Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

Lactation insufficiency affects many women worldwide. During lactation, a large portion of mammary gland alveolar cells become polyploid, but how these cells balance the hyperproliferation occurring during normal alveologenesis with terminal differentiation required for lactation is unknown. Here, we show that DNA damage accumulates due to replication stress during pregnancy, activating the DNA damage response. Modulation of DNA damage levels in vivo by intraductal injections of nucleosides or DNA damaging agents reveals that the degree of DNA damage accumulated during pregnancy governs endoreplication and milk production. We identify a mechanism involving early mitotic arrest through CDK1 inactivation, resulting in a heterogeneous alveolar population with regards to ploidy and nuclei number. The inactivation of CDK1 is mediated by the DNA damage response kinase WEE1 with homozygous loss of Wee1 resulting in decreased endoreplication, alveologenesis and milk production. Thus, we propose that the DNA damage response to replication stress couples proliferation and endoreplication during mammary gland alveologenesis. Our study sheds light on mechanisms governing lactogenesis and identifies non-hormonal means for increasing milk production.


Undifferentiated Competent
Representative FACS histograms for DNA content analysis of the CK8 + population in PD17.5 (left) and LD2 MGs (right).b) Representative FACS histograms for DNA content analysis of undifferentiated, competent, primed and differentiated (DIP5) HC11 cells.Red dashed lines show a magnification of the histogram corresponding to the 4C and >4C DNA content populations.c) Percentage of undifferentiated, competent, primed and differentiated (DIP5) HC11 cells with 2C, 4C or > 4C DNA content or in the S phase, as detected by FACS analysis.Data presented as mean ± SD.Data analyzed by one-way ANOVA with Tukey's multiple comparison test.Data representative of n=3 biologically independent experiments.p values: * < 0.05, **< 0.01, *** < 0.001, **** < 0.0001.failure results in endoreplication and increased milk production.a) Representative FACS histograms for DNA content analysis (left) and quantification (right) of undifferentiated HC11 cells treated with DMSO or blebbistatin (Blebbi) for 6 hours.b) Representative FACS histograms for DNA content analysis (left) and quantification (right) of differentiated (DIP3) HC11 cells treated with DMSO or Blebbi.c) Representative phase contrast images of showing milk domes in differentiated (DIP3) HC11 cells, treated with DMSO or Blebbi.d) Csn2 expression in differentiated (DIP3) HC11 cells, treated with DMSO or Blebbi, as detected by RT-qPCR.e) Representative images of Perilipin2 (PLIN2, green) in differentiated (DIP3) HC11 cells, treated with DMSO or Blebbi.Nuclear DNA by Hoechst.f) Quantification of PLIN2 in differentiated (DIP3) HC11 cells, treated with DMSO or Blebbi, as detected by IHC.Data presented as mean ± SD (a, b and d) and mean ± S.E.M (f).Data analyzed by unpaired, two-tailed Student's t-test.Data representative of n=3 biologically independent experiments.p values: * < 0.05, **< 0.01, *cells undergo endoreplication through an early mitotic arrest involving Cdk1 inactivation.a and b) Representative western blot (a) and quantification (b) of CYCLIN E and CYCLIN B expression in undifferentiated (U), competent (C), primed (P) and differentiated (DIP 2 and 5) HC11 cells.Quantification (b) shown as CYCLIN E/CYCLIN B ratio.c) Representative FACS histograms for DNA content analysis of undifferentiated HC11 cells treated with DMSO or Ro-3306 for 6 hours.d) Representative FACS histograms for DNA content analysis of differentiated (DIP3) HC11 cells treated with DMSO or Ro-3306.Data presented as mean ± S.E.M. Data analyzed by one-way ANOVA with Tukey's multiple comparison test.Data representative of n=3 biologically independent experiments.p values: * < 0.05, **< 0.01.
