Melanoma progression and metastasis are complex processes, and the mechanisms that mediate the switch from primary to metastatic melanoma remain poorly understood (25).
TGF-β exerts important and pleiotropic effects in melanoma progression. It acts in autocrine and paracrine manners to control and shape tumour growth, invasion, escape from immune surveillance, and metastasis (11, 26).
Positive and negative feedback regulatory loops modulate TGF-β signalling (27, 28). TGF-β positively regulates its own expression and induces the upregulation of TGF-β receptors (23, 24), thus amplifying signalling and Smad-mediated gene responses. TGF-β also induces the expression of some transcription factors that may cooperate with Smad in the transcriptional regulation of TGF-β target genes (28, 29, 30).
Increasing evidence indicates a role for the zinc finger protein ZNF224 in carcinogenesis, demonstrating that this transcription factor behaves as a tumour suppressor or oncogene in different types of cancer (31).
Here, we provided compelling evidence that ZNF224 acts as a positive regulator of TGF-β signalling in melanoma, thus contributing to TGF-β induced tumour-promoting effects.
Moreover, we found that TGF-β induces ZNF224 expression in melanoma cell lines and that ZNF224 overexpression is accompanied by prolonged Smad2 phosphorylation. Furthermore, overexpression and silencing experiments showed that ZNF224 was able to enhance the effects of TGF-β on some TGF-β-inducible genes, such as the EMT-related transcription factors Slug and Snail, which are associated with cell invasion and development of metastases and some epithelial-mesenchymal transition markers.
Although the molecular mechanisms of the transcriptional regulatory functions of ZNF224 within the TGF-β pathway were not extensively investigated here, it is conceivable that the DNA-binding activity of ZNF224 may facilitate the recruitment of Smad transcriptional complexes to target promoter sites, favouring high-affinity and selective interactions with cognate DNA. Numerous DNA-binding transcription factors play a crucial role in Smad-controlled target gene selection (28). However, considering that TGF-β can also activate intracellular pathways other than Smad-mediated canonical signalling (30), we cannot rule out that ZNF224 could also modulate the transcription of some TGF-β target genes in a Smad-independent manner. Moreover, ZNF224 may promote TGF-β signalling, activating or repressing other transcriptional regulators of this pathway.
Here, we identified one of the possible mechanisms by which ZNF224 could exert a regulatory role in the TGF-β pathway. By chromatin immunoprecipitation experiments and the evaluation of mRNAs expression changes following ZNF224 overexpression or silencing, we demonstrated that TGF-βR1, TGF-βR2, and TGF-β itself are direct target genes of ZNF224 transcriptional activation. The augmented expression of TGF-β and its receptors and prolonged Smad2 phosphorylation induced by ZNF224 contributes to the constitutive activation of the pathway, thus resulting in enhanced induction of some TGF-β-responsive genes, associated with EMT and subsequent malignant progression.
Furthermore, our data highlight the existence of a positive regulatory loop between TGF-β and ZNF224 in melanoma. Indeed, we also showed that ZNF224 expression was, in turn, induced by TGF-β, thus further contributing to the deregulated activation of this pathway.
Altogether, our data show that ZNF224 is required for the proliferation, migration, and invasiveness of melanoma cells. Its overexpression could represent a critical event in the multi-step process that leads to tumour cell invasion and metastasis. Furthermore, ZNF224 could act by promoting the acquisition of a mesenchymal phenotype and the metastatic behaviour of melanoma by modulating different EMT cancer-related proteins.
Great efforts are being made to develop drugs targeting TGF-β in melanoma (32, 33, 34). However, these approaches have proven challenging for rapid application in clinical practice due to the numerous physiological functions in which this signalling pathway is involved (35, 36).
The identification of ZNF224 as one of the modulators of TGF-β signalling will provide a deeper knowledge of the molecular events involving this pathway in melanoma progression and invasion. Further experiments will be performed to characterise the molecular network between ZNF224 and TGF-β, thus contributing to find new strategies for targeting TGF-β signalling and, consequently, new molecular therapeutic targets to treat this deadly disease.