Inhibition of Cyclooxygenase‐2 Suppresses Lymph Node Metastasis via VEGF‐C

Most experimental work addressing cyclooxygenase‐2 (COX‐2) inhibitor has focused on suppressing hematogenic spread. Little is known about the mechanism by which this inhibitor can also block lymphatic metastasis. Here, the effects of COX‐2 inhibitor on vascular endothelial growth factor‐C (VEGF‐C) expression, lymphangiogenesis and lymph node metastasis were investigated. Utilizing the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83‐a, which has a low metastatic capacity, we found elevated VEGF‐C and COX‐2 immunoreactivity in Anip973 cells compared with AGZY83‐a cells. Celecoxib down‐regulated expression of VEGF‐C mRNA and protein in Anip973 cells while PGE₂ up‐regulated expression of VEGF‐C mRNA and protein in AGZY83‐a cells in a concentration‐dependent manner. The expression of COX‐2 and VEGF‐C was significantly increased in xenografted Anip973 tumors compared with AGZY83‐a tumors. The Anip973 tumors showed more lymphatic vessels and lymph node metastasis than the AGZY83‐a tumors. In vivo, celecoxib decreased VEGF‐C expression in Anip973 tumor‐treated mice to a similar level to that in the AGZY83‐a tumor‐treated mice. Consistent with this decrease in VEGF‐C expression, the density of lymphatic vessels and lymph node metastasis in Anip973 tumor‐treated mice were suppressed to approximately that found in the AGZY83‐a tumor‐treated ones. Taken together, our results suggest that the differential expression of COX‐2 and VEGF‐C might help explain the different metastasis phenotype of lung adenocarcinoma cancer, and that COX‐2 inhibitor mediates VEGF‐C to block lymphangiogenesis and lymph node metastasis. Thus, COX‐2 may be a potential therapeutic target for blocking lymph node metastasis in lung adenocarcinoma. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

应用mRNA差异显示技术克隆人肺腺癌转移相关基因

肿瘤转移是恶性肿瘤最基本的生物学特征。为了探讨恶性肿瘤的侵袭和转移的机理，选择两株具有相同的细胞来源、但具有不同转移能力的人肺腺癌细胞系ＡＧＺＹ８３ａ和Ａｎｉｐ９７３作为研究材料，采用ｍＲＮＡ差异显示（ｍＲＮＡｄｉｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ）技术，对这两个细胞系的基因表达差异情况进行了分析。结果显示，在这两个细胞系之间存在明显的基因表达差异，说明在低转移的ＡＧＺＹ８３ａ向高转移的Ａｎｉｐ９７３演变过程中涉及多个基因的激活与失活。研究结果还提示，在高转移的Ａｎｉｐ９７３细胞系中，某些基因的表达或高表达与人类逆转录病毒基因ＬＴＲ的整合有关。另一个克隆与人类线粒体基因ＮＤ４具有高度同源性，其高度表达与Ａｎｉｐ９７３的转移表型有关。表明肿瘤的转移是一个多基因参与的过程，由于这些基因的相互作用及细胞内外环境因素的影响，最终决定了肿瘤细胞的转移表型。     

