Introduction
Breast cancer consists of multiple subtypes, and it has been postulated that the difference between subtypes arises in part from the type of mammary epithelial cell that transforms [
1,
2]. The molecular circuitry of a particular cell type determines how it responds to activation of a signaling pathway and likely dictates the sensitivity of that cell to particular oncogenic mutations [
3]. For instance,
Wip1-knockout mice have a delay in tumorigenesis in the MMTV-
neu model of breast cancer, but not in the MMTV-
wnt1 model [
4].
Wip1 is overexpressed in ~20% of human breast cancer cases, which belong mostly to the luminal and HER2
+ subtypes [
5]. Together, this suggests that the target cells for transformation by HER2/neu activation are dependent on Wip1, whereas those that can be transformed by Wnt1 are not.
Wip1 is a serine/threonine phosphatase of the PP2C (protein phosphatase 2C) family, and its oncogenic function has been attributed to, for instance, its role as a negative regulator of p53 by dephosphorylating key members of DNA-damage signaling, including ATM, Chk2, and p53 itself [
6]. In addition, Wip1 dephosphorylates and thereby inactivates the stress kinase p38MAPK, and inhibition of p38MAPK in Wip1-knockout mice partially restored sensitivity to MMTV-
neu-induced tumorigenesis [
7]. In this study, we examined the role of Wip1 in mammary epithelium to identify the cell types that are dependent on Wip1 activity and therefore may be involved in the early stages of HER2/neu-induced tumorigenesis.
Mammary epithelium consists of an outer basal layer of mainly contractile myoepithelial cells and an inner luminal layer that contains both steroid-receptor-positive cells and steroid-receptor-negative cells in a spatially ordered pattern [
8]. Mammary gland development during puberty is orchestrated by the steroid sex hormones estrogen and progesterone, which trigger proliferation indirectly in steroid-receptor-negative cells through paracrine factors produced by steroid-receptor-positive cells. Interestingly, steroid-receptor-positive cells act mainly as a conduit for proliferative signals, as they rarely divide themselves [
9,
10]. The luminal steroid-receptor-negative cells contain different progenitor subsets, including alveolar progenitor cells that are primed for milk production [
11‐
13]. During the initial phase of pregnancy, progesterone, together with the peptide-hormone prolactin, triggers a massive expansion of the alveolar cell population in a process termed lobulo-alveologenesis, followed by terminal differentiation of the alveolar cells later in pregnancy [
14,
15]. Both processes are strictly dependent on prolactin signaling, as any mutant in the prolactin receptor-JAK2-STAT5 signaling cascade has a defect in alveolar development [
16‐
18], and even after alveologenesis has been completed, lactation remains dependent on STAT5 expression [
19]. Activation of the prolactin receptor results in activation of the associated JAK2, which subsequently phosphorylates STAT5, allowing STAT5 to translocate to the nucleus and activate gene transcription [
20]. STAT5 directly binds to the promoter of milk genes, suggesting that in mammary epithelium, alveolar cells are the principal responders to prolactin [
21].
The cells most likely to be sensitive to transformation by Wnt1 are stem or progenitor cells that are part of the basal layer [
22,
23]. In contrast, compelling evidence suggests that the target cell for transformation in the MMTV-
neu model belongs to the alveolar lineage. Whey acidic protein (WAP) is one of the components of milk that is expressed late during alveolar differentiation. Lineage tracing with a WAP-promoter-driven Cre recombinase, together with a Rosa-lox-stop-lox-
LacZ reporter, showed that early lesions in MMTV-
neu mammary glands are all LacZ-positive, indicating that these cells expressed milk genes at some point [
24]. These LacZ-marked cells are also referred to as parity-identified mammary epithelial cells (PI-MECs) or lobule-restricted progenitors [
25]. Strikingly, mice with a cyclin D1 point mutation generate normal mammary ducts, but no PI-MECs, and are completely resistant to MMTV-
neu tumorigenesis [
26].
In line with the presumptive alveolar origin of HER2/neu-driven tumors and the attenuation of tumorigenesis in the absence of Wip1, we found delayed alveolar development during pregnancy in Wip1-knockout mammary glands. Unexpectedly, we identify a role for Wip1 in steroid-receptor-positive cells rather than adjacent alveolar progenitor cells. We show that in the virgin state, only steroid-receptor-positive cells activate STAT5, and this is strictly dependent on Wip1. Unlike alveolar cells that transcribe milk genes after STAT5 activation, hormone-sensing cells transcribe paracrine stimulators of alveolar proliferation (RANKL and IGF2), elucidating a role for steroid-receptor-positive cells in the growth-promoting rather than differentiation-inducing effects of prolactin. MMTV-neu tumors are estrogen-receptor negative but we show that before tumor formation, ERK activation by HER2/neu is most pronounced in steroid-receptor-positive cells, and this is dependent on Wip1. Finally, in virgin Wip1-knockout mice, HER2/neu activates STAT5 in alveolar progenitors but not steroid-receptor-positive cells, and paracrine signaling remains attenuated. This suggests that the target cells for transformation in the MMTV-neu model rely on Wip1-dependent signaling in neighboring cells, highlighting the instructive role of hormone-sensing cells in early pregnancy and premalignant development.
