Functional Restoration of Irradiated Salivary Glands Through Modulation of aPKCζ and Nuclear Yap in Salivary Progenitors

Abstract

Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 60,000 annual diagnoses in the U.S. and approximately 500,000 worldwide. About 90% of these individuals receive radiation therapy, and salivary hypofunction and xerostomia occur in 60-85% of these patients due to irreversible damage to the salivary glands. Current preventative and palliative care fail to improve quality of life, accentuating the need for regenerative therapies. Stem/progenitor-cell based therapies have been proposed to regenerate the irradiated glands; however, the identity of stem and progenitor cells in the adult salivary glands has remained somewhat elusive. Moreover, it is unclear how salivary progenitors respond to radiation and whether they can be stimulated to effectively reinstate salivary function. The second chapter of the present study describes the development of a label-retaining assay in salivary glands using EdU. The label-retaining cells (LRCs) identified in murine salivary glands have proliferative potential in vitro and expressed markers of putative salivary progenitors, such as Keratin 5, Keratin 14, and c-Kit. Interestingly, LRCs were still present 30 days following radiation, when chronic loss of saliva is evident. The significance of these findings lies in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining salivary gland homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. In the following chapter, we show that a unique population of murine salivary gland LRCs undergo compensatory proliferation in response to radiation. The initiation of compensatory proliferation is tightly associated with inactivation of the kinase aPKCζ and increased nuclear localization of YAP. This part of the study provides novel insights into the regulation of function of salivary gland progenitors, which can be utilized for the development of therapeutic agents to treat salivary hypofunction. Finally, the last chapter describes how the mechanisms found to initiate compensatory proliferation in acinar LRCs as a response to radiation are involved in the regeneration of salivary glands with IGF-1. Administration of IGF-1 post-radiation restores salivary function in mice, but the mechanisms of regeneration are still unknown. Here, we show that IGF-1 requires aPKCζ to restore saliva production. Further, IGF-1 inhibits nuclear translocation of Yap in an aPKCζ-dependent fashion. We propose that a tightly regulated balance in the levels of aPKCζ and Yap in acinar LRCs has to be maintained in order to restore function following radiation. In conclusion, the findings from this study provide new knowledge in regards to the regulation of function of salivary progenitors during a state of injury (by radiation) and during regeneration (with IGF), and offer potential targets of study for the development of new therapeutics for salivary gland dysfunction. Future studies will determine whether aPKCζ and Yap can be effectively targeted in salivary progenitors to restore salivary function in head and neck cancer patients who receive radiation therapy.