Francis L. McCabe

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)

CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models

Thromboembolic complications are frequently associated with advanced cancer. Interestingly, one of the major initiators of blood coagulation, tissue factor (TF), is reported to be overexpressed in several tumor types and can be found on both tumor cells and tumor vasculature. Although the exact mechanisms have yet to be elucidated, TF expressed on tumor cells can trigger intracellular signaling events through various pathways that can lead to tumor angiogenesis, proliferation, and metastasis. There exists preclinical evidence that disruption of TF dependent signaling can effectively inhibit tumor cell migration, metastasis, and angiogenesis. Here, we report for the first time that an antibody to tissue factor can also prevent tumor growth in vivo. Prophylactic administration of CNTO 859, a humanized anti‐human TF antibody, was shown to inhibit experimental lung metastasis of MDA‐MB‐231 human breast carcinoma cells by over 99% compared to a control antibody. Furthermore, therapeutic doses of CNTO 859 were shown to reduce tumor incidence and growth of orthotopically implanted MDA‐MB‐231 cells.

Tumor-Associated Macrophages Promote Invasion while Retaining Fc-Dependent Anti-Tumor Function

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b+CD14+ TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

αv Integrin-Targeted Immunoconjugates Regress Established Human Tumors in Xenograft Models

Purpose: Targeted delivery of cytotoxic agents to solid tumors through cell surface antigens can potentially reduce systemic toxicity and increase the efficacy of the targeted compounds. The purpose of this study was to show the feasibility of treating solid tumors by targeting αv integrins with antibody-maytansinoid conjugates and to test the relative in vivo activities of several linker-maytansinoid chemistries. Experimental Design: CNTO 364, CNTO 365, and CNTO 366 are targeted cytotoxic agents created by conjugating the CNTO 95 anti-αv integrin antibody with three distinct maytansinoid-linker structures. These structures were designed to have varying degrees of chemical substitution surrounding the disulfide bond linking the cytotoxic agent to the antibody. A model conjugate was shown to be specifically cytotoxic in vitro and highly active against established human tumor xenografts in immunocompromised rats. The in vivo antitumor activities of CNTO 364, CNTO 365, and CNTO 366 were compared in rat xenograft models. Results: CNTO 365, with a linker chemistry of expected intermediate stability, was shown to be substantially more active than the other two conjugates with lesser or greater substitution around the disulfide linkage. Conclusion: CNTO 95–maytansinoid immunoconjugates are potent antitumor agents against αv integrin–expressing human carcinomas. These studies show for the first time the feasibility of targeting αv integrins on solid tumors with tumor-activated prodrugs. The DM4 linker-maytansinoid configuration of CNTO 365 was substantially more active in the models tested here when compared with alternative configurations with greater or lesser chemical substitution surrounding the linker.

Comparative Activity of Oral and Parenteral Topotecan in Murine Tumor Models: Efficacy of Oral Topotecan

Studies were performed using several tumor models to determine the oral efficacy of topotecan. These studies were direct comparisons of oral administration with parenteral treatment by the intravenous, intraperitoneal, or subcutaneous routes. Treatment schedules included bolus treatments at 4- or 7-day intervals and a split-dose regimen (q3hx4) repeated at 4- or 7-day intervals. On the various schedules, the maximally tolerated dose of topotecan was either equivalent to or at most 1.7-fold that of parenteral administration, indicative of excellent oral bioavailability in the mouse. Orally administered topotecan was comparable in efficacy to parenteral treatment in four of five tumor models tested (i.v. L1210 leukemia, i.v. B16 melanoma, i.v. and s.c. Lewis lung carcinoma). The M5076 reticulum cell sarcoma implanted i.p. responded to i.p. and s.c. but not to orally administered topotecan. These studies provide convincing support for the clinical evaluation of orally administered topotecan.

High-dose recombinant interleukin-18 induces an effective Th1 immune response to murine MOPC-315 plasmacytoma.

