Frances Burke

Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.

Given the associations between chronic inflammation and epithelial cancer we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-α (refs. 5,6). TNF-α–/– mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-α–/– mice developed 5–10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-α could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-α–/– mice. TNF-α was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-α production. Deletion of a TNF-α inducible chemokine also conferred some resistance to skin tumor development. TNF-α has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-α–/– mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-α is important to the early stages of tumor promotion. Strategies that neutralize TNF-α production may be useful in cancer treatment and prevention.

The cytokine network

The availability of pure recombinant cytokines and molecular probes for their genes has generated an avalanche of scientific information. These data show that cytokines have a broad and overlapping range of cell regulatory activity both in vitro and in vivo. New factors are added to the cytokine list, and new functions reported for existing cytokines, with such frequency that it is difficult to retain an overall picture. With this problem in mind, a large wallchart was designed and was displayed at the second meeting of the British Cytokine Group* whose members pooled their collective knowledge, to list the known biological activities of these cytokines. This wallchart of cytokine activity, now referenced, is reproduced for Immunology Today. It is not a final list: new information and cytokines are continually reported and space has been left for readers to make their own additions. A neutrophil-activating peptide variously named monocyte-derived neutrophil chemotactic factor (MDNCF), neutrophil-activating factor (NAF), lymphocyte-derived neutrophil-activating peptide (LYNAP), which has been suggested as a candidate for interleukin 8 (IL-8), is included.

EVIDENCE FOR TUMOUR NECROSIS FACTOR/CACHECTIN PRODUCTION IN CANCER

Labile tumour-necrosis-factor-like (TNF) activity was detected by means of an enzyme-linked immunosorbent assay in 50% of 226 freshly obtained serum samples from cancer patients with active disease. In contrast, only 3% of 32 samples from normal subjects and 18% of 39 samples from cancer patients with no clinically evident disease were positive for this factor, with low levels of activity. Greater proportions of serum samples from patients with ovarian or oat-cell carcinoma were positive (69% and 63%) than those from patients with lymphoma (26%). RNA preparations from peripheral-blood mononuclear cells and solid tumours were probed with TNF complementary DNA; evidence of TNF messenger RNA was found in 8 of 11 samples of peripheral-blood mononuclear cells from cancer patients, but only 1 of 8 normal subjects, and in 2 of 6 colorectal tumours. As yet the inducing stimulus and the clinical significance of TNF production in cancer are not understood.

Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype.

Tumor-associated macrophages (TAM) may have tumor-promoting activity, but it is not clear how their phenotype is achieved. In this study, we demonstrate that ovarian cancer cells switch cocultured macrophages to a phenotype similar to that found in ovarian tumors. Tumor cells caused dynamic changes in macrophage cytokine, chemokine, and matrix metalloprotease mRNA, and protein-inducing mediators that are found in human cancer. Macrophage mannose, mannose receptor, and scavenger receptors (SR-As) were also up-regulated by coculture, but not by conditioned medium. To further validate the model, we studied SR-A regulation on TAM in vitro and in vivo. Coculture of murine macrophages from mice deficient in TNF-α or its receptors revealed that TNF-α was key to SR-A induction via its p75 receptor. SR-A expression was also reduced in TAM from ovarian cancers treated with anti-TNF-α Abs or grown in TNF-α−/− mice. Chemical communication between tumor cells and macrophages may be important in regulating the cancer cytokine microenvironment.

The detection and localization of monocyte chemoattractant protein-1 (MCP-1) in human ovarian cancer.

