Julia Wilson

Macrophages Induce Invasiveness of Epithelial Cancer Cells Via NF-κB and JNK

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-α- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-α-dependent activation of JNK and NF-κB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-κB activity exhibited enhanced invasiveness. Inhibition of the NF-κB pathway by TNF-α neutralizing Abs, an NF-κB inhibitor, RNAi to RelA, or overexpression of IκB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-κB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-κB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-α, via NF-κB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.

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 Inflammatory Cytokine Tumor Necrosis Factor-α Generates an Autocrine Tumor-Promoting Network in Epithelial Ovarian Cancer Cells

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-alpha, constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.

The Inflammatory Cytokine Tumor Necrosis Factor-α Regulates Chemokine Receptor Expression on Ovarian Cancer Cells

Epithelial ovarian cancer cells express the chemokine receptor, CXCR4, which may be associated with increased survival and metastatic potential, but the regulation of this receptor is not understood. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is found in ovarian cancer biopsies and is associated with increased tumor grade. In this report, we show that CXCR4 expression on human epithelial ovarian cancer cells is associated with, and can be modulated by, TNF-alpha. Ovarian cancer cells with high endogenous expression of TNF-alpha expressed higher levels of CXCR4 mRNA and protein than cells with low TNF-alpha expression. Stimulation of ovarian cancer cell lines and primary epithelial cancer cells with TNF-alpha resulted in increased CXCR4 mRNA and protein. The TNF-alpha-stimulated increase in CXCR4 mRNA was due partly to de novo synthesis, and up-regulation of CXCR4 cell surface protein increased migration to the CXCR4 ligand CXCL12. CXCR4 mRNA and protein was down-regulated by anti-TNF-alpha antibody or by targeting TNF-alpha mRNA using RNAi. TNF-alpha stimulation activated components of the nuclear factor kappaB pathway, and overexpression of the inhibitor of kappaB also reduced CXCR4 expression. Coculture of macrophages with ovarian cancer cells also resulted in cancer cell up-regulation of CXCR4 mRNA in a TNF-alpha-dependent manner. Finally, there was a correlation between the levels of TNF-alpha and CXCR4 mRNA in clinical biopsies of ovarian cancer, and TNF-alpha protein was expressed in CXCR4-positive tumor cells. TNF-alpha is a critical mediator of tumor promotion in a number of experimental cancers. Our data suggest that one mechanism may be through nuclear factor kappaB-dependent induction of CXCR4.

Endothelin-2 is a macrophage chemoattractant: implications for macrophage distribution in tumors

Endothelins (ET‐1, ET‐2 and ET‐3) are 21‐amino acid vasoactive peptides that bind to G‐protein‐linked transmembrane receptors, ET‐RA and ET‐RB. As well as modulating vasoconstriction, endothelins regulate growth in several cell types and may also affect differentiation, inflammation and angiogenesis. Both macrophages and endothelins are found in areas of hypoxia in solid tumors and ET‐2 expression may be modulated by hypoxia in some tumors. As the peptide structure of mature endothelins is similar to that of CXC chemokines, we asked if endothelins contribute to control of macrophage distribution in tumors. We found that ET‐2 is a chemoattractant for macrophages and THP‐1 monocytic cells, but not for freshly isolated monocytes. The chemotactic response to ET‐2 shows a typical bell‐shaped response curve. Experiments with endothelin receptor antagonists showed that migration to ET‐2 is mediated via the ET‐RB receptor. Moreover, monocytes do not express ET‐RB. Chemotaxis towards ET‐2 is via the MAPK pathway: p44 and p42 are phosphorylated when THP‐1 cells are stimulated with ET‐2, and the MAPKK inhibitor PD98059 stops chemotaxis. As with ‘classical’ chemokines, migration toET‐2 is also inhibited by hypoxia and by pertussis toxin. As well as its chemotactic properties, ET‐2 leads to activation of macrophages. In human breast tumors that express ET‐2, endothelins and ET‐RB expressing macrophages often co‐localized. While shorter than ‘classical’ chemokines, ET‐2 shares a similar peptide sequence with chemokines and may signal via a similar receptor and MAPK‐mediated pathway. Furthermore, ET‐2 expression by tumors may modulate the behavior of macrophages such that activated cells accumulate in areas of hypoxia.

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]

Analysis of CC chemokine and chemokine receptor expression in solid ovarian tumours.

