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Mammary morphogenesis is a complex process. Here the authors describe how stem cells build a three-dimensional self-organizing multi-lineage tissue by showing that positional signals from the extracellular matrix through the collagen receptor DDR1 lead stem cells to differentiate into multi-lineage committed multi-layered progeny.

The NavDx® blood test analyzes tumor tissue modified viral (TTMV)-HPV DNA to provide a reliable means of detecting and monitoring HPV-driven cancers. The test has been clinically validated in a large number of independent studies and has been integrated into clinical practice by over 1000 healthcare providers at over 400 medical sites in the US. This Clinical Laboratory Improvement Amendments (CLIA), high complexity laboratory developed test, has also been accredited by the College of American Pathologists (CAP) and the New York State Department of Health. Here, we report a detailed analytical validation of the NavDx assay, including sample stability, specificity as measured by limits of blank (LOBs), and sensitivity illustrated via limits of detection and quantitation (LODs and LOQs). LOBs were 0–0.32 copies/μL, LODs were 0–1.10 copies/μL, and LOQs were 2 = 1) across a broad range of analyte concentrations. These results demonstrate that NavDx accurately and reproducibly detects circulating TTMV-HPV DNA, which has been shown to aid in the diagnosis and surveillance of HPV-driven cancers.

Summary: The epithelial compartment of the mammary gland contains basal and luminal cell lineages, as well as stem and progenitor cells that reside upstream in the differentiation hierarchy. Stem and progenitor cell differentiation is regulated to maintain adult tissue and mediate expansion during pregnancy and lactation. The genetic factors that regulate the transition of cells between differentiation states remain incompletely understood. Here, we present a genome-scale method to discover genes driving cell-state specification. Applying this method, we identify a transcription factor, BCL11B, which drives stem cell self-renewal in vitro, by inhibiting differentiation into the basal lineage. To validate BCL11B's functional role, we use two-dimensional colony-forming and three-dimensional tissue differentiation assays to assess the lineage differentiation potential and functional abilities of primary human mammary cells. These findings show that BCL11B regulates mammary cell differentiation and demonstrate the utility of our proposed genome-scale strategy for identifying lineage regulators in mammalian tissues. : Miller et al. describe a strategy to identify candidate master regulators of cell lineage specification. This approach identified BCL11B as a key regulator of human mammary stem cell self-renewal in in vitro progenitor and differentiation assays. Using a combination of 2D and 3D primary cell culture techniques, they show that BCL11B drives stem cell self-renewal by inhibiting basal lineage commitment. Keywords: human mammary gland biology, mammary epithelial stem cells, BCL11B, epigenetics, single-cell genomics, epithelial differentiation, three-dimensional cell culture

PERK stress signaling is an important driver of cancer invasion and metastasis, but chemical inhibitors of PERK cause side effects. Here, the authors find that CREB3L1 is required for PERK's pro-metastatic function in breast cancer, and its inhibition suppresses cancer invasion and metastasis.

Summary: DNA damage activates checkpoints that limit the replicative potential of stem cells, including differentiation. These checkpoints protect against cancer development but also promote tissue aging. Because mice lacking Slug/Snai2 exhibit limited stem cell activity, including luminobasal differentiation, and are protected from mammary cancer, we reasoned that Slug might regulate DNA damage checkpoints in mammary epithelial cells. Here, we show that Slug facilitates efficient execution of RPA32-mediated DNA damage response (DDR) signaling. Slug deficiency leads to delayed phosphorylation of ataxia telangiectasia mutated and Rad3-related protein (ATR) and its effectors RPA32 and CHK1. This leads to impaired RAD51 recruitment to DNA damage sites and persistence of unresolved DNA damage. In vivo, Slug/Snai2 loss leads to increased DNA damage and premature aging of mammary epithelium. Collectively, our work demonstrates that the mammary stem cell regulator Slug controls DDR checkpoints by dually inhibiting differentiation and facilitating DDR repair, and its loss causes unresolved DNA damage and accelerated aging. : Gross et al. show that Slug/Snai2 controls stemness checkpoints in mammary epithelium by dually inhibiting differentiation and facilitating DNA damage response (DDR) repair. Slug was shown to regulate RPA32-mediated repair of DNA damage in mammary epithelial cells, and Slug loss led to unresolved DNA damage and accelerated aging within mammary epithelium in vivo. Keywords: stem cells, mammary gland, Slug, DNA damage, DNA damage checkpoints, aging

Phenotypic heterogeneity in cancers is associated with invasive progression and drug resistance. This heterogeneity arises in part from the ability of cancer cells to switch between phenotypic states, but the dynamics of this cellular plasticity remain poorly understood. Here we apply DNA barcodes to quantify and track phenotypic plasticity across hundreds of clones in a population of cancer cells exhibiting epithelial or mesenchymal differentiation phenotypes. We find that the epithelial-to-mesenchymal cell ratio is highly variable across the different clones in cancer cell populations, but remains stable for many generations within the progeny of any single clone—with a heritability of 0.89. To estimate the effects of combination therapies on phenotypically heterogeneous tumours, we generated quantitative simulations incorporating empirical data from our barcoding experiments. These analyses indicated that combination therapies which alternate between epithelial- and mesenchymal-specific treatments eventually select for clones with increased phenotypic plasticity. However, this selection could be minimized by increasing the frequency of alternation between treatments, identifying designs that may minimize selection for increased phenotypic plasticity. These findings establish new insights into phenotypic plasticity in cancer, and suggest design principles for optimizing the effectiveness of combination therapies for phenotypically heterogeneous tumours.

Perturbations in stem cell activity and differentiation can lead to developmental defects and cancer. We use an approach involving a quantitative model of cell-state transitions in vitro to gain insights into how SLUG/SNAI2, a key developmental transcription factor, modulates mammary epithelial stem cell activity and differentiation in vivo. In the absence of SLUG, stem cells fail to transition into basal progenitor cells, while existing basal progenitor cells undergo luminal differentiation; together, these changes result in abnormal mammary architecture and defects in tissue function. Furthermore, we show that in the absence of SLUG, mammary stem cell activity necessary for tissue regeneration and cancer initiation is lost. Mechanistically, SLUG regulates differentiation and cellular plasticity by recruiting the chromatin modifier lysine-specific demethylase 1 (LSD1) to promoters of lineage-specific genes to repress transcription. Together, these results demonstrate that SLUG plays a dual role in repressing luminal epithelial differentiation while unlocking stem cell transitions necessary for tumorigenesis.

Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. We identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors that are able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal and/or myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin-remodeling complexes.

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)-scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy.

Proteins and small molecules are the effectors of physiological action in biological systems and comprehensive methods are needed to analyze their modifications, expression levels and interactions. Systems-scale characterization of the proteome requires thousands of components in high-complexity samples to be isolated and simultaneously probed. While protein microarrays offer a promising approach to probe systems-scale changes in a high-throughput format, they are limited by the need to individually synthesize tens of thousands of proteins. We present an alternative technique, which we call diffusive gel (DiG) stamping, for patterning a microarray using a cellular lysate enabling rapid visualization of dynamic changes in the proteome as well protein interactions. A major advantage of the method described is that it requires no specialized equipment or in-vitro protein synthesis, making it widely accessible to researchers. The method can be integrated with mass spectrometry, allowing for the discovery of novel protein interactions. Here, we describe and characterize the sensitivity and physical features of DiG-Stamping. We demonstrate the biologic utility of DiG-Stamping by (1) identifying the binding partners of a target protein within a cellular lysate and by (2) visualizing the dynamics of proteins with multiple post-translational modifications.

Fibroblasts are important in orchestrating various functions necessary for maintaining normal tissue homeostasis as well as promoting malignant tumor growth. Significant evidence indicates that fibroblasts are functionally heterogeneous with respect to their ability to promote tumor growth, but markers that can be used to distinguish growth promoting from growth suppressing fibroblasts remain ill-defined. Here we show that human breast fibroblasts are functionally heterogeneous with respect to tumor-promoting activity regardless of whether they were isolated from normal or cancerous breast tissues. Rather than significant differences in fibroblast marker expression, we show that fibroblasts secreting abundant levels of prostaglandin (PGE2), when isolated from either reduction mammoplasty or carcinoma tissues, were both capable of enhancing tumor growth in vivo and could increase the number of cancer stem-like cells. PGE2 further enhanced the tumor promoting properties of fibroblasts by increasing secretion of IL-6, which was necessary, but not sufficient, for expansion of breast cancer stem-like cells. These findings identify a population of fibroblasts which both produce and respond to PGE2, and that are functionally distinct from other fibroblasts. Identifying markers of these cells could allow for the targeted ablation of tumor-promoting and inflammatory fibroblasts in human breast cancers.