IntroductionThe endogenous adenine nucleotides and adenosine are normally present at low concentrations in the extracellular milieu. However, metabolically stressful conditions, including inflammation and hypoxia characteristic of asthma, solid tumors, and other pathologic conditions, result in dramatic increases in extracellular concentrations of adenosine. [1][2][3] There are also mechanisms of nonlytic secretion of adenosine during hypoxic conditions.There is growing evidence that adenosine can actively modulate differentiation and function of myeloid cells. 4 Circulating cells of myeloid lineage, including monocytes and dendritic cell (DC) precursors, migrate to tissues where they differentiate into macrophages or DCs. DCs show impressive interaction with the adjacent microenvironment, 5,6 which regulates formation of DC subtypes and their functional properties, including expression of cytokines and growth factors. Because of rapid growth, solid tumors routinely experience severe hypoxia and necrosis, which causes adenine nucleotide degradation and adenosine release. Therefore, high levels of extracellular adenosine contribute to the local tumor microenvironment and may greatly influence differentiation of DCs from monocyte/macrophages and DC precursors migrating into tumor tissue. Adenosine acts through 4 subtypes of adenosine receptors, A 1 , A 2A , A 2B , and A 3 , which are members of the G-protein-coupled family of receptors. 7,8 A 2A adenosine receptors are generally anti-inflammatory, whereas A 2B and A 3 receptors are implicated in proinflammatory action of adenosine. Adenosine receptors are expressed abundantly on monocytes, and through these receptors adenosine exerts substantial modulatory effects on monocyte function and further differentiation. A 1 receptors were shown to stimulate formation of giant multinucleated cells from monocytes, whereas A 2 receptors inhibited this process. 9 A 2B receptors were implicated in mediating the inhibitory effect of adenosine on macrophage proliferation induced by M-CSF. 10 Exogenous adenosine can prevent monocytes from differentiating into macrophages, leading them to an intermediate differentiation stage between immature DCs and monocytes. 11 Cyclic nucleotides, including cAMP, which intracellular level increases in response to stimulation of adenosine A 2 receptors, regulate certain steps of monocyte differentiation and promote their differentiation toward a CD1a low CD14 ϩ/low CD209 ϩ intermediate cell but impair differentiation into functional DCs. 12 Up-regulation of DC-specific ICAM-3-grabbing nonintegrin (CD209) was not affected by cyclic nucleotides, 12 indicating that DC development was not blocked at the monocyte stage. The expression of all 4 adenosine receptor subtypes has been reported in human monocytes and myeloid DCs. 9,13-15 However, the effects of adenosine on differentiation of myeloid DCs from monocytes, macrophages, and hematopoietic progenitor cells (HPCs) and the roles of specific adenosine receptor subtypes involved in this process hav...

Exposure of carcinoma cell lines to the antibiotic geldanamycin induces the degradation of ErbB-2, a coreceptor tyrosine kinase that is frequently overexpressed in certain tumors. Using ErbB-2 mutants expressed as chimeric receptors or green fluorescent protein fusion proteins, we report that the kinase domain of ErbB-2 is essential for geldanamycin-induced degradation. The kinase domain of the related epidermal growth factor receptor was not sensitive to this drug. The data further indicate mechanistic aspects of ErbB-2 degradation by geldanamycin. The data show that exposure to the drug induces at least one cleavage within the cytoplasmic domain of ErbB-2 producing a 135-kDa fragment and a 23-kDa fragment. The latter represents the carboxyl-terminal domain of ErbB-2, whereas the former represents the ectodomain and part of the cytoplasmic domain. Degradation of the carboxylterminal fragment is prevented by proteasome inhibitors, whereas degradation of the membrane-anchored 135-kDa ErbB-2 fragment is blocked by inhibitors of the endocytosis-dependent degradation pathway. Confocal microscopy studies confirm a geldanamycin-induced localization of ErbB-2 on intracellular vesicles.ErbB-2 is a Type 1 transmembrane tyrosine kinase that functions as a co-receptor by forming dimers with other members of the ErbB receptor family (ErbB-1 (EGF 1 receptor), ErbB-3, and ErbB-4; Refs. 1 and 2). Although ErbB-2 has a potential ligand-binding ectodomain, no direct ligand has yet been identified. In its role as a co-receptor, ErbB-2 enhances the signaling capacity of its dimerization partners. The association of ErbB-2 with these various receptors is, however, entirely ligand-dependent. In the absence of growth factor ErbB-2 is reported to interact with CD44, an adhesion receptor, in ovarian carcinoma cell lines (3) and with a large plasma membrane glycoprotein complex in microvilli of a mammary adenocarcinoma cell line (4). ErbB-2 has also been demonstrated to form ligand-dependent complexes with the IL-6 receptor component gp130 (5) and Trk A (6), the nerve growth factor receptor.ErbB-2 was originally identified as the transforming oncogene neu in which a point mutation in the transmembrane domain is responsible for its oncogenic potential (7,8). ErbB-2 also functions as an oncogene when overexpressed (9, 10) and in humans is frequently overexpressed in breast and ovarian tumors (11). ErbB-2 overexpression in breast cancer is associated with a poor prognosis (12), and hence it is a target for therapeutic reagents, including monoclonal antibodies and drugs (13). Frequently, antibodies that decrease the growth of ErbB-2-expressing tumors also reduce the level of ErbB-2 by a mechanism that is unclear. Hence, the transforming activity of ErbB-2 is related to structural changes or changes in its level of expression.The benzoquinoid ansamycin antibiotics geldanamycin and herbimycin were first isolated from the culture broths of several actinomycete species (14, 15) and described as inhibitors of tyrosine kinase-dependent growth (16...

