Patricia Rafferty

Extracellular Matrix Metalloproteinase Inducer Stimulates Tumor Angiogenesis by Elevating Vascular Endothelial Cell Growth Factor and Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that play pivotal roles in promoting tumor disease progression, including tumor angiogenesis. In many solid tumors, MMP expression could be attributed to tumor stromal cells and is partially regulated by tumor-stroma interactions via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). The role of EMMPRIN during tumor angiogenesis and growth was explored by modulating EMMPRIN expression and activity using recombinant DNA engineering and neutralizing antibodies. In human breast cancer cells, changes in EMMPRIN expression influenced vascular endothelial growth factor (VEGF) production at both RNA and protein levels. In coculture of tumor cells and fibroblasts mimicking tumor-stroma interactions, VEGF expression was induced in an EMMPRIN- and MMP-dependent fashion, and was further enhanced by overexpressing EMMPRIN. Conversely, VEGF expression was inhibited by suppressing EMMPRIN expression in tumor cells, by neutralizing EMMPRIN activity, or by inhibiting MMPs. In vivo, EMMPRIN overexpression stimulated tumor angiogenesis and growth; both were significantly inhibited by antisense suppression of EMMPRIN. Expression of both human and mouse VEGF and MMP, derived from tumor and host cells, respectively, was regulated by EMMPRIN. These results suggest a novel tumor angiogenesis mechanism in which tumor-associated EMMPRIN functionally mediates tumor-stroma interactions and directly contributes to tumor angiogenesis and growth by stimulating VEGF and MMP expression.

Regulation of vascular endothelial growth factor expression by EMMPRIN via the PI3k-Akt signaling pathway

Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) is a cell surface glycoprotein overexpressed in many solid tumors. In addition to its ability to stimulate stromal MMP expression, tumor-associated EMMPRIN also induces vascular endothelial growth factor (VEGF) expression. To explore the underlying signaling pathways used by EMMPRIN, we studied the involvement of phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), JUN, and p38 kinases in EMMPRIN-mediated VEGF regulation. Overexpression of EMMPRIN in MDA-MB-231 breast cancer cells stimulated the phosphorylation of only Akt and MAPKs but not that of JUN and p38 kinases. Conversely, inhibition of EMMPRIN expression resulted in suppressed Akt and MAPK phosphorylation. Furthermore, the PI3K-specific inhibitor LY294002 inhibited VEGF production by EMMPRIN-overexpressing cells in a dose- and time-dependent manner. On the other hand, the MAPK inhibitor U0126 did not affect VEGF production. In vivo, EMMPRIN-overexpressing tumors with elevated VEGF expression had a high level of phosphorylation of Akt and MAPK. Finally, when fibroblast cells were treated with recombinant EMMPRIN, Akt kinase but not MAPK was phosphorylated concomitant with an increase in VEGF production. Both the activation of Akt kinase and the induction of VEGF were specifically inhibited with a neutralizing antibody to EMMPRIN. Our results show that in both tumor and fibroblast cells EMMPRIN regulates VEGF production via the PI3K-Akt pathway but not via the MAPK, JUN, or p38 kinase pathways. (Mol Cancer Res 2006;4(6):371–8)

Loss of toll-like receptor 3 function improves glucose tolerance and reduces liver steatosis in obese mice

OBJECTIVE Emerging evidence suggests a link between innate immunity and development of type 2 diabetes mellitus (T2D); however, the molecular mechanisms linking them are not fully understood. Toll-like Receptor 3 (TLR3) is a pathogen pattern recognition receptor that recognizes the double-stranded RNA of microbial or mammalian origin and contributes to immune responses in the context of infections and chronic inflammation. The objective of this study was to determine whether TLR3 activity impacts insulin sensitivity and lipid metabolism. MATERIALS AND METHODS Wild type (WT) and TLR3 knock-out (TLR3(-/-)) mice were fed a high fat diet (HFD) and submitted to glucose tolerance tests (GTTs) over a period of 33 weeks. In another study, the same group of mice was treated with a neutralizing monoclonal antibody (mAb) against mouse TLR3. RESULTS TLR3(-/-) mice fed an HFD developed obesity, although they exhibited improved glucose tolerance and lipid profiles compared with WT obese mice. In addition, the increase in liver weight and lipid content normally observed in WT mice on an HFD was significantly ameliorated in TLR3(-/-) mice. These changes were accompanied by up-regulation of genes involved in cholesterol efflux such as PPARδ, LXRα, and LXRα-targeting genes and down-regulation of pro-inflammatory cytokine and chemokine genes in obese TLR3(-/-) mice. Furthermore, global gene expression profiling in liver demonstrated TLR3-specific changes in both lipid biosynthesis and innate immune response pathways. CONCLUSIONS TLR3 affects glucose and lipid metabolism as well as inflammatory mediators, and findings in this study reveal a new role for TLR3 in metabolic homeostasis. This suggests antagonizing TLR3 may be a beneficial therapeutic approach for the treatment of metabolic diseases.

