Exploring PAR2 Trafficking in Oral Cancer: Implications for Pain Signaling and Drug Delivery

PROJECT SUMMARY Opioids remain the most efficacious, but insufficient, analgesic regimen for treating patients with oral cancer pain. Oral cancer pain is generated at the primary cancer site and is associated with high levels of proteases released in the cancer microenvironment. These proteases cleave distinct regions of the protease-activated

receptor 2 (PAR2) to activate PAR2 and mediate cleavage site-specific intracellular trafficking and signaling of the receptor. Mice lacking PAR2 do not develop oral cancer pain, highlighting a central role for neuronal PAR2 in oral cancer pain. The impact of oral cancer on PAR2 signaling and trafficking in trigeminal (TG) neurons is not

known. The long-term goal is to improve management of oral cancer patients by identifying components of the PAR2 signaling cascade that are viable targets for development of location-selective drug delivery systems to alleviate oral cancer pain. The overall objectives for this application are to augment my expertise in signaling

and nanoparticle drug development by gaining training in oral cancer pain and research with complex biological systems, as well as undertaking career development activities to foster transition to an independent investigator. The central hypothesis for the proposed research is that oral cancers release proteases that activate PAR2 and

induce sustained pain via PAR2 signaling from multiple cellular compartments in neurons. The rationale for this project is that identification of cancer-induced PAR2 signaling and trafficking affords the opportunity to develop drug delivery systems that specifically target these signaling pathways to treat cancer pain. The central

hypothesis will be tested by three Aims: K99, Determine oral cancer-induced changes in the 1) trafficking and 2) signaling of PAR2 in TG neurons, and 3) R00, Develop nanoparticles to target PAR2 located at the plasma membrane, endosomes and Golgi to alleviate oral cancer pain. In Aim 1, cancer induced changes in PAR2

trafficking will be investigated by confocal microscopy using TG neuron cultures from PAR2-muGFP mice bearing oral cancers and controls. Aim 2 will evaluate the signaling of neuronal PAR2 by measuring cAMP and Ca2+ in TG neuron cultures from cancer bearing and naïve mice expressing PAR2-muGFP in sensory (Nav1.8) neurons.

For aims 1 and 2, TG cultures will be challenged with (a) four oral cancer proteases, (b) a mixture of these proteases, and (c) conditioned media (CM) from human oral cancers that contain pain mediators (proteases) released by the cancers. The focus of the R00 phase will be on the development and in vitro and in vivo

evaluation of location-selective nanoparticles targeting PAR2 at the plasma membrane, endosomes and Golgi. The proposed studies are innovative because they are motivated by previously unappreciated oral cancer- induced PAR2 trafficking and signaling, and the drug targeting and delivery approach. The work is significant

because these studies will lay the foundation for repurposing drugs to target PAR2 at specific intracellular sites.