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EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival

Abstract

Expansion and remodelling of the mammary epithelium requires a tight balance between cellular proliferation, differentiation and death. To explore cell survival versus cell death decisions in this organ, we deleted the pro-survival gene Mcl-1 in the mammary epithelium. Mcl-1 was found to be essential at multiple developmental stages including morphogenesis in puberty and alveologenesis in pregnancy. Moreover, Mcl-1-deficient basal cells were virtually devoid of repopulating activity, suggesting that this gene is required for stem cell function. Profound upregulation of the Mcl-1 protein was evident in alveolar cells at the switch to lactation, and Mcl-1 deficiency impaired lactation. Interestingly, EGF was identified as one of the most highly upregulated genes on lactogenesis and inhibition of EGF or mTOR signalling markedly impaired lactation, with concomitant decreases in Mcl-1 and phosphorylated ribosomal protein S6. These data demonstrate that Mcl-1 is essential for mammopoiesis and identify EGF as a critical trigger of Mcl-1 translation to ensure survival of milk-producing alveolar cells.

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Figure 1: Dynamic expression of Mcl-1 protein levels during mammary gland development and involution.
Figure 2: Mcl-1 deletion impairs expansion of the mammary epithelium during puberty.
Figure 3: Mcl-1-dependent survival is essential for stem and progenitor cell activity.
Figure 4: Mcl-1 deletion impairs alveolar expansion during pregnancy.
Figure 5: Ablation of Mcl-1 in early lactation results in precocious involution.
Figure 6: EGF mediates upregulation of Mcl-1 protein at the onset of lactation.
Figure 7: An EGF–mTOR axis mediates upregulation of Mcl-1 protein through increased translation during lactation.

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Acknowledgements

We are grateful to J. Takeda and M. Takaishi for the gift of K5–cre transgenic mice and S. Glaser for provision of mice. We thank D. Huang for advice. We also thank J. L. Clancy, S. K. Archer and G. Duan for assistance with the polysome experiments, B. Helbert for genotyping, and the Animal, FACS, Imaging and Histology facilities at WEHI. This work was supported by the Australian National Health and Medical Research Council (NHMRC) grants no. 1016701, no. 1024852, no. 1086727; NHMRC IRIISS; the Victorian State Government through VCA funding of the Victorian Breast Cancer Research Consortium and Operational Infrastructure Support; and the Australian Cancer Research Foundation. N.Y.F. and A.C.R. are supported by a National Breast Cancer Foundation (NBCF)/Cure Cancer Australia Fellowship; A.T.L.L. by an Elizabeth and Vernon Puzey Scholarship from the University of Melbourne; S.A.B. by a NHMRC Postgraduate Scholarship no. 1017256; P.B., G.K.S. and G.J.L. by NHMRC Fellowships no. 1042629, no. 1058892, no. 637307; and J.E.V. by an Australia Fellowship.

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N.Y.F. generated and analysed all mouse models, acquired and interpreted main data sets. N.Y.F., G.J.L. and J.E.V. designed experiments. A.C.R. performed confocal microscopy imaging; B.P. qPCR analysis; A.T.L.L. and G.K.S. bioinformatic analysis; R.S. and T.P. polysome fractionation experiments; K.L. assisted with immunohistochemistry; T.B. assisted with western blot analysis; S.A.B. assisted with histological analysis; F.V. performed transplantation assays; P.B. and A.S. provided Mcl-1 mice and discussions. J.E.V. and N.Y.F. wrote the paper.

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Correspondence to Jane E. Visvader.

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Integrated supplementary information

Supplementary Figure 1 Expression of Mcl-1 in the mammary gland.

(a) Western blots showing the rapid decrease of Mcl-1 at the onset of involution. pY705 Stat3 represents a marker for involution (n = 2 independent experiments). (b) General strategy for flow cytometric analysis and sorting of mammary epithelial cells. Representative FACS plots (n = 10) showing the MaSC/basal (LinCD29hiCD24+) and luminal (LinCD29loCD24+) subsets. The luminal population was further subdivided into three distinct subpopulations based on CD14 and CD61 expression: CD14+CD61, CD14+CD61+ and CD14CD61. (c) Representative FACS plots (n = 8) showing the MaSC/basal (LinCD29hiCD24+) and luminal (LinCD29loCD24+) subsets isolated from the mammary glands of 5 week-old (puberty), 10 week-old adult (virgin), 18.5 day pregnant (18.5 dP) and 2 day lactating (2 dL) mice. (d) Western blots showing the decrease in Bcl-2 levels in luminal cells that occurs during pregnancy and lactation relative to virgin glands (n = 1). (e) Representative Western blots showing Mcl-1 levels in luminal and MaSC/basal cells in puberty versus adult and 2 day lactating mammary glands, with short (SE) and long exposures (LE) indicated. (f) Quantitative RT-PCR analysis of Mcl-1 mRNA in the luminal or MaSC/basal population of mammary glands from virgin, 18.5 day pregnant (18.5 dP) or 2 day lactating (2 dL) mice. Mean ± SEM for n = 3 independent samples. (g) Western blot analysis of Bcl-2 family protein expression in the four distinct mammary epithelial cell subsets of virgin mammary glands. Approximately 250,000 cells were used for each sample (n = 1). (h) Quantitative RT-PCR analysis of Mcl-1 expression in luminal (progenitor and mature) or MaSC/basal populations isolated from virgin mammary glands. Mean ± SEM, n = 3 independent samples.