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor‐α transgenic mice

TNF‐α is a pleitropic cytokine that expresses both pro‐ and anti‐inflammatory activity and transgenic mice expressing human tumor necrosis factor‐α (TNF‐α) exhibit a progressive polyarthritis that models rheumatoid arthritis (RA). One of the common comorbidities of RA is anemia of chronic disease (ACD). The purpose of these experiments was to study the changes in the bone marrow and peripheral blood that accompany polyarthritis in TNF‐α transgenic mice in an effort to better understand the pathogenesis of myelodysplasia and ACD. Polychromatic cytometry, hematology and serum cytokine analysis were used to study the pathogenesis of ACD in human TNF‐α transgenic mice. Our hematological evaluation revealed a mild, compensated, microcytic hypochromic anemia, and monocytosis. In the bone marrow, we observed alterations in cell kinetics, decreased relative expression of transferrin receptor and increased apoptosis and cell death in several late precursor cell populations. Although significant levels of human TNF‐α were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL‐β, IL‐5, IL‐6, IL‐10, IL‐12(p70), IL‐17, TNF‐α, IFNγ, GM‐CSF, MIP‐1αJE, MCP‐5 was observed. Tg197 mice develop a compensated, microcytic, hypochromic anemia, and a functional iron deficiency by 9 weeks of age. Changes in peripheral blood are reflected in alterations in cell kinetics, transferrin receptor expression and markedly increased apoptosis and cell death in the bone marrow indicating that TNF‐α may contribute to myelodysplasia in ACD. Moreover, since human TNF‐α can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD

Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice

Many immunosuppressive drugs are associated with an increased risk of neoplasia, principally non-melanoma skin cancers and B-cell lymphomas. However, only 6 of the 13 immunosuppressive drugs tested in 2 year bioassays increased the incidence of neoplasia. For example, the 2-year bioassays conducted with cyclosporine (CSA), an International Agency for Research on Cancer (IARC) Group 1 human carcinogen, were negative. The purpose of these investigations was to use transplanted tumor models in immunocompetent, syngeneic mice to gain insight into the failure of the 2-year bioassay to show an increased incidence of neoplasia with CSA. C3H HeN mice were used in a battery of assays with a transplanted squamous cell carcinoma (SCC VII cells) or a B-cell, lymphoma (38C13 cells) cells to study effects of CSA on local growth and metastases, experimental metastases, and progression of established metastases. Mice received CSA twice weekly by subcutaneous (SC) injection at doses of 0.5, 5, or 50 mg/kg; controls received the CSA vehicle. CSA had a modest inhibitory effect on SC tumors initiated by 38C13 cells and on intramuscular tumors initiated by SCC VII cells. CSA also decreased the number of lung colonies and decreased the size, growth fraction and vascularity of established lung metastases initiated by SCC VII cells. In contrast, CSA increased progressive growth of metastases to the sentinel lymph node from an intramuscular SCC VII tumor, but had no effect cellular traffic to the node. In conclusion, CSA at doses up to 50 mg/kg did not facilitate tumor progression and it partially inhibited tumor growth, suggesting that suppression of tumor progression may partially explain the failure of CSA to act as a carcinogen in 2 year bioassays.

Development of a murine model of lymph node metastases suitable for immunotoxicity studies.

INTRODUCTION Immunosuppressive drugs are associated with an increased risk of infections and in some cases neoplasia, particularly non-melanoma skin cancers. This paper describes the development of a model to test the effects of immunosuppressive drugs on local invasion and metastases of a squamous cell carcinoma in syngeneic, immunocompetent mice. METHODS SCC VII cells were labeled with 655 quantum dots (QDs), injected intramuscularly into C3H HEN mice and traffic and progressive growth in the draining popliteal lymph node were evaluated. RESULTS SCC VII cells express RAE-1, an NKG2D ligand, and were sensitive to natural killer (NK) cells in vitro. QDs were stable in SCC VII cells and showed no evidence of toxicity to the cells. In vivo, confocal microscopy showed that QD-labeled SCC VII cells could migrate to the draining node and microfluorimetry showed progressive traffic of QDs to the node. There was no evidence of systemic toxicity of QDs. Primary immunosuppression in SCID and SCID-beige mice and treatment of normal mice with immunosuppressive agents (anti-asialoGM1 and cyclophosphamide) can enhance traffic of QDs and/or metastases to the draining lymph node. In contrast, cyclosporine had no effect on traffic or metastases. CONCLUSION This model of local invasion and metastases may be useful in immunotoxicology for identifying and characterizing the hazard posed by selective immunosuppressive drugs.

