Hippo Inactivation in the Mammary Epithelium Drives the Evolution of a Tumor-Associated Niche. Hippo Inactivation in the Mammary Epithelium Drives the Evolution of a Tumor-Associated Niche

Basal-like breast cancers exhibit distinct intratumor cell heterogeneity that contributes to disease pathology. In this study we use a genetic mouse model of basal-like breast cancer driven by epithelial-specific inactivation of the Hippo pathway-regulating Lats1 and Lats2 kinases to elucidate epithelial-stromal interactions in the basal-like tumor niche. We demonstrate that basal-like carcinoma initiation is accompanied by the accumulation of distinct cancer-associated fibroblasts, tumor-associated macrophage recruitment, and dramatic extracellular matrix remodeling, phenocopying the stromal diversity observed in human triple-negative breast tumors.Epithelial-stromal signaling dysregulation was observed, including increased TGF-β, PDGF, and CSF intercellular communication. Autonomous activation of the transcriptional effector TAZ was observed in Lats1/2-deleted cells along with non-autonomous activation within the evolving tumor niche. We further demonstrate that small molecule inhibition of the YAP/TAZ-associated TEAD family of transcription factors can block the development of the carcinomas and tumor-associated microenvironment. These observations demonstrate that carcinomas resulting from Hippo pathway dysregulation in the mammary epithelium are sufficient to drive cellular events that promote a basal-like tumor-associated niche and suggest that targeting dysregulated YAP/TAZ-TEAD activity provides an opportunity for therapeutic intervention of basal-like mammary tumors. Overall design: We utilized a genetic mouse model allowing conditional deletion of Lats1/2 in Krt8+cells to study the impact of epithelial-specific Hippo pathway inactivation on mammary epithelial and stromal cell populations. These mice included Lats1 and Lats2-floxed alleles, a Tamoxifen-inducible Cre recombinase under the control of the Krt8 promoter, and a loxP-stop-loxP(lsl)-EYFP lineage trace to mark Cre+ cells (Lats1/2ff;lsl-EYFP; Krt8CreERT2). After administration of Tamoxifen, mammary glands from these and control (Lats1/2ff;lsl-EYFP) mice were dissociated and live cells were subsequently sorted for single-cell RNA-sequencing. Two mice were used for each condition.

基底样乳腺癌具有独特的瘤内细胞异质性，该特性参与疾病的病理发生过程。本研究构建了由上皮特异性失活调控Hippo通路(Hippo pathway)的Lats1与Lats2激酶驱动的基底样乳腺癌基因工程小鼠模型，旨在阐明基底样肿瘤微环境中的上皮-基质相互作用机制。

研究发现，基底样癌的发生伴随特异性癌症相关成纤维细胞的聚集、肿瘤相关巨噬细胞的招募以及显著的细胞外基质重塑，该表型与人类三阴性乳腺癌中观察到的基质多样性相吻合。团队同时检测到上皮-基质信号通路失调，具体表现为转化生长因子-β(transforming growth factor-β, TGF-β)、血小板衍生生长因子(platelet-derived growth factor, PDGF)以及集落刺激因子(colony-stimulating factor, CSF)介导的细胞间通讯增强。在Lats1/2缺失的细胞中，转录效应因子TAZ的自主性激活被检出，同时在不断演化的肿瘤微环境中也存在非自主性激活现象。本研究进一步证实，靶向YAP/TAZ相关TEAD家族转录因子的小分子抑制剂可阻断癌灶的发生与肿瘤相关微环境的形成。

上述研究结果表明，乳腺上皮中Hippo通路失调所引发的癌灶，足以驱动一系列促基底样肿瘤相关微环境形成的细胞事件；同时提示，靶向失调的YAP/TAZ-TEAD活性可为基底样乳腺肿瘤的治疗提供全新干预策略。

整体实验设计：本研究利用可在Krt8+细胞中条件性敲除Lats1/2的基因工程小鼠模型，探究上皮特异性Hippo通路失活对乳腺上皮及基质细胞群的影响。该小鼠模型包含Lats1和Lats2 flox等位基因、受Krt8启动子调控的他莫昔芬诱导型Cre重组酶，以及用于标记Cre+细胞的loxP-stop-loxP(lsl)-EYFP谱系示踪系统（基因型为Lats1/2ff;lsl-EYFP; Krt8CreERT2）。向小鼠注射他莫昔芬后，分离对照组（Lats1/2ff;lsl-EYFP）与实验组小鼠的乳腺组织，制备单细胞悬液并对活细胞进行分选，随后开展单细胞RNA测序。每组实验使用2只小鼠。