The receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in breast and other cancers. Here we tested the role of targeting RAGE by multiple approaches in the tumor and tumor microenvironment, to inhibit the metastatic process. We first tested how RAGE impacts tumor cell-intrinsic mechanisms using either RAGE overexpression or knockdown with short hairpin RNAs (shRNAs). RAGE ectopic overexpression in breast cancer cells increased MEK-EMT (MEK-epithelial-to-mesenchymal transition) signaling, transwell invasion and soft agar colony formation, and in vivo promoted lung metastasis independent of tumor growth. RAGE knockdown with multiple independent shRNAs in breast cancer cells led to decreased transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, targeting RAGE shRNA knockdown in human and mouse breast cancer cells, decreased orthotopic tumor growth, reduced tumor angiogenesis and recruitment of inflammatory cells, and markedly decreased metastasis to the lung and liver in multiple xenograft and syngeneic mouse models. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected breast cancer cells in wild-type and RAGE-knockout C57BL6 mice. RAGE-knockout mice displayed striking impairment of tumor cell growth compared with wild-type mice, along with decreased mitogen-activated protein kinase signaling, tumor angiogenesis and inflammatory cell recruitment. To test the combined inhibition of RAGE in both tumor cell-intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of xenografted tumors with FPS-ZM1 (1 mg/kg, two times per week). Compared with vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment and, most importantly, prevented metastasis to the lung and liver. These data demonstrate that RAGE drives tumor progression and metastasis through distinct tumor cell-intrinsic and -extrinsic mechanisms, and may represent a novel and therapeutically viable approach for treating metastatic cancers.

BackgroundGlioblastoma is the most common primary brain cancer in adults with an incidence of 3.4 per 100,000, making up about 15% of all brain tumors. Inconsistent results have been published in regard differences in survival between white and black glioblastoma patients. The objective of this to study the association between race and in Glioblastoma patients in the USA during 2010–2014.Methods and findingsThe National Cancer Institute’s Surveillance Epidemiology and End Results (SEER) database were used to evaluate race/ethnicity (White non-Hispanic, Black non-Hispanic, Asian/Pacific Islanders non-Hispanic (API)) and Hispanic) adults patients with first-time diagnosis of glioblastoma (International Classification of Diseases for Oncology, 3rd Edition [ICD-O-3], codes C711-C714, and histology type 9440/3) from 2010–2014. The primary outcome was 3-year overall survival which was defined as months from diagnosis to death due to any cause and cancer, Kaplan-Meier (KM) and log-rank test were used to compare overall survival times across race groups. Cox proportional hazard models were used to determine the independent effect of race on 3-year survival. Age, gender, health insurance coverage, primary site, tumor size, extent of surgery and year of diagnosis were included in the adjusted model. The 3-year overall survival for API-non Hispanic (NH) patients decreased by 25% compared with White NH glioblastoma patients (hazard ratio (HR) 0.75; 95% confidence interval (CI) 0.62–0.90)) after adjusting for age, gender, health insurance, primary site, tumor size, and extent of the surgery. Black NH (HR 0.95; 95% CI 0.80–1.13) and Hispanic (HR 1.01, 95% CI 0.84–1.21) exhibited similar mortality risks compared with White NH patients.ConclusionCompared with White NH, API NH with glioblastoma have a better survival. The findings from this study can help increase the accuracy of the prognostic outlook for white, black and API patients with GBM.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that can undergo proteolysis at the cell surface to release a soluble ectodomain. Here we observed that ectodomain shedding of RAGE is critical for its role in regulating signaling and cellular function. Ectodomain shedding of both human and mouse RAGE was dependent on ADAM10 activity and induced with chemical activators of shedding (ionomycin, phorbol 12-myristate 13-acetate, and 4-aminophenylmercuric acetate) and endogenous stimuli (serum and RAGE ligands). Ectopic expression of the splice variant of RAGE (RAGE splice variant 4), which is resistant to ectodomain shedding, inhibited RAGE ligand dependent cell signaling, actin cytoskeleton reorganization, cell spreading, and cell migration. We found that blockade of RAGE ligand signaling with soluble RAGE or inhibitors of MAPK or PI3K blocked RAGE-dependent cell migration but did not affect RAGE splice variant 4 cell migration. We finally demonstrated that RAGE function is dependent on secretase activity as ADAM10 and ␥-secretase inhibitors blocked RAGE ligand-mediated cell migration. Together, our data suggest that proteolysis of RAGE is critical to mediate signaling and cell function and may therefore emerge as a novel therapeutic target for RAGE-dependent disease states.Receptor for advanced glycation end products (RAGE) 3 is a transmembrane, multiligand receptor expressed by most cells but is characterized by its up-regulation in a range of inflammatory disease states including diabetes, various cancers, and cardiovascular disease (1). RAGE activation through ligand binding transduces intracellular signaling and in turn induces cell migration, invasion, and adhesion (1). Work from multiple groups has demonstrated using rodent models that blocking RAGE signaling impairs the development of numerous pathologic states and therefore highlights RAGE as an attractive therapeutic target (2-5). The most widely used means for blocking RAGE signaling is with soluble RAGE (sRAGE), the recombinantly produced extracellular domain of RAGE. Soluble RAGE acts as a decoy receptor and therefore neutralizes RAGE ligands (2, 6). Endogenous sRAGE isoforms, which have been identified in human and mouse sera, are generated through two distinct biological mechanisms: the alternative splicing of the transmembrane region of RAGE leading to a secreted isoform (esRAGE/RAGEv1) and the cleavage of extracellular domain (ectodomain or ECD) at the cell surface by ectodomain shedding (1, 7-10). Ectodomain shedding is a tightly regulated process and is mediated by various metalloproteinases (11, 12). Shedding is essential for normal physiological function but can be deregulated in various pathological disorders where altered levels of metalloproteinases are found (11, 12). In fact, many transmembrane proteins are known to undergo ectodomain shedding including cell adhesion molecules, ligands for growth factor receptors, immunoglobulins, and various enzymes (10 -13). Ectodomain shedding of cell sur...

PurposeMacrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.Materials and MethodsMIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.ResultsUROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.ConclusionsUrothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.

Background. An intraorbital injury with a blunt penetrating intraorbital foreign body (IOFB) is an unusual cause of penetrating trauma. This type of trauma is considered a surgical emergency given the risk to vision in addition to potential intracranial injuries such as vascular injury, dural laceration, and neurologic injury. A thorough history and physical exam, along with careful radiographic and multidiscipline intervention, is crucial in providing the patient the most appropriate care. Case Presentation. A 66-year-old male presented to the emergency room (ER) after falling down the stairs and suffering an orbitocranial penetrating injury. He underwent urgent fluoroscopy-guided foreign body removal with a multidisciplinary team after a workup revealed no significant ocular or intracranial injuries. The foreign body was removed with an anterior approach without any complications. Conclusion. In this study, we demonstrated that IOFB in proximity to orbitocranial structures requires a careful multidisciplinary team approach. An interventional radiology- (IR-) guided approach in extracting the foreign body is essential to prevent further injury. A high dose of intravenous steroid was not used due to initial suspicion of intracranial involvement. Prompt removal decreased risk of further vision loss.