高低转移肺腺癌细胞系中肿瘤抑制基因p16、p21和p53表达研究

目的 探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用。方法 利用FuGene转染方式分别将p21和p16基因的表达质粒转入－对肺腺癌细胞系Anip973和AGZY83－a中,同时用含野生型p53 基因的腺病毒感染p16基因转染前后的这一对细胞系。对P16和P21蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞仪分析。结果 p16基因的过表达只能使细胞系的G1期细胞比例提高,但细胞生长曲线,克隆形成率均未出现改变,未检测到凋亡信号。P21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降,并出现明显的G1期阻滞,但未检测到凋亡信号。p53基因感染AGZY83－a,Anip973及经过p16基因转染的细胞AGZY83－ap16和Anip973p16后呈现时间依赖性表达,细胞生长曲线和四唑盐比色法分析提高,野生型p53基因的大量表达明显抑制以上4种细胞的生长,Anip973和Anip973p16的生长抑制率高于AGZY83－a和AGZY83－ap16;Anip973p16和AGZY83-ap16的生长抑制率高于Anip973和AAGZY83－a。这4种细胞在感染p53后出现典型的凋亡信号。结论p16基因的过表达并不能抑制细胞系的生长,而p21基因的过表达通过G1期阻滞抑制这1对肺腺癌细胞的生长;野生型p53基因在AGZY83－a和Anip973中高效表达可产生明显的细胞生长抑制效应;野生型p53基因对肺腺癌高转移细胞系Anip973抑制作用更为明显。     

Differential expression of RAB5A in human lung adenocarcinoma cells with different metastasis potential

For the sake of better understanding the molecular mechanism of neoplasia, we have used the mRNA differential display technique to analyze two human lung adenocarcinoma cell lines, AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. We found that a significant differential cDNA fragment in Anip973 was over-expressed, then over-expressed cDNA fragment was cloned and sequenced. It showed that the over-expressed cDNA in Anip973 was RAB5A cDNA. And the RAB5A cDNA sequence was corresponding between the two cells. To determine whether RAB5A may be differentially expressed in the two human lung adenocarcinoma cells at protein level, we further detected RAB5A protein in the two cells by using immunofluorescent method. RAB5A protein was upregulated in highly metastatic Anip973. We also detected the difference in RAB5A gene expression at RNA level in human non-small cell lung carcinoma by RT-PCR. Using immunohistochemical staining, we also examined RAB5A change at protein level in 45 cases human non-small cell lung carcinoma paraffin sections. The results proved the evidence of upregulation of RAB5A in malignant tumor, indicated over-expression of RAB5A gene was correlated with the malignant degree and metastatic potential of lung cancer(² test, p <0.01). The RAB5A gene is a member of RAS superfamily, which can transcribe GTP-binding protein that plays an important role in signal transduction of protein trafficking at the cell surface and GDP/GTP cycle in the regulation of endocytotic membrane traffic. Thus our results indicated that over-expression of the RAB5A gene was involved in the process of transformation from AGZY83-a to the higher metastatic cell line Anip973. The result may be a powerful experimental evidence that over-expression of RAB5A gene associated with neoplasia metastasis.     

BRI基因在一对转移能力不同的肺腺癌细胞系中的序列分析与表达研究

目的 确认淀粉样纤维蛋白基因(amyloid fibrils，BRI)基因在l对同源但转移能力不同的肺腺癌细胞系AGZY83-a和Anip973中的序列并分析其表达。方法 采用测序技术，Northern印迹杂交。G显带后荧光原位杂交分析BRI基因在肺腺癌细胞系的序列与表达。结果 BRI基因在高转移肺腺癌细胞系Anip973中高表达，在其低转移母系AGZY83-a中低表达，两细胞系BRI基因染色体定位区均存在断裂重排。该基因染色体定位区在Anip973中出现扩增。已知BRI基因的-116bp～-5bp处碱基序列和-115bp～-5bp处碱基序列在AGZY83-a和Anip973中分别突变为CTCAGCAGCCCGC和TCAGCCGC。结论 BRI基因在转移能力不同的肺腺癌细胞系差异表达与该基因的染色体定位区域的断裂重排无关，与该基因染色体定位区拷贝数增加及5′非翻译区存在不同的突变可能相关。     

Triggering of DC migration by dengue virus stimulation of COX‐2‐dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation

A term “bone‐breaking fever” is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX‐prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX‐2 and the production of prostaglandin E₂ (PGE₂) in DC, and stimulated the DNA binding of NF‐κB and the kinase activity of both IκBα kinase (IKK) α and β. DV infection also activated MAPK and AP‐1 signaling. Both IκBα kinase‐NF‐κB and MAPK‐AP‐1 were upstream of COX‐2 activation. Our investigation into the significance of COX‐2‐PGE₂ pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX‐2 or MAPK activity suppresses DV‐induced DC migration. Our data also suggest that PGE₂ can induce CCR7 expression on DC and that antagonists of the PGE₂ receptors EP2 and EP4 suppress DV‐induced DC migration. We further show that the increased CCR7 expression was observed in both DV‐infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX‐2 synthesis in DV‐infected DC but also the autocrine action of PGE₂ on the migration of DV‐infected DC.     

