AbstractBackgroundLymphocyte antigen 6 complex locus K (LY6K), a glycosylphosphatidylinositol‐anchored protein, plays a dynamic role in cancer metastasis. In the current study, we deciphered the effects of LY6K on transforming growth factor‐β (TGF‐β) and epidermal growth factor (EGF) signaling through clathrin‐ and caveolin‐1 (CAV‐1)‐mediated endocytosis.MethodsAnalysis of the TCGA and GTEx dataset were performed to explore the expression and survival of LY6K in cancer patients. Short interfering RNA (siRNA) was used to knockdown the expression of LY6K in human cervical cancer patients. The effect of lack of LY6K on cell proliferation, migration, and invasion was performed, and RT‐qPCR and immunoblotting were performed to identify LY6K‐affected TGF‐β and EGF signaling pathways. Additionally, Immunofluorescence (IF) and transmission electron microscope (TEM) were performed to identify the role of LY6K in CAV‐1‐ and Clathrin‐mediated endocytosis.ResultsLymphocyte antigen 6 complex locus K expression level is elevated in higher grade cervical cancer patients correlating with poor overall survival, progression‐free survival, and disease‐free survival. LY6K‐depletion in HeLa and SiHa cancer cells suppressed EGF‐induced proliferation and TGF‐β‐enhanced migration and invasion. Both TGF‐β receptor‐I (TβRI) and EGF receptor (EGFR) localized at the plasma membrane regardless of LY6K expression, and LY6K bound TβRI irrespective of the presence of TGF‐β; however, LY6K did not bind EGFR. LY6K‐depleted cells showed impaired Smad2 phosphorylation upon TGF‐β treatment and lower proliferation rates following long‐term treatment with EGF. We revealed the atypical movement of TβRI and EGFR from plasma membrane upon ligand stimulation in LY6K‐depleted cells and an impaired movement of the endocytic proteins clathrin and CAV‐1.ConclusionsOur study demonstrates the key role of LY6K in both clathrin‐ and CAV‐1‐mediated endocytic pathways regulated by TGF‐β and EGF, and it suggests a correlation between LY6K overexpression in cervical cancer cells and poor overall survival.