USP21-EGFR signaling axis is functionally implicated in metastatic colorectal cancer

USP21-EGFR signaling axis is functionally implicated in metastatic colorectal cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article USP21-EGFR signaling axis is functionally implicated in metastatic colorectal cancer Ki-Young LEE, Ji Hye Shin, Mi-Jeong Kim, Ji Young Kim, Bongkum Choi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4594251/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Cell Death Discovery → Version 1 posted 11 You are reading this latest preprint version Abstract The emerging significance of ubiquitin-specific peptidase 21 (USP21) in stabilizing Fra-1 (FOSL1) has shed light on their involvement in promoting colorectal cancer (CRC) metastasis. Additionally, EGFR signaling has been linked reciprocally with Fra-1 activation in an MMP-dependent manner. However, the functional implications of the USP21-EGFR signaling axis in metastatic CRC (mCRC) remain incompletely understood. RNA-Seq data from tumor tissues ( n = 27) and matched normal tissues ( n = 27) from 27 mCRC patients were analyzed to investigate the clinical correlation between USP21 and EGFR expression. Functional studies including CRISPR/Cas9 gene editing method to generate USP21 -knockout ( USP21 -KO) CRC cells, in vitro cancer progression and tumor formation assays, in vivo xenograft assays in NSG mice, and therapeutic assays with the USP21 inhibitor, BAY-805, were conducted. Elevated levels of USP21 and EGFR expression in mCRC patients correlated with poorer survival outcomes. Mechanistically, USP21 was found to enhance EGFR stability by deubiquitinating EGFR, resulting in reduced EGFR levels in USP21 -KO colon cancer cells. USP21 -KO colon cancer cells exhibited significantly attenuated cell proliferation, migration, colony formation, and 3D tumor spheroid formation in response to EGF. Furthermore, tumorigenic activity in vivo was notably diminished in NSG mice xenografted with USP21 -KO colon cancer cells. Notably, the USP21 inhibitor, BAY-805, demonstrated a remarkable inhibitory effect on the formation of 3D tumor spheroids in colorectal cancer cells stimulated with EGF. These findings provide valuable insights into the potential of USP21 as both a therapeutic target and a predictive biomarker for intervening in mCRC induced by EGF. Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer Health sciences/Biomarkers/Prognostic markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC) stands as a pervasive and deadly malignancy worldwide, driven by a myriad of factors encompassing lifestyle choices, environmental exposures, viral infections, and smoking habits [ 1 – 3 ]. Recent advancements in omics data analysis of CRC patients have unearthed a wealth of genetic information crucial for delineating the pathophysiological landscape of CRC progression and for crafting targeted therapeutic interventions [ 4 , 5 ]. Dysregulation of epidermal growth factor receptor (EGFR)-mediated signaling pathways emerges as a potent instigator of CRC initiation and advancement [ 2 ]. EGFR, a pivotal member of the ErbB protein family, exerts profound influence over CRC cellular processes including proliferation, angiogenesis, migration, invasion, and tumorigenicity [ 2 , 6 ]. Despite gaps in understanding the precise molecular and cellular mechanisms governing EGFR upregulation in CRC, omics data from CRC patient cohorts consistently highlight elevated EGFR expression levels in tumor tissues, correlating with dismal prognosis [ 7 ]. Consequently, concerted research endeavors have been directed towards unraveling the intricate cellular regulatory mechanisms modulating EGFR expression [ 2 , 6 , 7 ]. The regulation of cellular EGFR expression and activation hinges on intricate EGFR trafficking pathways, exerting significant influence over biological outcomes driven by EGFR signaling in cancer [ 8 ]. EGFR fate is governed by two principal pathways: the recycling pathway and the degradative multivesicular bodies (MVBs)-lysosome pathway [ 8 – 10 ]. Various cellular factors, including Rab4, Rab35, calcium-modulating cyclophilin ligand (CAML), and Eps15S, play crucial roles in the EGFR-recycling pathway, orchestrating prolonged EGFR signaling [ 8 – 10 ]. Notably, ubiquitin-specific peptidases (USPs) have emerged as key regulators of EGFR signaling, with several members of the USP family implicated in the modulation of EGFR degradation [ 11 – 17 ]. Among these, USP21 has garnered attention for its role in stabilizing Fra-1 and PD-L1, promoting CRC metastasis [ 18 – 22 ]. Given the interconnectedness between EGFR signaling, Fra-1 activation, and PD-L1 expression [ 18 , 22 ], targeting USP21 holds promise as a strategy for combating metastatic CRC driven by EGFR signaling. However, the functional and clinical relevance of USP21 in metastatic CRC, particularly in association with EGFR expression, remains largely unexplored. This study aims to elucidate the relationship between USP21 and EGFR in metastatic CRC, unraveling their clinical significance and functional implications. Through comprehensive biochemical and functional analyses, we demonstrate that USP21 plays a pivotal role in stabilizing EGFR and driving CRC progression triggered by EGF. Conversely, knockout of USP21 in CRC cells impedes CRC progression and attenuates tumor formation in response to EGF stimulation. Importantly, pharmacological inhibition of USP21 with BAY-805 exerts a potent inhibitory effect on CRC tumor spheroid formation, highlighting the therapeutic potential of targeting USP21 in mCRC. Collectively, our findings underscore the promise of USP21 as both a therapeutic target and a predictive biomarker for mitigating mCRC induced by EGFR signaling. Material and methods CRC patient specimens Primary tumor tissues and adjusted matched normal tissues of mCRC patients ( n = 27) were collected at Samsung Medical Center (SMC, Seoul, Korea). Licensed pathologists confirmed histologic diagnoses and estimated all formalin-fixed paraffin-embedded samples with purity ≥ 40% according to H&E staining. Written informed consent was obtained from all participants. All methods, including authorization for utilization of patients’ specimens, were carried out in accordance with relevant guidelines and regulations. Experiments conducted on patient samples were approved by the Institutional Review Board (IRB) of Samsung Medical Center (IRB# 2010-04-004). RNA sequencing was conducted for all samples, as previously described [ 23 ]. Xenografted NSG mouse model NOD/SCID/IL-2Rγ null (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained under specific pathogen-free conditions in accordance with ethical guidelines for the care of these mice at the Bioanalysis Center Animal Facility, GenNBio Inc. (Seongnam, Korea). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Bioanalysis Center Animal Facility (IACUC #: 23-10-01). NSG mice (6–8 weeks old) were used to generate xenografted NSG mice. Control (Ctrl) HCT-15 (5 × 10 6 cells per mouse, n = 10) or USP21 -knockout (KO) HCT-15 cells (5 × 10 6 cells per mouse, n = 10) were injected under NSG mice skin (back area) within serum-free PBS. The final injection volume was 100 µL/mouse containing a 1:1 v/v mixture of ice-chilled Matrigel (BD Biosciences, La Jolla, CA, USA), which was kept on ice until injection. Five days after cancer cell injection, tumor volume was measured with a caliper every 4 days until 37 days after injection. Tumor volume (mm 3 ) was calculated as (length x width) × 0.5. Tumor growth curves are presented as average tumor volume ± SEM for each group in this study. All studies involving mice were approved by IACUC. Cells HCT-15 (human colorectal cancer cell line; CCL-225, American type culture collection (ATCC), Manassas, VA, USA), SW480 (human colon cancer cell line; CCL-228, ATCC), and HT-29 (human colorectal adenocarcinoma cell line; HTB-38, ATCC) were maintained in a medium recommended by ATTC, supplemented with 10% fetal bovine serum (FBS), penicillin (100 µg/mL), and streptomycin (100 µg/mL) in a 5% CO 2 humidified