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.

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.

Abnormal B-cell development in TIMP-deficient bone marrow.

Bone marrow (BM) is the primary site of hematopoiesis and is responsible for a lifelong supply of all blood cell lineages. The process of hematopoiesis follows key intrinsic programs that also integrate instructive signals from the BM niche. First identified as an erythropoietin-potentiating factor, the tissue inhibitor of metalloproteinase (TIMP) protein family has expanded to 4 members and has widely come to be viewed as a classical regulator of tissue homeostasis. By virtue of metalloprotease inhibition, TIMPs not only regulate extracellular matrix turnover but also control growth factor bioavailability. The 4 mammalian TIMPs possess overlapping enzyme-inhibition profiles and have never been studied for their cumulative role in hematopoiesis. Here, we show that TIMPs are critical for postnatal B lymphopoiesis in the BM. TIMP-deficient mice have defective B-cell development arising at the pro-B-cell stage. Expression analysis of TIMPless hematopoietic cell subsets pointed to an altered B-cell program in the Lineage-Sca-1+c-Kit+ (LSK) cell fraction. Serial and competitive BM transplants identified a defect in TIMP-deficient hematopoietic stem and progenitor cells for B lymphopoiesis. In parallel, reverse BM transplants uncovered the extrinsic role of stromal TIMPs in pro- and pre-B-cell development. TIMP deficiency disrupted CXCL12 localization to LepR+ cells, and increased soluble CXCL12 within the BM niche. It also compromised the number and morphology of LepR+ cells. These data provide new evidence that TIMPs control the cellular and biochemical makeup of the BM niche and influence the LSK transcriptional program required for optimal B lymphopoiesis.

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.

Metalloproteases: On the Watch in the Hematopoietic Niche.

Hematopoietic stem cells (HSCs) self-renew or differentiate into blood cell lineages following extrinsic cues propagated in specialized niches. Support cells and soluble factors in the niche respond to stress and enable progenitor activity. Metalloproteases (MMPs, ADAMs, ADAMTSs) and their inhibitors (TIMPs) control certain physical and biochemical features of the niche by altering protease-dependent bioavailability of local niche factors (e.g., CXCL12, SCF, TGFß, VEGF), matrix turnover, and cellular interactions. With over 40 examples of diverse metalloprotease substrates known to trigger fate-changing decisions, the spatially confined activity of this multi-member protease family is ideally positioned to constitute a higher order control over hematopoiesis. Comprehension of regulated proteolysis in the bone marrow may fuel innovative strategies to harness HSC fate and function.

Metalloprotease inhibitor TIMP proteins control FGF-2 bioavailability and regulate skeletal growth.

Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia. We identify TIMP/FGF-2/IHH as a novel nexus underlying bone lengthening where TIMPs negatively regulate the release of FGF-2 from chondrocytes to allow IHH expression. Using a knock-in approach that combines MMP-resistant or ADAMTS-resistant aggrecans with TIMP deficiency, we uncouple growth plate activity in axial and appendicular bones. Thus, natural metalloprotease inhibitors are crucial regulators of chondrocyte maturation program, growth plate integrity, and skeletal proportionality. Furthermore, individual and combinatorial TIMP-deficient mice demonstrate the redundancy of metalloprotease inhibitor function in embryonic and postnatal development.

Identifying the murine mammary cell target of metformin exposure.

The heterogeneity of breast cancer makes current therapies challenging. Metformin, the anti-diabetic drug, has shown promising anti-cancer activities in epidemiological studies and breast cancer models. Yet, how metformin alters the normal adult breast tissue remains elusive. We demonstrate metformin intake at a clinically relevant dose impacts the hormone receptor positive (HR+) luminal cells in the normal murine mammary gland. Metformin decreases total cell number, progenitor capacity and specifically reduces DNA damage in normal HR+ luminal cells, decreases oxygen consumption rate and increases cell cycle length of luminal cells. HR+ luminal cells demonstrate the lowest levels of mitochondrial respiration and capacity to handle oxidative stress compared to the other fractions, suggesting their intrinsic susceptibility to long-term metformin exposure. Uncovering HR+ luminal cells in the normal mammary gland as the major cell target of metformin exposure could identify patients that would most benefit from repurposing this anti-diabetic drug for cancer prevention/therapy purposes.

