Disruption of basal cell identity by Rank signaling drives mammary tumorigenesis

Rank signaling regulates mammary gland development and epithelial cell differentiation. Rank receptor is expressed by mammary basal and luminal populations, but, unlike luminal Rank, the contribution of basal Rank signaling to MG homeostasis remains poorly studied. We have combined timely regulated basal specific Rank expression with lineage tracing models and unveiled that basal Rank signaling controls basal cell identity in postnatal mammary glands. Ectopic basal Rank disrupts basal but also luminal cell identity, resulting in aberrant luminal-like differentiation of basal cells and impaired lactogenesis. Mechanistically, overactivation of basal Rank signaling leads to basal cell lineage infidelity, illustrated by the appearance of premalignant lesions composed by a basal-derived hybrid population with alveolar features which ultimately generates basal and luminal breast adenocarcinomas. Proteomic, transcriptomic and chromatin analyses support that the loss of tumor suppressive epigenetic regulators driven by basal Rank contributes to epithelial cell dedifferentiation and tumorigenesis. The basal Rank signature generated associates to poor prognosis particularly in human adenocarcinomas of the luminal subtype stressing the clinical relevance of our findings. Interestingly, our results reinforce the idea that luminal breast tumors might originate from basal cells that have suffered a luminal-like aberrant dedifferentiation triggered by Rank signaling Overall design: RNA sequencing (ATAC-seq) of mammary gland luminal and basal epithelial cells from K14-Ctrl and K14-Rank (Rank overexpression in basal cells) and K14-RankD/D (Rank depletion in basal cells) female mice We induce Rank overexpression by treating 4 week old K14-Rank, K14-RankD/D and K14-Ctrl females with 1 mg/ml doxycycline + 20mg/ml sucrose, ad libitum in the drinking water for 4 weeks Then, we collect MGs from adult females and FACS isolate luminal and basal cells for RNA-seq.

Rank信号通路(Rank signaling)调控乳腺发育与上皮细胞分化。Rank受体(Rank receptor)表达于乳腺基底细胞与腔面细胞群，但与腔面Rank通路不同，目前针对基底Rank信号通路在乳腺(mammary gland, MG)稳态维持中的作用研究仍较为匮乏。
我们结合时序调控的基底细胞特异性Rank表达系统与谱系示踪模型(lineage tracing models)，揭示了产后乳腺中基底Rank信号通路调控基底细胞身份的功能。异位表达的基底Rank不仅破坏基底细胞身份，同时也干扰腔面细胞身份，导致基底细胞异常向腔面样细胞分化，并损害泌乳功能。
从机制上看，基底Rank信号通路过度激活会引发基底细胞谱系保真度丧失，表现为出现具有肺泡特征的基底来源混合细胞群构成的癌前病变，最终诱发基底细胞型与腔面细胞型乳腺腺癌。
蛋白质组学、转录组学与染色质分析结果证实，由基底Rank通路驱动的抑癌表观调控因子丢失，参与了上皮细胞去分化与肿瘤发生过程。
我们构建的基底Rank信号特征与患者不良预后显著相关，尤其在腔面亚型人类腺癌中更为突出，凸显了本研究发现的临床价值。
有趣的是，我们的研究结果进一步支持了这一观点：腔面型乳腺肿瘤可能起源于受Rank信号通路触发发生腔面样异常去分化的基底细胞。
实验设计方案：对K14-Ctrl、K14-Rank(基底细胞过表达Rank)与K14-RankD/D(基底细胞Rank敲降)雌性小鼠的乳腺腔面与基底上皮细胞进行RNA测序(ATAC-seq)。我们通过给4周龄的K14-Rank、K14-RankD/D与K14-Ctrl雌性小鼠饮用含1mg/ml多西环素(doxycycline)+20mg/ml蔗糖(sucrose)的自由饮水，诱导Rank过表达，持续4周。随后收集成年雌性小鼠的乳腺组织，通过荧光激活细胞分选术(Fluorescence-Activated Cell Sorting, FACS)分离腔面与基底细胞，用于RNA测序。