damage increases endoreplication and milk production in vitro.a) Representative images of gH2AX (magenta) in undifferentiated HC11 cells treated with DMSO or Doxo for 24 hours.Nuclear DNA by Hoechst.b) Quantification of nuclear gH2AX in HC11 cells 24 hours after treatment with DMSO or Doxo, as detected by ICC.c) Representative FACS histogram for DNA content analysis of undifferentiated HC11 cells 24 hours after treatment with DMSO or Doxo.d) Percentage of undifferentiated HC11 cells with 2C, 4C or > 4C DNA content, or in the S phase, as detected by FACS analysis, 24 hours after treatment with DMSO or Doxo.e) Representative FACS histograms for DNA content analysis of differentiated (DIP3) HC11 cells treated with DMSO or Doxo.f) Percentage of differentiated (DIP3) HC11 cells with 2C, 4C or > 4C DNA content, or in the S phase, as detected by FACS analysis, after treatment with DMSO or Doxo.g) Quantification of differentiated (DIP3) HC11 cells treated with DMSO or Doxo and sorted based on DNA content (4C or > 4C), that are mono-, bi-or multinucleated (more than 2 nuclei).h) Representative Western Blot (left) and quantification (right) of CSN2 expression in differentiated (DIP3) HC11 cells after DMSO or Doxo treatment.i) Representative FACS histograms for DNA content analysis showing the sub-G1 population of HC11 cultures treated with DMSO or doxorubicin (Doxo) for 24h (top histograms) or after 3 days of differentiation (DIP3, bottom histograms).j) Percentage of HC11 cells within the sub-G1 population, as detected by FACS analysis, 24h after treatment with DMSO or Doxo, or 3 days after differentiation (DIP3).For (b) data show values of 100 cells per treatment (gray or red dots).Data presented as mean ± SEM (b) and mean ± SD (c, d, f-h and j).Data analyzed by Mann-Whitney u-test (b) and unpaired, two-tailed Student's t-test (c, d, f-h and j).Data representative of n=3 biologically independent experiments.p values: * < 0.05, **< 0.01, *** < 0.001, **** < 0.0001.DNA damage response to replication stress is activated during pregnancy and lactation.a) Representative images of RPA, pATR, pCHK1 or pATM (green) in optically-cleared sections of nulliparous, PD10.5, PD15.5, PD18.5 and LD2 MGs.CK8 shown in magenta.Nuclear DNA by propidium iodide.b and c) Representative western blot (b) and quantification (c) of pATR and ATR expression in nulliparous, PD10.5, PD15.5, PD18.5 and LD2 MGs.Quantification (c) shown as pATR/ATR ratio.d and e) Representative western blot (d) and quantification (e) of pATM and ATM expression in nulliparous, PD10.5, PD15.5, PD18.5 and LD2 MGs.Quantification (e) shown as pATM/ATM ratio.Data presented as mean ± SD.Data analyzed by one-way ANOVA with Tukey's multiple comparison test.Data representative of n=3 biologically independent experiments.p values: * < 0.05, **< 0.01, *** < 0.001.stress activates the DNA damage response and results in increased endoreplication and milk production in vitro.a) Detection of CYCLIN E by Western blot in wildtype (WT) or Cyclin E overexpressing (CCNE1) HC11 cells.b) Representative FACS histograms showing BrdU incorporation in WT and CCNE1 undifferentiated HC11 cells.Red histogram represents the negative isotype control (IgG).Black gate represents the identification of the BrdU + cells according to negative isotype control.Right histogram shows the quantification of WT or CCNE1 HC11 cells that are BrdU + .c and d) Representative images (c) and quantification (d) of nuclear gH2AX (green) in undifferentiated WT or CCNE1 HC11 cells.Nuclear DNA by Hoechst.e and f) Representative images (e) and quantification (f) of nuclear pATR (red) in undifferentiated WT or CCNE1 HC11 cells.Nuclear DNA by Hoechst.g) Representative FACS histograms for DNA content analysis of WT or CCNE1 differentiated (DIP3) HC11 cells.h) Percentage of differentiated (DIP3) WT or CCNE1 HC11 cells with 2C, 4C or >4C DNA content, or in the S phase, as detected by FACS analysis.i) Representative western blot (left) and quantification (right) of CSN2 expression in differentiated (DIP3) WT or CCNE1 HC11 cells.For (d and f), data show values of 100 cells per treatment (gray, green or red dots).Data presented as mean ± SD (b, h and i) and mean ± SEM (d and f).Data analyzed by unpaired, two-tailed Student's t-test (b, h and i) and Mann-Whitney u-test (d and f).Data representative of n=3 biologically independent experiments.p values: * < 0.05, *** < 0.001, **** < 0.0001.

