E301: AP-2α Regulates Uveal Melanoma Proliferation In Vitro

Uveal melanoma is a rare disease (accounting for 3-5% of new melanoma diagnoses) that often presents in advanced stages.  Up to 50% of patients with this condition develop metastases, and once metastatic, five-year survival is only 15%.  To better understand the mechanism of uveal melanoma metastases, we studied transcription factor AP-2α (encoded by the TFAP2A gene), which has previously been shown to drive cutaneous melanoma metastasis by activating downstream EZH2 through inhibition of the Nucleosome Remodeling and Deacetylase (NuRD) complex.


Methods:

TFAP2A knockout (KO) was generated with a double guide RNA CRISPR/Cas9 system in uveal melanoma lines Mel202 and 92.1, using electroporation with a nontargeting (NT) guide RNA as a control.  KO was confirmed by western blot analysis.  Cell proliferation and anchorage-independent growth phenotypes were assessed in vitro with MTT and soft agar assays, respectively.  To evaluate the role of the AP-2α/EZH2 pathway in uveal melanoma, western blot for EZH2 expression was performed after TFAP2A KO.  Then, uveal melanoma cells were treated with tazemetostat, a small molecular EZH2 inhibitor.  Effectiveness of tazemetostat treatment was confirmed by western blot and cell proliferation after treatment was assessed with the MTT assay.

Results:

Targeting of TFAP2A with CRISPR/Cas9 effectively knocked out AP-2α protein expression in uveal melanoma cell lines 92.1 and Mel202.  TFAP2A KO significantly decreased cellular proliferation in comparison to NT control (p < 0.0001) (Figure 1).  Anchorage-independent colony formation was also significantly reduced, though this may be explained by the suppressed cellular proliferation.  Furthermore, as observed in cutaneous melanoma, EZH2 protein expression was decreased in uveal melanoma TFAP2A KO cells.  Tazemetostat treatment was found to effectively decrease in the cellular content of H3K27me3, confirming pharmacologic inhibition of EZH2.  As a result, 92.1 cells had decreased proliferation after tazemetostat treatment, suggesting the AP-2α/EZH2 pathway is functional in uveal melanoma.


Conclusions:

The AP-2α/EZH2 pathway regulates uveal melanoma proliferation and may affect anchorage-independent growth.  By contrast, in cutaneous melanoma lines, inhibiting the AP-2α/EZH2 pathway reduced metastasis but did not alter proliferation or tumorigenesis.  Early studies suggest that the AP-2α/EZH2 pathway is functional in uveal melanoma and may be a therapeutic target, though further pre-clinical studies are needed.