Materials and methods
Mice
Wip1 KO (
Ppm1d
-/-
) mice were previously described [
27] (129Sv-C57BL/6-FVB background). We observed no difference between
Wip1 wild-type or heterozygote animals in the context of alveolar development, STAT5 activation or qPCR data, and therefore the "wild-type" control groups presented here consist of a mixture of wild-type and heterozygote animals. MMTV-
neu mice used for this study (and [
4,
26]) express the activated rat
ErbB2 (
c-neu) oncogene under control of the mouse mammary tumor virus promoter (strain TG.NK) [
28] and were purchased from the Jackson Laboratory (Jax#5038, FVB background). All animal protocols were approved by the SingHealth Institute Animal Care and Use Committee.
Timed mating and carmine staining of whole-mounted mammary glands
Female mice were placed in the cage of a male after 5 PM and checked for vaginal plugs at 9 AM the following morning (Day 0). Mice were killed by carbon dioxide inhalation and one number 3 (thoracic) gland was fixed in methacarn (60% methanol, 30% chloroform, 10% acetic acid) for 24 hours. Subsequently, the gland was placed in 70% ethanol for 24 hours, and then immersed in 0.2% carmine (Sigma C1022, St. Louis, MO, USA)/0.5% aluminum potassium sulfate (Sigma-Aldrich #23,708-6, St. Louis, MO, USA) stain for 18 hours. Next, glands were transferred to 70%, 90%, and 100% ethanol for 1 hour each, followed by 100% ethanol for 18 hours. Finally, glands were transferred to methyl salicylate (Sigma M2047, St. Louis, MO, USA) for visualization and photography with an Olympus SZX12 microscope.
Isolation of primary mammary epithelial cells
Mammary epithelial cells were isolated [
29], with minor modifications. Mice were killed by carbon dioxide inhalation and the number 4 (inguinal) and 5 mammary glands were excised after removal of mammary lymph nodes. Glands were chopped 3 times by using a McIlwain tissue chopper (Mickle Laboratory Engineering, Guildford, UK) on the finest setting, with a 90-degree rotation of the base plate between each round of chopping. Chopped glands from one animal were then placed in 10 ml digestion mix containing 3 mg/ml of collagenase A (Roche 11088793001, Mannheim, Germany) and 0.67 mg/ml trypsin (Becton Dickinson (BD) 215240, Sparks, MD, USA) at 37°C for 45 minutes with agitation every 15 minutes. Digested glands were subsequently centrifuged at 1,300 rpm (340 rcf) for 6 minutes at 4°C, and the fat layer and supernatant removed. The pellet (containing mammary epithelial organoids) was resuspended in 10 ml of L15 media (Sigma L1518, St. Louis, MO, USA) containing 6% fetal calf serum (Hyclone SV30160.03, Cramlington, UK) and centrifuged at 1,500 rpm (453 rcf) at room temperature. Supernatant was removed, and the pellet was resuspended in 5 ml of red blood cell lysis buffer (Sigma R7757, St. Louis, MO, USA) and incubated at room temperature for 5 minutes before centrifugation at 1,500 rpm for 5 minutes at 4°C. From this point, all centrifugation steps were performed at 1,500 rpm at 4°C. Pellet was then resuspended in DMEM +10% FCS and incubated for 30 minutes at 37°C in a T75 flask to allow the selective adherence of fibroblasts. Media containing organoids were collected and centrifuged. Supernatant was removed, and organoids were resuspended in L15 + 6% FCS (L15+) and kept overnight at 4°C. The next day, organoids were pelleted, washed twice in Ca
2+/Mg
2+-free PBS/0.02% wt/vol EDTA and incubated in 2 ml of Joklik MEM (Sigma M8028, St. Louis, MO, USA) for 15 minutes at 37°C. Organoids were centrifuged and resuspended in 2 ml of 0.25% trypsin-0.04% EDTA solution (Gibco 25200, Grand Island, NY, USA) and placed at 37°C for 2 minutes to generate single cells. Next, 5 ml of 5 μg/ml DNase I (type II) in serum-free L15 (Sigma D4527, St. Louis, MO, USA) was added for a further 5 minutes at 37°C to disperse cellular clumps. Then, 7 ml of L15+ was added (henceforth, all resuspensions were performed by using L15+), and the cell solution was passed through a 40-μm cell strainer (BD 352340, Sparks, MD, USA). The resultant single cells were pelleted, resuspended in L15+, and counted by using trypan blue and a hemocytometer. Cells were brought to a concentration of 1 × 10
6/ml and kept on ice.
