Droplet-based proteomics reveals CD36 as a marker for progenitors in mammary basal epithelium.

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS (droplet-based one-pot preparation for proteomic samples), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, identifying CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate that DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.

Insulin receptor loss impairs mammary tumorigenesis in mice.

Breast cancer (BC) prognosis and outcome are adversely affected by obesity. Hyperinsulinemia, common in the obese state, is associated with higher risk of death and recurrence in BC. Up to 80% of BCs overexpress the insulin receptor (INSR), which correlates with worse prognosis. INSR's role in mammary tumorigenesis was tested by generating MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC mouse models, respectively, with coordinate mammary epithelium-restricted deletion of INSR. In both models, deletion of either one or both copies of INSR leads to a marked delay in tumor onset and burden. Longitudinal phenotypic characterization of mouse tumors and cells reveals that INSR deletion affects tumor initiation, not progression and metastasis. INSR upholds a bioenergetic phenotype in non-transformed mammary epithelial cells, independent of its kinase activity. Similarity of phenotypes elicited by deletion of one or both copies of INSR suggest a dose-dependent threshold for INSR impact on mammary tumorigenesis.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages.

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.

Mammary epithelial cells have lineage-rooted metabolic identities.

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.

Author Correction: Human somatic cell mutagenesis creates genetically tractable sarcomas.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mammary stem cells and progenitors: targeting the roots of breast cancer for prevention.

Breast cancer prevention is daunting, yet not an unsurmountable goal. Mammary stem and progenitors have been proposed as the cells-of-origin in breast cancer. Here, we present the concept of limiting these breast cancer precursors as a risk reduction approach in high-risk women. A wealth of information now exists for phenotypic and functional characterization of mammary stem and progenitor cells in mouse and human. Recent work has also revealed the hormonal regulation of stem/progenitor dynamics as well as intrinsic lineage distinctions between mammary epithelial populations. Leveraging these insights, molecular marker-guided chemoprevention is an achievable reality.

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology.

A Progesterone-CXCR4 Axis Controls Mammary Progenitor Cell Fate in the Adult Gland.

Progesterone drives mammary stem and progenitor cell dynamics through paracrine mechanisms that are currently not well understood. Here, we demonstrate that CXCR4, the receptor for stromal-derived factor 1 (SDF-1; CXC12), is a crucial instructor of hormone-induced mammary stem and progenitor cell function. Progesterone elicits specific changes in the transcriptome of basal and luminal mammary epithelial populations, where CXCL12 and CXCR4 represent a putative ligand-receptor pair. In situ, CXCL12 localizes to progesterone-receptor-positive luminal cells, whereas CXCR4 is induced in both basal and luminal compartments in a progesterone-dependent manner. Pharmacological inhibition of CXCR4 signaling abrogates progesterone-directed expansion of basal (CD24+CD49fhi) and luminal (CD24+CD49flo) subsets. This is accompanied by a marked reduction in CD49b+SCA-1- luminal progenitors, their functional capacity, and lobuloalveologenesis. These findings uncover CXCL12 and CXCR4 as novel paracrine effectors of hormone signaling in the adult mammary gland, and present a new avenue for potentially targeting progenitor cell growth and malignant transformation in breast cancer.

Human somatic cell mutagenesis creates genetically tractable sarcomas.

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis.

The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection.

Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.