atmosphere at 37°C. Human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM; Welgene, LM-001-05) supplemented with 10% fetal bovine serum (FBS). Generation of USP21 -knockout (KO) colon cancer cell lines with CRISPR/Cas9 two vector system To generate USP21- KO colon cancer cells with CRISPR/Cas9 gene editing method, we used two vector systems, single guide RNA (sgRNA) and CRISPR-associated protein 9 (Cas9) vectors. sgRNA and Cas9 vectors were kindly provided by Dr. Daesik Kim (Sungkyunkwan University School of Medicine, Suwon, Korea). Guide RNA sequences for CRISPR/Cas9 were designed on the CRISPR design website ( http://crispr.mit.edu/ ) provided by the Feng Zhang Lab. Insert oligonucleotides for human USP21 gRNA were 5’-ATGACCGAGCCAACCTAATG-3’ (gRNA-1) / 5’- GTTTCCACATTAGGTTGGCT-3’ (gRNA-2) / 5’-CTTCTCTGGATACAGCCAGC-3’ (gRNA-3). Complementary oligonucleotides to guide RNAs (gRNAs) were annealed and cloned into a sgRNA vector. The sgRNA vector expressing gRNA of USP21 and Cas9 vector expressing Cas9 were transfected into HCT-15, SW480, and HT-29 colon cancer cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. At two weeks after transfection, colonies were isolated and single-cell selection was performed. The expression of USP21 in USP21 -KO colon cancer cells was analyzed by western blotting assay with an anti-USP21 antibody. Antibodies and reagents Anti-Myc (sc40), anti-USP21 (sc-515911), Anti-HA (sc-7392), and anti-GAPDH (sc-47724) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Flag (F3165) antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-EGFR antibody (2232S) was purchased from Cell Signaling Technology (Danvers, MA, USA). TrueBlot® secondary antibody (18-8817-33) were purchased from Rockland Immunochemicals (Pottstown, PA, USA). Goat anti-rabbit IgG (HRP) (GTX213110-01) antibody was purchased from GeneTex Inc. (Irvine, CA, USA). Rabbit anti-mouse IgG H&L (HRP) (ab6728) antibody was purchased from Abcam (Cambridge, MA, USA). Dimethyl sulfoxide (DMSO; D4540), Phosphate-buffered saline (PBS; CBP007A), glutaraldehyde (G6257-100ml), crystal violet (C6158-50g), cycloheximide (CHX; C1988), EGF (SRP3027), and thiazolyl blue tetrazolium bromide (MTT; M5655) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000 (11668019) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). BAY-805 (HY-153045) was purchased form MedChemExpress (Monmouth Junction, NJ, USA). Plasmid constructs EGFR-GFP (32751) and Flag-HA-USP21 (22574) vectors were purchased from Addgene (Watertown, MA, USA). pCMV-3Tag-7 (240202) and pCMV-3Tag-6 (240200) vectors were purchased from Agilent technologies (Santa Clara, CA, USA). Using Flag-HA-USP21 plasmid, full-length USP21 was cloned into the pCMV-3Tag-6 vector to generate a Flag-USP21 vector. Using the EGFR-GFP plasmid, full-length EGFR was cloned into the pCMV-3Tag-7 vector to generate Myc-EGFR vector. Flag-USP21 C221A mutant was generated by site-directed mutagenesis using Flag-USP21 wild-type (WT) plasmid as previously described [ 24 ]. Western blotting assay Western blotting and immunoprecipitation (IP) assays were performed as previously described [ 25 – 28 ]. Briefly, cell lysates were prepared from control (Ctrl) HCT-15, Ctrl SW480, Ctrl HT-29, USP21 -KO HCT-15, USP21 -KO SW480, and USP21 -KO HT-29 cells. They were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8–12%) and immune-probed with an anti-USP21 or anti-GAPDH antibody. HEK-293T cells were transfected with a mock control vector, Myc-EGFR, or Flag-USP21 vector. Cells were then incubated at 37°C for 24 h. After collecting cells, cell lysates were prepared and immunoprecipitated with an anti-Myc antibody. IP complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8–12%) and immune-probed with an anti-Myc or anti-Flag antibody. Ubiquitination and deubiquitination assay HEK-293T cells were transiently transfected with mock, Myc-EGFR, HA-Ub, Flag-USP21 wild type (WT), or Flag-USP21 C221A mutant vector. Cells were then incubated at 37°C for 24 h. After collecting cells, cell lysates were prepared