Manipulating the NF-κB pathway in macrophages using mannosylated, siRNA-delivering nanoparticles can induce immunostimulatory and tumor cytotoxic functions.

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.

Effects of vitamin A deficiency in the postnatal mouse heart: role of hepatic retinoid stores.

To determine whether hepatic depletion of vitamin A (VA) stores has an effect on the postnatal heart, studies were carried out with mice lacking liver retinyl ester stores fed either a VA-sufficient (LRVAS) or VA-deficient (LRVAD) diet (to deplete circulating retinol and extrahepatic stores of retinyl esters). There were no observable differences in the weights or gross morphology of hearts from LRVAS or LRVAD mice relative to sex-matched, age-matched, and genetically matched wild-type (WT) controls fed the VAS diet (WTVAS), but changes in the transcription of functionally relevant genes were consistent with a state of VAD in LRVAS and LRVAD ventricles. In silico analysis revealed that 58/67 differentially expressed transcripts identified in a microarray screen are products of genes that have DNA retinoic acid response elements. Flow cytometric analysis revealed a significant and cell-specific increase in the number of proliferating Sca-1 cardiac progenitor cells in LRVAS animals relative to WTVAS controls. Before myocardial infarction, LRVAS and WTVAS mice had similar cardiac systolic function and structure, as measured by echocardiography, but, unexpectedly, repeat echocardiography demonstrated that LRVAS mice had less adverse remodeling by 1 wk after myocardial infarction. Overall, the results demonstrate that the adult heart is responsive to retinoids, and, most notably, reducing hepatic VA stores (while maintaining circulating levels of VA) impacts ventricular gene expression profiles, progenitor cell numbers, and response to injury.

Aberrant activation of NF-κB signaling in mammary epithelium leads to abnormal growth and ductal carcinoma in situ.

BACKGROUND: Approximately 1 in 5 women diagnosed with breast cancer are considered to have in situ disease, most often termed ductal carcinoma in situ (DCIS). Though recognized as a risk factor for the development of more invasive cancer, it remains unclear what factors contribute to DCIS development. It has been shown that inflammation contributes to the progression of a variety of tumor types, and nuclear factor kappa B (NF-κB) is recognized as a master-regulator of inflammatory signaling. However, the contributions of NF-κB signaling to tumor initiation are less well understood. Aberrant up-regulation of NF-κB activity, either systemically or locally within the breast, could occur due to a variety of commonly experienced stimuli such as acute infection, obesity, or psychological stress. In this study, we seek to determine if activation of NF-κB in mammary epithelium could play a role in the formation of hyperplastic ductal lesions. METHODS: Our studies utilize a doxycycline-inducible transgenic mouse model in which constitutively active IKKß is expressed specifically in mammary epithelium. All previously published models of NF-κB modulation in the virgin mammary gland have been constitutive models, with transgene or knock-out present throughout the life and development of the animal. For the first time, we will induce activation at later time points after normal ducts have formed, thus being able to determine if NF-κB activation can promote pre-malignant changes in previously normal mammary epithelium. RESULTS: We found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures. Short-term activation created hyperproliferative, enlarged ducts with filled lumens. Increased expression of inflammatory markers was concurrent with the down-regulation of hormone receptors and markers of epithelial differentiation. Furthermore, the oncoprotein mucin 1, known to be up-regulated in human and mouse DCIS, was over-expressed and mislocalized in the activated ductal tissue. CONCLUSIONS: These results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer. Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

Biocompatible mannosylated endosomal-escape nanoparticles enhance selective delivery of short nucleotide sequences to tumor associated macrophages.

Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create a pro-tumor niche. Manipulation of the TAM phenotype is a novel, potential therapeutic approach to engage anti-cancer immunity. siRNA is a molecular tool for knockdown of specific mRNAs that is tunable in both strength and duration. The use of siRNA to reprogram TAMs to adopt an immunogenic, anti-tumor phenotype is an attractive alternative to ablation of this cell population. One current difficulty with this approach is that TAMs are difficult to specifically target and transfect. We report here successful utilization of novel mannosylated polymer nanoparticles (MnNP) that are capable of escaping the endosomal compartment to deliver siRNA to TAMs in vitro and in vivo. Transfection with MnNP-siRNA complexes did not significantly decrease TAM cell membrane integrity in culture, nor did it create adverse kidney or liver function in mice, even at repeated doses of 5 mg kg(-1). Furthermore, MnNP effectively delivers labeled nucleotides to TAMs in mice with primary mammary tumors. We also confirmed TAM targeting in the solid tumors disseminated throughout the peritoneum of ovarian tumor bearing mice following injection of fluorescently labeled MnNP-nucleotide complexes into the peritoneum. Finally, we show enhanced uptake of MnNP in lung metastasis associated macrophages compared to untargeted particles when using an intubation delivery method. In summary, we have shown that MnNP specifically and effectively deliver siRNA to TAMs in vivo.

Tracking NF-κB activity in tumor cells during ovarian cancer progression in a syngeneic mouse model.

BACKGROUND: Nuclear 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. METHODS: ID8 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. RESULTS: In 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. CONCLUSIONS: Our 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.

Adenosine receptors in regulation of dendritic cell differentiation and function.