PDGFRα+ stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland.

The mammary gland experiences substantial remodeling and regeneration during development and reproductive life, facilitated by stem cells and progenitors that act in concert with physiological stimuli. While studies have focused on deciphering regenerative cells within the parenchymal epithelium, cell lineages in the stroma that may directly contribute to epithelial biology is unknown. Here we identify, in mouse, the transition of a PDGFRα+ mesenchymal cell population into mammary epithelial progenitors. In addition to being adipocyte progenitors, PDGFRα+ cells make a de novo contribution to luminal and basal epithelia during mammary morphogenesis. In the adult, this mesenchymal lineage primarily generates luminal progenitors within lobuloalveoli during sex hormone exposure or pregnancy. We identify cell migration as a key molecular event that is activated in mesenchymal progenitors in response to epithelium-derived chemoattractant. These findings demonstrate a stromal reservoir of epithelial progenitors and provide insight into cell origins and plasticity during mammary tissue growth.

Cross-species genomics identifies DLG2 as a tumor suppressor in osteosarcoma.

Leveraging the conserved cancer genomes across mammals has the potential to transform driver gene discovery in orphan cancers. Here, we combine cross-species genomics with validation across human-dog-mouse systems to uncover a new bone tumor suppressor gene. Comparative genomics of spontaneous human and dog osteosarcomas (OS) expose Disks Large Homolog 2 (DLG2) as a tumor suppressor candidate. DLG2 copy number loss occurs in 42% of human and 56% of canine OS. Functional validation through pertinent human and canine OS DLG2-deficient cell lines identifies a regulatory role of DLG2 in cell division, migration and tumorigenesis. Moreover, osteoblast-specific deletion of Dlg2 in a clinically relevant genetically engineered mouse model leads to acceleration of OS development, establishing DLG2 as a critical determinant of OS. This widely applicable cross-species approach serves as a platform to expedite the search of cancer drivers in rare human malignancies, offering new targets for cancer therapy.

Proliferative heterogeneity of murine epithelial cells in the adult mammary gland.

Breast cancer is the most common cancer in females. The number of years menstruating and length of an individual menstrual cycle have been implicated in increased breast cancer risk. At present, the proliferative changes within an individual reproductive cycle or variations in the estrous cycle in the normal mammary gland are poorly understood. Here we use Fucci2 reporter mice to demonstrate actively proliferating mammary epithelial cells have shorter G1 lengths, whereas more differentiated/non-proliferating cells have extended G1 lengths. We find that cells enter into the cell cycle mainly during diestrus, yet the expansion is erratic and does not take place every reproductive cycle. Single cell expression analyses feature expected proliferation markers (Birc5, Top2a), while HR+ luminal cells exhibit fluctuations of key differentiation genes (ER, Gata3) during the cell cycle. We highlight the proliferative heterogeneity occurring within the normal mammary gland during a single-estrous cycle, indicating that the mammary gland undergoes continual dynamic proliferative changes.

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.

TIMPs: versatile extracellular regulators in cancer.

A compelling long-term goal of cancer biology is to understand the crucial players during tumorigenesis in order to develop new interventions. Here, we review how the four non-redundant tissue inhibitors of metalloproteinases (TIMPs) regulate the pericellular proteolysis of a vast range of matrix and cell surface proteins, generating simultaneous effects on tumour architecture and cell signalling. Experimental studies demonstrate the contribution of TIMPs to the majority of cancer hallmarks, and human cancers invariably show TIMP deregulation in the tumour or stroma. Of the four TIMPs, TIMP1 overexpression or TIMP3 silencing is consistently associated with cancer progression or poor patient prognosis. Future efforts will align mouse model systems with changes in TIMPs in patients, will delineate protease-independent TIMP function, will pinpoint therapeutic targets within the TIMP-metalloproteinase-substrate network and will use TIMPs in liquid biopsy samples as biomarkers for cancer prognosis.

RANKL blockade prevents and treats aggressive osteosarcomas.

