Introduction
There is a critical balance between proliferation, differentiation, and apoptosis in the cellular composition of every tissue. In the hematopoietic system,
MYB clearly plays a role in maintaining this balance.
MYB is essential for hematopoiesis [
1], is highly expressed in immature hematopoietic cells and its expression is down-regulated upon differentiation [
2,
3]. Moreover, enforced expression of normal and activated forms of MYB can suppress differentiation and maintain proliferation of hematopoietic cells [
4‐
6].
For these reasons, most work on
MYB has focused on its role in normal and leukemic hematopoiesis. However, there is increasing evidence for a role of
MYB in colonic epithelial cell differentiation and homeostasis, and notably, in colon cancer [
7]. The pattern of
MYB expression in normal colonic crypts suggests that, similarly to the hematopoietic system, expression is high in immature, rapidly proliferating cells, and decreases with differentiation and maturation [
8,
9], whereas reduced
MYB activity perturbs colonic epithelial proliferation, differentiation and viability [
8,
9]. The involvement of
MYB in epithelial tumors was first suggested by the amplification of
MYB in certain colon carcinoma-derived cell lines [
10], and by its expression in a substantial proportion of tumors [
7,
11]. Moreover
MYB expression in colon tumors correlates with poor clinical prognosis [
12], and an important transcriptional regulatory region of
MYB is frequently mutated in this disease [
13,
14]. Furthermore,
MYB is required for colon carcinoma cell proliferation [
7,
15], and is down-regulated during differentiation of these cells [
9] while, conversely, ectopic
MYB expression can suppress their differentiation [
16].
By contrast, much less is known about the functions of
MYB in mammary epithelial cells. Nevertheless, it has been shown that
MYB is expressed at relatively high levels in estrogen receptor (ER) positive breast cancers and tumor cell lines [
17]. Moreover we, and others [
18] have previously shown that
MYB is a direct target of estrogen/ER signaling, and that
MYB expression in breast cancer cells is regulated by transcriptional attenuation within its first intron [
19]. Importantly, we have also recently shown that
MYB is required for the proliferation of ER positive, but not ER negative, breast cancer cell lines [
19], identifying for the first time a functional role for
MYB in breast cancer. In addition, Fang
et al [
20] reported a prolactin-inducible association between MYB and Stat5a, and that a number of Stat5a-responsive promoters such as that of the
CISH gene are further stimulated by MYB. Their results suggested that MYB may act as a coactivator for Stat5a, and also supported a proliferative function for
MYB in human breast cancer.
To further understand the function of MYB in breast cancer and in mammary epithelial cells (MECs) generally, we have now investigated its role in the differentiation of these cells. Differentiation of human breast cancer cell lines (MCF-7 and ZR-75-1) and non-tumorigenic MEC (HC11) can be induced by chemical agents or lactogenic hormones, respectively, and results in morphological and molecular properties that are characteristic of mature ductal epithelial cells. We have found that mammary carcinoma cell lines in which MYB expression was 'knocked-down' by shRNA show changes that indicate differentiation has occurred in some of these cells. Moreover, these MYB knock-down cells are more sensitive to differentiation after exposure to low doses of differentiation-inducing agents, and can be driven into apoptosis with doses that would normally only induce differentiation. Conversely, ectopic expression of MYB suppressed differentiation and apoptosis induced by differentiation-inducing agents (DIAs). Taken together, our data show that MYB plays an important role in regulating the balance between proliferation, differentiation, and apoptosis in both normal and malignant mammary epithelial cells, and that this role is remarkably similar to that it plays in hematopoietic and colonic epithelial cells. Finally, our observation that DIAs and MYB inhibition synergize in killing breast tumor cells suggests an approach to developing new treatments for ER/MYB positive breast cancer.
Materials and methods
Cell culture
The breast cancer cell lines MCF-7 and ZR-75-1 were cultured in DMEM (Invitrogen, Mount Waverley, Vic, Australia) supplemented with 10% FBS, l-glutamine, penicillin G, and streptomycin sulfate (All from GIBCO/BRL, Grand Island, New York, USA). All cell lines were maintained at 37°C in a humidified 5% carbon dioxide/95% air incubator. Prior to reaching confluence, cells were trypsinized with a 0.05% trypsin/0.53 mM EDTA solution and resuspended in fresh growth medium before plating onto a new growth surface.
Sodium butyrate, vitamin E succinate, and 12-O-tetradecanoylphorbol-13-acetate were purchased from Invitrogen (Mount Waverley, Vic, Australia).
HC11 cells were grown in Roswell Park Memorial Institute medium (RPMI)-1640 (Invitrogen, Mount Waverley, Vic, Australia) medium containing 10% FBS, 5 μg/ml insulin, and 10 ng/ml epidermal growth factor (both from Sigma-Aldrich, Castle Hill, NSW, Australia). Differentiation was induced three days after reaching confluence in medium containing 5 μg/ml insulin, 1 μg/ml hydrocortisone and 5 μg/ml prolactin (Sigma-Aldrich, Castle Hill, NSW, Australia).
For lipid droplet staining, cells grown on glass cover slips were rinsed twice with PBS and fixed for 20 minutes with PBS containing 4% paraformaldehyde at 20°C. After another PBS rinse and staining for 15 minutes with Nile Red and 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Castle Hill, NSW, Australia), the cells were PBS washed and mounted. Fluorescence imaging was performed by using automated excitation and emission filter wheels of a Fluorescent AxioSkop 2 plus Microscope (Carl Zeiss Pty Ltd, New South Wales, Australia). For flow cytometric analysis, cells not grown on glass cover slips were washed in PBS, and resuspended in PBS containing 0.01% Nile Red for 15 minutes at 37°C. After three washes in PBS, they were analyzed by flow cytometry using a FACS Calibur instrument (Becton Dickinson, San Jose, CA, USA), and the primary data were then processed using CellQuest software (Becton Dickinson, San Jose, CA, USA).