and immunoprecipitated with an anti-Myc antibody. IP complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8–12%) and immune-probed with an anti-Myc, anti-Flag, or anti-HA antibody. Cycloheximide (CHX) chase assay Cycloheximide (CHX) chase assay was performed to determine the half-life of EGFR following previous protocols [ 29 ]. Briefly, Control (Ctrl) HCT-15, Ctrl SW480, Ctrl HT-29, USP21 -KO HCT-15, USP21 -KO SW480, and USP21 -KO HT-29 cells were treated with CHX (20–30 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) for different time periods. EGFR was then detected using western blotting assay with an anti-EGFR antibody. Wound-healing migration assay A wound-healing migration assay was performed following previous protocols [ 25 , 30 – 33 ]. Briefly, Control (Ctrl) HCT-15, Ctrl SW480, Ctrl HT-29, USP21 -KO HCT-15, USP21 -KO SW480, and USP21 -KO HT-29 cells were seeded into 12-well plates and cultured to reach confluence. Cell monolayers were gently scratched and washed with a culture medium. After floating cells and debris were removed, cells attached to culture plates were treated with vehicle (DMSO, 0.01% v/v concentration) or EGF (20 ng/mL) for different time periods. Cell images were captured after culturing for different time periods as indicated in each experiment. Transwell migration assay Ctrl HCT-15, Ctrl SW480, Ctrl HT-29, USP21 -KO HCT-15, USP21 -KO SW480, and USP21 -KO HT-29 cells were suspended in a culture medium (250 µL) and added to the upper compartment of a 24-well Transwell® chamber (8 µm pore; Corning, 3422). Ctrl HCT-15, Ctrl SW480, Ctrl HT-29, USP21 -KO HCT-15, USP21 -KO SW480, and USP21 -KO HT-29 cells and culture medium (250 µL) were mixed with vehicle (DMSO, 0.1% v/v concentration) or EGF (20 ng/mL) and incubated at 37°C for 24 h. Migratory cells would pass through polycarbonate membrane and cling to the bottom side. Non-migratory cells would stay in the upper chamber. After removing non-migratory cells, migratory cells were fixed using 2.5% glutaraldehyde (Sigma-Aldrich, G6257-100 mL) and then stained with 0.1% crystal violet (Sigma-Aldrich, C6158-50g). Anchorage-independent soft agar colony formation assay Anchorage-independent soft agar colony formation assay was performed following previous protocols [ 25 , 30 – 33 ]. Briefly, Ctrl HCT-15, Ctrl HT-29, USP21 -KO HCT-15, and USP21 -KO HT-29 cells (1 × 10 4 cells /well) mixed with 0.3% Agarose (BioShop Canada, AGA001.500) in complete medium were plated onto the bottom of a 0.5% agar layer in a 6-well plate with a complete medium. Growth medium (2 mL) with vehicle (DMSO, 0.01% v/v concentration) or EGF (20 ng/mL) was added to the top of the layer and cells were incubated at 37°C for 28 days. Anchorage-dependent colony formation assay The ability of a single cell to grow into a colony was assessed by the colony formation assay as previously described [ 25 , 30 – 33 ]. Briefly, Ctrl HCT-15, Ctrl HT-29, USP21 -KO HCT-15, and USP21 -KO HT-29 cells were harvested with trypsin-EDTA and resuspended as single cells. Cells (1×10 3 cells per well) were plated into 6-well plates and treated with vehicle (DMSO, 0.01% v/v concentration) or EGF (20 ng/mL). Cells were incubated for ~ 12 days. Colonies were stained with 0.5% crystal violet (Sigma-Aldrich, C6158-50g) for 30 min at room temperature. The number of colonies was counted using ImageJ software. Three-dimensional (3D) spheroids formation assay using agarose-coated plates 3D spheroids formation assay was performed following previous protocols [ 34 , 35 ]. Briefly, 1.5% agarose hydrogel was added to each well of a 96-well culture plate. The plate was then incubated at room temperature (RT) for 30 min. Ctrl HCT-15, Ctrl HT-29, USP21 -KO HCT-15, and USP21 -KO HT-29 cells were seeded in 100 µl growth medium at a concentration of 500 cells per well. Plates were incubated at 37°C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (DMSO, 0.1% v/v concentration) or EGF (20 ng/mL) and incubated for additional time periods. Spheroid formation and growth were evaluated using