Cell labeling, flow-cytometric analysis, and fluorescence-activated cell sorting
Fluorochrome-conjugated antibodies were titrated on primary mammary epithelial cells to ensure maximal positive-to-background fluorescence ratio (see Additional file
1). Anti-mouse and/or anti-rat compensation beads (BD 552843 and 552845, respectively) were used for single-stain antibody controls. Compensation controls also included two cellular samples: unstained cells and cells with DAPI (Sigma D8417, St. Louis, MO, USA). Cells were incubated with antibodies on ice for 45 minutes with agitation each 15 minutes. Samples were then washed with twice the sample volume and resuspended in L15+ containing 200 ng/ml of DAPI, except non-DAPI compensation controls. All multiple-labeled samples were gated on FSC-A versus SSC-A and doublet discrimination (FSC-H versus FSC-W and SSC-H versus SSC-W) and DAPI negativity (see Additional file
2). Samples contained anti-CD45 to exclude lymphocytes from analysis. Cells were analyzed and sorted on a BD FACS-Aria II containing 355 nm UV, 488 nm blue, 561 nm yellow-green, and 633 nm red lasers. Sorting for culture or
in vivo assays was performed into L15+.
Generation of cDNA by direct reverse transcription and qPCR analysis
For analysis of transcript levels by quantitative polymerase chain reaction (qPCR), cells were sorted directly into lysis buffer (10 IU RNase inhibitor (Invitrogen 10777, Carlsbad, CA, USA), 2 m
M DTT, 0.15% Tween-20 (Biorad) in 12 μl of nuclease-free water) in PCR tubes. Then 500 cells were sorted into each tube (making approximately 14 μl total volume). Reverse transcription was performed by using Superscript VILO (Invitrogen 11754, Carlsbad, CA, USA), as per manufacturer's protocol. Primers were designed that span introns to exclude the detection of genomic DNA and selected for optimal melt curve and amplification profiles (for primer sequences (see Additional file
3). qPCR was performed by using SSo Fast Evagreen supermix reagent (Biorad 172-500, Hercules, CA, USA) as per manufacturer's protocol. Per subpopulation, two to three tubes were assayed, normalized with
HPRT (validated to be consistent between groups), averaged, and compared with matched WT samples according to the delta-delta c(t) method. The relative values from three to five sets of mice were assessed with paired
t test for statistical significance.
Mammary gland transplantation and immunofluorescence
The number 4 and 5 mammary glands were harvested from donor mice, and the mammary glands digested and sorted, as outlined earlier. Then 25,000 bulk epithelial cells were injected into cleared number 4 fat pads of 21-day-old WT-recipient mice and allowed to engraft for 8 weeks. Glands were then harvested, fixed, and stained with carmine alum, as outlined earlier. After whole-mount analysis, glands were removed from methyl salicylate and washed 5 times for 1 hour in 100% EtOH before immersion in xylene for 2 × 1 hour. Tissue was then embedded in paraffin and processed for immunofluorescence.
Confocal immunofluorescence
Fresh number 3 mammary glands were fixed for 18 hours in 4% buffered formaldehyde (ICM Pharma, Singapore), processed, and embedded in paraffin wax. The 5-μm sections were cut and adhered to Superfrost Plus-coated slides (Menzel-Glaser J1800AMNZ, Braunschweig, Germany) overnight at 37°C. Sections were deparaffinized in xylene (2 × 5 minutes) and 100% ethanol (2 × 5 minutes), before rehydration in graded ethanol (90%, 2 × 5 minutes; 70%, 2 × 5 minutes) and immersion in distilled H
2O. Antigen retrieval was performed in 600 ml of 1 m
M disodium-EDTA by heating in a microwave on high for 5 minutes, on 30% power for an additional 5 minutes, and then cooled at room temperature for 1 hour. Slides were immersed in distilled H
2O and washed in PBS for 5 minutes. Sections were encircled with a wax pen and primary antibody diluted in PBS (for dilutions and suppliers, see Additional file
1) + 10% normal serum from the species in which the secondary antibody was raised, was applied and incubated at 4°C overnight. Sections were washed in PBS (2 × 5 minutes) before the addition of secondary antibody (in PBS + 10% normal serum), for 30 minutes at room temperature. Sections were washed in PBS (2 × 5 minutes) before the addition of DAPI (1 μg/ml) for 2 minutes at room temperature. Sections were then washed in PBS and mounted in Vectashield fluorescence mounting media (Vector Laboratories H-1000, Burlingame, CA, USA) for visualization. Images were acquired on a Zeiss 710 confocal microscope with a pinhole aperture of 1 Airy unit. Negative controls can be found in Additional file
4. For cell enumeration, at least seven fields were randomly selected, and > 1,000 cells were counted per animal.
Competing interests
The authors declare that they do not have competing interests.
Authors' contributions
GT and DS carried out the mouse studies, including tissue and molecular analyses. DS, VH, and KK analyzed and sorted primary cells with FACS for qPCR analyses and participated in the design of the experiments. KG and BT took care of the mice and performed the genotyping. DB and AP conceived of the study. GT and AP designed and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript.