Loss of STING impairs lactogenic differentiation

Abstract

Heightened energetic and nutrient demand during lactogenic differentiation of the mammary gland elicits upregulation of various stress responses to support cellular homeostasis. Here, we identify the stimulator of interferon genes (STING) as an immune supporter of the functional development of mouse mammary epithelial cells (MECs). An in vitro model of MEC differentiation revealed that STING is activated in a cGAS-independent manner to produce both type I interferons and proinflammatory cytokines in response to the accumulation of mitochondrial reactive oxygen species. Induction of STING activity was found to be dependent on the breast tumor suppressor gene single-minded 2 (SIM2). Using mouse models of lactation, we discovered that loss of STING activity results in early involution of #3 mammary glands, severely impairing lactational performance. Our data suggest that STING is required for successful functional differentiation of the mammary gland and bestows a differential lactogenic phenotype between #3 mammary glands and the traditionally explored inguinal 4|9 pair. These findings affirm unique development of mammary gland pairs that is essential to consider in future investigations into normal development and breast cancer initiation.

Keywords: Breast cancer; Inflammation; Lactation; Mammary gland development; Mouse; STING.

Fig. 1.

cGAS-independent STING expression during mammary epithelial cell differentiation. (A) Immunostaining for STING in #4 mammary glands of WT mice across normal development; P, pregnancy day; L, lactation day. (B) Immunostaining for STING in cGas^−/− mammary gland tissue at lactation day (dL) 6. (C,D) Immunoblot analysis of cGAS (C) and STING (D) in undifferentiated CIT3 WT cells transfected with dsDNA. (E,F) Immunoblot analysis of STING (E) and cGAS (F) in CIT3 WT cells across MEC differentiation. Mouse embryonic fibroblasts (MEFs) transfected with cGAS-HA used as positive control for cGAS expression. hD, hours of differentiation. Scale bars: 50 µm.

Fig. 2.

STING inhibition reduces type I IFN gene expression during MEC differentiation. (A-F) qPCR analysis of Csn2 (A), Ifna4 (B), Ifnb1 (C), Il1b (D), Tnf (E) and Il6 (F) in CIT3 WT, undifferentiated (UnD) versus 48 h of differentiation (hD). (G) Immunoblot analysis of CIT3 WT cells transfected with siSting at 24 hD. (H-K) qPCR analysis of Csn2 (H), Ifna4 (I), Ifnb1 (J) and Il1b (K) in CIT3 WT cells following siSting transfection at 24 hD. *P≤0.05, **P≤0.01 (paired, two-sided Student's t-test). Data are mean±s.d.

Fig. 3.

Loss of Sim2 decreases STING expression. (A) Immunostaining for STING in control and Sim2^fl/fl #4 mammary glands at lactation day (dL) 10. (B) Microscopy images of undifferentiated (UnD) versus 24 h of differentiation (hD) CIT3 WT and Sim2^−/− MECs. (C) Immunoblot analysis of STING in CIT3 WT and Sim2^−/− MECs. (D-I) qPCR analysis of Csn2 (D), Ifna4 (E), Ifnb1 (F), Il1b (G), Tnf (H) and Il6 (I) in CIT3 WT and Sim2^−/− cells. (J) Immunoblot analysis of cGAS-STING in SUM159 cells following dsDNA transfection. (K) qPCR analysis of Sting in CIT3 WT cells. (L) Immunoblot analysis of CIT3 WT cells following CHX treatment across early differentiation. (M) qPCR analysis of STING in SUM159 cells. (N,O) STING protein expression following CHX treatment in SUM159 Empty (N) and SIM2-overexpressing (O) cells. *P≤0.05, **P≤0.01, ***P≤0.001 (paired, two-sided Student's t-test). Data are mean±s.d. Scale bars: 50 µm.

Fig. 4.

Mitoquinol treatment decreases STING-dependent gene expression. (A) Relative fluorescence intensity of cellular reactive oxygen species (ROS) in CIT3 WT versus Sim2^−/− cells at 2, 8 and 24 h of differentiation (hD). (B) Relative fluorescence intensity of cellular ROS in CIT3 WT cells following MitoQ treatment. In A,B, boxes represent mean±s.d. (C) Immunoblot analysis of SIM2 and STING protein expression in CIT3 WT cells at 24hD, with and without MitoQ treatment. Numbers above the blot represent densitometry analysis for SIM2. (D-I) qPCR analysis of Csn2 (D), Ifna4 (E), Ifnb1 (F), Il1b (G), Il6 (H) and Tnf (I) in CIT3 WT cells at 48 hD, with and without MitoQ treatment. (J) Immunoblot analysis of SUM159 Empty and SIM2-FLAG cells following 24 h MitoQ treatment. *P≤0.05, **P≤0.01, ***P≤0.001 (paired, two-sided Student's t-test). Data are mean±s.d.

Fig. 5.

Loss of Sting negatively impacts lactational performance. (A) Average weight of cross-fostered pups nursed by WT and Sting^gt/gt mice (n=3 WT, n=3 Sting^gt/gt). (B) Weight of cross-fostered pups nursed by WT and Sting^gt/gt mice. Arrows indicate death of pup. (C) H&E-stained #4 mammary tissue sections from WT and Sting^gt/gt mice at lactation day (dL) 10. (D) Quantification of cellularity (number of nuclei per sample area) for WT and Sting^gt/gt mice. (E-J) Immunostaining and quantification of percentage of staining at dL10 for CSN2 (E) pSTAT5 (F) E-cadherin (G) K14 (H) pSTAT3 (I) and c-CASP3 (J). *P≤0.05, **P≤0.01 (paired, two-sided Student's t-test). Data are mean±s.d. Scale bars 50 µm.

Fig. 6.

Loss of Sting impairs functional differentiation in #3 mammary glands. (A) H&E-stained #3 mammary tissue sections from WT and Sting^gt/gt mice at lactation day (dL) 10. (B) Quantification of cellularity (number of nuclei per sample area) for WT and Sting^gt/gt mice. (C-H) Immunostaining at dL10 for c-CASP3 (C), pSTAT3 (D), pSTAT5 (E), CSN2 (F), E-cadherin (G) and K14 (H). (I,J) H&E-stained #3 mammary tissue sections from WT and Sting^gt/gt mice at 10-week virgin (I) and pregnancy day (P)16 (J). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (paired, two-sided Student's t-test). Data are mean±s.d. Scale bars: 50 µm.