2.1 Cell culture

HeLa, SiHa, and HEK 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM; Cytiva) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C in 5% CO₂ humidified atmosphere. MDA-MB-468, U-937, and A549 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (HyClone) supplemented with 10% FBS under same conditions.

2.2 Reagents and antibodies

The following primary antibodies were used for immunoblotting: mouse monoclonal anti-LY6K (Santa Cruz Biotechnology; sc-393560), sheep polyclonal anti-LY6K (R&D Systems; AF6648), rabbit monoclonal anti-caveolin-1 (Cell Signaling Technology; #3267), rabbit polyclonal anti-TGFβRI (Santa Cruz Biotechnology, sc-398), rabbit polyclonal anti-TGFβRI (Santa Cruz Biotechnology, sc-402), rabbit monoclonal anti-EGF receptor (Cell signaling Technology, #4267), rabbit monoclonal anti-clathrin heavy chain (Cell Signaling Technology, #4796), goat polyclonal anti-Smad2/3 (Santa Cruz Biotechnology, sc-6033), rabbit polyclonal anti-pSmad2 (Ser465/467) (Cell Signaling Technology, #3101), rabbit monoclonal anti-pSmad3 (Ser423/425) (Cell Signaling Technology, #9520), rabbit monoclonal anti-AKT (pan) (Cell Signaling Technology, #4691), rabbit monoclonal anti-pAKT (Ser473) (Cell Signaling Technology, #4060), rabbit polyclonal anti-p44/42 MAPK (ERK1/2) (Cell Signaling Technology, #9102), rabbit polyclonal anti-p44/42 MAPK (ERK1/2) (Cell Signaling Technology, #9102), mouse monoclonal anti-pp44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell signaling Technology, #9106). The following primary antibodies were used for immunoprecipitation: mouse monoclonal anti-LY6K (Santa Cruz Biotechnology, sc-393560), rabbit polyclonal anti-TGFβRI (Santa Cruz Biotechnology, sc-398), rabbit monoclonal anti-EGF receptor (Cell signaling Technology, #4267), mouse IgG isotype control (Santa Cruz Biotechnology, sc-2025). The following primary antibodies were used for immunofluorescence: mouse monoclonal anti-LY6K (Santa Cruz Biotechnology, sc-393560), rabbit polyclonal anti-TGFβRI (Santa Cruz Biotechnology, sc-398), rabbit monoclonal anti-EGF receptor (Cell signaling Technology, #4267), rabbit monoclonal anti-EEA1 (C45B10) (Cell Signaling Technology, #3288), rabbit monoclonal anti-caveolin-1 (Cell Signaling Technology, #3267), rabbit monoclonal anti-clathrin heavy chain (Cell Signaling Technology, #4796). HRP-conjugated goat anti-mouse or anti-rabbit IgG (Fab) secondary antibodies were purchased from Enzo Life Sciences, HRP-conjugated mouse anti-goat IgG from Santa Cruz Biotechnology, and HRP-conjugated donkey anti-sheep from R&D Systems. Recombinant human TGF-β1 protein was purchased from R&D Systems.

2.3 Plasmids and RNA interference

Human TGFβR1 cloned in mCherry-N1 vector (#54969) was purchased from Addgene. pCMV6-LY6K was a gift from Prof. Jong Hoon Park, Sookmyung Women's University. Plasmid transfections were performed using Jetprime (Polyplus-transfection) reagent according to the manufacturer's protocol. To silence LY6K expression, cells were transfected with 20 ng/L siRNA using Lipofectamine RNAiMAX transfection reagent (Invitrogen). siRNAs were synthesized by Bioneer with sequence: 5′-UUUCUCUCUCACAAACAUG-3′ (siLY6K #2). 5′-AAGGAGGUGCAAAUGGACAGA-3′ (siLY6K #3).

2.4 Reverse transcription quantitative polymerase chain reaction

Total RNA was isolated using RNAiso Plus (TaKaRa), where 500 μL of the reagent was added to each cell culture in a 6-well plate. The cell suspensions were mixed with 100 μL chloroform in Eppendorf tubes, centrifuged at 18,000 × g for 15 min at 4°C, and 200 μL supernatant was harvested. It was then mixed with an equal volume of isopropanol and centrifuged at 18,000 × g for 15 min at 4°C. The pellets were dissolved in ultrapure water (Invitrogen). The extracted RNA was reverse transcribed using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific), and RT-qPCR was performed using 2× Q-PCR Master Mix (SMOBIO) and LY6K specific primers (forward: 5′-AGCCCATGCCCTTCTTTTACCTCA-3′; reverse: 5′-CCAGCCACAGCCCACAG-3′). The expression levels were normalized with β-actin level.

