Increased Metabolites Of5-lipoxygenase From Hypoxic Ovarian Cancer Cells Promote Tumor-associated Macrophage Infiltration

BackgroundThe microenvironment of most solid tumors is comprised of a variety of nonmalignant stromal cells which play critical roles in tumor progression and metastasis. Among them, tumor-associated macrophages (TAMs) are the most notable infiltrating inflammatory leukocytes. Many observations have indicated that TAMs usually exhibit M2-like phenotype, secreting a vast diversity of growth factors, proteolytic enzymes, and pro-angiogenic cytokines and immunosuppressive cytokines. TAMs could promote tumor progression by favoring angiogenesis and lymphoangiogenesis, ECM remodelling and the taming of adaptive immunity. Clinical and epidemiological studies have shown that a high abundance of TAMs is strongly correlated with the poor prognosis in several types of cancer, such as breast, prostate, ovarian and cervical cancers. Recruitment of TAMs is one of the primary events in tumor development and metastasis. TAMs are often thought to be derived from peripheral blood monocytes that are recruited into the tumor from the local circulation, and strongly influenced by the microenvironmental factors present within the developing tumor. However, TAMs are found not only in well-vascularized stromal areas but also in avascular and hypoxic areas, where they are often in high density and in close proximity to tumor cells. The latter phenomenon has been reported in breast and bladder tumors.5-lipoxygenase (5-LOX) is a member of the lipoxygenase gene family and expressed in the ovarian cancer and ovarian cancer cell line. It is also a key enzyme assisting the conversion of arachidonic acid to5-HETE and leukotrienes (LTs, LTB4, LTC4, etc.). Studies indicated that5-HETE and its derived products were potent inducers of neutrophil chemotactic migration through endothelial and epithelial barriers. LTB4could attract leucocytes to the sites of inflammation and activate them for host defence. Consequently, the metabolites of5-LOX may play a critical role in TAM recruitment in the hypoxic regions of tumors. PurposeTumor-associated macrophages (TAM) play a critical role in the progression and metastasis of many tumors, including ovarian tumor. TAMs were often found in a high density in the hypoxic areas of tumors. However, the relevant mechanisms have not been studied explicitly until now. In this study, we aimed to investigate potential role of5-LOX metabolites from hypoxic ovarian cancer cells in TAM infiltration in vitro and in vivo.Experimental Design1. Immunochemical staining was used to detect the expression of5-LOX, CD68(a marker of human macrophages) and HIF-la in human primary ovarian tumor specimens.2. In human ovarian tumor tissues, the expression of5-LOX, CD68and HIF-la were detected, and clinicopathological variables were analyzed.3. Western blotting, qRT-PCR and ELISA were used to detect the expression of key proteins in5-LOX pathway and the production of metabolites.4. The migration and invasion of macrophages in response to5-LOX metabolites were detected by transwell.5. Western blotting, qRT-PCR, transwell, immunofluorescence and immunohistochemistry staining were used to detect the expression of MMP-7in response to5-LOX metabolites.6. Western blotting was used to detect signaling pathway in5-HETE/LTB4-enhanced MMP-7expression.7. Release of TNF-α and HB-EGF by macrophages were examined by ELISA in response to metabolites of5-LOX.8. We next investigated whether administration of Zileuton inhibited MMP-7expression and decreased macrophages infiltration in xenograft. Nude mice bearing SKOV-3ovarian tumor xenografts were dosed with Zileuton. The expression of F4/80,5-LOX, HIF-la and MMP-7were detected by immunofluorescence and immunohistochemistry assay.Results1.5-LOX level correlates with the density of TAM and HIF-la level in human ovarian tumor tissues High density of TAMs has been found in hypoxic area of ovarian cancer. To examine whether the expression of5-LOX correlate with the density of TAM and HIF-1α level in human ovarian cancer, we detected the expression of5-LOX, CD68(a marker of human macrophages) and HIF-la in71human primary ovarian tumor