Project Summary Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key lipid-signaling molecule that regulates a vast array of biological activities. However, the function of PIP2-regulated signaling in mammalian cell-fate decision and tumorigenesis has received less attention. Research from our laboratory indicates that an endosome-localized PIP2 producing enzyme, type I gamma phosphatidylinositol phosphate kinase i5 (PIPKIγi5), is crucial for the regulation of Epidermal Growth Factor Receptor (EGFR) and Hippo signaling, two essential signaling pathways in the control of cell proliferation and Cancer Stem Cell (CSC) regeneration. The dysregulation of EGFR or Hippo signaling has been found in many types of cancers including head and neck squamous cell carcinoma (HNSCC). Here is the current hypothesis: By interacting with the small GTPase Rab7a, PIPKIγi5 modulates EGFR endosomal trafficking and degradation; By interacting with the E3 ubiquitin ligase Neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4), PIPKIγi5 controls the stability of Hippo pathway core components WW45 and LATS2; Via modulating the EGFR and Hippo signaling, PIPKIγi5 controls HNSCC CSC regeneration, tumorigenesis, and metastasis. Aim 1 will characterize the PIPKIγi5-Rab7a interaction and determine whether this interaction controls EGFR signaling by modulating endosome maturation. Aim 1 will also demonstrate whether PIPKIγi5 controls the Hippo core components WW45/LATS2 ubiquitination and degradation by associating with NEDD4. In Aim 2, both in vitro cell line models and in vivo xenograft mouse models will be used to determine the effects of PIPKIγi5 on HNSCC CSC self-renewal and metastasis. Furthermore, Aim 2 will further validate the function of PIPKIγi5 in HNSCC tumorigenesis by using PIPKIγi5-knockout mouse models. The successful completion of this application will reveal a novel PIP2- regulated pathway that controls HNSCC progression by modulating EGFR and Hippo signaling.