Chemokines may control the macrophage infiltrate found in many solid tumors. In human ovarian cancer, in situ hybridization detected mRNA for the macrophage chemokine monocyte chemoattractant protein-1 (MCP-1) in 16/17 serous carcinomas, 4/4 mucinous carcinomas, 2/2 endometrioid carcinomas, and 1/3 borderline tumors. In serous tumors, mRNA expression mainly localized to the epithelial areas, as did immunoreactive MCP-1 protein. In the other tumors, both stromal and epithelial expression were seen. All tumors contained variable numbers of cells positive for the macrophage marker CD68. MCP-1 mRNA was also detected in the stroma of 5/5 normal ovaries. RT-PCR demonstrated mRNA for MCP-1 in 7/7 serous carcinomas and 6/6 ovarian cancer cell lines. MCP-1 protein was detected by ELISA in ascites from patients with ovarian cancer (mean 4.28 ng/ml) and was produced primarily by the cancer cells. Human MCP-1 protein was also detected in culture supernatants from cell lines and in ascites from human ovarian tumor xenografts which induce a peritoneal monocytosis in nude mice. We conclude that the macrophage chemoattractant MCP-1 is produced by epithelial ovarian cancer and that the tumor cells themselves are probably a major source. MCP-1 may contribute to the accumulation of tumor-associated macrophages, which may subsequently influence tumor behavior.

Expression and regulation of tumor necrosis factor alpha in normal and malignant ovarian epithelium.

Epidemiologic studies implicate inflammatory stimuli in the development of ovarian cancer. The proinflammatory cytokine tumor necrosis factor α (TNF-α) and both its receptors (TNFRI and TNFRII) are expressed in biopsies of this malignancy. Here, we tested the hypothesis that TNF-α is a regulator of the proinflammatory microenvironment of ovarian cancer. A cancer profiling array showed higher expression of TNF-α in ovarian tumors compared with normal ovarian tissue, and cultured ovarian cancer cells expressed up to 1,000 times more TNF-α mRNA than cultured normal ovarian surface epithelial cells; TNF-α protein was only detected in the supernatant of tumor cell cultures. Treatment with TNF-α induced TNF-α mRNA via TNFRI in both malignant and normal cells with evidence for enhanced TNF-α mRNA stability in tumor cells. TNF-α induced TNF-α protein in an autocrine fashion in tumor but not in normal ovarian surface epithelial cells. The TNF-α neutralizing antibody infliximab reduced the constitutive levels of TNF-α mRNA in tumor cell lines capable of autocrine TNF-α production. Apart from TNF-α mRNA expression, several other proinflammatory cytokines were constitutively expressed in malignant and normal ovarian surface epithelial cells, including interleukin (IL)-1α, IL-6, CCL2, CXCL8, and M-CSF. TNF-α treatment further induced these cytokines with de novo transcription of IL-6 mRNA contrasting with the increased stability of CCL2 mRNA. RNA interference directed against TNF-α was highly effective in abolishing constitutive IL-6 production by ovarian tumor cells. In summary, we show that TNF-α is differentially regulated in ovarian cancer cells compared with untransformed cells and modulates production of several cytokines that may promote ovarian tumorigenesis. Infliximab treatment may have a role in suppressing the TNF-α-driven inflammatory response associated with ovarian cancer. [Mol Cancer Ther 2006;5(2):382-90]

Hypoxia down-regulates MCP-1 expression: implications for macrophage distribution in tumors

Monocyte chemoattractant protein 1 (MCP‐1) is likely to contribute to the macrophage infiltrate in human ovarian carcinomas. Although MCP‐1 is predominantly expressed by the tumor parenchyma, macrophages accumulate at highest density in necrotic regions, which are associated with low oxygen tensions. Tumor necrosis factor α (TNF‐α) can stimulate MCP‐1 production and is also present within ovarian carcinomas. We have investigated the effect of hypoxia both on MCP‐1 expression in ovarian cancer cell lines and monocyte migration. Hypoxia down‐regulated TNF‐α‐induced MCP‐1 mRNA and protein production by ovarian cancer cells. The effect was mimicked by cobalt chloride and desferrioxamine, consistent with a specific oxygen‐sensing mechanism. Unlike antioxidants, hypoxia did not inhibit nuclear factor κB mobilization. Monocyte migration in response to MCP‐1 was also diminished under hypoxic conditions. Down‐regulation of MCP‐1 expression and the inhibition of monocyte migration are independent effects of hypoxia that may contribute to the distribution of macrophages within ovarian tumors. J. Leukoc. Biol. 63: 758–765; 1998.

