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The placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family, which shares with VEGF-A the tyrosine kinase receptor VEGFR-1 and the co-receptor neuropilin-1 (NRP-1). In melanoma models, PlGF enhances tumour growth and neovessel formation, whereas NRP-1 promotes the metastatic process. Increased secretion of PlGF and expression of NRP-1 have also been involved in intrinsic or acquired resistance to anti‑angiogenic therapies. In this study we investigated whether PlGF and NRP-1 cooperate in promoting melanoma aggressiveness independently of VEGFR-1. For this purpose, the melanoma cell clones M14-N, expressing NRP-1 and lacking VEGFR-1, and M14-C, devoid of both receptors, were used. M14-N cells are characterized by an invasive phenotype and vasculogenic mimicry, whereas M14-C cells possess a negligible invasive capacity. The results indicated that M14-N cells secrete higher levels of PlGF than M14-C cells and that PlGF is involved in the invasion of the extracellular matrix (ECM) and vasculogenic mimicry of M14-N cells. In fact, neutralizing antibodies against PlGF reverted ECM invasion in response to PlGF and markedly reduced the formation of tubule-like structures. Moreover, M14-N cells migrated in response to PlGF and chemotaxis was specifically abrogated by anti-NRP-1 antibodies, demonstrating that PlGF directly activates NRP-1 in the absence of VEGFR-1. We also compared the levels of PlGF in the plasma of patients affected by metastatic melanoma with those of healthy donors and evaluated whether PlGF levels could be affected by a bevacizumab-containing chemotherapy regimen. Melanoma patients showed a 20-fold increase in plasma PlGF and the bevacizumab-containing regimen induced a reduction of VEGF-A and in a further increase of PlGF. In conclusion, our studies suggest that the activation of NRP-1 by PlGF directly contributes to melanoma aggressiveness and represents a potential compensatory pro-angiogenic mechanism that may contribute to the resistance to therapies targeting VEGF-A.

Placenta growth factor (PlGF) and its receptor vascular endothelial growth factor receptor-1 (VEGFR-1) are co-expressed in a large number of human melanoma cell lines. Moreover, a correlation between in vivo PlGF production and melanoma progression has been suggested. To investigate whether PlGF might have a role in protecting melanoma cells from the cytotoxic effects of the anticancer agent temozolomide (TMZ), which is used for the treatment of this malignancy, we stably transfected a doxycycline-inducible PlGF antisense mRNA into a human melanoma cell clone that secretes VEGF-A and PlGF and expresses receptors for both growth factors. Induction of PlGF antisense mRNA in the transfected cells (13443/ASP3 subclone) halved TMZ IC(50), and exogenous addition of PlGF to the culture medium 24 h before TMZ treatment, partially restored IC(50) values to that of control cells. The increased sensitivity of 13443/ASP3 cells upon PlGF antisense mRNA expression was not due to down-regulation of O6-methylguanine-DNA methyltransferase, a DNA repair protein that represents the main mechanism of resistance to TMZ. Since the activity of the transcription factor nuclear factor-κB (NF-κB) has been correlated to melanoma chemoresistance, we investigated whether NF-κB was involved in PlGF-induced melanoma cell resistance to TMZ. Induction of PlGF antisense mRNA in 13443/ASP3 cells halved the levels of active NF-κB and the specific inhibition of this transcription factor increased sensitivity of 13443/ASP3 cells to TMZ. In conclusion, our data strongly suggest that PlGF plays a role in melanoma cell resistance to TMZ through a pathway that involves NF-κB activation.

Poly(ADP-ribose) polymerase (PARP) is a family of nuclear proteins which regulate a number of cell functions, such as DNA repair, transcription, remodelling of chromatin structure, cell division and cell death. We and others have recently demonstrated that down-regulation of cellular PARP activity, using pharmacological inhibitors, impairs a number of endothelial functions and angiogenesis. In the present study, we investigated the potential mechanisms underlying the anti-angiogenic effect exerted by the potent PARP inhibitor GPI 15427, analyzing gene expression in human endothelial cells shortly after treatment with this compound. Analysis of gene and protein expression indicated that a 2-h exposure of human endothelial cells to GPI 15427 induced a rapid decrease of syndecan-4 (SDC-4), a transmembrane protein involved in modulation of cell signalling during angiogenesis that plays a role in endothelial cell migration and adhesion. Moreover, treatment with the PARP inhibitor induced a reduction of a helix-loop-helix transcription factor, the inhibitor of DNA binding-1 (Id-1), also implicated in the control of endothelial functions. We suggest that the inhibitory effect exerted by GPI 15427 on the angiogenic process is likely due to the reduced activity of specific transcription factors, such as Oct-1 and CREB that contribute to the regulation of SDC-4 and Id-1 expression, respectively. In conclusion, these results strongly suggest that PARP activity is capable of modulating molecules required for endothelial cell migration, adhesion, proliferation or differentiation during the angiogenic process.

The vascular endothelial growth factor-A (VEGF-A) is a cytokine that promotes angiogenesis through the activation of two tyrosine kinase receptors, VEGFR-1 and VEGFR-2, on vascular endothelial cells. Moreover, several experimental evidences indicate that VEGF-A may also play a role in tumor progression by acting on neoplastic cells expressing VEGFRs. In this study we show that human melanoma cells that simultaneously produce VEGF-A and express VEGFRs exhibit a higher spontaneous ability to invade the extracellular matrix (ECM) than melanoma cells not expressing either VEGF-A or VEGFRs. Exposure of VEGFR expressing melanoma cells to exogenous VEGF-A further increases their ability to invade the ECM. Moreover, an inhibitor of VEGFR tyrosine kinase activity is able to abrogate VEGF-A-induced stimulation of ECM invasion. A cell clone (13443/N2) derived from a VEGF-A responsive melanoma cell line and expressing high levels of VEGFR-2 invades the ECM eight-fold more efficiently than a cell clone derived from the same cell line and expressing extremely low levels of the receptor. Exposure of 13443/N2 cells to VEGF-E, which selectively binds and activates VEGFR-2, increases their ability to invade the ECM. Finally, the expression of the VEGF-A mRNA antisense sequence in 13443/N2 cells markedly reduces the release of VEGF-A and ECM invasion. In conclusion, our data show for the first time that a VEGF-A-driven autocrine loop promotes human melanoma cell ability to invade the ECM, and strongly support the hypothesis that activation of VEGFR-2 plays a primary role in this process.