Supplementary Figure 2 Mcl-1 protein is induced in ductal and alveolar luminal cells at the switch to lactation.

Whole-mount 3D confocal images of mammary glands at 2 days of lactation (2 dL) or 18.5 days of pregnancy, showing ducts and/or alveoli. The tissues were stained for F-actin (red), Mcl-1 (green) and E-cadherin (blue) expression. An outline of the ducts is depicted in the merged images. The white arrow depicts the mesh of elongated myoepithelial cells that surround each alveolus. Representative of 3 experiments. Scale bars, 50 μm.

Supplementary Figure 3 No apparent change in the stability of Mcl-1 based on cycloheximide pulse-chase experiments and no induction of Mcl-1 by prolactin.

(a) Luminal cells (LinCD29loCD24+) were sorted from the mammary glands of 18.5 day pregnant or 2 day lactating mice, plated in ultra-low adherence plates and incubated with cycloheximide (CHX) for the indicated times. Lysates were subjected to western blot analysis for Mcl-1 and Actin protein levels (n = 2 experiments). (b,c) Mammary epithelial cells (LinCD24+) were sorted from virgin (b) or 18.5 day pregnant (c) mammary glands, starved of serum and EGF for 36 h and exposed to the lactogenic hormone prolactin for 3 days. Western blot analysis was performed to determine Mcl-1, milk and actin protein expression (n = 1).

Supplementary Figure 4 EGF is dramatically upregulated in alveolar cells at the onset of lactation.

(a) Heatmaps of the 100 most differentially expressed genes in the luminal population between late pregnancy and early lactation, clustered according to their expression values. Luminal and MaSC/basal cell populations were isolated from virgin, 18.5 day pregnant (18.5 dP) and 2 day lactating (2 dL) mammary glands (FVB/N). (c) Heatmap of gene expression for members of the EGF receptor/ligand family that are differentially expressed between late pregnancy and early lactation for the luminal population. 18.5 dP or 2 dL mice. dP, days pregnancy; dL, days lactation.

Supplementary Figure 5 Increased phospho-S6 expression in alveolar luminal cells at the onset of lactation.

(a) Immunostaining of phospho-S6 protein in the mammary glands of FVB/N mice (n = 2 mice per stage) through development. Scale bar: 50 μm. (b) 3D whole-mount confocal analysis of pS6 in the mammary glands of FVB/N mice (n = 3) at 4 days of lactation. Tissues were incubated with Phalloidin to observe the entire tissue at the cellular level. Scale bar: 50 μm. (c) Immunostaining for cleaved caspase 3 in the mammary glands isolated from mothers administrated RAD001 or vehicle at 2 days of lactation (n = 3 dams). Scale bar: 50 μm. Representative images (ac) are shown for n = 3 experiments.

Supplementary Figure 6 Polysome profiles and RNA expression data for mammary tissue from 17 days pregnant (dP), and 2 or 4 days lactating mice (dL).

Data from two independent biological repeat experiments are shown: Experiment 1 (ad) and Experiment 2 (eh). (a,e) Polysome fractionation profiles (A254: absorbance at 254 nm, dominated by ribosomal RNA) from mammary gland tissue; (b,f) Representative graphs from one gradient per experiment showing RNA integrity across gradient fractions as determined on the Agilent Bioanalyzer; (c,g) Distribution of Mcl-1, Bcl-x and Actin mRNA amongst gradient fractions isolated from mammary glands at 17 dP, 2 dL and 4 dL; (d,h) Quantitative RT-PCR analysis of Mcl-1, Bcl-x and Actin mRNA steady-state levels in the unfractionated cell lysates used for polysome fractionation (mean of 3 technical replicates).

Supplementary Figure 7 Uncropped scans of western blots.

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Fu, N., Rios, A., Pal, B. et al. EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival. Nat Cell Biol 17, 365–375 (2015). https://doi.org/10.1038/ncb3117

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