Tumor-associated macrophages (TAMs) are critically important in the context of solid tumor progression. Counterintuitively, these host immune cells can often support tumor cells along the path from primary tumor to metastatic colonization and growth. Thus, the ability to transform protumor TAMs into antitumor, immune-reactive macrophages would have significant therapeutic potential. However, in order to achieve these effects, two major hurdles would need to be overcome: development of a methodology to specifically target macrophages and increased knowledge of the optimal targets for cell-signaling modulation. This study addresses both of these obstacles and furthers the development of a therapeutic agent based on this strategy. Using ex vivo macrophages in culture, the efficacy of mannosylated nanoparticles to deliver small interfering RNA specifically to TAMs and modify signaling pathways is characterized. Then, selective small interfering RNA delivery is tested for the ability to inhibit gene targets within the canonical or alternative nuclear factor-kappaB pathways and result in antitumor phenotypes. Results confirm that the mannosylated nanoparticle approach can be used to modulate signaling within macrophages. We also identify appropriate gene targets in critical regulatory pathways. These findings represent an important advance toward the development of a novel cancer therapy that would minimize side effects because of the targeted nature of the intervention and that has rapid translational potential.

BackgroundNuclear factor-kappa B (NF-kappaB) signaling is an important link between inflammation and peritoneal carcinomatosis in human ovarian cancer. Our objective was to track NF-kappaB signaling during ovarian cancer progression in a syngeneic mouse model using tumor cells stably expressing an NF-kappaB reporter.MethodsID8 mouse ovarian cancer cells stably expressing an NF-kappaB-dependent GFP/luciferase (NGL) fusion reporter transgene (ID8-NGL) were generated, and injected intra-peritoneally into C57BL/6 mice. NGL reporter activity in tumors was non-invasively monitored by bioluminescence imaging and measured in luciferase assays in harvested tumors. Ascites fluid or peritoneal lavages were analyzed for inflammatory cell and macrophage content, and for mRNA expression of M1 and M2 macrophage markers by quantitative real-time RT-PCR. 2-tailed Mann-Whitney tests were used for measuring differences between groups in in vivo experiments.ResultsIn ID8-NGL cells, responsiveness of the reporter to NF-kappaB activators and inhibitors was confirmed in vitro and in vivo. ID8-NGL tumors in C57BL/6 mice bore histopathological resemblance to human high-grade serous ovarian cancer and exhibited similar peritoneal disease spread. Tumor NF-kappaB activity, measured by the NGL reporter and by western blot of nuclear p65 expression, was markedly elevated at late stages of ovarian cancer progression. In ascites fluid, macrophages were the predominant inflammatory cell population. There were elevated levels of the M2-like pro-tumor macrophage marker, mannose-receptor, during tumor progression, and reduced levels following NF-kappaB inhibition with thymoquinone.ConclusionsOur ID8-NGL reporter syngeneic model is suitable for investigating changes in tumor NF-kappaB activity during ovarian cancer progression, how NF-kappaB activity influences immune cells in the tumor microenvironment, and effects of NF-kappaB-targeted treatments in future studies.

Increased expression of the epidermal growth factor (EGF) receptor (EGFR) and ErbB-2 is implicated into the development and progression of breast cancer. Constant ligand-induced activation of EGFR and ErbB-2 receptortyrosine kinases is thought to be involved in the transformation of fibroblasts and mammary epithelial cells. Data herein show that ligand stimulation of cells that express both the EGFR and the ErbB-2 may result either in cell proliferation or apoptosis depending on the expression levels of EGFR and ErbB-2. Mammary tumor cells that express low levels of both receptors or high levels of ErbB-2 and low levels of EGFR survive and proliferate in the presence of EGF. In contrast, fibroblastic cells or mammary tumor cells, which co-express high levels of EGFR and ErbB-2 invariably undergo apoptosis in response to EGF. In these cells persistent activation of p38 MAPK is an essential element of the apoptotic mechanism. Also, the data implicate a p38-dependent change in mitochondrial membrane permeability as a downstream effector of apoptosis. Ligand-dependent apoptosis in cells co-expressing high levels of EGFR and ErbB-2 could be a natural mechanism that protects tissues from unrestricted proliferation in response to the sustained activation of receptor-tyrosine kinases. EGFR1 /ErbB-1 and ErbB-2 are type I receptor-tyrosine kinases characterized by the presence of an extracellular ligand binding domain, a single transmembrane domain, and a cytoplasmic region that includes a tyrosine kinase domain. The extracellular domain of the EGFR is recognized by seven distinct ligands that are related in primary sequence, which results in tyrosine kinase activation (1). In response to ligand stimulation, the EGFR forms homo-or heterodimers as part of the activation and signaling mechanism (2, 3). ErbB-2 has no known direct ligand but can be activated through heterodimerization with EGFR or other members of the ErbB family. Studies of the interactions between ErbB receptors have shown that ErbB-2 is the preferred partner for heterodimerization and thereby potentiates transformation and pro-survival signaling pathways (2-6).Ligand binding induces the rapid internalization of the EGFR and its subsequent lysosomal degradation (7). In contrast, ErbB-2 transactivation by the activated EGFR does not stimulate rapid internalization of ErbB-2 (8 -11). Removal of transactivated ErbB-2 from the cell surface proceeds slowly through a combination of intracellular degradation mechanisms, including lysosomes, the proteasome, and other intracellular proteases (10, 12-13). Heterodimerization of ErbB-2 and the EGFR actually impedes the rapid ligand-dependent internalization of the EGFR and, thereby, results in increased activation of signaling pathways (4, 9, 14).Increased EGFR or ErbB-2 expression or structural alterations in either receptor are frequent in human malignancies (2, 15-17). Overexpression of the EGFR is observed in a significant proportion of brain tumors (18 -20). Also, glioblastomas and other tumors often express a del...