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. 2023 Jul 19;15(14):3671.
doi: 10.3390/cancers15143671.

SELENOF Controls Proliferation and Cell Death in Breast-Derived Immortalized and Cancer Cells

Roudy C Ekyalongo  1 , Brenna Flowers  1 , Tanu Sharma  1 , Alexandra Zigrossi  1 , An Zhang  1 , Anaisa Quintanilla-Arteaga  1 , Kanishka Singh  1 , Irida Kastrati  1
Affiliations

SELENOF Controls Proliferation and Cell Death in Breast-Derived Immortalized and Cancer Cells

Roudy C Ekyalongo et al. Cancers (Basel). .

Abstract

SELENOF expression is significantly lower in aggressive breast tumors compared to normal tissue, indicating that its reduction or loss may drive breast tumorigenesis. Deletion of SELENOF in non-tumorigenic immortalized breast epithelial MCF-10A cells resulted in enhanced proliferation, both in adherent culture and matrix-assisted three-dimmensional (3D) growth. Modulation of SELENOF in vitro through deletion or overexpression corresponded to changes in the cell-cycle regulators p21 and p27, which is consistent with breast tumor expression data from the METABRIC patient database. Together, these findings indicate that SELENOF affects both proliferation and cell death in normal epithelial and breast cancer cells, largely through the regulation of p21 and p27. In glandular cancers like breast cancer, the filling of luminal space is one of the hallmarks of early tumorigenesis. Loss of SELENOF abrogated apoptosis and autophagy, which are required for the formation of hollow acini in MCF-10A cells in matrix-assisted 3D growth, resulting in luminal filling. Conversely, overexpression of SELENOF induced cell death via apoptosis and autophagy. In conclusion, these findings are consistent with the notion that SELENOF is a breast tumor suppressor, and its loss contributes to breast cancer etiology.

Keywords: acinar growth; breast cancer; cell death; proliferation; selenoprotein F (SELENOF); tumor suppressor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Loss of SELENOF increases proliferation of MCF-10A cells in adherent culture. (A) SELENOF levels in whole-cell extracts were determined through Western blotting of MCF-10A wild-type (WT) or single-cell-derived lines from SELENOF knockout cells (KO clones 1 and 2). β-Actin is shown as a loading control. The uncropped blots are shown in File S1. (B) Cell proliferation was measured over 6 days in culture. Cells were fixed, stained with crystal violet, solubilized in 1% SDS, and quantified by absorbance at 570 nm. (C) Relative intensity of EdU-488-stained cells 48 h post-seeding was analyzed using FACS. (D) mRNA expression of Ki67 was examined using RT-QPCR. **** p < 0.0001.
Figure 2
Figure 2
Loss of SELENOF increases three-dimensional acinar growth in the extracellular matrix. (A) Representative pictures of acini from MCF-10A WT (first column) or SELENOF KO lines (second and third columns) grown in 5%, 10%, or 20% matrigel for 20 days. Scale bar indicates 100 µm. (B) Average number of acini (>100 µm in diameter) per field (n = 3 wells, 7 fields each) plotted for each cell line. (C) Average acini diameter size per field (n = 3 wells, 7 fields each) calculated for each cell line. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
SELENOF controls proliferation and cell death via p21 and p27. (A) Cell cycle distribution was determined in MCF-10A WT and SELENOF KO cells stained with EdU and PI and analyzed using FACS. (B) The levels of SELENOF, p21, and p27 were determined using Western blotting. β-Actin is shown as a loading control. (C) Cell viability of MCF-10A WT vs. SELENOF KO cells was determined through crystal violet staining after 72 h of treatment with −/+ 2 µM K03681. Data were normalized to vehicle controls and shown as 100% for each cell line. (D) Cell death was estimated using PI staining and FACS analysis in cells treated for 48 h with −/+ 2 µM K03681. (E) Levels of SELENOF, p21, and p27 were determined through Western blotting. β-Actin is shown as a loading control. MCF-7 SELENOF cells were treated with −/+Dox 1 µg/mL for 3 days. The uncropped blots are shown in File S1. (F) Cell viability was determined using crystal violet staining. MCF-7 SELENOF cells were transfected with siNeg, sip21, or sip27, 10 nM each, for 48 h, and treated with −/+ 1 µg/mL Dox for an additional 72 h. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
SELENOF expression and its related gene signatures correlate with cell cycle regulators in breast tumors. (A) Correlation plots generated to analyze the expression of SELENOF versus Ki67, p21, or p27 in breast tumors from the METABRIC database, n = 1904 samples. Spearman and Pearson coefficients and their respective p-values are indicated. (B) The top 400 genes co-expressed (negative correlation in the left panel versus positive correlation in the right panel) with SELENOF in breast tumors were analyzed for pathway enrichment using the Metascape gene annotation software (Metascape.org). The −log of p-values are shown in the graphs.
Figure 5
Figure 5
Loss of SELENOF renders MCF-10A cells resistant to apoptosis and autophagy. (A) Fluorescence images of 3D acini at 4× generated from MCF-10A WT or SELENOF KO cells. All cells were seeded in 5% matrigel for 5 days prior to treatment with −/+ 10 µM ZVAD and −/+ 5 µM CQ, then grown for another 15 days. The first row shows representative images of Hoechst 33,342 staining of nuclei in blue. The second row shows representative images of Calcein AM staining of live cells in green. The third row shows representative images of propidium iodide (PI) staining of dead cell in red. The last row shows a merged image of all three stainings. (B) Cell death was estimated using FACS of cells stained with PI after 24 or 48 h of serum deprivation. ** p < 0.01, # p < 0.0001.
Figure 6
Figure 6
SELENOF overexpression induces apoptosis and autophagy in breast cancer cells. (A) Transmission electron microscopy analysis of MCF-7 SELENOF cell treated with −/+ 1 µg/mL Dox for 72 h. Examples of the observed mitochondrial morphology (top row) and phagosome formation (bottom row) are indicated by arrows. Serum starvation (last column) for 24 h was used as a positive control for autophagy. (B) DNA fragmentation was estimated using Hoechst 33,342 staining in cells treated as described in (A). (C) The levels of SELENOF and LC3-II were determined using Western blotting in whole cell extracts from MCF-7 SELENOF cells treated with 1 µg/mL Dox at the indicated times. Serum starvation for 24 h was used as a positive control for autophagy. β-Actin is shown as a loading control. The uncropped blots are shown in File S1. (D) Cell viability was determined using crystal violet staining of cells treated with −/+ 1 µg/mL Dox, −/+10 µM ZVAD, and −/+5 µM chloroquine (CQ) for 5 days. Data are normalized to vehicle control and shown as 100%. ** p < 0.01, **** p < 0.0001.
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