Alterations in epidermal growth factor (EGF) expression are known to be of prognostic relevance in human melanoma, but EGF-mediated effects on melanoma have not been extensively studied. As lymph node metastasis usually represents the first major step in melanoma progression, we were trying to identify a potential role of primary tumor–derived EGF in the mediation of melanoma lymph node metastases. Stable EGF knockdown (EGFkd) in EGF-high (M24met) and EGF-low (A375) expressing melanoma cells was generated. Only in EGF-high melanoma cells, EGFkd led to a significant reduction of lymph node metastasis and primary tumor lymphangiogenesis in vivo, as well as impairment of tumor cell migration in vitro. Moreover, EGF-induced sprouting of lymphatic but not of blood endothelial cells was abolished using supernatants of M24met EGFkd cells. In addition, M24met EGFkd tumors showed reduced vascular endothelial growth factor-C (VEGF-C) expression levels. Similarly, in human primary melanomas, a direct correlation between EGF/VEGF-C and EGF/Prox-1 expression levels was found. Finally, melanoma patients with lymph node micrometastases undergoing sentinel node biopsy were found to have significantly elevated EGF serum levels as compared with sentinel lymph node–negative patients. Our data indicate that tumor-derived EGF is important in mediating melanoma lymph node metastasis. Abbreviations: A375 Ctrl, A375 (wild-type cell line); A375 EGFkd, A375 stable pLenti6/V5-GW/+EmGFP-Hmi405014; A375 mirNeg, A375 stable pLenti6/V5-GW/+EmGFP−with nontargeting miRNA; EGFkd, EGF knockdown; EGFR, EGF receptor; FFPE, formalin-fixed paraffin-embedded; HUVEC, human umbilical vein endothelial cells; LECT, immortalized (TERT) lymphatic endothelial cells; M24 Ctrl, M24met (wild-type cell line); M24 EGFkd, M24met stable pLenti6/V5-GW/+EmGFP-Hmi405014; M24 mirNeg, M24met stable pLenti6/V5-GW/+EmGFP− with nontargeting miRNA; mRNA, messenger RNA; SCID, severe combined immunodeficient; SLN, sentinel lymph node; VEGF, vascular endothelial growth factor

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