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

Abstract

Although overexpression of the low-affinity p75 neurotrophin receptor (p75NTh) 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 p75NTR 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 p75NTR as determined by Northern blotting and immunoprecipitation analysis of 1251I-labeled cell surface proteins correlated with NGFstimulated invasion. The MeWo melanoma sublines used in this study did not express pl4OPrt°otr" mRNA or any p140Proto-trk variant transcripts including p70tr`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 pl4OProto-tr cytoplasmic substrates. These cells did express authentic pl45trkc transcripts. However, NGF did not catalytically activate pl45t receptors via increased tyrosine phosphorylation as would be expected if pl45trkc participated in the signaling established by NGF. Furthermore, a NGF-stimulated wurineanalogue-sensitive kinase activity was found to coimmunoprecipitate with p75N . This p75 -associated kinase may coordinate initial signaling events evoked by p75NTR ligand interaction. Addition of 2.5S NGF, at concentrations that should saturate cell surface p75NTR, 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 pl40Proto-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 p75NTR receptor and its associated purine-analogue-sensitive kinase in signaling enhanced matrix penetration of NGF-rich stromal microenvironments such as the brain.

Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells

“…These results were related to protein NTR content in these cells by performing NTR (p75 NTR and TrkC) immunoprecipitation studies, as previously reported (Herrmann et al, 1993). Figure 1 shows the results of a representative Western blot analysis using c-Src and c-Yes antibodies designed to determine c-Src and c-Yes levels in human parental MeWo and its two variants, 3 S 5 and 70W, possessing di erent and contrasting metastatic capabilities (Ishikawa et al, 1988a,b).…”

“…In the present report, we show that the activity and level of c-Yes, but not c-Src, are upregulated in human brain-metastatic 70W cells, that such activity correlates with the metastatic potential of human melanoma cells, that stimulation of c-Yes by select NT correlates with their capacity to act as brain-invasive factors and with increased expression of p75 NTR and TrkC present in these cells (Marchetti et al, , 1995Herrmann et al, 1993). These results suggest a speci®c role of NRPTKs activation in melanomas, and the potential involvement of NRPTKs in the development of brain metastases.…”

“…We have previously demonstrated that NT are important factors a ecting invasive processes of brain-metastatic melanoma cells: NGF and NT-3 but not BDNF and NT-4/5 stimulate invasive processes in these cells as well as the production of heparanase, an important ECM-degrading enzyme involved in tumor metastasis (Marchetti et al, , 1995(Marchetti et al, , 1996Nicolson et al, 1994a,b). Correlative with selective NT-driven upregulation of invasion, the presence of aberrant levels of speci®c NT receptors in metastatic melanoma human samples has been reported (Marchetti et al, 1995), with corresponding in vivo analyses using probes recently developed of NT/NTR biological system (Bothwell, 1990;Lamballe et al, 1991;Chao, 1992;Marchetti et al, 1995;Marchetti and Nicolson, 1997a,b): only NT receptors corresponding to the neurotrophins NGF and NT-3 were found to be overexpressed in metastatic melanoma (Marchetti at al., , 1995Herrmann et al, 1993). Recently Loganzo et al (1993) demonstrated that the non receptor tyrosine kinase (NRPTK) c-Yes is increased in expression and activity in malignant melanoma.…”

“…Cells were then harvested and analysed for NTR (p75 NTR , TrkC) receptor content through immunoprecipitation as previously reported (Herrmann et al, 1993). pp60 c-Src /pp62 c-Yes in human melanoma cells were analysed by Western blotting.…”

“…NT including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/ 5) (Chao, 1992) can bind not only to the low-a nity p75 neurotrophin receptor (p75 NTR ) (Bothwell, 1990;Chao, 1992;Barbacid, 1993) but also to the higha nity NTR represented by the TRK family of tyrosine kinase receptors (Bothwell, 1990;Lamballe et al, 1991;Chao, 1992;Barbacid, 1993). Malignant melanoma cells express p75 NTR in direct relation to their malignancy and ability to metastasize to the brain (Marchetti et al, , 1995Herrmann et al, 1993) within regions that synthesize and respond to NT (Marchetti et al, 1995). We have found that NT a ect invasive processes in brain-metastatic melanoma and production of extracellular matrix (ECM-degrading enzymes .…”