Prostaglandin E2 and its receptor EP2 trigger signaling that contributes to YAP‐mediated cell competition

Cell competition is a biological process by which unfit cells are eliminated from “cell society.” We previously showed that cultured mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying the elimination of active YAP‐expressing cells was unknown. Here, we used high‐throughput chemical compound screening to identify cyclooxygenase‐2 (COX‐2) as a key molecule triggering cell competition. Our work shows that COX‐2‐mediated PGE₂ secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase‐cyclic AMP‐PKA pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion. Thus, COX‐2‐induced PGE₂ appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.     

Calcium/calmodulin‐dependent protein kinase II regulates cyclooxygenase‐2 expression and prostaglandin E2 production by activating cAMP‐response element‐binding protein in rat peritoneal macrophages

Prostaglandin E₂ (PGE₂) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE₂ production is regulated by arachidonic acid release and cyclooxygenase‐2 (COX‐2) expression. In the present study, we found that COX‐2 expression can be achieved by activating Ca²⁺/Calmodulin (CaM)‐dependent protein kinase II (CaMKII) and cAMP‐response element‐binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca²⁺]_i), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX‐2 expression and PGE₂ production in a chronological order. These results suggested that Ca²⁺/CaM‐dependent CaMKII plays an important role in mediating COX‐2 expression and PGE₂ production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE₂ production, which plays an essential role in inflammation and cancers.     

A regulatory cross‐talk between Vγ9Vδ2 T lymphocytes and mesenchymal stem cells

The physiological functions of human TCRVγ9Vδ2⁺ γδ lymphocytes reactive to non‐peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVγ9Vδ2⁺ lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B‐cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of γδ‐cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX‐2‐dependent production of prostaglandin E2 (PGE₂) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vγ9Vδ2 T lymphocytes. COX‐2 expression and PGE₂ production by MSC were not constitutive, but were induced by IFN‐γ and TNF‐α secreted by activated Vγ9Vδ2 T cells. This regulatory cross‐talk between MSC and Vγ9Vδ2 T lymphocytes involving PGE₂ could be of importance for the antitumor and antimicrobial activities of γδ T cells.     

Reciprocal modulation of C/EBP‐α and C/EBP‐β by IL‐13 in activated microglia prevents neuronal death

In response to aggravation by activated microglia, IL‐13 can significantly enhance ER stress induction, apoptosis, and death via reciprocal signaling through CCAAT/enhancer‐binding protein alpha (C/EBP‐α) and C/EBP‐beta (C/EBP‐β). This reciprocal signaling promotes neuronal survival. Since the induction of cyclooxygenase‐2 (COX‐2) and peroxisome proliferator‐activated receptor gamma/heme oxygenase 1 (PPAR‐γ/HO‐1) by IL‐13 plays a crucial role in the promotion of and protection from activated microglia, respectively; here, we investigated the role of IL‐13 in regulating C/EBPs in activated microglia and determined its correlation with neuronal function. The results revealed that IL‐13 significantly enhanced C/EBP‐α/COX‐2 expression and PGE₂ production in LPS‐treated microglial cells. Paradoxically, IL‐13 abolished C/EBP‐β/PPAR‐γ/HO‐1 expression. IL‐13 also enhanced ER stress‐evoked calpain activation by promoting the association of C/EBP‐β and PPAR‐γ. SiRNA‐C/EBP‐α effectively reversed the combined LPS‐activated caspase‐12 activation and IL‐13‐induced apoptosis. In contrast, siRNA‐C/EBP‐β partially increased microglial cell apoptosis. By NeuN immunochemistry and CD11b staining, there was improvement in the loss of CA3 neuronal cells after intrahippocampal injection of IL‐13. This suggests that IL‐13‐enhanced PLA2 activity regulates COX‐2/PGE₂ expression through C/EBP‐α activation. In parallel, ER stress‐related calpain downregulates the PPAR‐γ/HO‐1 pathway via C/EBP‐β and leads to aggravated death of activated microglia via IL‐13, thereby preventing cerebral inflammation and neuronal injury.     

Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

Altered monocyte cyclo‐oxygenase response in non‐obese diabetic mice

Monocytes infiltrate islets in non‐obese diabetic (NOD) mice. Activated monocyte/macrophages express cyclo‐oxygenase‐2 (COX‐2) promoting prostaglandin‐E₂ (PGE₂) secretion, while COX‐1 expression is constitutive. We investigated in female NOD mice: (i) natural history of monocyte COX expression basally and following lipopolysaccharide (LPS) stimulation; (ii) impact of COX‐2 specific inhibitor (Vioxx) on PGE₂, insulitis and diabetes. CD11b⁺ monocytes were analysed for COX mRNA expression from NOD (n = 48) and C57BL/6 control (n = 18) mice. NOD mice were treated with either Vioxx (total dose 80mg/kg) (n = 29) or methylcellulose as control (n = 29) administered by gavage at 4 weeks until diabetes developed or age 30 weeks. In all groups, basal monocyte COX mRNA and PGE₂ secretion were normal, while following LPS, after 5 weeks of age monocyte/macrophage COX‐1 mRNA decreased (P < 0·01) and COX‐2 mRNA increased (P < 0·01). However, diabetic NOD mice had reduced COX mRNA response (P = 0·03). Vioxx administration influenced neither PGE₂, insulitis nor diabetes. We demonstrate an isoform switch in monocyte/macrophage COX mRNA expression following LPS, which is altered in diabetic NOD mice as in human diabetes. However, Vioxx failed to affect insulitis or diabetes. We conclude that monocyte responses are altered in diabetic NOD mice but COX‐2 expression is unlikely to be critical to disease risk.     

The fish oil ingredient,docosahexaenoic acid,activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti‐inflammatory role in macrophages

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti‐inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A₂ (cPLA₂) and lipid mediators produced from cPLA₂ activation are involved in the anti‐inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA₂ and cyclooxygenase 2 (COX‐2), and cause prostaglandin E₂ (PGE₂) release in a murine macrophage cell line RAW264.7 and in human primary monocyte‐derived macrophages. DHA and GW9508 activate cPLA₂ via GPR120 receptor, G protein Gαq and scaffold protein β‐arrestin 2. Extracellular signal‐regulated kinase 1/2 activation is involved in DHA‐ and GW9508‐induced cPLA₂ activation, but not p38 mitogen‐activated protein kinase. The anti‐inflammatory role of DHA and GW9508 is in part via activation of cPLA₂, COX‐2 and production of PGE₂ as a cPLA₂ inhibitor or a COX‐2 inhibitor partially reverses the DHA‐ and GW9508‐induced inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. The cPLA₂ product arachidonic acid and PGE₂ also play an anti‐inflammatory role. This effect of PGE₂ is partially through inhibition of the nuclear factor‐κB signalling pathway and through the EP4 receptor of PGE₂ because an EP4 inhibitor or knock‐down of EP4 partially reverses DHA inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. Hence, DHA has an anti‐inflammatory effect partially through induction of PGE₂.     

