PEDF is an emerging agent for the targeted anti-cancer therapies. The combination of the potent and diverse effects on tumor cells, as well as its anti-angiogenic action; make it especially compelling, particularly for advanced melanoma, a highly aggressive cancer which responds poorly to the currently available treatments. Despite the growing interest in the therapeutic applications of PEDF, very few studies used global expression analysis to seek molecular mediators underlying its multiple biological actions [54
]. In this study we describe genome-wide changes in the expression profile of A375 aggressive human melanoma cells caused by PEDF.
In general, PEDF decreased the expression of numerous genes involved in key tumor-specific functions critical for melanoma progression and whose products are likely to contribute to PEDF known anti-cancer properties.
Firstly, several factors that contribute to angiogenesis and chemotaxis were diminished due to PEDF expression. This is consistent with previous observations by us and others describing PEDF-dependent inhibition of angiogenic potential and motility of the melanoma cells [20
]. Decreased expression of these genes weakened the ability of melanoma cells to induce neovascularization. The decrease of VEGF mRNA in PEDF-expressing cells was less than two-fold; hence, it was not included in our master list. However, VEGF protein levels were decreased to a larger extent than mRNA [20
], suggesting additional post-transcriptional events. A recent expression profiling study in prostate adenocarcinoma has also shown downregulation of angiogenesis-related genes upon treatment with recombinant PEDF, which included fibroblast growth factor 3, neuropilin 1, brain-specific angiogenesis inhibitor 2 and endothelial PAS domain protein 1 [55
]. In our study, neuropilin and fibroblast growth factor family members were also regulated by PEDF in A375 melanoma cells. IL8, whose expression is also diminished in the presence of PEDF, falls in the same category. IL8 is a multifunctional cytokine, which stimulates angiogenesis, proliferation and migration [36
]. IL8 levels directly correlate with the metastatic potential and aggressiveness of melanoma [37
]. IL8 protein and mRNA were significantly decreased due to the transient or stable PEDF over-expression in A375 melanoma cells. However, IL8 regulation by PEDF in A375 melanoma cells seemed to be mainly post-transcriptional as we were unable to register a decrease in the activity of IL8 reporter constructs upon PEDF over-expression, despite the presence of multiple binding sites for the transcription factors potentially regulated by PEDF in A375 melanoma cells and other cell types, and involved in IL8 regulation, like NFATC2 [50
]. The promoter region used in our study has been previously shown to respond to a number of IL8 stimulating factors including hypoxia [62
], acidosis [63
], TNFα and IL1 [64
Altered expression of adhesion and extracellular matrix–related molecules by PEDF may, in addition to the chemokine expression, also contribute to PEDF-dependent inhibition of melanoma migration and invasion. In our study, PEDF decreased collagen IV (COL4A2
) expression, an essential component of the basement membrane, which is important for angiogenesis, adhesion and migration [38
]. PEDF also enhanced expression of fibronectin, an extracellular matrix protein involved in adhesion, proliferation and migration through its binding to integrins [39
]. Increased fibronectin could enhance cell adhesion which may impair invasiveness. A recent report shows that silencing of fibronectin in thyroid carcinoma cells enhances tumor growth and metastasis [66
PEDF over-expression in A375 cells modified the expression of a number of genes attributed in other studies to the malignant progression of human melanoma, including activin A, insulin-like growth factor binding protein 3 and galectin 3, several members of the SOX transcription factor family, and the genes that belong to the Notch (NOTCH2NL
] or Wnt (DKK1
] pathways. Importantly, PEDF downregulated several melanoma markers currently utilized in clinico-pathological diagnosis of melanoma, such as S100β [68
]; or used as prognostic factors for relapse and metastasis, like S100β or MIA [69
]. Collectively, these changes in the expression pattern of the genes related to melanoma progression may reflect the switch to a less aggressive phenotype by PEDF-expressing melanoma cells and supports the therapeutic potential of PEDF in this type of cancer.
Interestingly, PEDF over-expression in A375 cells altered the expression of genes involved in functions specific for melanocyte lineage, upregulating MLPH
and downregulating RAB27A.
These genes are involved in melanosome trafficking and therefore play a key role in melanin transfer [52
]. PEDF has been detected in immature melanosomes of melanoma cells [70
], and prior reports have proposed its participation in pigment production through induction of tyrosinase [71
] and promotion of melanosome maturation in retinal pigment epithelium cells [72
]. We have previously described that PEDF is expressed at high levels in melanocytes, and restricts their proliferative and migratory potential [26
]. Modulation of MLPH
expression by PEDF in A375 cells suggests that it could also mediate melanocyte-specific processes, such melanin distribution in the skin. In keeping with this new role, we found that PEDF increases the expression of SOX9, which was recently implicated in control of melanocyte differentiation and pigmentation, acting upstream of microphthalmia-associated transcription factor (MITF) [73
In summary, our study points to novel molecular targets and signaling pathways that may potentially contribute to determine PEDF’s ability to restrict the aggressiveness of A375 and other human melanoma cells. We also stumbled upon unanticipated PEDF targets involved in melanocytic lineage-specific functions. Our results presented set the stage for further in-depth studies of the molecular mechanisms underlying PEDF effects in melanoma, which would be essential for the clinical translation of this factor.