Osteosarcoma (OS) is the most common primary bone cancer, which occurs primarily in children and adolescents, severely affecting survivors' quality of life. Despite its chemosensitivity and treatment advances, long-term survival rates for OS patients have stagnated over the last 20 years. Thus, it is necessary to develop new molecularly targeted therapies for this metastatic bone cancer. Mutations in TP53 and RB are linked to OS predisposition and to the evolution of spontaneous OS. We established receptor activator of nuclear factor κB ligand (RANKL) as a therapeutic target for suppression and prevention of OS. Combined conditional osteoblast-specific deletions of Rb, p53, and the protein kinase A (PKA) regulatory subunit Prkar1α genes in genetically engineered mouse models (GEMMs) generate aggressive osteosarcomas, characterized by PKA, RANKL, and osteoclast hyperactivity. Whole-body Rankl deletion completely abrogates tumorigenesis. Although osteoblastic Rank deletion has little effect, osteoclastic Rank deletion delays tumorigenesis and prolongs life span. The latter is associated with inactivation of osteoclastogenesis and up-regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Further, we use these GEMMs as preclinical platforms to show that RANKL blockade with RANK-Fc arrests tumor progression and improves survival and also inhibits lung metastasis. Moreover, preemptive administration of RANK-Fc completely prevents tumorigenesis in mice highly predisposed to this aggressive cancer. Denosumab, a fully human monoclonal antibody against RANKL, is currently used to treat patients with osteoporosis or bone metastases. Our studies provide a strong rationale to consider RANKL blockade for the treatment and prevention of aggressive RANKL-overexpressing OS in humans.

Timp3 loss accelerates tumour invasion and increases prostate inflammation in a mouse model of prostate cancer.

BACKGROUND: Altered expression and activity of proteases is implicated in inflammation and cancer progression. An important negative regulator of protease activity is TIMP3 (tissue inhibitor of metalloproteinase 3). TIMP3 expression is lacking in many cancers including advanced prostate cancer, and this may facilitate invasion and metastasis by allowing unrestrained protease activity. METHODS: To investigate the role of TIMP3 in prostate cancer progression, we crossed TIMP3-deficient mice (Timp3(-/-)) to mice with prostate-specific deletion of the tumor suppressor Pten (Pten(-/-)), a well-established mouse model of prostate cancer. Tumor growth and progression were compared between Pten(-/-), Timp3(-/-) and control (Pten(-/-), Timp3(+/+)) mice at 16 weeks of age by histopathology and markers of proliferation, vascularity, and tumor invasion. Metalloproteinase activity within the tumors was assessed by gelatin zymography. Inflammatory infiltrates were assessed by immunohistochemistry for macrophages and lymphocytes whereas expression of cytokines and other inflammatory mediators was assessed by quantitative real time PCR and multiplex ELISA. RESULTS: Increased tumor growth, proliferation index, increased microvascular density, and invasion was observed in Pten(-/-), Timp3(-/-) prostate tumors compared to Pten(-/-), Timp3(+/+) tumors. Tumor cell invasion in Pten(-/-), Timp3(-/-) mice was associated with increased expression of matrix metalloprotease (MMP)-9 and activation of MMP-2. There was markedly increased inflammatory cell infiltration into the TIMP3-deficient prostate tumors along with increased expression of monocyte chemoattractant protein-1, cyclooxygenase-2, TNF-α, and interleukin-1ß; all of which are implicated in inflammation and cancer. CONCLUSIONS: This study provides important insights into the role of altered protease activity in promoting prostate cancer invasion and implicates prostate inflammation as an important promoting factor in prostate cancer progression.

RANK Signaling Amplifies WNT-Responsive Mammary Progenitors through R-SPONDIN1.

Systemic and local signals must be integrated by mammary stem and progenitor cells to regulate their cyclic growth and turnover in the adult gland. Here, we show RANK-positive luminal progenitors exhibiting WNT pathway activation are selectively expanded in the human breast during the progesterone-high menstrual phase. To investigate underlying mechanisms, we examined mouse models and found that loss of RANK prevents the proliferation of hormone receptor-negative luminal mammary progenitors and basal cells, an accompanying loss of WNT activation, and, hence, a suppression of lobuloalveologenesis. We also show that R-spondin1 is depleted in RANK-null progenitors, and that its exogenous administration rescues key aspects of RANK deficiency by reinstating a WNT response and mammary cell expansion. Our findings point to a novel role of RANK in dictating WNT responsiveness to mediate hormone-induced changes in the growth dynamics of adult mammary cells.

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

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.

Loss of the Timp gene family is sufficient for the acquisition of the CAF-like cell state.

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.

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.