Deletion of Cdkn1b in ACI rats leads to increased proliferation and pregnancy-associated changes in the mammary gland due to perturbed systemic endocrine environment

Abstract

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27 + quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b + mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b -/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b -/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b +/+ and Cdkn1b -/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.

CDKN1B mutation and copy number variation are associated with tumor aggressiveness in luminal breast cancer

“…Of the 13 somatic mutations (Figure 1A, see long lines) that we identified in LBC solid biopsies, four generated a stop codon and resulted in the loss of p27 C‐terminus. This observation confirmed the higher frequency of truncating/frame shift mutations among the different mutations that were observed in sporadic breast cancer [7,8] (Figure 1A, see short lines) and supported the possibility that truncation of p27 C‐terminus directly contributes to onset and/or progression of breast cancer, as we and others have recently proposed [12,28,36–38].…”

“…Consistent with this, p27 deficiency in mice was associated with hypoplasia, impaired ductal branching and lobulo‐alveolar differentiation [40], a phenotype consistent with a putative role for p27 in regulating the number and proliferation of mammary epithelial progenitors. Of note, we and others have observed that an absence of p27 in Cdkn1b −/− female animals induces dramatic changes in mammary epithelial cells, probably due to altered hormonal signaling and perturbed endocrine environment [38] (G Mungo, personal communication October 2020).…”

“…This possibility is indirectly sustained by literature data showing that p27 expression in normal breast tissue may be used as a read out of the fraction of actively cycling cells and this, in turn, may represent a marker for breast cancer risk assessment in premenopausal women [37]. Furthermore, it is well accepted that mammary epithelial progenitors are the normal cell‐of‐origin of breast cancer and it has been clearly shown in animal models that p27 regulates the number of hormone‐responsive luminal progenitors that eventually impact on breast cancer risk [38]. In another study, the human normal mammary gland was profiled and a subset of p27+ cells that might represent quiescent hormone‐responsive progenitors was identified [39].…”

Desensitization of human breast progenitors by a transient exposure to pregnancy levels of estrogen

“…While the sequence of events and the interaction between the individual molecular players await further scrutiny, other studies lend support to the present findings. Thus, the regulation of p27 + hormone-responsive progenitors in human normal breast has been suggested as a marker of breast cancer risk 11 , and more specifically, deletion of Cdkn1b in rats leads to generation of a higher proportion of mature mammary luminal cells 23 , further implicating the pool size of hormone-responsive luminal progenitors in breast cancer risk. The role of S100A7 in normal breast also remains to be elucidated.…”

90 YEARS OF PROGESTERONE: Progesterone receptor signaling in the normal breast and its implications for cancer

“…Here on, we will describe what has been revealed about the importance in mediating distinct biological aspects of PR signaling function in vivo. Of note, the rat model was used to identify PR targets by ChIP-seq and revealed that networks relating to cell cycle and FGF and ErbB signaling were enriched (Ding et al, 2019).…”