Cyclooxygenase-2 up-regulates CCR7 via EP2/EP4 receptor signaling pathways to enhance lymphatic invasion of breast cancer cells

Recent studies demonstrate that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis. However, the mechanism by which COX-2 increases the invasion of cancer cells to lymph node is unclear. CCR7 is a chemokine receptor that plays important roles in the mediation of migration of leukocytes and dendritic cells toward lymphatic endothelial cells (LECs) that express receptor ligand CCL21. We found that treatment of prostaglandin E(2) or ectopic expression of COX-2 in MCF-7 cells up-regulated CCR7 expression. On the contrary, knockdown of COX-2 by small hairpin RNA reduced CCR7 in COX-2-overexpressing MDA-MB-231 cells. Interaction of CCR7 and CCL21 was important for the migration of breast cancer cells toward LECs because antibodies against these two molecules inhibited the migration. We also found that COX-2 increased CCR7 expression via the EP2 and EP4 receptor in breast cancer cells. EP2 and EP4 agonists stimulated CCR7 in MCF-7 cells, whereas antagonists or small hairpin RNA of EP2 and EP4 attenuated CCR7 in MDA-MB-231 cells. Protein kinase A and AKT kinase were involved in COX-2-induced CCR7. Pathological analysis demonstrated that COX-2 overexpression was associated with CCR7, EP2, and EP4 expressions in breast tumor tissues. In addition, CCR7 expression in COX-2-overexpressing tumors was significantly correlated with lymph node metastasis. Collectively, we suggest that CCR7 is a down-stream target for COX-2 to enhance the migration of breast cancer cells toward LECs and to promote lymphatic invasion.     

Expressions of ER,PR, HER-2, COX-2, and VEGF in Primary and Relapsed/Metastatic Breast Cancers

In the present study, we evaluated expressions of estrogen receptor (ER), progestin receptor (PR), human epidermal growth factor receptor-2 (HER-2), cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) in primary and relapsed/metastatic breast cancers to elucidate the clinical significance of these markers. The markers were evaluated by immunohistochemistry in specimens of 50 patients with primary or metastatic breast cancer. Positive rates of ER were significantly (p = 0.002) higher in primary versus relapsed/metastatic breast cancer (70 vs. 38 %, respectively). The VEGF positive expression rates were also significantly higher in primary versus metastatic cancer (82 vs. 38 %, respectively; p < 0.001). By contrast, positive rates of HER-2 and COX-2 were not significantly different between different types of cancer. COX-2 correlated with HER-2 expression in both primary and relapsed/metastatic focuses of breast cancer. COX-2 also correlated with VEGF expression in primary breast cancer. Expressions of ER, PR, HER2, and COX-2 did not correlate between primary and relapsed/metastatic breast cancers, indicating that the treatment decision should be made according to the status of these markers in relapsed/metastatic focuses. The total change rates of ER, PR, HER-2, COX-2, and VEGF were 26, 18, 10, 30, and 58 %, respectively. In conclusion, HER-2 and COX-2, along with VEGF, appear to play a role in the development and progression of breast cancer. In addition, all of the studied markers may serve as indicators of prognosis.     

Progesterone receptor inhibits aromatase and inflammatory response pathways in breast cancer cells via ligand-dependent and ligand-independent mechanisms