Tissue Factor (TF) Expression and Angiogenesis in Tumor Progression and Inhibition of Tumor Growth by Anti-TF Antibodies in Human TissueFactor Knock-In Mice

Background: Tissue factor (TF) serves as the primary initiator of the extrinsic pathway of blood coagulation and mediates signaling via the protease activated receptor-2 (PAR2). TF is over-expressed in several tumor types and may facilitate tumor progression and angiogenesis. To test the hypothesis that inhibition of TF may have an anti-tumor effect, we induced pulmonary adenomas (PA) in human TF knock in (huTF-KI) mice with urethane and studied the relationship between expression of TF and mutations in K-ras with tumor progression and the effect of a monoclonal anti-TF antibody on growth of a transplantable lymphoma. Methods: huTF-KI mice received 10 weekly intraperitoneal (i.p.) injections of urethane and samples of lung were collected at six week intervals between weeks 10 and 28. Expression of TF and von Willibrand factor (vWF) in PA were studied by immunohistochemistry (IHC) and mutations in K-ras were studied by laser capture microdissection and polymerase chain reaction (LCM-PCR). Results: IHC showed that expression of TF and vWF increased as pulmonary epithelial hyperplasias and PA progressed. Dual staining TF and vWF showed that areas of high expression of TF correlated with the tumor vasculature and LCM-PCR showed that mutation of K-ras correlated with expression of TF and the angiogenic switch in PA. Finally, an anti-huTF monoclonal antibody slowed the growth of transplantable urethane-induced lymphomas. Conclusion: Taken together, these data suggest that expression of TF plays an important role in tumor progression and the angiogenic switch. These data also suggest that anti-TF antibodies may be a viable tumor immunotherapy

Development of a squamous cell carcinoma mouse model for immunotoxicity testing

Abstract An important component of safety assessment of new pharmaceuticals is evaluation of their potential to increase the risk of developing cancer in humans. The traditional 2-year rodent bioassay often is not feasible or scientifically applicable for evaluation of biotherapeutics. Additionally, it has poor predictive value for non-genotoxic immunosuppressive compounds. Thus, there is a need for alternative testing strategies. A novel 3-stage tumor model in syngeneic C3H/HeN mice was evaluated here to study the effects of immunosuppressive drugs on tumor promotion and progression in vivo. The model employed a skin squamous cell carcinoma cell line (SCC VII) due to the increased prevalence of squamous cell carcinoma (SCC) in humans associated with immunosuppression after transplants. Local invasion, colonization and tumor progression were evaluated. The validation set of immunosuppressive drugs included: Cyclosporin (CSA), cyclophosphamide (CTX), azathioprine, etanercept, abatacept and prednisone. Local invasion was evaluated by histological assessment as well as fluorescence trafficking from Qdot®-labeled tumor cells from the site of inoculation to the draining lymph node. Colonization was evaluated by lung colony counts following intravenous inoculation. Tumor progression was assessed by morphometric analysis of lesion area, angiogenesis and growth fraction of established metastatic neoplasia. Immunosuppressive drugs in the validation set yielded mixed results, including decreased progression. The methods and results described herein using an in vivo syngeneic mouse tumor model can provide insight about the assessment of immunosuppressive drugs in carcinogenicity risk assessment.

Neutralizing CCL2 Inhibits Breast Tumor Growth Via Impact on the Tumor/Stroma Microenvironment.

CCL2, (CC-chemokine ligand 2 or monocyte chemoattractant protein-1 (MCP-1)), is overexpressed in many human tumors and is believed to exert pro-tumor effects by recruiting monocytes to the tumor, where these cells become tumor associated macrophages (TAMs). TAMs secrete growth factors that stimulate angiogenesis and tumor growth, as well as proteases to promote tumor invasion and metastasis. CCL2 expression levels in primary breast tumors have been correlated with macrophage infiltration and blood vessel density, which in turn is correlated with disease stage and prognosis. These correlations indicate that CCL2 is a key player in tumor macrophage infiltration and/or tumor growth/invasion, and suggest that neutralizing CCL2 could be an effective form of therapy for breast cancer patients.The objective of these studies was to investigate whether CCL2 blockade could inhibit tumor growth in mice bearing human breast tumors. The human breast tumor cell lines MDA-MB-231 (ER-, PR-, Her2-) and MDA-MB-361 (ER+, PR+, Her2+) were implanted orthotopically in immunocompromised mice, and in both models the primary tumors metastasized to lungs and brain. Neutralizing antibodies to human CCL2 (CNTO 888) and to the mouse orthologs, MCP-1 and MCP-5, were administered therapeutically, either as a cocktail (termed CCL2 blockade) or individually to study the relative roles of host vs tumor derived CCL2 in promoting tumor growth.In both tumor models, CCL2 blockade significantly inhibited the growth of established primary tumors in the mammary fat pad. In addition, CCL2 blockade inhibited metastasis to distant sites. As measured by Taqman, visual inspection and immunohistochemistry, mice with MDA-MB-361 tumors treated with CCL2 blockade showed significantly reduced metastasis to lungs and brain, while mice bearing MDA-MB-231 tumors showed significantly reduced metastasis to lungs.To define the relative roles of human tumor-derived CCL2 vs mouse host-derived MCP-1/MCP-5, in vivo monotherapy tumor studies were conducted using the individual neutralizing antibodies. These studies included the mammary fat pad model and a tail vein metastasis model. In both cases, only the treatment with the anti-mouse MCP-1 antibody significantly inhibited primary tumor growth and distant metastasis, indistinguishable from the effect of CCL2 blockade treatment. In the tail vein metastasis model, the antibody treatment resulted in significantly fewer detectable lesions with these lesions showing a significant reduction in both tumor size and growth fraction, suggesting antibody treatment inhibits tumor seeding and growth. Mechanistic studies are in progress to further understand the basis of the anti-tumor effect mediated by the antibody treatment. These results demonstrate that host-derived MCP-1, produced from the tumor microenvironment, plays the critical role in tumor growth and metastasis in these models of human breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6095.

Anti-CXCL13 and Anti-TNFα Monoclonal Antibodies Combinatorial Treatment Inhibits Autoimmune Disease in a Murine Model of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the involvement of multiple organ systems with alternating clinical exacerbations and remissions. Circulating immune complexes and autoantibodies can cause tissue damage and organ dysfunction with manifestations involving the skin, serosal surfaces, central nervous system, and kidneys (Rahman & Isenberg, 2008). B cells are believed to play an important role in SLE. B cells can function as APCs, produce cytokines and chemokines contributing to lymphoid regulation, and can respond to stimuli in the microenvironment at local tissues (Ramanujam & Davidson, 2008). Pathogenic autoantibodies produced by autoreactive B cells are believed to play an important role in the pathogenesis of SLE. CXCL13 has been shown to be a key mediator of organization of lymphoid tissues. CXCL13 is a B cell chemoattractant that is expressed by peritoneal macrophages and follicular dendritic cells in secondary lymphoid organs, such as the follicles of Peyer’s patches, the spleen and lymph nodes. Through interaction with CXCR5, a G-protein coupled receptor, CXCL13 attracts B lymphocytes and promotes migration of small numbers of T helper follicular cells and macrophages (Gunn et al., 1998). CXCL13 is critical for B cell homing and follicle formation in lymph node and spleen, and it is required for the development of lymph nodes and Peyer’s patches (Ansel et al., 2000). CXCL13 protein level is elevated in ectopic B cell follicles formed in the inflamed tissues of multiple chronic diseases, and plays an important role in maintaining inflammation by actively recruiting B cells (Carlsen et al., 2004; Magliozzi et al., 2004; Salolonsson et al., 2002; Shi et al., 2001;). CXCL13 has been shown to have increased expression in the thymus and kidney of aged NZB/W F1 mice, and may play a role in breaking immune tolerance in the thymus of autoimmune prone mice (Ishikawa et al., 2001). Treatment with anti-CXCL13 has shown efficacy in animal models of RA and EAE (Bagaeva et al. 2006; Zheng et al., 2005). Because of its function and presence in various pathological conditions, CXCL13 and CXCL13 dependent pathways are thought to be instrumental in the pathogenesis of a variety of diseases where B cells may play a significant role, including RA, OA, UC, and SLE, and could be potential targets for autoimmune therapy (Table 1).