2.5 Immunoblotting

Cells were lysed using GST -IP (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% (v/v) NP-40, and a protease inhibitor cocktail tablet [Roche]) and incubated for 15 min on ice. After centrifugation at 18,000 × g, the supernatant was mixed with 5× SDS and boiled at 99°C for 10 min. The proteins were separated by SDS polyacrylamide gel electrophoresis and transferred onto a 0.45 μm nitrocellulose membrane (Cytiva). The membranes were blocked with 3% (w/v) BSA in TBS-T for 20 min and incubated overnight with the appropriate primary antibodies at 4°C. The membranes were then washed with TBS-T and incubated with HRP-conjugated secondary antibodies in 5% (w/v) skim milk prepared in TBS-T at room temperature. The protein signal was detected with the ECL solution and analyzed in a Fusion Solo-S image analyzer (Vilber Lourmat).

2.6 Proliferation and wound healing assay

HeLa and SiHa cells were seeded into 96-well plates after transfection with siRNA. The cells were photographed using a Lionheart FX Automated Microscope (Agilent) at 37°C/5% CO₂ for 48 h. The cell area was calculated using the Gen5 software.

For the wound healing assay, cells were seeded into 6-well plates. After 48 h post-transfection, the cell monolayer was manually wounded with a 200 μL micropipette tip. The cells were imaged using Lionheart FX Automated Microscope (Agilent) at 37°C/5% CO₂ for 48 h. The wound closure was calculated using the Gen5 software.

2.7 Invasion assay

The cells were seeded into the upper chamber of inserts (8 μm pore size, 6.5 mm insert; SPL) coated with Matrigel (Corning). After 24 h, the cells and medium were removed from the upper chamber and washed with PBS. The invasive cells were then fixed by placing the inserts into 4% paraformaldehyde in a 24-well plate. After drying the inserts, 1% crystal violet (Sigma-Aldrich) was added to the 24-well plate to stain the cells. The inserts were then carefully washed with distilled water and dried using a cotton swab.

2.8 Immunoprecipitation

Cells were lysed with the lysis buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl, 1% v/v TritonX-100, 1 mM EDTA, and 10% v/v glycerol). Following centrifugation, 1 mg cell lysate protein was incubated with 1 μg antibody for 2 h at 25°C and then with 30 μL protein G beads (Amicogen) overnight at 4°C. The immunocomplexes were washed (4 times) with lysis buffer and mixed with 5× SDS buffer to perform immunoblotting as described above (Section 2.5).

2.9 Endosome isolation

Endosome compartments were isolated from HeLa and SiHa cells using a Minute™ Endosome Isolation and Cell Fractionation Kit (Invent Biotechnologies; ED-028) as indicated. Briefly, cells were resuspended in buffer A and incubated for 10 min. The filter cartridge filled with cell extracts was centrifuged and the pellet was obtained as a plasma membrane. The supernatant added buffer B was incubated overnight at 4°C. The endosome pellet was resuspended with RIPA buffer.

2.10 Immunofluorescence

HeLa and SiHa cells were cultured onto glass coverslips and fixed with 4% (v/v) formaldehyde (Sigma-Aldrich) diluted in PBS for 15 min at room temperature. The cells were then washed with PBS, permeabilized, and blocked with 0.1% Triton X-100 and 3% BSA in PBS for 15 min at room temperature. The cells fixed onto coverslips were incubated with the primary antibody at 4°C overnight and then with secondary antibody at room temperature for 2 h. DAPI (Sigma-Aldrich) was utilized for staining nuclei.

2.11 Transmission electron microscopy

Cells were fixed with 2% paraformaldehyde and 2.5% (v/v) glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer pH 7.2 at 4°C overnight. The fixed cells were washed with 0.05 M sodium cacodylate buffer, postfixed with osmium tetroxide (OsO4) at 4°C for 1 h, and then stained with 0.5% uranyl acetate in distilled water at 4°C overnight. The samples were serially dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) and embedded in Spurr's resin. For TEM imaging, ultrathin sections (70 nm) of solid resin were prepared using an ultramicrotome (UC7; Leica), and the specimens were photographed under a transmission electron microscope (LIBRA 120; Carl Zeiss).

2.12 Bioinformatic analysis

LY6K mRNA expression levels in human cervical cancer patients and healthy individuals were obtained from the UCSC Xena Project (http://xena.ucsc.edu/) while its expression levels at the cervical cancer stage and survival outcomes were obtained from cBioportal (https://www.cbioportal.org/). Its expression levels during human cervical cancer progression from normal to grade 3 stage were obtained from GSE63514. LY6K mRNA expression in various cancer patients was acquired from GEPIA (http://gepia.cancer-pku.cn/).

