Stem Cell-Like Properties of CK2β-down Regulated Mammary Cells

Abstract

The ubiquitous protein kinase CK2 has been demonstrated to be overexpressed in a number of human tumours. This enzyme is composed of two catalytic α or α' subunits and a dimer of β regulatory subunits whose expression levels are probably implicated in CK2 regulation. Several recent papers reported that unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition, a process involved in cancer invasion and metastasis. Herein, through transcriptomic and miRNA analysis together with comparison of cellular properties between wild type and CK2β-knock-down MCF10A cells, we show that down-regulation of CK2β subunit in mammary epithelial cells induces the acquisition of stem cell-like properties associated with perturbed polarity, CD44^high/CD24^low antigenic phenotype and the ability to grow under anchorage-independent conditions. These data demonstrate that a CK2β level establishes a critical cell fate threshold in the control of epithelial cell plasticity. Thus, this regulatory subunit functions as a nodal protein to maintain an epithelial phenotype and its depletion drives breast cell stemness.

Keywords: EMT; breast cancer; epithelial plasticity; protein kinase CK2; stem cell.

Figure 1

Modulation of miRNAs in ΔCK2β-MCF10A cells. (A) Log2 fold change of the main miRNAs modulated in CK2β-depleted versus parental MCF10A cells measured by miRNA array analysis; (B) Changes of miRNA expression between CK2β-depleted and Mock-MCF10A cells were confirmed by using the indicated TaqMan probes. The relative amount of each miRNAs was determined by cross-normalization to ΔCK2β samples using the comparative method and miR-720 as an internal reference; (C) Two targets of miR-200 and miR-30 families, respectively Zeb1 and integrin β3, were analyzed by Western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was determined (3.5 and 2.3 for Zeb1 and integrin β3 respectively). Arrows indicate integrin β3 localization; (D) Integrin β1 and β4, targets of miR-21 were analyzed by western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was 0.4 for integrin β1. F-actin in green, nuclei in blue, and integrin β in red. Scale bar, 10 μm.

Figure 2

ΔCK2β-MCF10A cells have properties of cancer stem cells(CSCs) and are drug resistant. (A) FACS analysis of CD24 and CD44 markers in in Mock- and ΔCK2β-cells (Blue line, unlabelled cells; green line, Mock-cells; red line, ΔCK2β-cells). Results are representative of three independent experiments; (B) E-cadherin and Vimentin expression levels measured by RT-qPCR. The fold changes compare Mock- to ΔCK2β-cells. p < 0.05; (C) Cell proliferation kinetic of ΔCK2β- (■) and Mock-MCF10A (●); (D) Anoikis: ΔCK2β- (■) and Mock-MCF10A (●) were grown on Poly-HEMA for 48 h. Cell viability was measured with the cell viability-GLO^® assay, and apoptosis was visualized by Western blot using anti-PARP antibody; (E) Dose-response curves of ΔCK2β- (■) and Mock-MCF10A cells (●) treated with Paclitaxel. Bars denote the standard error (n = 5); (F) Representative images (top) and quantification (bottom) of mammosphere formation from Mock- and ΔCK2β-cells after first (grey bar) and second (black bar) dissociation steps (scale bar 50 μm).

Figure 3

ΔCK2β- and Mock-MCF10A cell positioning and polarity. (A) Mock and ΔCK2β- MCF10A cells cultured as monolayer (a,b,e,f) or as doublets on H-shaped micropatterns (c,d,g–j) were stained for DNA (blue, a,b,e,f), E-cadherin (red, a–d), F-actin (green, e–h) or paxillin (far red, i,j). Average staining over 20 images on pattern is shown (c,d,g–j). Scale bar, 10 μm; (B) (a) Representative image of doublet cells stained for α-catenin (red), centrosome (green), and DNA (blue) on curved H-shaped micropattern; (b) Time-lapse acquisition of control- and ΔCK2β-MCF10A cell doublets on micropattern was performed. Automated movie analysis of Hoechst-stained cells provided the angular distribution of the nucleus–nucleus axis orientation that is represented by graph; (c) The X coordinate of the normalized nucleus-centrosome vector toward the cell-cell junction was calculated. Horizontal bar graph shows quantification of polarity index as previously described [47]; (C) Confocal images of 3D culture in Matrigel^® for nine days, of Mock- and ΔCK2β-MCF10A cells stained for DNA in blue, F-actin in green, and Golgi apparatus in red. Scale bar, 20 μm.

Figure 4

IHC analysis of ΔCK2β- and Mock-MCF10A cells injected in inguinal mammary fate pad. (A) Three months after injection of GFP-transfected Mock- or ΔCK2β-MCF10A cells in mammary fat pad, the glands were harvested, fixed, paraffin included, and sections were stained with anti-GFP (a and b, respectively); (B) High magnification views (40×) of the boxed regions show the staining of GFP (a,e), cytokeratin 5-6 (b,f), cytokeratin 18 (c,g) and αSMA (d,h). Sections were counterstained with hematoxylin; (C) Six weeks post-injection of Mock-cells or ΔCK2β-cells, mammary gland sections were immunostained with human specific Cytokeratin 8/18. Sections were counterstained with Hematoxylin. Pictures are representative of different mammary gland sections injected with Mock-cells (a) or ΔCK2β-cells (b–d). Thin arrows indicate mouse mammary epithelial cells and thick arrows human stained luminal cells. Scale bars, 50 μm.