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. 2023 Jun 6;24(12):9806.
doi: 10.3390/ijms24129806.

p53 Affects Zeb1 Interactome of Breast Cancer Stem Cells

Sergey E Parfenyev  1 , Sergey V Shabelnikov  1 , Elena N Tolkunova  1 , Nickolai A Barlev  1   2 , Alexey G Mittenberg  1
Affiliations

p53 Affects Zeb1 Interactome of Breast Cancer Stem Cells

Sergey E Parfenyev et al. Int J Mol Sci. .

Abstract

P53 is a critical tumor suppressor that protects the integrity of genome and prevents cells from malignant transformation, including metastases. One of the driving forces behind the onset of metastases is the epithelial to mesenchymal transition (EMT) program. Zeb1 is one of the key transcription factors that govern EMT (TF-EMT). Therefore, the interaction and mutual influence of p53 and Zeb1 plays a critical role in carcinogenesis. Another important feature of tumors is their heterogeneity mediated by the presence of so-called cancer stem cells (CSCs). To this end, we have developed a novel fluorescent reporter-based approach to enrich the population of CSCs in MCF7 cells with inducible expression of Zeb1. Using these engineered cell lines, we studied the effect of p53 on Zeb1 interactomes isolated from both CSCs and regular cancer cells. By employing co-immunoprecipitations followed by mass spectrometry, we found that the composition of Zeb1 interactome was affected not only by the p53 status but also by the level of Oct4/Sox2 expression, indicating that stemness likely affects the specificity of Zeb1 interactions. This study, together with other proteomic studies of TF-EMT interactomes, provides a framework for future molecular analyses of biological functions of Zeb1 at all stages of oncogenesis.

Keywords: Zeb1; breast cancer; cancer stem cells; epithelial-to-mesenchymal transition (EMT); metastasis; p53.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Different expression levels of stemness factors Oct4 and Sox2 in MCF-7/Zeb1 Oct4/Sox2 cells according to the activity of SORE6-mCherry reporter plasmid under light (left panel) and fluorescence microscopy (right panel): L, H, denote low and high expression levels of Oct4/Sox2 stemness factors, respectively.
Figure 2
Figure 2
MCF-7-Zeb1 cells with high expression of mCherry exhibit traits of cancer stem cells. RT-PCR with oligonucleotides specific to stemness factors ((a): CD44, (b): Oct4, (c): Sox2) were performed on MCF-7/Zeb1 cells with low (L) and high (H) expression of the SORE6-mCherry reporter. Cells were non-treated or treated with doxycycline to induce the expression of Zeb1. These cells (wt p53, left) were treated identically to the cells with knocked down expression of p53 (shRNA-p53, right).
Figure 3
Figure 3
Western blot analysis of MCF-7/Zeb1 cells with different status of p53 (wt and shp53) divided into two groups according to low (L) or high (H) expression of the SORE6-mCherry reporter. (a) Immunoblots of total lysates prepared from MCF-7/Zeb1 cells (p53+ or p53-) stained for p53. Actin was used as the loading control. (b) Immunoblots of MCF-7/Zeb1 Oct4/Sox2 cells: wild-type p53 (left panel), shp53 (right panel) stained with Zeb1, Nanog, Oct4 and Sox2 antibodies, respectively. The actin signal was used as the loading control. L, H, − denote low and high level Oct4/Sox2-expressing clones, respectively.
Figure 4
Figure 4
Western blot analysis of Zeb1 interactome components co-immunoprecipitated from MCF-7/Zeb1 cells with different status of p53 (wt and shp53) divided into two groups according to low or high expression of the SORE6-mCherry reporter.
Figure 5
Figure 5
STRING database analysis of protein networks, showing proteins connected to the Zeb1 node. Zeb1 interactomes of MCF-7/Zeb1 cells with different status of p53 (wt and shp53) divided into two groups according to low (L) or high (H) expression of the SORE6-mCherry reporter. (a): MCF-7/Zeb1 wt-p53 cell clones with low Oct4/Sox2 expression levels; (b): MCF-7/Zeb1 wt-p53 cell clones with high Oct4/Sox2 expression levels; (c): MCF-7/Zeb1 shp53 cell clones with low Oct4/Sox2 expression levels; (d): MCF-7/Zeb1 shp53 cell clones with high Oct4/Sox2 expression levels.
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