Interleukin (IL)-18 has profound antitumor activity when administered at high doses as a single agent for prolonged periods in BALB/c mice bearing late, well-established MOPC-315 tumors. Management with a qD x 27 schedule resulted in regression of tumors in all animals receiving 5 mg/kg/d. A protracted daily management regimen appears to be necessary to induce regression in this advanced tumor model. Biologic markers were assessed and appear to be potentially useful in evaluating the immunologic and antitumor activity of IL-18. The biomarkers of IL-18s immunologic activity include, but are not limited to, IL-1&agr;, IL-2, IL-8, IL-10, IL-12, IL-13, interferon-&ggr;, tumor necrosis factor-&agr;, and granulocyte-macrophage colony-stimulating factor. The profile of these circulating cytokines and their expression levels at baseline, and after IL-18 delivery, can be measured in the serum, as well as from splenocytes of mice or human peripheral blood mononuclear cells derived from either normal subjects or patients with cancer. We compared IL-18 and IL-12 alone or in combination for their ability to induce cytokine production and natural killer cytolytic activity. Our data support the notion that IL-18 induces a predominantly Th1 response, and that the mechanism of IL-18 activity differs from that of IL-12. The biologic activity of IL-18 management revealed by increases in serum levels of cytokines and enhancement of natural killer cytolytic activity will be useful as clinical trials initiate in 2002. Expression of interferon-&ggr; and granulocyte-macrophage colony-stimulating factor serum levels correlates directly over a broad dose escalation with the level of IL-18. Therefore, this provides a convenient pharmacodynamic reference to the biologic response to IL-18 that may serve to guide the conduct of clinical trials.

Circulating Human Interleukin-8 as an Indicator of Cancer Progression in a Nude Rat Orthotopic Human Non–Small Cell Lung Carcinoma Model

Clinically relevant animal models of human cancer are necessary for the evaluation of putative therapeutics. We hypothesized that circulating human lung cancer–associated proteins would correlate with physiologic measurements from an orthotopic H460 human non–small cell lung carcinoma model that we developed in immunodeficient rats. Physiologic measurements and serum samples were collected over time. Serum interleukin-8 (IL-8), p53, vascular endothelial growth factor, and matrix metalloproteinase-9 were quantitated for correlation with physiologic measurements. Matrix metalloproteinase-9 and p53 were not significantly detectable. Circulating vascular endothelial growth factor was detected at high levels in some tumor-bearing animals. Human IL-8 was detectable in all tumor-bearing animals and correlated positively with markers of respiratory acidosis (pH, P = 0.012; TCO2, P = 0.024; pCO2, P = 0.007; and HCO3−, P = 0.029) and with surface body temperature (P = 0.001) beginning on day 16 after implantation. IL-8 levels negatively correlated with survival (P < 0.001), indicating an association with tumor burden. Circulating human IL-8 might be a useful, clinically relevant circulating tumor protein marker due to its positive correlation with multiple physiologic variables associated with lung cancer progression. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2180–7)

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

Original paperSynthesis and antineoplastic activity of alanyl-2-glycyl peptide derivatives of nocodazoleSynthèse et activité antinéoplastique de dérivés alanyl-2-glycyl peptide de nocodazole

Abstract To provide the antineoplastic agent nocodazole, 1, with improved solubility and drug-transport properties, The (S)-alanyl-2-glycyl moiety was introduced as a benzimidazole-nitrogen-substituent which would be expected to be enzymatically cleaved in vivo to regenerate 1 and achieve enhanced antineoplastic effectiveness. Synthesis produced 2 pairs of diastereo-isomers (2a–d) which were separated and purified by solubility and chromatography. Site of substitution (N1 vs. N3) and chirality were established by 1H NMR nOe difference spectra and CD spectra on 2a–d and derivatives. The compounds were evaluated in cytotoxicity, leukemia, and solid tumor models and as inhibitors of tubulin binding. These test results indicated that these compounds were not functioning as prodrugs for 1. However, the solid tumor model study showed that 2 compounds had equivalent to enhanced antitumor effectiveness over 1 itself.To provide the antineoplastic agent nocodazole, 1, with improved solubility and drug-transport properties, The (S)-alanyl-2-glycyl moiety was introduced as a benzimidazole-nitrogen-substituent which would be expected to be enzymatically cleaved in vivo to regenerate 1 and achieve enhanced antineoplastic effectiveness. Synthesis produced 2 pairs of diastereo-isomers (2a–d) which were separated and purified by solubility and chromatography. Site of substitution (N1 vs. N3) and chirality were established by 1H NMR nOe difference spectra and CD spectra on 2a–d and derivatives. The compounds were evaluated in cytotoxicity, leukemia, and solid tumor models and as inhibitors of tubulin binding. These test results indicated that these compounds were not functioning as prodrugs for 1. However, the solid tumor model study showed that 2 compounds had equivalent to enhanced antitumor effectiveness over 1 itself.