phase-contrast microscopy. Sizes of spheroids and non-spherical cells were assessed using ImageJ Software (National Institutes of Health, Bethesda, MD, USA). For the determination of IC 50 value of BAY-805 in wild-type HCT-15 or HT-29 spheroids, WT HCT-15 or WT HT-29 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37°C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (0.01% DMSO) or different concentrations of BAY-805. Spheroids were incubated for different time periods. IC 50 value of BAY-805 was calculated by GraphPad Prism 8 software. To evaluate the inhibitory effect of BAY-805 on HCT-15 or HT-29 spheroids induced by EGF, WT HT-29 or WT HCT-15 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37°C for an additional 48 hours to allow the formation of 3D spheroids in culture, and spheroids were treated with vehicle (0.01% DMSO) or 7.5 µM BAY-805 in HT-29 spheroids and 1.6 µM BAY-805 in HCT-15 spheroids. After 24 hr, spheroids were further treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). Tumor spheroid formation and growth were evaluated using phase-contrast microscopy. Sizes of spheroids were assessed using the Image J Software. MTT Assay Ctrl HCT-15, Ctrl SW480, Ctrl HT-29, USP21 -KO HCT-15, USP21 -KO SW480, and USP21 -KO HT-29 cells were seeded into 96-well culture plates at a density of 1 × 10 3 cells/well, treated with vehicle (DMSO, 0.01% v/v concentration) or EGF (2.5 ng/mL), and grown in a culture medium supplemented with 10% FBS for different time periods. Cell viability was measured using an MTT reagent (Sigma-Aldrich, M5655) dissolved in PBS (1 mg/mL). On the day when measurements were taken, the medium was carefully replaced with fresh RPMI + 10% FBS, added with diluted MTT (1:10, 10% MTT), and incubated at 37°C for 3 h. After removing the incubation medium, formazan crystals were dissolved in a 100 µL solution of DMSO. MTT reduction was quantified by measuring light absorbance at 595 nm using a Bio-Rad Model 680 microplate reader (Bio-Rad, CA, USA). Each test was repeated at least four times in quadruplicate. RNA sequencing Primary tumor tissues and adjusted matched normal tissues were obtained from CRC patients ( n = 27) and RNA sequencing was done using the Illumina TruSeq RNA Sample Preparation Kit v2, as described in the previous report [ 23 ]. The experiments conducted on patient samples were approved by the institutional review board of Samsung Medical Center (IRB# 2010-04-004). Written informed consents were obtained from all participating patients. All experiments and analysis procedures were performed in accordance with the relevant guidelines and regulations. Statistical analysis All data are expressed as mean ± SD (standard deviation) or mean ± SEM. Statistical significance was determined by Student’s t-test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). P -values are marked by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001, and ****, P < 0.0001). Results USP21 stabilizes EGFR by deubiquitinating EGFR in colon cancer cells. In the context of colorectal cancer (CRC) metastasis, USP21 plays a pivotal role by stabilizing Fra-1 through deubiquitination, thereby facilitating the expression of metastasis-related genes like MMPs [ 18 ]. Notably, the activation of Fra-1 is intertwined with EGFR signaling in an MMP-dependent manner [ 18 – 21 ]. Despite the recognized interconnectedness of USP21-Fra-1 and Fra-1-EGFR, the regulatory axis of USP21-EGFR in metastatic CRC remains largely unexplored, as depicted in Fig. 1A. Our study began by elucidating the biochemical relationship between USP21 and EGFR. We observed a direct interaction between USP21 and EGFR (Fig. 1B, lane 3), a finding further validated through semi-endogenous immunoprecipitation (Fig. 1C). Remarkably, we found that USP21 induced the deubiquitination of EGFR in a dose-dependent manner (Fig. 1D, lane 1 vs. lanes 2–4). To probe whether this deubiquitination activity hinged on the enzymatic function of USP21, we generated a catalytically inactive mutant, USP21 C221A, and conducted