specimens by immunochemical staining. As shown in Figure1α and b, the expression of5-LOX was strongly correlated with the density of TAM and HIF-la level. Consistent with this finding, the density of TAM was also associated with HIF-la level. Taken together, these results demonstrate that the expression of5-LOX correlates with the density of TAM and HIF-1α level in ovarian tumor.2.5-LOX level and the density of TAM are associated with metastasis and stage of human ovarian tumorTo investigate the correlation of5-LOX level and the density of TAM with human ovarian tumor development and metastasis, we detected the expression of5-LOX, CD68, HIF-la and analyzed clinicopathological variables in human ovarian tumor tissues. We found that the expression of HIF-1α,5-LOX and CD68were positively correlated with metastasis and Figo stage. Furthermore, CD68level was positively associated with lymph nodes metastasis. These data suggest that5-LOX level and the density of TAM are associated with metastasis and stage of human ovarian tumor.3. Expression of key proteins and metabolites in5-LOX pathway are enhanced in hypoxic ovarian cancer cellsThe correlation of the expression of5-LOX with HIF-1α level suggested that5-LOX and its metabolites might be raised in hypoxic ovarian cancer cells. Hypoxia increased the transcript of5-LOX, FLAP, LTA4H in ovarian cancer cell lines SKOV-3and OVCAR-3. We further examined the protein levels of5-LOX and FLAP by Western blotting, and the upregulation was observed on hypoxic treatment. The production of5-HETE and LTB4were higher under hypoxia condition than normoxia. Knockdown of HIF-1α using siRNA abolished the induction of5-LOX and FLAP, indicating that the induction of5-LOX and FLAP were HIF-la-dependent. These results indicate that hypoxia enhances expression of key proteins in5-LOX pathway and the production of metabolites.4. Increased5-LOX metabolites from hypoxic ovarian cancer cells promote macrophages migration and invasionThe correlation of TAM density with5-LOX expression suggested that5-LOX metabolites from ovarian cancer cells may contribute to the migration and invasion of macrophages. Compared with normoxic conditioned medium, the number of migrating macrophages in hypoxic conditioned medium was obviously increased. Similarly, the invasion of macrophages was enhanced much more in hypoxic conditioned medium. Besides, to determine whether5-LOX metabolites from hypoxic ovarian cancer cells contribute to macrophages migration and invasion, we used MK886(an inhibitor of FLAP) to block5-LOX pathway in SKOV-3cells. Normoxic and hypoxic conditioned medium from MK886-treated SKOV-3cells decreased migrating and invading macrophages compared to corresponding conditioned medium from untreated tumors (DMSO). And there was no significant difference between normoxic and hypoxic conditioned medium from MK886-treated SKOV-3cells. Furthermore, after treatment with5-HETE/LTB4, the migration and invasion of macrophages were enhanced. In addition, similarly, compared with normoxic conditioned medium from OVCAR-3cells, the migration and invasion of macrophages were enhanced upon hypoxic conditioned medium. Together, these results indicate that increased5-LOX metabolites from hypoxic ovarian cancer cells promote macrophages migration and invasion.5. Metabolites of5-LOX promote macrophages invasion through upregulation of MMP-7MMPs were thought to predominantly degrade the extracellular matrix (ECM) and facilitate macrophages invasion. Thus, we investigated transcript levels of MMPs in macrophages cultured in conditioned medium from normoxic and hypoxic human ovarian SKOV-3cells. Significantly higher transcript levels of MMP-2, MMP-7and MMP-9were detected in normoxic and hypoxic conditioned medium compared to medium alone (Con). However, compared to normoxic conditioned medium, only MMP-7level was significantly higher in hypoxic conditioned medium. Invasion of macrophages upon normoxic and hypoxic conditioned medium was obviously increased. After neutralizing MMP-7function with anti-MMP-7antibody, the increased invasion was suppressed. These results suggested metabolites from hypoxic SKOV-3cells may promote macrophages invasion through upregulation of MMP-7. To