Inhibition of Radiation-Induced Salivary Gland Fibrosis by Targeting Copper Metabolism

ABSTRACT A first-line treatment for head and neck cancer is radiation therapy, but ionizing radiation can lead to chronic oral complications such as fibrosis of the salivary glands (SG) and xerostomia. Therapeutic strategies to restore SG function include gene therapy, stem cell transplantation and various bioengineering approaches; however, they

are dependent on the presence of residual functional SG tissue, a condition not met with full radiation treatment due to extensive fibrotic coverage of the SG. Although a role for Lysyl Oxidase (LOX) in radiation-induced fibrosis has not been investigated in SG, it is notable that each LOX family member has been implicated in various

fibrotic disorders affecting a wide range of organs. This has prompted efforts to develop monoclonal antibodies targeting specific LOX enzymes; however, recent Phase II clinical trials of a monoclonal antibody against Lysyl Oxidase-Like (LOXL2) failed to show efficacy against primary sclerosing cholangitis. These findings underscore

the importance of considering functional redundancy when targeting individual LOX and since the catalytic site of all LOX family members possesses a conserved binding site for copper (Cu), focusing on inhibition of Cu delivery to these enzymes is a novel antifibrotic strategy. Preliminary studies have identified a novel compound,

MKV3, as a potent inhibitor of the ATP7A copper transporter. Furthermore, ATP7A trafficking to the plasma membrane enhances LOX activity in cancer cells which is blocked by MKV3. Ionizing radiation was found to stimulate Cu-dependent ATP7A trafficking in rat parotid Par-C10 cells and that copper modulators, such as

tetrathiomolybdate (TTM) and MKV3, block this process. To extend these findings to in vivo studies, preliminary results indicate that radiation treatment not only triggers Cu-dependent ATP7A trafficking in mouse submandibular glands (SMG) from the perinuclear region to the plasma membrane but also causes collagen

deposition of extracellular matrix, with these events being reduced by TTM. Finally, ATP7A localization was found to be altered in the fibrotic SMG of irradiated patients, thus indicating a likelihood for clinical applications of these findings. Based on the above, it is hypothesized that ionizing radiation triggers Cu-stimulated ATP7A

trafficking in SG, which facilitates the metalation of LOX enzymes leading to increasing collagen crosslinking and fibrosis. Therefore, the following Aims are proposed: Aim 1 will elucidate the molecular mechanisms of altered Cu homeostasis in irradiated SG. Aim 2 will evaluate the therapeutic potential of SMG-specific Atp7a gene

silencing on radiation-induced fibrosis and saliva secretion. Aim 3 will evaluate the therapeutic effects of pharmacological targeting of copper transport on radiation-induced SMG fibrosis. Together, these studies will demonstrate that targeting of Cu metabolism can be used as a novel treatment for radiation-induced SG fibrosis.