IL-1ß promotes stemness and invasiveness of colon cancer cells through Zeb1 activation.

BACKGROUND: IL-1ß is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1ß may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1ß in colon cancer stem cell (CSC) development. METHODS: Using self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1ß on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116. RESULTS: We found that IL-1ß can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1ß-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1ß-induced spheres, indicating that there might be a close association between EMT and IL-1ß-induced CSC self-renewal. Indeed, IL-1ß treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1ß-induced EMT and stem cell formation. CONCLUSION: Our findings indicate that IL-1ß may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1ß and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Pro-inflammatory cytokine interleukin-1ß promotes the development of intestinal stem cells.

OBJECTIVE: We investigated the effect of IL-1ß on the development of intestinal epithelial stem cells. MATERIALS AND METHODS: Normal intestinal epithelial cell line IEC-18 cells were cultured in the presence or absence of 200 pM of IL-1ß in serum-free medium (SFM) for various time periods. The effects of IL-1ß on intestinal stem cell self-renewal and IEC-18 cell proliferation were evaluated by a colony formation assay, MTT assay, and a focus formation assay. The expression of stemness genes including Bmi-1, Lgr-5, c-myc, Nanog, and ß-catenin in IEC-18 cells were measured by quantitative PCR and western blot analysis. RESULTS: IEC-18 cells grew as a monolayer in SFM in the absence of IL-1ß. Cellular spheres were formed when IEC-18 cells were grown in SFM in the presence of IL-1ß. IL-1ß induced the development of large colonies in the soft-agar as well as the formation of foci when IEC-18 cells were cultured in type-I collagen-coated plates. The expression of Bmi-1, Lgr-5, c-myc, Nanog, and ß-catenin were significantly increased in IEC-18 cells treated with IL-1ß. CONCLUSION: Our studies provide direct evidence the IL-1ß may play an important role in the self-renewal of intestinal epithelial stem cells and the development of cancer stem cells.

Interleukin-1ß and transforming growth factor-ß cooperate to induce neurosphere formation and increase tumorigenicity of adherent LN-229 glioma cells.

INTRODUCTION: Glioma stem cells (GSCs) have the property of self-renewal and appear to be a driving force for the initiation and recurrence of gliomas. We recently found that the human tumorigenic LN-229 glioma cell line failed to form neurospheres in serum-free conditions and generated mostly small tumors in vivo, suggesting that either LN-229 GSCs are not active in these conditions or GSCs are absent in the LN-229 cell line. METHODS: Using self-renewal assay, soft-agar colony assay, cell proliferation assay, invasion assay, real time PCR analysis, ELISA and in vivo tumorigenic assay, we investigated the effects of interleukin (IL)-1ß and transforming growth factor (TGF)-ß on the development of GSCs from LN-229 cells. RESULTS: Here, we demonstrate that the combination of IL-1ß and TGF-ß can induce LN-229 cells to form neurospheres in serum-free medium. IL-1ß/TGF-ß-induced neurospheres display up-regulated expression of stemness factor genes (nestin, Bmi-1, Notch-2 and LIF), and increased invasiveness, drug resistance and tumor growth in vivo: hallmarks of GSCs. These results indicate that IL-1ß and TGF-ß cooperate to induce a GSC phenotype in the LN-229 cell line. Induction of nestin, LIF and Notch-2 by IL-1ß/TGF-ß can be reverted after cytokine withdrawal. Remarkably, however, up-regulated Bmi-1 levels remained unchanged after cytokine withdrawal; and the cytokine-withdrawn cells maintained strong clonogenicity, suggesting that Bmi-1 may play a crucial role in tumorigenesis. CONCLUSIONS: Our finding indicates that glioma cells without self-renewal capability in standard conditions could also contribute to glioma malignancy when cytokines, such as IL-1ß and TGF-ß, are present in the tumor environment. Targeting GSC-promoting cytokines that are highly expressed in glioblastomas may contribute to the development of more effective glioma therapies.

A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant.

In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid-plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an N-terminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean.

Blockade of thrombospondin-1-CD47 interactions prevents necrosis of full thickness skin grafts.

