CXCL5 Facilitates Melanoma Cell-Neutrophil Interaction and Lymph Node Metastasis.

Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.

Opposing Roles of JNK and p38 in Lymphangiogenesis in Melanoma.

In primary melanoma, the amount of vascular endothelial growth factor C (VEGF-C) expression and lymphangiogenesis predicts the probability of metastasis to sentinel nodes, but conditions boosting VEGF-C expression in melanoma are poorly characterized. By comparative mRNA expression analysis of a set of 22 human melanoma cell lines, we found a striking negative correlation between VEGF-C and microphthalmia-associated transcription factor (MITF) expression, which was confirmed by data mining in GEO databases of human melanoma Affymetrix arrays. Moreover, in human patients, high VEGF-C and low MITF levels in primary melanoma significantly correlated with the chance of metastasis. Pathway analysis disclosed the respective c-Jun N-terminal kinase and p38/mitogen-activated protein kinase activities as being responsible for the inverse regulation of VEGF-C and MITF. Predominant c-Jun N-terminal kinase signaling results in a VEGF-C(low)/MITF(high) phenotype; these melanoma cells are highly proliferative, show low mobility, and are poorly lymphangiogenic. Predominant p38 signaling results in a VEGF-C(high)/MITF(low) phenotype, corresponding to a slowly cycling, highly mobile, lymphangiogenic, and metastatic melanoma. In conclusion, the relative c-Jun N-terminal kinase and p38 activities determine the biological behavior of melanoma. VEGF-C and MITF levels serve as surrogate markers for the respective c-Jun N-terminal kinase and p38 activities and may be used to predict the risk of metastasis in primary melanoma.

Activin A is anti-lymphangiogenic in a melanoma mouse model.

Melanoma spreads primarily to the sentinel lymph nodes, and its risk correlates with lymphangiogenesis, which is mainly driven by vascular endothelial growth factor (VEGF)-C. However, anti-lymphangiogenic factors are poorly characterized. We have shown in a melanoma model that Wnt1 reduces lymphangiogenesis by reducing VEGF-C expression. Screening this model for additional potentially anti-lymphangiogenic factors identified increased activin A expression and reduced expression of the antagonist, follistatin (FST), in Wnt1(+) cells. Activin A is known to reduce blood vessel formation, but the effects on lymphangiogenesis are unknown. Here we show that human primary melanoma expresses significantly higher levels of activin A and lower levels of FST compared with nevi and melanoma metastasis. Using our mouse model with melanoma cells overexpressing Wnt1, FST, Wnt1/FST, or the inhibin ßA subunit (INHBA, resulting in activin A expression), we found both activin A and Wnt1 to reduce lymphangiogenesis. Whereas Wnt1 also reduced metastasis, this was not seen with activin A. In vitro, activin A phosphorylated SMAD2 in both melanoma and lymphatic endothelium but, although it reduced sprouting of lymphatic endothelium, it enhanced the migration of melanoma cells. In conclusion, activin A is an anti-lymphangiogenic factor, but because of its pleiotropic effects on cell mobility it appears not suitable as a pharmacological target.

Wnt1 is anti-lymphangiogenic in a melanoma mouse model.

Wnt signals contribute to melanoma progression by boosting their proliferation and survival. Initially, we expected that activated Wnt signaling also improves their proficiency to recruit blood and lymph vessels. To assess this, we added cell culture supernatants (SNs) of Wnt1(+) and Wnt1(-) melanoma to endothelial spheroids. Whereas SNs of Wnt1(-) melanoma cells induced lymphatic sprouts, those of Wnt1(+) cells were unable to do so and this was restored by vascular endothelial growth factor C (VEGF-C). Subsequent testing of several human melanoma lines revealed that Wnt1 suppressed their VEGF-C expression. This Wnt1 effect did not depend on glycogen synthase kinase-3ß (GSK3ß), ß-catenin, or activator protein-1, but was blocked by cyclosporine A (CsA). To analyze Wnt1 effects in melanoma in vivo, we selected Wnt1(-) melanoma cell lines, overexpressed Wnt1, and injected them subepidermally into severe combined immunodeficient (SCID) mice. We found reduced VEGF-C expression, reduced lymphangiogenesis, and delayed metastasis to sentinel nodes in Wnt1(+) as compared with Wnt1(-) melanoma (P<0.05). Concomitant overexpression of VEGF-C or feeding of animals with CsA restored lymphangiogenesis and metastasis in Wnt1(+) melanoma. In conclusion, Wnt1 is anti-lymphangiogenic by suppressing melanoma-derived VEGF-C expression.