Semaphorin 4D expression is associated with a poor clinical outcome in cervical cancer patients.

Lymphangiogenesis is thought to be essential for cancer progression, making it an important target in cancer therapy. Lymphangiogenic factors (VEGF-C and VEGF-D) are upregulated in various tumors/cancers, and play an important role in lymphangiogenesis and lymph node metastasis. Similarly, semaphorin 4D (Sema4D) is a potent inducer of angiogenesis, and its overexpression is associated with tumor progression and poor prognosis in a variety of malignancies. However, little is known regarding the functional relationship between Sema4D and VEGF-C/VEGF-D and in the mediation of lymphangiogenesis and lymph node metastasis and clinical outcome. The current study aimed to evaluate the effect of Sema4D expression on outcome in patients with cervical cancer, and to explore the molecular mechanism of Sema4D in tumor progression. We evaluated Sema4D expression, density of lymphatic vessels, and invasion of lymphatic vessels with immunohistochemical methods in 232 human cervical cancers with long-term follow-up. Sema4D expression was correlated with patho-clinical parameters and patients' outcome. A cervical cancer cell line was used to investigate the contribution of sema4D to tumor progression by studying the role of Sema4D in VEGF-C/-D and cell migration using reverse transcription-polymerase chain reaction and Western blotting. We observed that Sema4D expression was higher in metastatic cervical cancer than in nonmetastatic cervical cancer (P<0.001). CD34-positive or D2-40-positive lymphatic vessel density was significantly increased in cases with lymph node metastasis compared with those without lymph node metastasis. The increased Sema4D expression was associated with VEGF-C/-D, the presence of lymphatic invasion, the occurrence of lymph node metastasis, and FIGO stage. We also observed a novel association between Sema4D upregulation and poor prognosis in cervical cancer. In vitro, the Sema4D inhibitory antibody and Sema4D-shRNA suppressed VEGF-C and VEGF-D in the human cervical carcinoma cell lines HeLa, Siha, and Caski cells. Invasiveness assay demonstrated that Sema4D could augment the invasive potential of the tumor cells in the cervical cancer lines and induction of cellular invasiveness by Sema4D stimulation could be inhibited by knockdown of plexinB1 by siRNA. Further mechanistic investigations of tumor cell invasiveness showed that Sema4D could induce activation of GTPase Ras homolog gene family, member A (RhoA), MAPK and AKT. In addition, plexinB1 knockdown by siRNA could suppress the Sema4D signal transmitted to MAPK and Akt. Taken together, these results suggest that Sema4D autocrine within tumor cells contributes to enhanced invasion and tumor progression through increased motility of cervical cancer and VEGF-C/-D-mediated lymphangiogenesis. Sema4D might be useful as a molecular marker of poor prognosis in cervical cancer.

Liu H ，Yang Y ，Xiao J ，Yang S ，Liu Y ，Kang W ，Li X ，Zhang F ... -  《-》

Semaphorin 4D, a lymphocyte semaphorin, enhances tumor cell motility through binding its receptor, plexinB1, in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor, for which the development of new biomarkers and therapeutic targets has become critical. The main cause of poor prognosis in PDAC patients is the high invasive and metastatic potential of the cancer. In the present study, we report a new signaling pathway that was found to mediate the enhanced tumor cell motility in pancreatic cancer. Semaphorin 4D (Sema4D) is a ligand known to be expressed on different cell types, and has been reported to be involved in the regulation of immune functions, epithelial morphogenesis, and tumor growth and metastasis. In this study, we revealed for the first time that the cancer tissue cells expressing Sema4D in PDAC are tumor-infiltrating lymphocytes. The overexpression of Sema4D and of its receptor, plexinB1, was found to be significantly correlated with clinical factors, such as lymph node metastasis, distant metastasis, and poor prognosis in patients with PDAC. Through in vitro analysis, we demonstrated that Sema4D can potentiate the invasiveness of pancreatic cancer cells and we identified the downstream molecules. The binding of Sema4D to plexinB1 induced small GTPase Ras homolog gene family, member A activation and resulted in the phosphorylation of MAPK and Akt. In addition, in terms of potential therapeutic application, we clearly demonstrated that the enhanced-cell invasiveness induced by Sema4D could be inhibited by knockdown of plexinB1, suggesting that blockade of plexinB1 might diminish the invasive potential of pancreatic cancer cells. Our findings provide new insight into possible prognostic biomarkers and therapeutic targets in PDAC patients.

Kato S ，Kubota K ，Shimamura T ，Shinohara Y ，Kobayashi N ，Watanabe S ，Yoneda M ，Inamori M ，Nakamura F ，Ishiguro H ，Nakaigawa N ，Nagashima Y ，Taguri M ，Kubota Y ，Goshima Y ，Morita S ，Endo I ，Maeda S ，Nakajima A ，Nakagama H ... -  《-》

COX-2 expression is correlated with VEGF-C, lymphangiogenesis and lymph node metastasis in human cervical cancer.

Lymphangiogenesis has been shown to promote lymph node metastasis in cancers, making it an important target in cancer therapy. Vascular endothelial growth factor (VEGF)-C is upregulated in various tumors/cancers and is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis (LNM). Likewise, cyclooxygenase (COX)-2 plays major roles in carcinogenesis, tumor growth and metastasis via multiple mechanisms including inactivation of host antitumor immunity and promotion of tumor cell migration, tumor cell invasiveness and tumor-associated angiogenesis and lymphangiogenesis. We previously demonstrated an association between COX-2 and VEGF-C in an in vitro model of lung cancer. However, little is known about the regulation of VEGF-C by COX-2 in cervical cancer. In this study, we measured the COX-2 and VEGF-C expressions by immunohistochemistry in 23 LNM-positive and 20 LNM-negative cervical cancer specimens. We then examined the correlations among the expressions and the lymphatic microvessel density (LMVD) and ultrastructural changes to the lymphatic vessel walls by enzyme histochemical staining and electron microscopy. In addition, we used the HeLa cervical cancer cell line to explore the in vitro regulation of VEGF-C by COX-2 and its metabolite, PGE(2), using siRNA-mediated gene silencing and EP receptor blockade. The LNM-positive specimens exhibited significantly higher VEGF-C expression, COX-2 expression and LMVD than the LNM-negative specimens. Furthermore, there were strong correlations between the levels of COX-2 expression and the levels of VEGF-C expression and secretion and a significant positive association between the LMVD and LNM. siRNA-mediated knockdown of COX-2 expression inhibited VEGF-C mRNA expression while EP1 and EP4 receptor antagonists reduced the VEGF-C protein level and tyrosine phosphorylation of Src kinase. Moreover, inhibition of Src kinase with the tyrosine kinase inhibitor PP1 attenuated VEGF-C expression. Collectively, our data provide evidence for a clinical association between COX-2 and VEGF-C expressions in cervical cancer. EP1 and EP4 receptors may be involved in the COX-2-mediated regulation of VEGF-C protein and mRNA expressions. Src may be a downstream mediator of EP1 and EP4 receptors. COX-2 inhibition may diminish LNM by suppressing VEGF-C-mediated lymphangiogenesis.

Liu H ，Xiao J ，Yang Y ，Liu Y ，Ma R ，Li Y ，Deng F ，Zhang Y ... -  《-》

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.

Feng Y ，Wang W ，Hu J ，Ma J ，Zhang Y ，Zhang J ... -  《-》

Tumour expression of lymphangiogenic growth factors but not lymphatic vessel density is implicated in human cervical cancer progression.

Sotiropoulou N ，Bravou V ，Kounelis S ，Damaskou V ，Papaspirou E ，Papadaki H ... -  《-》