Supplementary Figure 8. Replication stress regulates endoreplication and milk production in vivo. a)
Representative FACS DNA content analysis histograms from CK8 + LD2 MGs after contralateral IDI with PBS or hydroxyurea(Hu).Red dashed lines show a magnification of the histogram corresponding to the 4C and >4C DNA content populations.b) Percentage of CK8 + cells with 2C or 4C DNA content in LD2 MGs, as detected by FACS analysis, after contralateral IDI with PBS or Hu.Colored data points and dashed lines represent paired samples.c) Representative FACS DNA content analysis histograms from CK8 + LD2 MGs after contralateral IDI with PBS or nucleosides (Nucs).Red dashed lines show a magnification of the histogram corresponding to the 4C and >4C DNA content populations.d) Percentage of CK8 + cells with > 4C DNA content in LD2 MGs, as detected by FACS analysis, after contralateral IDI with PBS or Nucs.Colored data points and dashed lines represent paired samples.e) Representative FACS DNA content analysis histograms from CK8 + LD5 MGs after contralateral IDI with PBS or nucleosides (Nucs).Red dashed lines show a magnification of the histogram corresponding to the 4C and >4C DNA content populations.f) Percentage of CK8 + cells with 2C or 4C DNA content in LD5 MGs, as detected by FACS analysis, after contralateral IDI with PBS or Nucs.Colored data points and dashed lines represent paired samples.g) Milk protein gene expression, Lalba, Wap, Csn2, Plin2, Xdh1 and Btn1, in LD2 MGs after contralateral IDI with PBS or Nucs, as detected by RT-qPCR.Colored data points and dashed lines represent paired samples.
Data presented as mean ± SD (b, d and f) and mean ± SEM (g).Data analyzed by paired, two-tailed Student's t-test.Data representative of n=4 biologically independent experiments except (e and f), representative of n=3.p values: * < 0.05, **< 0.01.
and LD2 MGs, as detected by RT-qPCR.c) Wee1 expression in whole MG tissue after contralateral IDI with DMSO/Doxo, PBS/Hu or PBS/Nucs, as detected by RT-qPCR.Colored data points and dashed lines represent paired samples.d) Representative FACS DNA content analysis histograms of CK8 + cells from LD2 MGs after contralateral IDI with DMSO or Mk-1775.Red dashed lines show a magnification of the histogram corresponding to the 4C and >4C DNA content populations.e) Percentage of CK8 + cells with 2C, 4C or > 4C DNA content in LD2 MGs, as detected by FACS analysis, after contralateral IDI with DMSO or Mk-1775.Colored data points and dashed lines represent paired samples.f) Representative FACS DNA content analysis histograms of CK8 + cells from LD5 MGs after contralateral IDI with DMSO or nucleosides Mk-1775.Red dashed lines show a magnification of the histogram corresponding to the 4C and >4C DNA content populations.g) Percentage of CK8 + cells with 2C or 4C DNA content in LD5 MGs, as detected by FACS analysis, after contralateral IDI with DMSO or Mk-1775.Colored data points and dashed lines represent paired samples.h) Milk protein gene expression, Lalba, Wap, Csn2, Plin2, Xdh1 and Btn1, expression in LD2 MGs after contralateral IDI with DMSO or Mk-1775, as detected by RT-qPCR.Colored data points and dashed lines represent paired samples.Data presented as mean ± SEM (

Supplementary Figure 10. Tetraploid and polyploid cells emerge in a CK8 high luminal subpopulation at LD5. a)
Representative dot plots showing the CK8 negative (black), CK8 low ; SSC low (green), CK8 high ; SSC low (blue) and CK8 high ; SSC high (red) populations, as detected by FACS analysis, from PD17.5 (left) or LD5 (right) MGs.b) CK8 expression in PD17.5 (blue) or LD5 (red) whole mammary gland (MG) cell preps.CK8 + cells were detected by using an IgG negative control (grey).c) Percentage of CK8 low ; SSC low , CK8 high ; SSC low and CK8 high ; SSC high cells from PD17.5 (blue) or LD5 (red) MGs, as detected by FACS analysis.d-f) Percentage of cells with 2C, 4C and >4C DNA content within the CK8 low ; SSC low (d), CK8 high ; SSC low (e) and CK8 high ; SSC high (f) populations, as detected by FACS analysis, from PD17.5 (blue) or LD5 (red) MGs.g and h) Representative FACS histograms for DNA content analysis of the CK8 low ; SSC low , CK8 high ; SSC low and CK8 high ; SSC high