Aromatase (product of CYP19 gene), the critical enzyme in estrogen biosynthesis, is up-regulated in 70% of all breast cancers and is highly correlated with cyclooxygenase 2 (COX-2), the rate-determining enzyme in prostanoid biosynthesis. Expression of COX-2 also is correlated with the oncogene HER-2/neu. The efficacy of current endocrine therapies for breast cancer is predicted only if the tumor contains significant amounts of estrogen receptor. Because the progesterone receptor (PR) is an estrogen-induced target gene, it has been suggested that its presence may serve as an indicator of estrogen receptor functional capacity and the differentiation state of the tumor. In the present study, we tested the hypothesis that PR serves a crucial protective role by antagonizing inflammatory response pathways in the breast. We observed that progesterone antagonized the stimulatory effects of cAMP and IL-1beta on aromatase, COX-2, and HER-2/neu expression in T47D breast cancer cells. These actions of progesterone were associated with increased expression of the nuclear factor-kappaB inhibitor, IkappaBalpha. In 28 breast cancer cell lines, IkappaBalpha expression was positively correlated with PR mRNA levels; overexpression of a phosphorylation-defective mutant of IkappaBalpha inhibited expression of aromatase, COX-2, and HER-2/neu. Moreover, in breast cancer cell lines cultured in the absence of progesterone, up-regulation of endogenous PR caused decreased expression of aromatase, COX-2, and HER-2/neu expression, whereas down-regulation of endogenous PR resulted in a marked induction of aromatase and HER-2/neu mRNA. Collectively, these findings suggest that PR plays an important antiinflammatory role in breast cancer cells via ligand-dependent and ligand-independent mechanisms.     

Age-dependent expression of glucocorticoid- and mineralocorticoid receptors on neural precursor cell populations in the adult murine hippocampus

Steroid hormones are regulators of adult hippocampal neurogenesis and are central to hypotheses regarding adult neurogenesis in age-related and psychiatric disturbances associated with altered hippocampal plasticity--most notably dementias and major depression. Using immunohistochemistry, we examined the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors during adult hippocampal neurogenesis. In young mice only 27% of dividing cells in the subgranular zone expressed GR, whereas 4 weeks after division 87% had become positive for GR and MR. GR was expressed by 50% of the radial glia-like type-1 and type-2a progenitor cells, whereas MR was expressed only by mature calbindin-positive granule cells. Doublecortin-positive neuronal progenitor cells (type-2b) and early postmitotic calretinin-positive neurons were devoid of GR and MR expression. Fifty per cent of the intermediate type-3 cells showed GR expression, possibly reflecting cells terminating maturation. Thus, all subpopulations of dividing precursor cells showed an identical receptor profile (50% GR, no MR), except for type-2b cells, which expressed neither receptor. There was also no overlap between calretinin and GR early postnatally (P8) or after physical activity or exposure to an enriched environment, both of which are potent neurogenic stimuli. In contrast, in old age calretinin-positive young neurons became GR and MR positive, suggesting increased steroid sensitivity. Age also increased the expression of GR in type-1 and type-2a precursor cells. Other intermediates were so rare in old age that they could not be studied. This course and variability of receptor expression in aging might help to explain differential vulnerability of adult neural precursor cells to corticoid-mediated influences.     

Expression of the 11beta-hydroxysteroid dehydrogenase type II enzyme in breast tumors and modulation of activity and cell growth in PMC42 cells

Manipulating the metabolism of glucocorticoids may serve as a useful adjunct in the treatment of breast cancer. The 11β-hydroxysteroid dehydrogenase type 2 enzyme (11βHSD2) potently inactivates glucocorticoids thereby protecting the non-selective mineralocorticoid receptor (MR) in fluid transporting tissues. In the present study, Western blot analysis showed the presence of 11βHSD2 in 66% of the breast tumor samples. The 11βHSD2 and MR are also present in the breast tumor cell line PMC42. Glycyrrhetinic acid abolished glucocorticoid metabolism and inhibited cell growth by 40%, the latter at concentrations consistent with glucocorticoid receptor (GR) and MR binding studies. Metabolism was increased by glucocorticoids, the anti-glucocorticoid RU 38486 and anti-mineralocorticoid spironolactone, while aldosterone had no effect. Neither cortisol nor aldosterone affected cell proliferation, but both RU 38486 and spironolactone caused a significant decrease in cell number. The effects of RU 38486 were only observed at micromolar concentrations and are inconsistent with an action via GR or progesterone receptor (PR). This study shows that 11βHSD2 activity and cell proliferation of PMC42 cells can be modulated via steroid receptors.     

