A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

Abstract

Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells.

Materials and methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells.

Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC.

Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

Keywords: Immortalization; luminal differentiation; myoepithelial differentiation; self-renewal; stem cell.

Figure 1

Analyses of stem / progenitor cell markers in parental and immortalized human mammary epithelial cells isolated and cultured in the DFCI-1 medium, (a) Western blotting of parental and immortal cells. A total of 50 μg of cell lysates were western blotted, using indicated antibodies. β-actin was used as loading control, ( b ) Immunofluorescence staining of normal and immortal hMECs using CD49f antibody (red), and DAPI (blue) used for nuclear staining is shown ×20

Figure 2

In vitro self-renewal and luminal differentiation of immortalized hMECs. (a) Cells cultured in a two-dimensional culture with MEGM media. Immunostaining of cells with rabbit-anti human K5 (green) and mouse anti-human MUC1 (red) ×20, (b) Cells cultured in three-dimensional Matrigel with DFCI-2 medium. The acini were co-stained with rabbit-anti human K5 (red) and FITC conjugated anti-human MUC1 antibody (green) ×63

Figure 3

In vitro self-renewal and myoepithelial cell differentiation of immortalized hMECs in MEGM medium, (a) Morphology of cells after beginning of differentiation, ×10 (b) Immunofluorescence staining of myoepithelial cells. The cells were co-stained with rabbit-anti human K5 (green) and a myoepithelial cell marker (α-SMA), with mouse anti-human α-SMA antibody (red) ×20

Figure 4

Effect of p53 mutant on 76N.TERT myoepithelial cell differentiation (a) Western blotting shows mutant p53 overexpression, (b) Lack of myoepithelial cell differentiation in 76N.TERT-p53R249S compared with 76N.TERT-V. Images were acquired under a Nikon inverted microscope using ×4 and ×10 objectives