To understand the chemokine network in a tissue, both chemokine and chemokine receptor expression should be studied. Human epithelial ovarian tumours express a range of chemokines but little is known about the expression and localisation of chemokine receptors. With the aim of understanding chemokine action in this cancer, we investigated receptors for CC–chemokines and their ligands in 25 biopsies of human ovarian cancer. CC–chemokine receptor mRNA was generally absent from solid tumours, the exception being CCR1 which was detected in samples from 75% of patients. CCR1 mRNA localised to macrophages and lymphocytes and there was a correlation between numbers of CD8+ and CCR1 expressing cells (P = 0.031). mRNA for 6 CC-chemokines was expressed in a majority of tumour samples. In a monocytic cell line in vitro, we found that CCR1 mRNA expression was increased 5-fold by hypoxia. We suggest that the CC-chemokine network in ovarian cancer is controlled at the level of CC-chemokine receptors and this may account for the phenotypes of infiltrating cells found in these tumours. The leukocyte infiltrate may contribute to tumour growth and spread by providing growth survival factors and matrix metalloproteases. Thus, CCR1 may be a novel therapeutic target in ovarian cancer.

Molecular profiling of cervical cancer progression

Most cancer patients die of metastatic or recurrent disease, hence the importance to identify target genes upregulated in these lesions. Although a variety of gene signatures associated with metastasis or poor prognosis have been identified in various cancer types, it remains a critical problem to identify key genes as candidate therapeutic targets in metastatic or recurrent cancer. The aim of our study was to identify genes consistently upregulated in both lymph node micrometastases and recurrent tumours compared to matched primary tumours in human cervical cancer. Taqman Low-Density Arrays were used to analyse matched tumour samples, obtained after laser-capture microdissection of tumour cell islands for the expression of 96 genes known to be involved in tumour progression. Immunohistochemistry was performed for a panel of up- and downregulated genes. In lymph node micrometastases, most genes were downregulated or showed expressions equal to the levels found in primary tumours. In more than 50% of lymph node micrometastases studied, eight genes (AKT, BCL2, CSFR1, EGFR1, FGF1, MMP3, MMP9 and TGF-β) were upregulated at least two-fold. Some of these genes (AKT and MMP3) are key regulators of epithelial–mesenchymal transition in cancer. In recurrent tumours, almost all genes were upregulated when compared to the expression profiles of the matched primary tumours, possibly reflecting their aggressive biological behaviour. The two genes showing a consistent downregulated expression in almost all lymph node metastases and recurrent tumours were BAX and APC. As treatment strategies are very limited for metastatic and recurrent cervical cancer, the upregulated genes identified in this study are potential targets for new molecular treatment strategies in metastatic or recurrent cervical cancer.

Endothelins Induce CCR7 Expression by Breast Tumor Cells via Endothelin Receptor A and Hypoxia-Inducible Factor-1

Endothelin expression is increased in breast tumors and is associated with invasion and metastasis, whereas CCR7 expression by breast tumor cells may have a role in the organ specificity of breast cancer spread. In this article, we have analyzed whether endothelins influence breast tumor cell expression of the chemokine receptor CCR7. Stimulation of human breast tumor cell lines with endothelins increased cell surface expression of CCR7 via endothelin receptor A. The iron chelators desferrioxamine and cobalt chloride, which induce hypoxia-inducible factor (HIF)-mediated transcription, also increased CCR7 expression; transfection of a dominant-negative version of the HIF regulatory subunit, HIF-1alpha, into MCF-7 cells abolished CCR7 induction by endothelins, indicating that increased expression is due to HIF-1 stabilization. Endothelin stimulation promoted invasion toward the CCR7 ligands CCL19 and CCL21. Endothelin-mediated chemokine-independent invasion itself is dependent on CCR7 activity and could be abolished using a CCR7-neutralizing monoclonal antibody. In human breast carcinomas, mRNA expression of endothelins correlated with the level of CCR7 expression, both of which were associated with the presence of lymph node metastases. Expression of the CCR7 ligands CCL19 and CCL21 was also higher in breast cancer patients with lymph node involvement compared with those without, but expression of these chemokines did not correlate with endothelin expression. These data show that CCR7 may be regulated by the breast tumor microenvironment and further support the use of endothelin receptor antagonists in the treatment of invasive and metastatic breast cancer.

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.