Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor: Melanoma p75 functions independently of trkA

Although overexpression of the low-affinity p75 neurotrophin receptor (p75^NTR) is frequently associated with advanced stages of human melanoma progression, the functional significance of this finding is unknown. We examined whether the degree of cell surface expression of p75^NTR in human melanoma cell variants determines their extent of invasion stimulated by nerve growth factor (NGF). Treatment of MeWo melanoma cells or a metastatic spontaneous wheat germ agglutinin-resistant variant subline (70W) of MeWo cells with 2.5S NGF resulted in a dose-dependent enhancement of invasion through a reconstituted basement membrane. This effect was most pronounced with the 70W subline that exhibits brain-metastasizing potential in nude mice but was not found with a poorly metastatic MeWo variant subline (3S5). The expression of p75^NTR as determined by Northern blotting and immunoprecipitation analysis of ¹²⁵I-labeled cell surface proteins correlated with NGF-stimulated invasion. The MeWo melanoma sublines used in this study did not express p140^proto-trkA mRNA or any p140^proto-trkA variant transcripts including p70^trkA as determined by Northern analysis and RT-PCR analysis. Thus, these melanoma cells would not be expected to form functional p75-p140 heterodimers or p140-p140 homodimers capable of transducing an NGF-generated signal to p140^proto-trkA cytoplasmic substrates. These cells did express authentic p145^trkC transcripts. However, NGF did not catalytically activate p145^trkC receptors via increased tyrosine phosphorylation as would be expected if p145^trkC participated in the signaling established by NGF. Furthermore, a NGF-stimulated purine-analogue-sensitive kinase activity was found to coimmunoprecipitate with p75^NTR. This p75^NTR-associated kinase may coordinate initial signaling events evoked by p75^NTR ligand interaction. Addition of 2.5S NGF, at concentrations that should saturate cell surface p75^NTR, to matrix-adherent cultures of human MeWo and 70W but not 3S5 melanoma cells suppressed the expression of 92-kDa type IV collagenase and stimulated the production of 72-kDa type IV collagenase in its fully active 68-kDa form. In the absence of p140^proto-trkA, the matrix-dependent effects of NGF on metalloproteinase expression of brain-metastatic 70W melanoma cells suggest a signaling role for the low-affinity melanoma p75^NTR receptor and its associated purine-analogue-sensitive kinase in signaling enhanced matrix penetration of NGF-rich stromal microenvironments such as the brain.