Integrin α4 Induces Lymphangiogenesis and Metastasis via Upregulation of VEGF‐C in Human Colon Cancer

Vascular endothelial growth factor‐C (VEGF‐C) is a key regulator in lymphangiogenesis, and is overexpressed in various malignancies. Integrin α4β1, a new member of the VEGF‐C/VEGF receptor pathway, was found to be overexpressed in melanoma tumors. However, little is known regarding the potential role of integrin α4β1 in lymphangiogenesis and other solid tumors. The aim of this study was to investigate the expression patterns of integrin α4 and VEGF‐C in relation to lymphangiogenesis and clinicopathological parameters in human colon cancer. The expression of integrin α4, VEGF‐C, and VEGFR‐3 was assessed in 71 human colon cancer tissues and 30 paracancerous normal tissues by immunohistochemical staining. Lymphatic microvessel density (LMVD) was measured after D2‐40‐labeling, and the correlations among different factors were statistically analyzed. The expression of integrin α4, VEGF‐C, VEGFR‐3, and LMVD was higher in colon cancer tissues compared with the normal paracancerous colon tissues. There was a positive correlation between the expression of integrin α4 and VEGF‐C. Integrin α4 and VEGF‐C were significantly associated with the clinicopathological parameters (LMVD, Duke's stage, and lymph node metastasis). Kaplan–Meier analyses indicated that patients with high integrin α4 or VEGF‐C expression had significantly shorter overall survival and tumor‐free survival time. Multivariate analyses suggested that integrin α4 and VEGF‐C may serve as independent prognostic factors for human colon cancer. Both integrin α4 and VEGF‐C are involved in lymphangiogenesis and lymphatic metastasis. Our results demonstrated that integrin α4 is a novel prognostic indicator for human colon cancer. Anat Rec, 299:741–747, 2016. © 2016 Wiley Periodicals, Inc.     

Expression of VEGF‐C and VEGF‐D as Significant Markers for Assessment of Lymphangiogenesis and Lymph Node Metastasis in Non‐Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF)‐C and VEGF‐D induce lymphangiogenesis through activation of VEGF receptor 3 (VEGFR‐3) and have been implicated in tumor spread to the lymphatic system. Lymph node dissemination critically determines clinical outcome and therapeutic options of patients with non‐small cell lung cancer (NSCLC). However, the relationship of VEGF‐C, VEGF‐D, and lymph node metastasis in cancers, including NSCLC, is still controversial. To evaluate the relationship between lymphangiogenesis and lymph node metastasis, the expression of VEGF‐C and VEGF‐D in NSCLC tumors were detected by immunohistochemistry and quantitative real‐time polymerase chain reaction (QRT‐PCR). QRT‐PCR revealed that in marginal region VEGF‐C and VEGF‐D mRNA was significantly higher than in tumor center, and VEGF‐D mRNA was also higher than that in peritumoral lung tissue. Immunohistochemically, we observed the same heterogeneous expression of VEGF‐C and VEGF‐D proteins. The group with high expression of VEGF‐C and VEGF‐D in marginal region had a higher incidence of lymph node metastasis compared with the group with low expression. Furthermore, the group with high expression of VEGF‐D in marginal region had a higher incidence of lymphatic invasion. The group with high peritumoral lymphatic vessel density (LVD) had higher expression of VEGF‐C and VEGF‐D mRNA compared with the group with low peritumoral LVD. Our studies suggested that the expression of VEGF‐C and VEGF‐D at invasive edge was significantly associated with lymph node metastasis or lymphatic invasion in patients with NSCLC and may be involved in regulation of lymphangiogenesis and lymph node metastasis in NSCLC. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

