Using Nonclassical Estrogen Signaling to Prevent Melanoma

Our laboratory discovered that the molecular mechanism by which melanocortin 1 receptor (MC1R) activation and cAMP signaling impacts nucleotide excision repair (NER) is by PKA-mediated phosphorylation of ATR, an event that is scaffolded by AKAP12, and that this post-translational modification of ATR enhances its association with XPA. We have already determined that GPER signaling accelerates the repair of UV photodamage and have strong preliminary evidence that the GPER signaling cascade intersects with MC1R-mediated events. Therefore, we will determine the kinetics of PKAmediated ATR phosphorylation (on S435, the critical site for enhancement of DNA repair) downstream of GPER signaling. We will directly interrogate the involvement of ATR-pS435 in GPER-mediated enhancement of NER by using phospho-mimetic and phospho-dead constructs already in use in our MC1R-focused studies. We will determine whether GPER-mediated NER enhancement is mediated through enhanced binding of XPA to UV-damaged DNA by measuring chromatin-associated XPA through Western blotting and by the oligonucleotide retrieval immunoprecipitation (ORiP) method that our lab developed using UV-damaged oligonucleotides as “bait”. We will determine whether GPER signaling enhances 5’ strand incision (as MC1R signaling does) by using well-established strand incision assays and determine the need for AKAP12 in estrogen-enhanced NER by using Crispr-deleted cells rescued with wilt type, PKA-binding defective and ATR binding-defective AKAP12 transfection rescue. As GPER signaling is a translationally appealing approach to optimize genomic stability in MC1R-defective (melanoma-prone) individuals, we will determine whether GPER signaling enhances NER in MC1Rdefective melanocytes by measuring clearance of UV photoproducts +/- GPER stimulation. Finally, we will determine the impact of GPER signaling on UV-induced mutagenesis to show in proof-of-principle experiments that GPER activation protects melanocytes against UV genomic instability. We have the critical experience and perspective and have all of the necessary reagents and experimental protocols “up and running” to carry out this work.