Differentiation of functional dendritic cells (DCs) critically depends on the microenvironment. DCs differentiate in hypoxic tumor sites and inflamed or damaged tissue. Because local concentrations of adenosine reach high physiologically relevant levels in these conditions, we assessed the expression of adenosine receptors and the effect of their activation on differentiation of human monocytes and mouse peritoneal macrophages and hematopoietic progenitor cells (HPCs) into myeloid DCs. Stimulation of adenosine receptors skews DC differentiation toward a distinct cell population characterized by expression of both DC and monocyte/macrophage cell surface markers. Pharmacologic analysis and experiments with cells from A(2B) adenosine receptor knockout mice identified A(2B) receptor as the mediator of adenosine effects on DCs. Unlike normal myeloid DCs, adenosine-differentiated DCs have impaired allostimulatory activity and express high levels of angiogenic, pro-inflammatory, immune suppressor, and tolerogenic factors, including VEGF, IL-8, IL-6, IL-10, COX-2, TGF-beta, and IDO. They promoted tumor growth if injected into tumors implanted in mice. Using adenosine desaminase knockout animals, we showed that DCs with proangiogenic phenotype are highly abundant under conditions associated with elevated levels of extracellular adenosine in vivo. Adenosine signaling through A(2B) receptor is an important factor of aberrant DC differentiation and generation of tolerogenic, angiogenic, and proinflammatory cells.

Bax activation and translocation to mitochondria mediate EGF-induced programmed cell death.

The ErbB family of receptor tyrosine kinases is involved in the regulation of cell proliferation, differentiation and apoptosis. Previous studies indicate that cells expressing elevated levels of the EGFR and ErbB-2 undergo programmed cell death in response to EGF or other EGFR ligands. However, the detailed mechanisms of EGF-induced apoptosis are unclear. This report demonstrates that in the cells undergoing EGF-dependent apoptosis Bax changes its conformation and forms multimeric aggregates, which accumulate on the mitochondrial membrane. Bax activation and translocation to the mitochondria induces a loss of mitochondrial transmembrane potential and cell death. Also, during EGF-induced apoptosis there is downregulation of Bcl-xL, an anti-apoptotic protein. Expression of Bcl-xL in cells susceptible to EGF-dependent apoptosis prevents cell death. The data indicate that addition of EGF does not result in a significant release of cytochrome c from mitochondria and EGF-induced apoptosis is mainly caspase independent.

Identification of proteolytic fragments from ErbB-2 that induce apoptosis.

The receptor tyrosine kinase ErbB-2 plays an important role in cell proliferation and differentiation as well as oncogenesis. We have found that ErbB-2 kinase domain fragmentation is important for the induction of apoptosis. Exogenous expression of peptides derived from the ErbB-2 kinase domain induces cells death with the hallmarks of apoptosis. In contrast, transfection of the ErbB-2 carboxy-terminal domain did not induce apoptosis. We have identified a 37-residue segment from the ErbB-2 kinase N-terminal lobe that can strongly induce apoptosis in transfected cells. Cell death was not blocked by the pan-caspase inhibitor z-VAD-FMK. Similar fragments derived from several other receptor tyrosine kinases also induce cell death. These data imply that proteolytic fragmentation of tyrosine kinases liberates apoptotic fragments that can accelerate cell death.

Ligand-induced, p38-dependent apoptosis in cells expressing high levels of epidermal growth factor receptor and ErbB-2.

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 receptor-tyrosine 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.

Ectodomain cleavage of ErbB-4: characterization of the cleavage site and m80 fragment.

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646-657 was incubated with recombinant tumor necrosis factor-alpha-converting enzyme, fragments representing residues 646-651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.

Identification of ErbB-2 kinase domain motifs required for geldanamycin-induced degradation.

The ansamycin antibiotic geldanamycin (GA) induces the intracellular degradation of ErbB-2/neu. Degradation of ErbB-2 proceeds through cleavage(s) within the kinase domain, resulting in the formation of a 135 kDa ectodomain fragment and a fragment(s) of approximately 50 kDa containing the COOH-terminal region. On the basis of independent means of identification, two adjacent sequence motifs have been identified in ErbB-2 that are required for GA-induced degradation. These motifs encompass residues 776-783 and 784-786 within the NH(2)-terminal lobe of the ErbB-2 kinase domain. This is also a region in which the epidermal growth factor receptor and ErbB-2 kinase domains differ significantly in sequence. Although mutations in this region abrogate GA-induced ErbB-2 degradation, the tyrosine kinase activity of ErbB-2 is not disrupted. Interestingly, these ErbB-2 mutants are specifically resistant to GA-induced degradation but retain sensitivity to other drugs, such as staurospore and curcumin, which are also able to provoke ErbB-2 degradation.

Local ferroelectricity in SrTiO3 thin films.

The temperature-dependent polarization of SrTiO3 thin films is investigated using confocal scanning optical microscopy. A homogeneous out-of-plane and an inhomogeneous in-plane ferroelectric phase are identified from images of the linear electro-optic response. Both hysteretic and nonhysteretic behavior are observed under a dc bias field. Unlike classical transitions in bulk ferroelectrics, local ferroelectricity is observed at temperatures far above the dielectric permittivity maximum. The results demonstrate the utility of local probe experiments in understanding inhomogeneous ferroelectrics.