Regulatory and functional connection of microphthalmia-associated transcription factor and anti-metastatic pigment epithelium derived factor in melanoma.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.

Hypoxia negatively regulates antimetastatic PEDF in melanoma cells by a hypoxia inducible factor-independent, autophagy dependent mechanism.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.

Functional impact of cancer-associated mutations in the tumor suppressor protein ING4.

Inhibitor of growth 4 (ING4) is a member of the ING family of tumor suppressor proteins. In this study, we have analyzed the impact of two mutations in ING4 associated with human tumors (Y121N and N214D), testing their behavior in a series of functional, biochemical and structural analyses. We report that the N214D mutation dramatically dampened the ability of ING4 to inhibit proliferation, anchorage-independent growth or cell migration or to sensitize to cell death. In turn, the Y121N mutant did not differ significantly from wild-type ING4 in our assays. Neither of the mutations altered the normal subcellular localization of ING4, showing predominantly nuclear accumulation. We investigated the molecular basis of the defect in the activity of the N214D mutant. The folding and ability to bind histone marks of ING4 was not significantly altered by this mutation. Instead, we found that the functional impairment of the N214D mutant correlates with reduced protein stability due to increased proteasome-mediated degradation. In summary, our data demonstrates that a point mutation of ING4 associated to human tumors leads to the loss of several essential functions of ING4 pertinent to tumor protection and highlight the importance of ING4 function to prevent tumorigenesis.