2.13 Statistical analysis

Values are presented as mean ± standard deviation (SD). One-way ANOVA and Two-way ANOVA were used for multiple comparisons within groups, and individual group mean differences were analyzed using two-tailed Student's t-test. Values of *p < 0.05, **p < 0.01, and ***p < 0.001 were considered benchmarks of significant differences. All statistical analyses were performed using GraphPad Prism 8.0.

3.1 Expression level of LY6K is elevated in cervical cancer

The plasma membrane-localized CTA was selected from 110 genes encoding membrane-localized proteins, obtained after GeneCards database analysis (http://www.genecards.org) of 1019 CTA gene candidates previously published as highly expressed CTA genes.²⁴ The expression levels of these 110 genes were compared among various human cancer and normal tissues (http://gepia.cancer-pku.cn/index.html). The workflow has been presented diagrammatically in Figure 1A. Finally, seven genes showing high expressional variation between normal and cancerous tissues were selected. Among these, LY6K was significantly upregulated in lung and cervical cancer tissues than in others (Figure 1B). Indeed, high LY6K expression was observed in human cervical cancer cells viz. HeLa and SiHa cells (Figure 1C). Although LY6K mRNA expression levels were lower in the U937 cells than in HeLa and SiHa cells (Figure 1D), LY6K protein expression was detected, suggesting translational regulation. Furthermore, the Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) cohort were used to analyze LY6K mRNA expression in association with the pathological characteristics, where LY6K was upregulated during higher grade in cervical cancer patients (Figure 1E). However, the LY6K expression level showed no significant difference between the cervical cancer metastatic stages in the TCGA database (Figure 1F). Cervical cancer patients with high levels of LY6K expression showed poor OS. Although the difference in both disease-free and progression-free survival rates was not statistically significant, they exhibited a tendency toward poor outcomes (Figure 1G–I).

3.2 Loss of LY6K inhibits proliferation, migration, and invasiveness of cervical cancer cells

The depletion of LY6K in breast cancer cells decreases their migration and invasion⁷; therefore, the effect of LY6K depletion in cervical cancer cells was examined on their proliferation, migration, and invasion. HeLa and SiHa cells were transfected with two siLY6Ks and the loss of LY6K expression was confirmed by RT-PCR and immunoblot assays 48 h post-transfection (Figure 2A). The selected siLY6K #2 was used for subsequent experiments. Inhibition in the basal level of proliferation was observed upon LY6K depletion in these cells, and the increase in proliferation exhibited upon EGF treatment was abolished. Additionally, the impairment of EGF-induced proliferation was greater than that observed upon TGF-β induction in both HeLa and SiHa cells (Figure 2B). Furthermore, the native cells showed higher migration and invasion rates 48 h post-treatment with TGF-β, and these effects were abolished upon LY6K depletion. Such impaired migration and invasiveness in the LY6K-depleted HeLa and SiHa cells was larger upon induction with TGF-β than with EGF (Figure 2C,D). Therefore, LY6K depletion suppresses the effects of EGF and TGF-β along with the basal levels of proliferation, migration, and invasiveness in cervical cancer cells. These results indicate the role of LY6K in the proliferation, migration, and invasiveness of cervical cancer cells, which enhance upon treatment with TGF-β and EGF.

3.3 Depletion of LY6K reduces TGF-β-mediated Smad activation but increases EGF signaling pathway

Since TGF-β- and EGF-induced proliferation, migration, and invasiveness are affected in LY6K-depleted cervical cancer cells, the effect of TGF-β and EGF on signaling pathways was further studied in such cells. TGF-β-induced Smad2 phosphorylation was significantly suppressed in LY6K-depleted HeLa and SiHa cells. However, Smad3 phosphorylation showed no appreciable alteration (Figure 3A). LY6K-depleted HeLa and SiHa cells also showed enhanced phosphorylation of EGFR and ERK 1 h after EGF treatment (Figure 3B). Since the proliferation propensity of LY6K-depleted cervical cancer cells is low, the enhanced EGF signaling pathway was further resolved. The phosphorylation of EGFR, AKT, and ERK was examined 1-day post-treatment with EGF rather than short-term treatment. The EGF signaling pathway was attenuated upon LY6K depletion, whereas no variations were observed in the TGF-β signaling pathway between short-term and long-term treatments with TGF-β (Figure 3B). To rule out potential off-target effect of the siRNA, we examined the TGF-β and EGF signaling pathways using another siLY6K#3. However, no significant differences were observed, indicating that the observed effect were specific to LY6K depletion (Figure S1).

