treated group.
Fig. 9
Fig. 9. Enhanced cell death in BCSCs treated with shRNA-C under oxidative stress conditions.
(A) Transfection efficiency images of shRNA-C expressing a lentiviral vector for the knock-down of the CD66c gene. (B) Cell viability of BCSCs treated with shRNA-C under oxidative stress conditions. Data are expressed as the mean ± SEM. **P < 0.02 for in the absence or presence of H2O2. ***P < 0.01 for H2O2-only group versus H2O2treated with shRNA-C group. (C) FACS analysis of cell death induced by treatment with H2O2 post-shRNA-C transfection in BCSCs. (D) Quantitative viable cell population of BCSCs treated with shRNA-C and H2O2 by FACS analysis. Data are expressed as the mean ± SEM. *P < 0.05 or **P < 0.02 for the comparison of no H2O2 treated groups and H2O2 treated groups or **P < 0.02 for shRNA-SC versus shRNA-C groups in the presence of H2O2. ***P < 0.01 for the comparison of NC and shRNA-C groups in the presence of H2O2. (E) Quantitative death cell population induced by shRNA-C treatment with H2O2. Data are expressed as the mean ± SEM. *P < 0.05 or **P < 0.02 for in the absence or presence of H2O2. **P < 0.02 for the comparison of shRNA-SC with H2O2 group and shRNA-C with H2O2 group. ***P < 0.01 for the comparison of H2O2 only group and shRNA-C with H2O2 group.

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