Uteroplacental insufficiency alters nephrogenesis and downregulates cyclooxygenase-2 expression in a model of IUGR with adult-onset hypertension

Clinical and animal studies indicate that intrauterine growth restriction (IUGR) following uteroplacental insufficiency (UPI) reduces nephron number and predisposes toward renal insufficiency early in life and increased risk of adult-onset hypertension. In this study, we hypothesized that the inducible enzyme cyclooxygenase-2 (COX-2), a pivotal protein in nephrogenesis, constitutes a mechanism through which UPI and subsequent glucocorticoid overexposure can decrease nephron number. We further hypothesized that UPI downregulates the key enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts corticosterone to inert 11-dehydrocorticosterone, thereby protecting both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) from the actions of corticosterone. Following bilateral uterine ligation on the pregnant rat, UPI significantly decreased renal COX-2, 11beta-HSD2, and GR mRNA and protein levels, but upregulated expression of MR at birth. At day 21 of life, 11beta-HSD2, GR, and also MR mRNA and protein levels were downregulated. UPI did not affect blood pressures (BP) at day 21 of life but significantly increased systolic BP in both genders at day 140. We conclude that in our animal model, UPI decreases fetal COX-2 expression during a period of active nephrogenesis in the IUGR rat, which is also characterized by decreased nephron number and adult-onset hypertension.     

Expression of glucocorticoid receptor isoforms in human adrenocortical adenomas

Introduction

Glucocorticoid receptor (GR) is expressed in the normal human adrenal gland, however, no study has been performed to evaluate the separate expression of α- and β-isoforms (GRα and GRβ) in normal human adrenals and in adrenocortical adenomas.

Experimental

GRα and GRβ mRNA expression was examined by quantitative real-time PCR in 31 adrenal tissues including 19 non-functioning adenomas (NFA), 6 cortisol-producing adenomas (CPA) and 6 normal adrenocortical tissues. In addition, the presence and cellular localization of GRα and GRβ proteins in adrenal tissues were studied by immunohistochemistry.

Results

Compared to normal adrenocortical tissues, both GRα and GRβ mRNAs were significantly increased in CPA but not in NFA. Using anti-GRα antibody a strong nuclear staining was observed in NFA and CPA, and a less remarkable immunoreactivity was detected in some nuclei of normal adrenocortical cells. GRβ immunostaining was absent in normal adrenal tissues and NFA, while a strong cytoplasmic and nuclear immunoreaction was found in CPA.

Conclusions

Altered expression of GRα and GRβ in CPA raises their possible role in the pathophysiology of these adrenal tumors, although further studies are needed to elucidate the potential significance of these findings.     

Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C(19) androgens to C(18) estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE(2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFbeta did not alter COX-2 levels at 24h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFbeta did not. In contrast to MCF-7 cells, TGFbeta and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFbeta, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFbeta and TPA after 24h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE(2) from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.     

Mannose-containing molecular patterns are strong inducers of cyclooxygenase-2 expression and prostaglandin E2 production in human macrophages

The induction of cyclooxygenase-2 (COX-2) and the production of PGE(2) in response to pathogen-associated molecular patterns decorated with mannose moieties were studied in human monocytes and monocyte-derived macrophages (MDM). Saccharomyces cerevisiae mannan was a robust agonist, suggesting the involvement of the mannose receptor (MR). MR expression increased along the macrophage differentiation route, as judged from both its surface display assessed by flow cytometry and the ability of MDM to ingest mannosylated BSA. Treatment with mannose-BSA, a weak agonist of the MR containing a lower ratio of attached sugar compared with pure polysaccharides, before the addition of mannan inhibited COX-2 expression, whereas this was not observed when agonists other than mannan and zymosan were used. HeLa cells, which were found to express MR mRNA, showed a significant induction of COX-2 expression upon mannan challenge. Conversely, mannan did not induce COX-2 expression in HEK293 cells, which express the mRNA encoding Endo180, a parent receptor pertaining to the MR family, but not the MR itself. These data indicate that mannan is a strong inducer of COX-2 expression in human MDM, most likely by acting through the MR route. Because COX-2 products can be both proinflammatory and immunomodulatory, these results disclose a signaling route triggered by mannose-decorated pathogen-associated molecular patterns, which can be involved in both the response to pathogens and the maintenance of homeostasis.     