Furthermore, the TGF-β signaling pathway is altered in LY6K-depleted cells; therefore, the interaction between LY6K and TGF-β receptor-I (TβRI) at the plasma membrane was assessed. Myc-tagged LY6K and mCherry-tagged TβRI were highly expressed in HEK293T cells. The LY6K was pulled down, where it co-immunoprecipitated with TβRI (Figure 3C). To further confirm their interaction, HeLa and SiHa cells were treated with TGF-β, and lysates were immunoprecipitated with anti-LY6K antibody to examine endogenous interaction. LY6K was observed to bind TβRI, regardless of the presence of TGF-β (Figure 3D). In contrast to TβRI, EGFR did not bind endogenous LY6K in HeLa and SiHa cells regardless of the presence of EGF (Figure 3E). Conclusively, LY6K-depletion associates with the inhibition and activation of TGF-β and EGF signaling pathways, respectively, during their short-term treatments.

3.4 Depletion of LY6K suppresses TβRI and EGFR-mediated endocytosis

The co-localization of TβRI and EGFR with LY6K in the plasma membrane and endosome was examined to explore the underlying mechanism by which LY6K affects TGF-β and EGF signaling pathways. After the treatment of TGF-β and EGF, the levels of TβRI and EGFR were examined at the plasma membrane and endosome. In LY6K-depleted cells, treatment of TGF-β and EGF resulted in reduced levels of TβRI and EGFR in endosome (Figure 4A), suggesting that endocytosis of TβRI and EGFR is mediated by LY6K. To further confirm, the localization of TβRI and EGFR was examined using immunofluorescence. Following treatment with TGF-β, TβRI and LY6K predominantly colocalized at the plasma membrane, while a fraction of them migrated to the endosome in HeLa and SiHa cells (Figure 4B). However, in the absence of LY6K, TβRI scattered at the plasma membrane, and TGF-β treatment did not affect its migration into the endosome from the plasma membrane (Figure 4B).

Contrastingly, LY6K did not colocalize with EGFR, and EGF treatment promoted endosome localization of EGFR; however, upon LY6K depletion, EGFR remained at the plasma membrane (Figure 4C). These results indicate that TβRI and EGFR localize at the plasma membrane, and receptor-mediated endocytosis in LY6K-depleted cells rarely occurs in response to TGF-β and EGF, suggesting the implication of LY6K in TGF-β- and EGF-mediated signaling pathways.

3.5 Depletion of LY6K decreases Clathrin- and CAV-1-mediated endocytosis

LY6K is involved in clathrin- and CAV-1-mediated signal transfer, and the signals of EGF and TGF-β are transferred by receptor endocytosis at the plasma membrane,^{25, 26} therefore, alterations in the endocytic proteins, clathrin and CAV-1, as an effect of LY6K depletion were examined. In the absence of LY6K, the clathrin levels decreased in both HeLa and SiHa cells, but CAV-1 levels only decreased in SiHa cells (Figure 5A). Furthermore, the localization of clathrin and CAV-1 was examined in the presence and absence of LY6K. CAV-1 clustered in the lipid rafts in both HeLa and SiHa cells and migrated into the endosome in response to TGF-β but not EGF treatment in HeLa cells, while in response to both in SiHa cells. Although HeLa and SiHa cells are cervical cancer cell lines, they are different types of cancer. HeLa is an adenocarcinoma cell line, while SiHa is a squamous cell carcinoma cell line. Although there is a report suggesting differential performance of CAV-1 among breast cancer cell lines,²⁷ the underlying reasons for this variation are yet to be elucidated. However, no such cytosolic migration was observed upon LY6K depletion, despite TGF-β and EGF treatment (Figure 5B), indicating that LY6K is required during CAV-1-mediated endocytosis. Concerning clathrin, both LY6K-depleted HeLa and SiHa cells showed lower clathrin levels in the plasma membrane and less perinuclear clustering compared with non-depleted cells. Even after treatment with TGF-β and EGF, the localization of clathrin remained unaltered in absence of LY6K (Figure 5C), indicating that clathrin-mediated endocytosis reduces in the absence of LY6K.

The proteins endocytosed via clathrin and CAV-1 are delivered to early endosome antigen-1 (EEA1)-positive endosomes.^{28, 29} The early endosomes undergo maturation and move to the perinuclear region after forming clusters.³⁰ Both HeLa and SiHa cells displayed scattered localization of EEA1-positive endosomes throughout cytosol, which moved to the perinuclear region following EGF and TGF-β treatment for 1 h. However, this perinuclear movement in response to EGF and TGF-β was impaired upon LY6K depletion (Figure 5D).

The involvement of LY6K in TGF-β-mediated endocytosis was further confirmed where endocytic compartments were observed by transmission electron microscopy. CAV-1-mediated endocytosis regulated TβRI levels, but clathrin-mediated endocytosis did not occur in the absence of TGF-β. However, in presence of TGF-β, clathrin-mediated endocytosis was high in both HeLa and SiHa cells, whereas it was impaired following LY6K depletion (Figure 5E). Conclusively, LY6K plays an important role in membrane endocytosis of cancerous cells, irrespective of the type of endocytosis.