Therapeutic antibodies that block the programmed death-ligand 1 (PD-L1)/programmed death-1 (PD-1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer (mUC)1–5. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here, we examined tumours from a large cohort of mUC patients treated with an anti–PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden (TMB). Lack of response was associated with a signature of transforming growth factor β (TGF-β) signalling in fibroblasts, particularly in patients with CD8+ T cells that were excluded from the tumour parenchyma and instead found in the fibroblast- and collagen-rich peritumoural stroma—a common phenotype among patients with mUC. Using a mouse model that recapitulates this immune excluded phenotype, we found that therapeutic administration of a TGF-β blocking antibody together with anti–PD-L1 reduced TGF-β signalling in stromal cells, facilitated T cell penetration into the centre of the tumour, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding outcome in this setting and suggests that TGF-β shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T cell infiltration.

Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.

Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+ Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.breast cancer | myeloid | CSF3 | prokineticin 2

Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24-deficient mice (Zmpste24 ؊/؊ ) have indicated a role for Zmpste24 in the processing of CAAX-type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24 ؊/؊ mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures-akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24 ؊/؊ mice. Zmpste24 ؊/؊ mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24 ؊/؊ mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.metalloproteinase ͉ knockout mice ͉ brittle bones ͉ CAAX motif T he mammalian zinc metalloproteinase Zmpste24 has attracted attention because it shares a high degree of sequence identity with Ste24p, a Saccharomyces cerevisiae enzyme required for the maturation of the farnesylated mating pheromone a-factor (1-3). Ste24p plays two distinct roles in a-factor biogenesis (2, 4). First, it acts as a CAAX endoprotease, clipping off the C-terminal three amino acids from the protein (i.e., the ϪAAX of the CAAX motif) (3). Release of the ϪAAX from a-factor can also be mediated by Rce1p, the CAAX endoprotease involved in Ras processing (3). The removal of the ϪAAX exposes a carboxyl-terminal farnesylcysteine, which is methylated by Ste14p (5). Second, Ste24p clips the amino-terminal extension of a-factor, rendering it susceptible to a final endoproteolytic cleavage by Axl1p or Ste23p (6). Aside from a-factor, no other substrates for Ste24p have been identified, but other substrates likely exist because genetic screens in yeast have demonstrated that STE24 mutations can reverse the topological orientation of membrane proteins (7) and can affect the viability of yeast with mutations in genes encoding actin cytoskeleton proteins (8).Zmpste24 faithfully carries out both of Ste24p's processing steps in a-factor biogenesis and thus is a bona fide Ste24p ortholog (2, 9). Although it would be tempting to speculate that Zmpste24 processes an ''a-factor-like'' peptide in mammals, no a-factor ortholog has yet been identified. We have previously speculated that prelamin A (a precursor to lamin A, a component of the nuclear lamina) might be a Zmpste24 substrate (2, 6) because prelamin A (like yeast a-factor) is a farnesylated CAAX protein that undergoes more than one proteolytic processing step (10). After the removal of the C-terminal ϪAAX, an additional 15 res...

No abstract

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.

No abstract

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.