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.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K-NRF2-regulated pathway.

Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1's tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase-AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Patient-reported outcomes after corrective osteotomy for a symptomatic malunion of the distal radius.

AIMS: The primary aim of this study was to describe patient satisfaction and health-related quality of life (HRQoL) following corrective osteotomy for a symptomatic malunion of the distal radius. METHODS: We retrospectively identified 122 adult patients from a single centre over an eight-year period who had undergone corrective osteotomy for a symptomatic malunion of the distal radius. The primary long-term outcome was the Patient-Rated Wrist Evaluation (PRWE) score. Secondary outcomes included the Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) score, the EQ-5D-5L score, complications, and the Net Promoter Score (NPS). Multivariate regression analysis was used to determine factors associated with the PRWE score. RESULTS: Long-term outcomes were available for 89 patients (72%). The mean age was 57 years (SD 15) and 68 were female (76%). The median time from injury to corrective osteotomy was nine months (interquartile range (IQR) 6 to 13). At a mean follow-up of six years (1 to 11) the median PRWE score was 22 (IQR 7 to 40), the median QuickDASH score was 11.4 (IQR 2.3 to 31.8), and the median EQ-5D-5L score was 0.84 (IQR 0.69 to 1). The NPS was 69. Multivariate regression analysis showed that the presence of an associated ulnar styloid fracture was the only significant independent factor associated with a worse PRWE score when adjusting for confounding variables (p = 0.004). CONCLUSION: We found that corrective osteotomy for malunion of the distal radius can result in good functional outcomes and high levels of patient satisfaction. However, the presence of an ulnar styloid fracture may adversely affect function. Level of Evidence: III (cohort study). Cite this article: Bone Joint J 2020;102-B(11):1542-1548.

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.

The Radiographic Union Score for HUmeral fractures (RUSHU) predicts humeral shaft nonunion.

AIMS: The primary aim of this study was to develop a reliable, effective radiological score to assess the healing of humeral shaft fractures, the Radiographic Union Score for HUmeral fractures (RUSHU). The secondary aim was to assess whether the six-week RUSHU was predictive of nonunion at six months after the injury. PATIENTS AND METHODS: Initially, 20 patients with radiographs six weeks following a humeral shaft fracture were selected at random from a trauma database and scored by three observers, based on the Radiographic Union Scale for Tibial fractures system. After refinement of the RUSHU criteria, a second group of 60 patients with radiographs six weeks after injury, 40 with fractures that united and 20 with fractures that developed nonunion, were scored by two blinded observers. RESULTS: After refinement, the interobserver intraclass correlation coefficient (ICC) was 0.79 (95% confidence interval (CI) 0.67 to 0.87), indicating substantial agreement. At six weeks after injury, patients whose fractures united had a significantly higher median score than those who developed nonunion (10 vs 7; p < 0.001). A receiver operating characteristic curve determined that a RUSHU cut-off of < 8 was predictive of nonunion (area under the curve = 0.84, 95% CI 0.74 to 0.94). The sensitivity was 75% and specificity 80% with a positive predictive value (PPV) of 65% and a negative predictive value of 86%. Patients with a RUSHU < 8 (n = 23) were more likely to develop nonunion than those with a RUSHU ≥ 8 (n = 37, odds ratio 12.0, 95% CI 3.4 to 42.9). Based on a PPV of 65%, if all patients with a RUSHU < 8 underwent fixation, the number of procedures needed to avoid one nonunion would be 1.5. CONCLUSION: The RUSHU is reliable and effective in identifying patients at risk of nonunion of a humeral shaft fracture at six weeks after injury. This tool requires external validation but could potentially reduce the morbidity associated with delayed treatment of an established nonunion. Cite this article: Bone Joint J 2019;101-B:1300-1306.

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.

The identification of topics for research that are important to people with ulcerative colitis.

OBJECTIVES: To identify topics for research that are important to people with ulcerative colitis, and to provide a framework by which their research priorities can be analysed. METHODS: This is a qualitative study using focus groups and interviews. Forty people with ulcerative colitis participated. Topics for research of importance to participants were identified and analysed using the Framework method. RESULTS: Topics were grouped into nine main categories: finding the cause of colitis, cure of colitis, prevention of colitis, living with colitis, treatment (conventional, complementary and surgical) and its complications, control of particular symptoms, information provision, communicating with health professionals and methods of service delivery. An initial framework to classify the research priorities of people with colitis is provided. CONCLUSIONS: This study suggests the potential to utilize patients' views to generate research topics that are rarely researched and to involve them in setting the research agenda. People with ulcerative colitis are able to identify many different areas of research that they feel are important to them. This may help to ensure that research is relevant to patients' needs.

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.

A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis.

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.

Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression.

Controversy over the role of antioxidants in cancer has persisted for decades. Here, we demonstrate that synthesis of the antioxidant glutathione (GSH), driven by GCLM, is required for cancer initiation. Genetic loss of Gclm prevents a tumor's ability to drive malignant transformation. Intriguingly, these findings can be replicated using an inhibitor of GSH synthesis, but only if delivered prior to cancer onset, suggesting that at later stages of tumor progression GSH becomes dispensable potentially due to compensation from alternative antioxidant pathways. Remarkably, combined inhibition of GSH and thioredoxin antioxidant pathways leads to a synergistic cancer cell death in vitro and in vivo, demonstrating the importance of these two antioxidants to tumor progression and as potential targets for therapeutic intervention.

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.

BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival.

Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.

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.