Parity-induced changes to mammary epithelial cells control NKT cell expansion and mammary oncogenesis

SummaryPregnancy reprograms the epigenome of mammary epithelial cells (MECs) in a manner that control responses to pregnancy hormone re-exposure and the rate of carcinoma progression. However, the influence of pregnancy on the tissue microenvironment of the mammary gland is less clear. Here, we used single-cell RNA sequencing to comparatively profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis revealed an expansion of γδ Natural Killer T (NKT) immune cells following pregnancy, in association with upregulation of immune signal molecules in post-pregnancy MECs. We show that expansion of NKT cells following pregnancy is due to elevated expression of the antigen presenting molecule CD1d protein, which is known to induce NKT activation. Accordingly, loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKT cells, accompanied the development of mammary oncogenesis in response to cMYC overexpression and loss of Brca1 function. Collectively, our findings illustrate how pregnancy-induced epigenetic changes modulate the communication between MECs and the mammary immune microenvironment, and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

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Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland

Development ◽

10.1242/dev.127.20.4493 ◽

2000 ◽

Vol 127 (20) ◽

pp. 4493-4509

Author(s):

H.P. Gardner ◽

G.K. Belka ◽

G.B. Wertheim ◽

J.L. Hartman ◽

S.I. Ha ◽

...

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelium ◽

Ovarian Hormones ◽

Mammary Development ◽

Mammary Epithelial ◽

Serine Threonine Kinase ◽

Proliferation And Differentiation ◽

Induced Changes

The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.

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Stress-Induced Suppression of Milk Protein Is Involved in a Noradrenergic Mechanism in the Mammary Gland

Endocrinology ◽

10.1210/en.2019-00300 ◽

2019 ◽

Vol 160 (9) ◽

pp. 2074-2084

Author(s):

Takeshi Chiba ◽

Tomoji Maeda ◽

Yu Fujita ◽

Rika Takeda ◽

Akihiko Kikuchi ◽

...

Keyword(s):

Epithelial Cells ◽

Milk Protein ◽

Mammary Epithelial Cells ◽

Mammary Epithelium ◽

Milk Proteins ◽

Mammary Epithelial ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

The Mean ◽

Induced Changes

Abstract Stress decreases milk components such as milk protein and milk yield. The objective of this study was to investigate whether noradrenaline (NA) in milk constituted a factor associated with stress-induced changes in milk proteins such as β-casein. Breast milk obtained from eight healthy, nursing women contained NA at concentrations ranging from 12.7 to 115.5 nM. The expression of tyrosine hydroxylase (TH), a rate-limiting enzyme of NA synthesis, was observed in primary normal human mammary epithelial cells (HMECs), and in MCF-12A and MCF-10A cell lines. The mean NA concentration in culture medium used by MCF-12A transfected with TH small interfering RNA (siRNA) was significantly lower than that of cells transfected with control siRNA. NA concentration in milk in restraint-stressed nursing mice was significantly higher than that in nonstressed nursing mice, owing to elevated TH expression in the mammary epithelium. The mean β-casein concentration in milk in restraint-stressed mice was significantly lower than that in nonstressed mice. NA treatment resulted in a concentration-dependent decrease in β-casein expression in HMECs. β2 adrenergic receptor (ADRB2) expression was observed in HMECs, MCF-12A, and MCF-10A, and immunohistochemical analysis of ADRB2 using mammary epithelium sections obtained from mice at day 10 of lactation showed that ADRB2 was expressed at the apical membrane of mammary epithelium. Treatment with salbutamol, an ADRB2 stimulant, decreased β-casein expression in a concentration-dependent manner in MCF-12A. Our results showed that endogenous NA derived from mammary epithelial cells likely comprises one of the factors involved in stress-induced changes in milk proteins such as β-casein.

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Phosphoinositide-3 Kinase–Rac1–c-Jun NH2-terminal Kinase Signaling Mediates Collagen I–induced Cell Scattering and Up-Regulation of N-Cadherin Expression in Mouse Mammary Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-12-1123 ◽

2006 ◽

Vol 17 (7) ◽

pp. 2963-2975 ◽

Cited By ~ 58

Author(s):

Yasushi Shintani ◽

Margaret J. Wheelock ◽

Keith R. Johnson

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Morphological Changes ◽

Three Dimensional ◽

Mammary Epithelial ◽

Collagen I ◽

Jnk Signaling ◽

Cell Scattering ◽

Phosphoinositide 3 Kinase ◽

Induced Changes

During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I–induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I–induced N-cadherin up-regulation. These data implicate PI3K–Rac1–JNK signaling in collagen I–induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I–induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I–induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K–Rac1–JNK signaling.

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