Role of prostaglandin E produced by osteoblasts in osteolysis due to bone metastasis

Prostaglandin E2 (PGE2) is produced in bone mainly by osteoblasts and stimulates bone resorption. Osteolytic bone metastasis of cancers is accompanied by bone resorption. In this study, we examined the roles of PGE2 in osteolysis due to bone metastasis of breast cancer. Injection of human breast cancer cells, MDA-MB-231 (MDA-231), into nude mice causes severe osteolysis in the femur and tibia. The expression of cyclo-oxygenase-2 (COX-2) and the receptor activator of NF-kappaB ligand (RANKL), a key molecule in osteoclast differentiation, mRNAs was markedly elevated in bone with metastasis. When MDA-231 cells were cocultured with mouse calvaria, COX-2-induced PGE2 production and bone resorption progressed. The contact with MDA-231 cells could induce the expression of COX-2 and RANKL in osteoblasts by mechanisms involving MAP kinase and NF-kappaB. The blockage of PGE2 signal by indomethacin and EP4 antagonist abrogated the osteoclast formation induced by the breast cancer cells. Here, we show a PGE-dependent mechanism of osteolysis due to bone metastasis.     

TREM-1 Is Induced in Tumor Associated Macrophages by Cyclo-Oxygenase Pathway in Human Non-Small Cell Lung Cancer

It is increasingly recognized that the tumor microenvironment plays a critical role in the initiation and progression of lung cancer. In particular interaction of cancer cells, macrophages, and inflammatory response in the tumor microenvironment has been shown to facilitate cancer cell invasion and metastasis. The specific molecular pathways in macrophages that immunoedit tumor growth are not well defined. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a member of the super immunoglobulin family expressed on a select group of myeloid cells mainly monocyte/macrophages. Recent studies suggest that expression of TREM-1 in tumors may predict cancer aggressiveness and disease outcomes in liver and lung cancer however the mechanism of TREM-1 expression in the setting of cancer is not defined. In this study we demonstrate that tumor tissue from patients with non-small cell lung cancer show an increased expression of TREM-1 and PGE₂. Immunohistochemistry and immunofluorescence confirmed that the expression of TREM-1 was selectively seen in CD68 positive macrophages. By employing an in vitro model we confirmed that expression of TREM-1 is increased in macrophages that are co-cultured with human lung cancer cells. Studies with COX-2 inhibitors and siCOX-2 showed that expression of TREM-1 in macrophages in tumor microenvironment is dependent on COX-2 signaling. These studies for the first time define a link between tumor COX-2 induction, PGE₂ production and expression of TREM-1 in macrophages in tumor microenvironment and suggest that TREM-1 might be a novel target for tumor immunomodulation.     

Tie2: a journey from normal angiogenesis to cancer and beyond

The tyrosine kinase receptor Tie2 was initially identified as a specific vascular growth factor that governed several properties of endothelial cells under both physiological and pathological conditions. It was subsequently found that angiopoietins, the natural ligands of Tie2, modulate Tie2-dependent signaling, which in turn regulates the survival and apoptosis of endothelial cells, controls vascular permeability, and regulates the capillary sprouting that occurs during normal angiogenesis such as through development and ovarian remodeling. Tie2 also seems to play a crucial role in several vascular abnormalities, such as familial venous malformations. Beyond its critical role in angiogenesis, Tie2 also appears to maintain the long-term population and quiescent status of hematopoietic stem cells in the bone marrow stem cell niche. In cancer, Tie2 was originally found to be overexpressed in tumoral vessels. More recently, our laboratory and others have found that Tie2 is also expressed outside the vascular compartment in several types of cancer, including leukemia and solid neoplasms such as gastric tumors, breast tumors, and gliomas. The role of Tie2 in these tumoral cells is currently being explored. In this regard, our group reported the importance of Tie2-expressing glioma cells in their adhesion to the tumoral microenvironment. Because cancer may be considered as a complex organ with several cellular lineages coexisting in the same tumor, the expression of Tie2 by different tumoral compartments makes this cellular receptor an attractive target for cancer therapy.     

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.