confirm whether the5-LOX metabolites increased MMP-7expression, Western blotting analysis was used to determine protein levels in macrophages incubated with5-HETE and LTB4. We found5-HETE and LTB4increased the expression of MMP-7in a dose dependent manner. Similarly, after neutralizing MMP-7function, the5-HETE and LTB4-stimulated invasion were suppressed. Furthermore, we investigated the expression of MMP-7in macrophages in human ovarian tumor specimens. Macrophages close to cancer cells with high5-LOX level had a high expression of MMP-7. These data indicate that metabolites of5-LOX promote macrophages invasion through upregulation of MMP-7expression.6.5-HETE/LTB4-enhanced MMP-7expression in macrophages is dependent on activation of p38pathwayTo further confirm signaling pathway in5-HETE/LTB4-enhanced MMP-7expression, we examined the effect of5-HETE/LTB4on MMP-7level in inhibitor-treated macrophages. However, the5-HETE/LTB4-enhanced MMP-7expression was significantly reduced by treating the cells with SB203580(an inhibitor of p38), suggesting that the effect is p38dependent. We next examined whether5-HETE/LTB4could activate these pathways in macrophages.5-HETE and LTB4both increased the phosphorylation of p38(T180/182), but5-HETE is more significantly than LTB4. Furthermore, p38inhibitor SB203580abolished5-HETE/LTB4-activated phosphorylation of p38(T180/182). Besides,5-HETE/LTB4didn’t significantly activate PI3K/Akt, mTOR, SAPK/JNK and ERK1/2pathway. Taken together, these data indicate that5-HETE/LTB4-enhanced MMP-7expression in macrophages is dependent on activation of p38pathway.7. Metabolites of5-LOX enhance release of TNF-a and HB-EGF through upregulation of MMP-7Release of TNF-a and HB-EGF by macrophages were examined in response to normoxic and hypoxic conditioned medium from SKOV-3cells. We observed that hypoxic conditioned medium enhanced release of TNF-a and HB-EGF much more than normoxic conditioned medium. Furthermore, hypoxic conditioned medium from MK886-treated SKOV-3cells decreased the release of TNF-a and HB-EGF compared to medium from untreated cells. It illustrates that increased5-LOX metabolites from hypoxic ovarian cancer cells could enhance the release of TNF-a and HB-EGF by macrophages. And after treatment with5-HETE/LTB4, the release of TNF-a and HB-EGF were also increased. However, the enhanced release was significantly reduced by neutralizing MMP-7function with anti-MMP-7antibody, suggesting that the effect was MMP-7-dependent. These results indicate that increased5-LOX metabolites from hypoxic ovarian cancer cells enhance the release of TNF-a and HB-EGF by macrophages through MMP-7-dependent way.8. Zileuton inhibits MMP-7expression and decreases macrophages infiltration in xenograftZileuton, a selective and specific5-LOX inhibitor, could block5-LOX metabolic pathway. We next investigated whether administration of Zileuton inhibits MMP-7expression and decreases macrophages infiltration in xenograft. Nude mice bearing SKOV-3ovarian tumor xenografts were dosed with Zileuton. Immunohistochemistry assay indicated that5-LOX level was associated with HIF-1α expression. Furthermore, murine macrophages (F4/80) density correlated with5-LOX and HIF-la level in tumor cells. Moreover, although there was no significant difference in the5-LOX level between two groups, Zileuton application significantly reduced macrophages infiltration in the hypoxic areas of xenografts. Immunofluorescence and immunohistochemistry assay revealed that the expression of MMP-7in macrophages correlated with the level of5-LOX in tumor cells. However, after dosed with Zileuton, the density of macrophages and the expression of MMP-7in macrophages were reduced in hypoxic areas of xenografts. Most importantly, the Zileuton therapy markedly inhibited ovarian tumor growth. These data suggest that Zileuton inhibits MMP-7expression and decreases macrophages infiltration in xenograft.ConclusionsTogether, these in vivo and in vitro observations provide evidences for the first time that the metabolites of5-LOX from hypoxic cancer cells promote TAM infiltration though the upregulation of MMP-7. These results have immediate translational implications for the therapeutic exploitation of TAM.