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.
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...
Purpose: BRAF and MEK inhibitors (BRAFi and MEKi) are actively used for the treatment of metastatic melanoma in patients with BRAF V600E mutation in their tumors. However, the development of resistance to BRAFi and MEKi remains a difficult clinical challenge with limited therapeutic options available to these patients. In this study, we investigated the mechanism and potential therapeutic utility of combination BRAFi and adoptive T-cell therapy (ACT) in melanoma resistant to BRAFi.Experimental Design: Investigations were performed in vitro and in vivo with various human melanoma cell lines sensitive and resistant to BRAFi as well as patient-derived xenografts (PDX) derived from patients. In addition, samples were evaluated from patients on a clinical trial of BRAFi in combination with ACT.Results: Herein we report that in human melanoma cell lines, senstitive and resistant to BRAFi and in PDX from patients who progressed on BRAFi and MEKi therapy, BRAFi caused transient upregulation of mannose-6-phosphate receptor (M6PR). This sensitized tumor cells to CTLs via uptake of granzyme B, a main component of the cytotoxic activity of CTLs. Treatment of mice bearing resistant tumors with BRAFi enhanced the antitumor effect of patients' TILs. A pilot clinical trial of 16 patients with metastatic melanoma who were treated with the BRAFi vemurafenib followed by therapy with TILs demonstrated a significant increase of M6PR expression on tumors during vemurafenib treatment.Conclusions: BRAF-targeted therapy sensitized resistant melanoma cells to CTLs, which opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease.
Background Women with breast cancer (BCa) experience heightened distress, which is related to greater inflammation and poorer outcomes. The s100 protein family facilitates the inflammatory response by regulating myeloid cell function through the binding of Toll‐like receptor 4 and the receptor for advanced glycation end products (RAGE). The heterodimer s100A8/A9 RAGE ligand is associated with hastened tumor development and metastasis. Previously, a 10‐week stress‐management intervention using cognitive behavioral therapy (CBT) and relaxation training (RT) was associated with less leukocyte inflammatory gene expression in patients with BCa; however, its impact on s100A8/A9 was not examined. Because a 10‐week intervention may be impractical during primary treatment for BCa, the authors developed briefer forms of CBT and RT and demonstrated their efficacy in reducing distress over 12 months of primary treatment. Here, the effects of these briefer interventions were tested effects on s100A8/A9 levels over the initial 12 months of BCa treatment. Methods Postsurgical patients with BCa (stage 0‐IIIB) were randomized to a 5‐week, group‐based condition: CBT, RT, or health education control (HE). At baseline and at 12 months, women provided sera from which s100A8/A9 levels were determined using any enzyme‐linked immunosorbent assay. Results Participants (mean age ± standard deviation, 54.81 ± 9.63 years) who were assigned to either CBT (n = 41) or RT (n = 38) had significant s100A8/A9 decreases over 12 months compared with those who were assigned to HE (n = 44; F[1,114] = 4.500; P = .036) controlling for age, stage, time since surgery, and receipt of chemotherapy or radiation. Greater increases in stress‐management skills from preintervention to postintervention predicted greater reductions in s100A8/A9 levels over 12 months (β = −0.379; t[101] = −4.056; P < .001). Conclusions Brief, postsurgical, group‐based stress management reduces RAGE‐associated s100A8/A9 ligand levels during primary treatment for BCa.
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