Cyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis

Abstract

Objective: The cyclin-dependent kinase inhibitors (CDKIs), p21(CIP1) and p27(KIP1) regulate growth and differentiation in diverse tissue types. We aimed to determine whether p21(CIP1) or p27(KIP1) could induce a terminally differentiated phenotype in breast cells, and to examine if CDKI expression is regulated by basement membrane interactions.

Materials and methods: Effects of increased CDKI expression on the phenotype of MCF-10A breast epithelial cells were examined by retroviral transduction of p21(CIP1) or p27(KIP1) cDNA.

Results: Overexpression of p21(CIP1) or p27(KIP1) reduced MCF-10A growth rates in monolayer cultures, altered cellular morphology and stimulated accumulation of neutral lipid droplets, suggesting partial lactational differentiation. However, markers of luminal differentiation (oestrogen and progesterone receptors, alpha-lactalbumin, beta-casein and adipophilin) were absent when examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Cell-basement membrane contacts are known to be essential for full mammary epithelial cell differentiation and therefore parental MCF-10A cells were cultured on a basement membrane preparation (Matrigel) in which they form acini. Immunocytochemistry showed that Ki67, the cell proliferation marker, was initially expressed at high levels and as growth decreased p27(KIP1) expression steadily increased. Surprisingly, p21(CIP1) was highest at the early stages of acinus growth and was detected in proliferating cells, as demonstrated by colocalization in dual Ki67/p21(CIP1) immunofluorescence. Overexpression of p21(CIP1) or p27(KIP1) impaired formation of acini, whereas their knockdown, using siRNA, increased acinus formation.

Conclusion: We conclude that both p21(CIP1) and p27(KIP1) induce partial secretory differentiation of mammary cells in monolayer, but during acinus morphogenesis in 3D culture they have a highly regulated temporal expression pattern.

Figure 1

Ectopic CDKI expression decreases proliferation. Proteins were extracted from FACS‐sorted cells. Twenty‐five micrograms of total proteins were separated on a 10% acrylamide gel and stained for either p21^CIP1 (a) or p27^KIP1 (b) immunoreactivity. Lanes are: non‐transduced cells (lane C), GFP‐vector transduced cells (lanes 1 and 3), p21^CIP1/GFP‐transduced cells (lane 2) and p27^KIP1/GFP‐transduced cells (lane 4). Cells were transduced with either empty vector (c and e), pKat‐p21^CIP1 (d) or pKat‐p27^KIP1 (f) and stained for either p21^CIP1 (c and d) or p27^KIP1 (e and f) immunoreactivity. (g) FACS‐sorted transduced cells were plated into 96‐well plates and cultured for up to 12 days. Cell protein was stained with sulforhodamine B and optical density measured at 540 nm p21^CIP1 () and p27^KIP1 () transduced cells showed retarded growth compared to control cells () or vector treated cells (). Results are expressed as average ± SEM.

Figure 2

Ectopic CDKIs do not induce lactation‐related genes. (a) RNA was extracted from non‐transduced MCF‐10A cells (Con), FACS‐sorted cells transduced with GFP, p21^CIP1 or p27^KIP1. Normal breast tissue was used as a positive control. RNA was reverse transcribed and the cDNA amplified by PCR using probes for human oestrogen receptor‐α (ER); human progesterone receptor (PR) and human acidic ribosomal phosphoprotein p0 (ARPP0) as a loading control. (b) RNA was extracted from non‐transduced MCF‐10A cells (Con), FACS‐sorted cells transduced with GFP, p21^CIP1 or p27^KIP1, after culture in the presence (+PRL) or absence of ovine prolactin (–PRL) (3 µg/mL) for 2 days. Normal breast tissue was used as a positive control. RNA was reverse transcribed and the cDNA amplified by PCR using probes for human α‐lactalbumin and human β‐casein. Human acidic ribosomal phosphoprotein p0 (ARPP0) was used as a loading control. Untreated cells (c), vector‐treated cells (d), p21^CIP1‐transduced cells (e) or high pKat‐p27^KIP1‐transduced cells (f) were FACS‐sorted, plated into chamber slides and cultured. Neutral lipids were stained with Oil Red O.

Figure 3

Basement membrane induces tightly controlled CDKI expression. (a) Untransduced MCF‐10A cells were cultured on Matrigel for up to 12 days formed acini. These structures were fixed and sectioned at regular intervals and examined for p27^KIP1, Ki67 or p21^CIP1 immunoreactivity. One thousand cells were scored for staining positivity in triplicate and results expressed as average percentage of positive cells ± SEM. Results are p27^KIP1 () (b), Ki67 () and p21^CIP1 () (c). (d) Representative photomicrographs showing colocalization of Ki67 and p21^CIP1 MCF‐10A cells were cultured on Matrigel, fixed and sectioned as described in Materials and Methods. Sections were subjected to dual immunofluorescence for p21^CIP1 (red) and Ki67 (green). Nuclei were counterstained with DAPI (blue). Dual‐labelled cells are indicated by arrows.

Figure 4

Ectopic CDKI expression inhibits acinus formation. FACS‐sorted MCF‐10A cells transduced with GFP‐containing vector (solid bar), p21^CIP1 (shaded bar) or p27^KIP1 (open bar) were cultured on Matrigel as described in Materials and Methods. The number (a) and diameter (b) of acini formed were assessed at days 6 and 12 during culture. Results are expressed as percentage GFP control ± SEM. Statistical significance was determined by Student's t‐test, with significance being indicated by a P‐value (P = 0.05). Significance was determined between GFP transduced and p21^CIP1 and p27^KIP1 (indicated by *) or p21^CIP1 and p27^KIP1 (indicated by ^†).

Figure 5

siRNA‐mediated knockdown of CDKIs alters acinus formation. (a) Optimization of siRNA knockdown using MCF‐7 cells. Cells were treated overnight with increasing concentrations of p21^CIP1 siRNA or p27^KIP1 siRNA in the presence of siPort transfection reagent (Ambion) at a ratio of 6 : 1 (ng siPort: ng siRNA). RNA was extracted from cells and subject to RT‐PCR for either p21^CIP1, p27^KIP1 or the house‐keeping gene ARPP0 as a loading and specificity control. Specific knockdown of both p21^CIP1 and p27^KIP1 could be observed using 30–60 nm siRNA (b–d). Effects of CDKI directed siRNAs on acinus formation by MCF‐10A cells. Cells were transfected with control siRNA (solid bars) or 60 nm siRNA for p21^CIP1 (b and d) or p27^KIP1 (c and e) and plated onto Matrigel as described in Materials and Methods. The number (b and c) and diameter (d and e) of acini formed were assessed at days 3 and 9 during culture. Results are expressed as percentage control siRNA transfected cells ± SEM. Statistical significance was determined by Student's t‐test, with significance being indicated by a P‐value (P = 0.05) (indicated by *). Significance was determined between control siRNA transfected and p21^CIP1 or p27^KIP1 siRNA.