Molecular Mechanisms of Flower Function During Epidermal Barrier Maturation and cSCC Development

Abstract

The epidermis functions as a critical barrier protecting the body from environmentalinsults, a function dependent upon proper execution of keratinocyte terminal differentiation. This process is tightly regulated by calcium (Ca²⁺)-dependent molecular events that enable dramatic alterations in keratinocyte structure and behavior. Despite decades of research, the molecular mechanisms that translate Ca²⁺ signals into the polarized secretion of lamellar bodies (LBs), specialized organelles essential for barrier formation, remain incompletely understood. This thesis identifies FLOWER (FWE), a small transmembrane protein with no prior known function in skin, as a novel regulator of Ca²⁺-dependent LB trafficking during epidermal differentiation and demonstrates its relevance to both homeostatic barrier formation and disease. Using computational modeling and experimental validation, the canonical human FWE isoform, hFWE4, was revealed to assume a four-transmembrane domain structure positioning N- and C-terminal tails cytosolically. This topology enabled interaction with vesicular trafficking machinery and facilitated rapid, AP-2-dependent endocytosis from the plasma membrane. In contrast, the non-canonical isoform hFWE3 was retained in the endoplasmic reticulum with no apparent trafficking function. Investigations into FWE expression in human epidermis revealed robust upregulation in terminally differentiating keratinocytes of the upper stratum spinosum and stratum granulosum, where FWE localized to LBs. Genetic ablation of FWE in epidermal organoids impaired barrier function and dysregulated expression of genes involved in cornification and LB biology. Proteomic profiling of hFWE4-positive vesicles identified extensive trafficking machinery including Rab GTPases, SNARE complexes, and diverse LB cargo. hFWE4 potentiation of intracellular Ca²⁺ release facilitated surface trafficking of LB cargo. FWE-positive LBs exhibited striking mislocalization in Darier and Grover disease, providing support for the clinical relevance of FWE-dependent vesicular trafficking. Investigation of FWE in cutaneous squamous cell carcinoma (cSCC) revealed expression in highly differentiated tumor regions. FWE knockout altered keratinization patterns and impaired terminal differentiation in xenografts, while ectopic hFWE4 expression induced cell cycle arrest and differentiation. FWE expression reliably distinguished well-differentiated from poorly-differentiated tumors, suggesting utility as a prognostic biomarker. Collectively, this work established FWE as a multifunctional regulator of Ca²⁺-dependent LB trafficking essential for epidermal barrier formation and positioned it as a promising therapeutic target and diagnostic marker in cutaneous disease.