Activity of human monocytes in IgE antibody-dependent surveillance and killing of ovarian tumor cells

We have previously shown that a chimeric IgE antibody against the folic acid receptor (MOv18 IgE) inhibits tumor growth in a SCID mouse model of ovarian carcinoma. MOv18 IgE gave greater protection than the corresponding chimeric MOv18 IgG1. We have now confirmed these effects in a nude‐mouse model of ovarian carcinoma and have demonstrated for the first time that human monocytes are active in IgE antibody‐dependent cell‐mediated cytotoxicity. Injection of tumor‐bearing mice with PBMC and MOv18 IgE led to infiltration of monocytes into the tumors and prolonged survival of the mice.Incubation of PBMC or purified monocytes and MOv18 IgE with ovarian tumor cells in vitro resulted in tumor cell killing proportional to the expression of unoccupied FcϵRI on monocytes.We observed phagocytosis of tumor cells by the monocytes in vitro. Our results suggest that tumor‐specific IgE antibodies may be exploited for immunotherapy of cancer.

Interferon gamma induces cell cycle arrest and apoptosis in a model of ovarian cancer: Enhancement of effect by batimastat

Locoregional human IFN-gamma may have activity against refractory ovarian cancer. We investigated this further in an ovarian cancer xenograft model. Administered at clinically relevant doses, intraperitoneal IFN-gamma prolonged the survival of mice bearing multiple established peritoneal tumours, with optimal treatment giving a 3-6-fold increase in median survival time. Daily dosing, which was superior to intermittent treatment, decreased DNA synthesis and induced apoptosis in tumour cells with maximal effects after 7-21 days treatment. This was preceded by an increase in p53 protein at 48 h. The effect of IFN-gamma was not enhanced by sequential treatment with carboplatin. However, the matrix metalloprotease inhibitor, batimastat, further increased mouse survival when given after IFN-gamma. Thus IFN-gamma is cytotoxic to ovarian epithelial cells in vivo and intensive locoregional dosing over short periods is effective. Sequential administration of novel agents that perturb the host/tumour relationship may be of benefit.

Aberrant regulation of argininosuccinate synthetase by TNF‐α in human epithelial ovarian cancer

The pro‐inflammatory cytokine, tumour necrosis factor‐α, TNF‐α, is dysregulated in malignant compared with normal ovarian surface epithelium (OSE). Several epidemiological studies have associated inflammation with ovarian tumorigenesis, with TNF‐α playing a key role in modulating invasion, angiogenesis and metastasis. Here, we show that TNF‐α also induces expression of arate‐limiting enzyme in arginine synthesis, argininosuccinate synthetase (AS), thereby linking inflammation with several arginine‐dependent metabolic pathways, implicated in accelerated carcinogenesis and tumour progression. Having identified AS mRNA induction in TNF‐α‐treated IGROV‐1 ovarian cancer cells, using RNA‐arbitrarily primed‐PCR, we then observed differential regulation of AS mRNA and protein in malignant, compared with normal, OSE cells. A cDNA cancer profiling array with matched normal ovarian and ovarian tumour samples revealed increased expression of AS mRNA in the latter. Moreover, AS protein co‐localised with TNF‐α in ovarian cancer cells, with significantly higher levels of AS in malignant compared with normal ovarian tissue. Increased co‐expression of AS and TNF‐α mRNA was also observed in 2 other epithelial tumours, non‐small cell lung and stomach cancer, compared with normal corresponding tissues. In summary, high levels of AS expression, which may be required for several arginine‐dependent processes in cancer, including the production of nitric oxide, proline, pyrimidines and polyamines, is regulated by TNF‐α and may provide an important molecular pathway linking inflammation and metabolism to ovarian tumorigenesis.