The role of
PTHrP in the highly metastatic human
melanoma disease is not known. This study investigates the mechanisms of action of this secreted factor through homozygous inactivation of the
Pthrp gene in A375 human
melanoma cells. In vitro,
Pthrp-ablated cells (knockout [KO]-A375, -/-) showed decreased motility and anchorage-independent growth, rounder morphology, and a significant reduction in invasion capacity compared with nonablated A375 cells (wild-type [WT]-A375, +/+).
PTHrP peptide 1-34 and
conditioned medium from WT-A375 cells partially restored the invasive phenotype in KO-A375.
Pthrp ablation substantially decreased actin polymerization, matrix
metallopeptidase 9 expression and
focal adhesion kinase phosphorylation. In vivo,
green fluorescent protein-transduced ablated and nonablated A375 cells were injected intracardially or sc into nude mice to study proliferation and multiorgan
metastasis. Dissemination of injected
Pthrp-ablated cells to lung and liver was reduced by 85% and 50%, respectively, compared with nonablated controls (120 hours after injection). The number of metastatic lesions and the percentage of animals with
metastasis were markedly lower in mice injected with
Pthrp-ablated A375, and 45% of these animals survived a 7-week period compared with 15% of mice injected with nonablated WT-A375. When mice injected with WT-A375 were treated with our blocking anti-
PTHrP monoclonal antibody raised against the first 33
amino acids of human
PTHrP,
tumor size was decreased by more than 80% over 4 weeks and survival was significantly improved over 8 months. This study provides direct evidence of the major role for
PTHrP in
melanoma invasion and
metastasis and suggests that agents that suppress
PTHrP may be beneficial against
melanoma progression.