VEGF‐C,VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours

Ramani P, Nash R, Radevsky L, Patel A, Luckett M & Rogers C
(2012) Histopathology
VEGF‐C, VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours Aims: More than 50% of neuroblastomas (NBs) present with haematogenous and/or lymphatic metastasis; however, little is known about the clinicopathological significance in NBs of the key lymphangiogenesis growth factors vascular endothelial growth factor (VEGF)‐C and VEGF‐D and the receptor VEGFR‐3. Methods and results: Ninety‐three NBs and nine ganglioneuromas (GNs) were immunostained for VEGF‐C, VEGF‐D and VEGFR‐3. VEGF‐C and VEGF‐D were present in 76% and 82% of the NBs, respectively. There was no significant difference in VEGF‐C expression between NBs and GNs. VEGF‐D expression was significantly higher in NBs compared with GNs and in MYCN‐amplified NBs. VEGFR‐3 tumoral cell expression (VEGFR‐3c), present in 48% of the NBs, was significantly higher in NBs from children ≥18 months at presentation and those belonging to a high‐risk group. VEGFR‐3 lymphovascular density was increased significantly in NBs compared with GNs and in NBs associated with adverse clinicopathological and biological factors. Lymphovascular invasion, assessed in VEGFR‐3‐stained vessels, was present in ～50% of NBs. Cox regression analyses demonstrated that VEGFR‐3c expression was associated with a significantly shorter event‐free survival and that its effect was independent of the important pathological variable, mitosis–karyorrhexis index. Conclusions: VEGF‐D and VEGFR‐3 up‐regulation support tumour progression in NB and VEGFR‐3c may provide a useful prognostic marker in NBs.     

Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model

We reported that cyclo-oxygenase (COX)-2 expression in human breast cancer stimulated cancer cell migration and invasiveness, production of vascular endothelial growth factor (VEGF)-C and lymphangiogenesis in situ, largely from endogenous PGE2-mediated stimulation of prostaglandin E (EP)1 and EP4 receptors, presenting them as candidate therapeutic targets against lymphatic metastasis. As human breast cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. We tested the roles of COX-2 and EP receptors in VEGF-C and -D production by a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors and produced VEGF-C and -D, both inhibited with COX-2 inhibitors or EP4 (but not EP1, EP2 or EP3) antagonists. C3H/HeJ mice, when implanted SC in both inguinal regions with C3L5 cells suspended in growth factor-reduced Matrigel, exhibited rapid tumor growth, tumor-associated angiogenesis and lymphangiogenesis (respectively measured with CD31 and LYVE-1 immunostaining), metastasis to the inguinal and axillary lymph nodes and the lungs. Chronic oral administration of COX-1/COX-2 inhibitor indomethacin, COX-2 inhibitor celecoxib and an EP4 antagonist ONO-AE3-208, but not an EP1 antagonist ONO-8713 at nontoxic doses markedly reduced tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in responding mice revealed reduced VEGF-C and -D proteins, AkT phosphorylation and increased apoptotic/proliferative cell ratios consistent with blockade of EP4 signaling. We suggest that EP4 antagonists deserve clinical testing for chemo-intervention of lymphatic metastasis in human breast cancer.     

Mechanisms of chorioamnionitis‐associated preterm birth: interleukin‐1β inhibits progesterone receptor expression in decidual cells

In chorioamnionitis (CAM), a major cause of preterm birth (PTB), maternal–fetal inflammation of the decidua and amniochorion cause the release of cytokines that elicit cervical ripening, fetal membrane rupture and myometrial activation. We posit that this inflammatory milieu triggers PTB by inhibiting progesterone receptor (PR) expression and increasing decidual prostaglandin (PG) production. Immunohistochemical staining of decidua detected significantly lower PR levels in decidual cells (DCs) from CAM‐complicated PTB. Incubation of DCs with IL‐1β decreased PR expression and significantly increased PGE₂ and PGF_2α production and COX‐2 expression. The addition of PGF_2α to DC cultures also suppressed PR expression. However, the COX inhibitor, indomethacin, did not reverse IL‐1β suppression of PR expression in DC cultures. Although IL‐1β treatment activated the NF‐K B, ERK1/2 and p38 MAPK signalling cascades in DCs, inhibition of ERK1/2 MAPK signalling alone was sufficient to completely reverse the suppression of PR levels by IL‐1β. These findings suggest that CAM‐associated PTB is induced at least in part by IL‐1β‐mediated functional progesterone withdrawal. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.