The human Flower isoform hFWE4 facilitates cornification in cutaneous squamous cell carcinoma

Background: Cutaneous squamous cell carcinoma (cSCC) arises from transformed keratinocytes that often retain partial differentiation potential. Because differentiation status correlates with prognosis, molecular markers that distinguish well- from poorly-differentiated (WD vs PD) tumors are of clinical value. Flower (FWE), a four-transmembrane protein that regulates lamellar body (LB) trafficking during epidermal barrier formation, was examined here as a candidate marker and regulator of terminal differentiation in cSCC. Methods: FWE expression was analyzed in human cSCC cell lines, xenografts, and clinical tumors. CRISPR/Cas9 was used to generate hFWE knockout (KO) SCC-13 cells for xenografting in NCG mice (n=12). RNA-seq, immunoblotting, and immunofluorescence quantified differentiation-associated changes in KO tumors. Lentiviral EGFP-2A-hFWE4 constructs were used to assess impact of hFWE4 overexpression on proliferation and differentiation outcomes. Results: FWE was induced during Ca²⁺-driven differentiation of cultured SCC cells and localized to suprabasal keratinocytes in SCC-13 xenografts. hFWE KO tumors exhibited a slight reduction in average mass (0.33 to 0.19g, p<0.05) and showed altered keratinization characterized by reduced keratohyalin granule-containing cells and solid parakeratosis. Immunofluorescence of KO tumors revealed 63% and 82% reductions in filaggrin- and loricrin-positive areas, respectively (p<0.01-0.0001), while RNA-seq identified 73 downregulated genes (FDR<0.05, |log₂FC|>1)-enriched for LB and cornification pathways-including KLK5 , KLK7 , SLURP1 , and LORICRIN . Ectopic hFWE4 expression induced G1 arrest (↑G1 by 5-17%, ↓S-phase by 6-10%; p < 0.0001) in SCC-13, COLO 16 and SCC-12b.2 cells, and reduced the fraction of cells that were Ki67⁺ (0.57% to 0.13%, p<0.05) and ITGB1⁺ (11.3% to 2.4%, p<0.05) while increasing the fraction that were filaggrin<⁺ (34.6% to 57.2%; p<0.01) in SCC-13 xenografts. In human tumors (WD n=9, PD n=5), FWE-positive area was significantly higher in WD than PD regions (6.2-fold increase; p<0.0001). Conclusions: In cSCC, loss of FWE disrupts LB-dependent cornification, while ectopic expression elicits G1-arrest and differentiation. As high FWE-positive area correlates with increased differentiation grade in human cSCC, we propose that FWE represents both a mechanistic regulator of cornification and a promising molecular marker for objective grading of cSCC differentiation.