Deletion of Cdkn1b in ACI rats perturbs mammary progenitor cell proliferation and differentiation through non-cell-autonomous mechanisms

Emerging data indicate that breast epithelial stem cells and progenitors, particularly those in the luminal epithelial cell lineage, are the cells-of-origin of breast carcinomas, and factors that influence breast cancer risk may alter the number and/or properties of these cells. We hypothesize that a subset of p27+cells represent hormone-responsive progenitors that are quiescent due to the high activity of TGFβ signaling in these cells. The Estrogen-induced mammary tumor model in ACI inbred rats is physiologically relevant rodent model of breast cancer. In the present study we successfully generatedCdkn1bknockout ACI rats and performed comprehensive phenotypic assessment and RNAseq profiling using FACS sorted basal (CD24+CD29high) and luminal (CD24+CD29low) cell populations to characterizeCdkn1b+/+andCdkn1b-/-females in prepubertal and adult cohorts. We found that p27KO rats exhibited mammary differentiation phenotype and reduced numbers of mammary epithelial progenitor pool, Interestingly, p27 ablation has the most pronounced effect on luminal progenitor cell gene expression, and milk protein genes and pStat5 were dramatically upregulated, while PR and FoxA1 were greatly downregulated inCdkn1b-/-luminal cells. Further characterization of mammary glands of prepubertalCdkn1bknockout rats by fat pad transplantation illustrated p27 deletion in the mammary cancer susceptible ACI rat strain induced mammary epithelial cell differentiation through cell non-autonomous mechanisms.现有新兴研究数据表明，乳腺上皮干细胞及其祖细胞（尤其是腔上皮细胞谱系中的这类细胞）是乳腺癌的起源细胞，而影响乳腺癌发病风险的相关因素可改变此类细胞的数量与/或功能特性。我们提出假说：一部分p27阳性细胞属于激素响应性祖细胞，这类细胞因自身转化生长因子β（Transforming Growth Factor β, TGFβ）信号通路活性过高而处于静息状态。ACI近交系大鼠的雌激素诱导乳腺肿瘤模型，是与生理状态高度相关的乳腺癌啮齿类动物模型。本研究成功构建了Cdkn1b基因敲除的ACI大鼠模型，并通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）分离得到基底细胞（CD24+CD29high）与腔上皮细胞（CD24+CD29low）群体，随后对其进行全面的表型分析与RNA测序（RNA sequencing, RNAseq）谱学分析，以表征青春期前与成年队列中Cdkn1b野生型（Cdkn1b+/+）及敲除型（Cdkn1b-/-）雌性大鼠的乳腺组织特征。研究结果显示，p27敲除（p27KO）大鼠呈现出乳腺分化异常表型，且乳腺上皮祖细胞池的数量有所减少。值得注意的是，p27缺失对腔上皮祖细胞的基因表达影响最为显著：在Cdkn1b-/-腔上皮细胞中，乳蛋白基因与磷酸化信号转导与转录激活因子5（phosphorylated Signal Transducer and Activator of Transcription 5, pStat5）的表达水平显著上调，而孕激素受体（Progesterone Receptor, PR）与叉头框蛋白A1（Forkhead box A1, FoxA1）的表达则大幅下调。进一步通过脂肪垫移植术对青春期前Cdkn1b基因敲除大鼠的乳腺进行特征分析后发现：在乳腺癌易感的ACI大鼠品系中，p27缺失可通过细胞非自主性机制诱导乳腺上皮细胞分化。