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. 2018 Oct 8;39(10):1264-1273.
doi: 10.1093/carcin/bgy096.

Caloric restriction inhibits mammary tumorigenesis in MMTV-ErbB2 transgenic mice through the suppression of ER and ErbB2 pathways and inhibition of epithelial cell stemness in premalignant mammary tissues

Zhikun Ma  1 , Amanda B Parris  1 , Erin W Howard  1 , Yujie Shi  2 , Shihe Yang  3 , Yunbo Jiang  3 , Lingfei Kong  2 , Xiaohe Yang  1   2   3
Affiliations

Caloric restriction inhibits mammary tumorigenesis in MMTV-ErbB2 transgenic mice through the suppression of ER and ErbB2 pathways and inhibition of epithelial cell stemness in premalignant mammary tissues

Zhikun Ma et al. Carcinogenesis. .

Abstract

Caloric intake influences the onset of many diseases, including cancer. In particular, caloric restriction (CR) has been reported to suppress mammary tumorigenesis in various models. However, the underlying cancer preventive mechanisms have not been fully explored. To this end, we aimed to characterize the anticancer mechanisms of CR using MMTV-ErbB2 transgenic mice, a well-established spontaneous ErbB2-overexpressing mammary tumor model, by focusing on cellular and molecular changes in premalignant tissues. In MMTV-ErbB2 mice with 30% CR beginning at 8 weeks of age, mammary tumor development was dramatically inhibited, as exhibited by reduced tumor incidence and increased tumor latency. Morphogenic mammary gland analyses in 15- and 20-week-old mice indicated that CR significantly decreased mammary epithelial cell (MEC) density and proliferative index. To understand the underlying mechanisms, we analyzed the effects of CR on mammary stem/progenitor cells. Results from fluorescence-activated cell sorting analyses showed that CR modified mammary tissue hierarchy dynamics, as evidenced by decreased luminal cells (CD24highCD49flow), putative mammary reconstituting unit subpopulation (CD24highCD49fhigh) and luminal progenitor cells (CD61highCD49fhigh). Mammosphere and colony-forming cell assays demonstrated that CR significantly inhibited mammary stem cell self-renewal and progenitor cell numbers. Molecular analyses indicated that CR concurrently inhibited estrogen receptor (ER) and ErbB2 signaling. These molecular changes were accompanied by decreased mRNA levels of ER-_targeted genes and epidermal growth factor receptor/ErbB2 family members and ligands, suggesting ER-ErbB2 signaling cross-talk. Collectively, our data demonstrate that CR significantly impacts ER and ErbB2 signaling, which induces profound changes in MEC reprogramming, and mammary stem/progenitor cell inhibition is a critical mechanism of CR-mediated breast cancer prevention.

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Figures

Figure1.
Figure1.
CR increases tumor latency in MMTV-ErbB2 transgenic mice. MMTV-ErbB2 transgenic mice were treated with control diet (n = 20) or 30% CR diet (n = 20) from 8 weeks of age until 60 weeks of age. (A) Body weight in mice fed control and CR diets was recorded every 2 weeks from 6 to 54 weeks of age. (B) Tumor development was monitored twice a week from 20 to 60 weeks of age. The percentage of tumor-free control and CR mice is shown in the Kaplan–Meier curves (P ≤ 0.0001).
Figure 2.
Figure 2.
CR modifies mammary morphogenesis and proliferative index in MMTV-ErbB2 mice. (A) Representative images of whole mounts from 15-week-old (left panels) and 20-week-old (right panels) mice are shown. (B) Mammary gland area was measured in 15-week-old mice (n = 6 for each group). Histopathological analysis of mammary tissues from 15-week-old mice was also performed with representative images of hematoxylin and eosin-stained mammary tissues from control and CR mice shown in (C). (D) Twenty-week-old control and CR mice were injected with BrdU 2 h before euthanasia (n = 6 for each group). Then mammary tissues were analyzed for nuclear incorporation of BrdU using IHC. Representative images of BrdU-stained tissues are shown with the percentage of BrdU+ cells graphed in the panel below. All values are graphed as the mean ± SE (**P ≤ 0.01).
Figure 3.
Figure 3.
CR alters MEC subpopulations in premalignant mammary tissues from MMTV-ErbB2 mice. Primary MECs were isolated from 20-week-old MMTV-ErbB2 mice that were fed control or CR diets for 12 weeks. Cells were labeled with CD24/CD49f or CD61/CD49f fluorescent antibodies for flow cytometry analysis of the different subpopulations. Representative plots of the CD24/CD49f-probed cells are shown in (A). (B) Luminal (left panel), MRU (middle panel) and myoepithelial (right panel) cell populations were compared between the control and CR mice. Representative plots of the CD61/CD49f-probed cells are shown in (C). (D) The percentage of CD61highCD49fhigh (Q2) cell populations were compared between the control and CR mice. All values are graphed as the mean ± SE (*P ≤ 0.05, **P ≤ 0.01).
Figure 4.
Figure 4.
CR inhibits MaSC self-renewal and colony formation of mammary progenitor cells from MMTV-ErbB2 mice. Primary MECs were isolated from 20-week-old MMTV-ErbB2 mice that were fed the control or CR diet. Then, the MECs were used for CFC (A), mammosphere (B) and 3D culture (C) assays according to the procedures as described in the ‘Materials and methods’. Representative images for each assay are shown above the corresponding graphs. All values are depicted in the appropriate graphs as the mean ± SE (*P ≤ 0.05, **P ≤ 0.01, as compared to corresponding controls).
Figure 5.
Figure 5.
CR attenuates ErbB2 and ER signaling pathways in MMTV-ErbB2 mice. Western blotting of MECs isolated from 15-week-old mice consuming control or 30% CR diets (n = 4 for each group) was performed to analyze the expression and activation of key molecules associated with RTK (A) and ER (B) signaling. (C) Quantitative real-time PCR analysis of mRNA from MECs was also performed to measure the expression of genes associated with ER and RTK signaling activation. Relative mRNA levels (as normalized to β-actin mRNA levels) are shown for the indicated genes ± SE (**P ≤ 0.01, as compared to corresponding controls).
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