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.

CDH4 is a novel determinant of osteosarcoma tumorigenesis and metastasis.

The era of cancer genomics now provides an opportunity to discover novel determinants of osteosarcoma (OS), the most common primary bone cancer in children and adolescents known for its poor prognosis due to lung metastasis. Here, we identify CDH4 amplification in 43.6% of human osteosarcoma using array CGH and demonstrate its critical role in osteosarcoma development and progression. Gain or loss-of-function of CDH4, which encodes R-cadherin, causally impacts multiple features of human OS cells including cell migration and invasion, osteogenic differentiation, and stemness. CDH4 overexpression activates c-Jun via the JNK pathway, while CDH4 knockdown suppresses both tumor xenograft growth and lung colonization. In OS patient specimens, high CDH4 expression associates with lung metastases and poor prognosis. Collectively, our bioinformatics, functional, molecular, and clinical analyses uncover an oncogenic function of CDH4 in osteosarcoma and its relationship with patient outcome.

Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies.

Bioassays of planarian neoplasia highlight the potential of these organisms as useful standards to assess whether environmental toxins such as cadmium promote tumorigenesis. These studies complement other investigations into the exceptional healing and regeneration of planarians - processes that are driven by a population of active stem cells, or neoblasts, which are likely transformed during planarian tumor growth. Our goal was to determine if planarian tumorigenesis assays are amenable to mechanistic studies of cadmium carcinogenesis. To that end we demonstrate, by examining both counts of cell populations by size, and instances of mitosis, that the activity of the stem cell population can be monitored. We also provide evidence that specific biomodulators can affect the potential of planarian neoplastic growth, in that an inhibitor of metalloproteinases effectively blocked the development of the lesions. From these results, we infer that neoblast activity does respond to cadmium-induced tumor growth, and that metalloproteinases are required for the progression of cancer in the planarian.

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.

Homotypic RANK signaling differentially regulates proliferation, motility and cell survival in osteosarcoma and mammary epithelial cells.

RANKL (receptor activator of NF-κB ligand) is a crucial cytokine for regulating diverse biological systems such as innate immunity, bone homeostasis and mammary gland differentiation, operating through activation of its cognate receptor RANK. In these normal physiological processes, RANKL signals through paracrine and/or heterotypic mechanisms where its expression and function is tightly controlled. Numerous pathologies involve RANKL deregulation, such as bone loss, inflammatory diseases and cancer, and aberrant RANK expression has been reported in bone cancer. Here, we investigated the significance of RANK in tumor cells with a particular emphasis on homotypic signaling. We selected RANK-positive mouse osteosarcoma and RANK-negative preosteoblastic MC3T3-E1 cells and subjected them to loss- and gain-of-RANK function analyses. By examining a spectrum of tumorigenic properties, we demonstrate that RANK homotypic signaling has a negligible effect on cell proliferation, but promotes cell motility and anchorage-independent growth of osteosarcoma cells and preosteoblasts. By contrast, establishment of RANK signaling in non-tumorigenic mammary epithelial NMuMG cells promotes their proliferation and anchorage-independent growth, but not motility. Furthermore, RANK activation initiates multiple signaling pathways beyond its canonical target, NF-κB. Among these, biochemical inhibition reveals that Erk1/2 is dominant and crucial for the promotion of anchorage-independent survival and invasion of osteoblastic cells, as well as the proliferation of mammary epithelial cells. Thus, RANK signaling functionally contributes to key tumorigenic properties through a cell-autonomous homotypic mechanism. These data also identify the likely inherent differences between epithelial and mesenchymal cell responsiveness to RANK activation.

Notch activation by the metalloproteinase ADAM17 regulates myeloproliferation and atopic barrier immunity by suppressing epithelial cytokine synthesis.

Epithelial cells of mucosal tissues provide a barrier against environmental stress, and keratinocytes are key decision makers for immune cell function in the skin. Currently, epithelial signaling networks that instruct barrier immunity remain uncharacterized. Here we have shown that keratinocyte-specific deletion of a disintegrin and metalloproteinase 17 (Adam17) triggers T helper 2 and/or T helper 17 (Th2 and/or Th17) cell-driven atopic dermatitis and myeloproliferative disease. In vivo and in vitro deficiency of ADAM17 dampened Notch signaling, increasing production of the Th2 cell-polarizing cytokine TSLP and myeloid growth factor G-CSF. Ligand-independent Notch activation was identified as a regulator of AP-1 transcriptional activity, with Notch antagonizing c-Fos recruitment to the promoters of Tslp and Csf3 (G-CSF). Further, skin inflammation was rescued and myeloproliferation ameliorated by delivery of active Notch to Adam17(-)(/-) epidermis. Our findings uncover an essential role of ADAM17 in the adult epidermis, demonstrating a gatekeeper function of the ADAM17-Notch-c-Fos triad in barrier immunity.

Prkar1a is an osteosarcoma tumor suppressor that defines a molecular subclass in mice.

Some cancers have been stratified into subclasses based on their unique involvement of specific signaling pathways. The mapping of human cancer genomes is revealing a vast number of somatic alterations; however, the identification of clinically relevant molecular tumor subclasses and their respective driver genes presents challenges. This information is key to developing more targeted and personalized cancer therapies. Here, we generate a new mouse model of genomically unstable osteosarcoma (OSA) that phenocopies the human disease. Integrative oncogenomics pinpointed cAMP-dependent protein kinase type I, alpha regulatory subunit (Prkar1a) gene deletions at 11qE1 as a recurrent genetic trait for a molecularly distinct subclass of mouse OSA featuring RANKL overexpression. Using mouse genetics, we established that Prkar1a is a bone tumor suppressor gene capable of directing subclass development and driving RANKL overexpression during OSA tumorigenesis. Finally, we uncovered evidence for a PRKAR1A-low subset of human OSA with distinct clinical behavior. Thus, tumor subclasses develop in mice and can potentially provide information toward the molecular stratification of human cancers.

SIRT1 acts as a nutrient-sensitive growth suppressor and its loss is associated with increased AMPK and telomerase activity.

SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice also showed increased growth capacity and decreased dependency on growth factors. Consistent with this, SIRT1 inhibition was associated with increased telomerase activity in human cells. We also observed a significant increase in AMPK levels up on SIRT1 inhibition under glucose limiting conditions. Although SIRT1 suppression cooperated with hTERT to promote cell growth, either overexpression or suppression of SIRT1 alone had no effect on life span of human diploid fibroblasts. Our findings challenge certain models and connect nutrient sensing enzymes to the immortalization process. Furthermore, they show that in certain cell lineages, SIRT1 can act as a growth suppressor gene.