For siRNA transfection experiments, MCF-7 cells were plated and transfected the following day with 100 nM of BCL2 specific, or and control siRNAs (Dharmacon Research, Lafayette, CO, USA) by using lipofectamine 2000 (Invitrogen, Mount Waverley, Vic, Australia). Briefly, all transfections were performed in a mixture of Opti-MEM and complete media without antibiotics, as described previously [
21]. The transfection incubation time for siRNA/lipofectamine 2,000 complexes was 24 hours.
Chromatin immunoprecipitation assay
MCF-7 cells were grown to 95% confluence in phenol-red-free DMEM supplemented with 10% charcoal-stripped FBS for 72 hours; at which time, 10 nM b-estradiol was added for 12 hours. Chromatin immunoprecipitation (ChIP) was performed as previously described [
22] by using rabbit immunoglobulin (Ig) G (Sigma, Castle Hill, NSW, Australia) and 1.1 anti-Myb/5.1 anti-Myb or 1.1 anti-Myb/Thelma anti-Myb monoclonal antibodies [
23]. The resultant samples were used as real-time PCR templates to quantify binding of MYB to the various regions of
BCL2, MYC or
GAPDH. Primer sequences used in the PCR were: Myb binding sites (
MBS) 1 F 5'-GCTCAGAGGAGGGCTCTTTC-3', MBS 1 R 5'-TTTCTCCTCCTCCTGGTCCT-3',
MBS 2 F 5'-CCCGCCTCTTCACCTTTCAG-3',
MBS 2 R 5'-CAATGGCACTTCAAGTCCCGA,
MBS 3 F-5'-GGTCAGGTGGACCACAGGT- 3',
MBS 3 R 5'-GTCCAAGAATGCAAAGCACA-3',
MBS 4 F 5'-CACAGCGCCAACAGAACTAC-3',
MBS 4 R 5'-ACAGGCCAGATGCCAGATAC-3',
MYC F 5'-GCCTGCGATGATTTATACTCACAG-3',
MYC R 5'-CGGAGATTAGCGAGAGAGGATC-3',
GAPDH F 5'-ATCAATGGAAATCCCATCACCATCT-3',
GAPDH R 5'-GGTTTTTCTAGACGGCAGGTCAG-3', Upstream Control F 5'-GCAGGTGCTCAACAGATGAA-3', Upstream Control R 5'-GGGATTGCCTTACAGGTGAA-3'.
TUNEL
Apoptosis-induced nuclear DNA fragmentation was detected using the TMR Red In Situ Cell Death Detection Kit (Roche Diagnostics Corp, Indianapolis, IN, USA) following the manufacturer's protocol. Briefly, 24 hours after DIA treatment, cells grown on glass cover slips were fixed. This was followed by incubation in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction mix for 60 minutes at 37°C. Slides were washed three times in PBS/Triton X-100/BSA (0.3%) and visualized on a fluorescent microscope. Cells not grown on glass cover slips were washed in PBS, and resuspended in PBS and TUNEL reaction mix for 60 minutes at 37°C. Cells were washed three times in PBS, and analysed by flow cytometry on the FACS Calibur as described earlier.
Quantitative RT-PCR
Total RNA was prepared by using a RNeasy MiniKit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed in a total of 20 μl by using SuperScript III (Invitrogen, Carlsbad, CA, USA). The resulting cDNA was then diluted to a total volume of 100 μl with sterile water. Each real-time PCR consisted of 1 μl of diluted reverse transcriptase product, iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and 50 nM forward and reverse primers (see below). Reactions were carried out on a RotorGene 3000 (Corbett Research, Sydney, NSW, Australia) at 95°C for 10 minutes, followed by 40 cycles of 95°C for 20 seconds, 56°C for 15 seconds, and 72°C for 20 seconds. Fluorescence measurements analyzed by using the RotorGene 3000 software. The fold-change expression of each gene was calculated by using the ΔΔCT method, with cyclophilin A as an internal control. Primers used for real-time PCR were: MYB F, 5'-GCCAATTATCTCCCGAATCGA-3'; MYB R, 5'-ACCAACGTTTCGGACCGT A-3'; ß-casein F, 5'-CCCTCAAATCCCAAAACTCA-3'; ß-casein R, 5'-GAGCAGAAGGGCTTGAACAG-3'; BCL2 F, 5'-GTTCGGTGGGGTCATGTGTGTGGAGAGCG-3'; BCL2 R 5'-TAGCTGATTCGACGTTTTGCCTGA-3'; Cyclophilin A F, 5'-GGCAAATGCTGGACCCAACACAAA-3'; Cyclophilin A R, 5'-CTAGGCATGGGAGGGAACAAGGAA-3'.
Western blotting
Western blot analysis was conducted as described previously [
24]. Briefly, extracts prepared in SDS loading buffer were resolved in SDS/10% PAGE gels and transferred to PVDF membranes. These were incubated overnight in the presence of anti-c-Myb antibody 1.1 [
25] and were developed by using ECL western blotting substrates (Pierce Biotechnology, Rockford, IL, USA).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YD performed laboratory experiment and data analyses. TJG performed data analyses. YD, RGR and TJG initiated and designed the study and were involved in writing the manuscript. All authors have read and approved the final manuscript.