BACKGROUND: Skin graft survival and healing requires rapid restoration of blood flow to the avascular graft. Failure or delay in the process of graft vascularization is a significant source of morbidity and mortality. One of the primary regulators of blood flow and vessel growth is nitric oxide (NO). The secreted protein thrombospondin-1 (TSP1) limits NO-stimulated blood flow and growth and composite tissue survival to ischemia. We herein demonstrate a role for TSP1 in regulating full thickness skin graft (FTSG) survival. METHODS AND RESULTS: FTSG consistently fail in wild type C57BL/6 mice but survive in mice lacking TSP1 or its receptor CD47. Ablation of the TSP1 receptor CD36, however, did not improve FTSG survival. Remarkably, wild type FTSG survived on TSP1 null or CD47 null mice, indicating that TSP1 expression in the wound bed is the primary determinant of graft survival. FTSG survival in wild type mice could be moderately improved by increasing NO flux, but graft survival was increased significantly through antibody blocking of TSP1 binding to CD47 or antisense morpholino oligonucleotide suppression of CD47. CONCLUSIONS: TSP1 through CD47 limits skin graft survival. Blocking TSP1 binding or suppressing CD47 expression drastically increases graft survival. The therapeutic applications of this approach could include burn patients and the broader group of people requiring grafts or tissue flaps for closure and reconstruction of complex wounds of diverse etiologies.

Blocking thrombospondin-1/CD47 signaling alleviates deleterious effects of aging on tissue responses to ischemia.

OBJECTIVE: Decreased blood flow secondary to peripheral vascular disease underlies a significant number of chronic diseases that account for the majority of morbidity and mortality among the elderly. Blood vessel diameter and blood flow are limited by the matricellular protein thrombospondin-1 (TSP1) through its ability to block responses to the endogenous vasodilator nitric oxide (NO). In this study we investigate the role TSP1 plays in regulating blood flow in the presence of advanced age and atherosclerotic vascular disease. METHODS AND RESULTS: Mice lacking TSP1 or CD47 show minimal loss of their resistance to ischemic injury with age and increased preservation of tissue perfusion immediately after injury. Treatment of WT and apolipoprotein E-null mice using therapeutic agents that decrease CD47 or enhance NO levels reverses the deleterious effects of age- and diet-induced vasculopathy and results in significantly increased tissue survival in models of ischemia. CONCLUSIONS: With increasing age and diet-induced atherosclerotic vascular disease, TSP1 and its receptor CD47 become more limiting for blood flow and tissue survival after ischemic injury. Drugs that limit TSP1/CD47 regulation of blood flow could improve outcomes from surgical interventions in the elderly and ameliorate vascular complications attendant to aging.

Increasing survival of ischemic tissue by targeting CD47.

Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.

Mast cell-mediated inflammatory responses require the alpha 2 beta 1 integrin.

Although the alpha 2 beta 1 integrin is widely expressed and has been extensively studied, it has not been previously implicated in mast cell biology. We observed that alpha 2 integrin subunit-deficient mice exhibited markedly diminished neutrophil and interleukin-6 responses during Listeria monocytogenes- and zymosan-induced peritonitis. Since exudative neutrophils of wild-type mice expressed little alpha 2 beta 1 integrin, it seemed unlikely that this integrin mediated neutrophil migration directly. Here, we demonstrate constitutive alpha 2 beta 1 integrin expression on peritoneal mast cells. Although alpha 2-null mice contain normal numbers of peritoneal mast cells, these alpha 2-null cells do not support in vivo mast cell-dependent inflammatory responses. We conclude that alpha 2 beta 1 integrin provides a costimulatory function required for mast cell activation and cytokine production in response to infection.

The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo.

The alpha 2 beta 1 integrin serves as a receptor for collagens, laminin, and several other nonmatrix ligands. Many studies have suggested that the alpha 2 beta 1 integrin is a critical mediator of platelet adhesion to collagen within the vessel wall after vascular injury and that the interactions of the platelet alpha 2 beta 1 integrin with subendothelial collagen after vascular injury are required for proper hemostasis. We have used the alpha 2 beta 1 integrin-deficient mouse to evaluate the contributions of the alpha 2 beta 1 integrin in 2 in vivo models of thrombosis. Studies using a model of endothelial injury to the carotid artery reveal that the alpha 2 beta 1 integrin plays a critical role in vascular thrombosis at the blood-vessel wall interface under flow conditions. In contrast, the alpha 2 beta 1 integrin is not required for the formation of thrombi and pulmonary emboli following intravascular injection of collagen. Our results are the first to document a critical in vivo role for the alpha 2 beta 1 integrin in thrombus formation at the vessel wall under conditions of shear following vascular injury.