deubiquitination assays comparing USP21 wild type (WT) and the USP21 C221A mutant. The deubiquitination of EGFR consistently occurred in the presence of USP21 WT, but not in the USP21 C221A mutant (Fig. 1E, lane 3 vs. lane 2), suggesting that the deubiquitination of EGFR might indeed be reliant on the catalytic activity of USP21. Subsequently, we investigated whether USP21-mediated deubiquitination of EGFR affected EGFR stability. To do so, we utilized CRISPR-Cas9 gene editing to generate three distinct USP21 -knockout (KO) colon cancer cell lines: USP21 -KO HCT-15, USP21 -KO HT-29, and USP21 -KO SW480 (Fig. 1F, CRISPR-Cas9 gene editing; Fig. 1G, USP21 -KO HCT-15; Fig. 1H, USP21 -KO HT-29; Fig. 1I, USP21 -KO SW480). Assessing EGFR's half-life via cycloheximide (CHX) chase assay in control (Ctrl) cells and USP21 -KO colon cancer cells revealed significantly reduced EGFR levels in USP21 -KO cells compared to their respective Ctrl counterparts (Fig. 1J, USP21 -KO HCT-15 vs. Ctrl HCT-15; Fig. 1K, USP21 -KO SW480 vs. Ctrl SW480; Fig. 1L, USP21 -KO HT-29 vs. Ctrl HT-29). These findings suggest that USP21 interacts with and stabilizes EGFR, potentially by preventing its ubiquitin-mediated degradation within multivesicular body (MVB)-lysosome vesicles, thus leading to elevated EGFR expression levels (Fig. 1M). USP21 plays a crucial role in the progression of colon cancer and impacts survival rates in patients with metastatic CRC. Given the above biochemical results, we investigated the involvement of USP21 in CRC progression through both in vitro and in vivo assays using USP21 -KO colon cancer cells. Notably, we observed a significant reduction in both transwell migration and wound healing in USP21 -KO HCT-15, USP21 -KO HT-29, and USP21 -KO SW480 cells as compared to Ctrl HCT-15, Ctrl HT-29, and Ctrl SW480 cells, respectively (Supplementary Fig. 1A-C, transwell migration; Supplementary Fig. 2A-C, wound healing). Moreover, cell proliferation was significantly reduced in USP21 -KO HCT-15, USP21 -KO HT-29, and USP21 -KO SW480 cells as compared to Ctrl HCT-15, Ctrl HT-29, and Ctrl SW480 cells, respectively (Supplementary Fig. 3A-C). Additionally, anchorage-dependent or -independent colony formation was markedly attenuated in USP21 -KO HCT-15 and USP21 -KO HT-29 cells as compared to Ctrl HCT-15 and Ctrl HT-29 cells, respectively (Fig. 2 A, B, anchorage-dependent; Fig. 2 C, D, anchorage-independent). To assess the tumorigenic potential of USP21, we performed in vitro three-dimensional (3D) tumor spheroid assays and in vivo xenograft assays using NSG mice. Tumor spheroids derived from USP21 -KO HCT-15 cells exhibited significantly smaller sizes compared to those from Ctrl HCT-15 cells (Fig. 2 E, F, Ctrl HCT-15 vs. USP21 -KO HCT-15). Additionally, the number of invasive cells originating from spheroids increased in Ctrl HCT-15 cells after day 10, whereas it significantly decreased in USP21 -KO HCT-15 cells (Fig. 2 G, H, Ctrl HCT-15 vs. USP21 -KO HCT-15). While the size of tumor spheroids progressively increased in Ctrl HT-29 cells, it was notably diminished in USP21 -KO HT-29 cells (Fig. 2 I, J, Ctrl HT-29 vs. USP21 -KO HT-29). Importantly, xenografting NSG mice with either Ctrl HCT-15 or USP21 -KO HCT-15 cells revealed a notable attenuation in tumor growth and size in mice harboring USP21 -KO HCT-15 cells compared to those with Ctrl HCT-15 cells (Fig. 2 K-M, Ctrl HCT-15 vs. USP21 -KO HCT-15), strongly indicating the critical role of USP21 expression in the tumorigenicity of colon cancer cells. Based on the insights gained from our biochemical and cellular studies into the functional role of USP21, we sought to examine its correlation with the survival outcomes of CRC patients. To achieve this, we analyzed a cohort of CRC patients with metastasis ( n = 27, Supplementary Table 1). Utilizing RNA sequencing data, we compared the differential magnitude of USP21 expression in tumor tissues ( n = 27) versus matched normal tissues ( n = 27) and categorized the 27 CRC patients into two groups: USP21-upregulated (USP21 up ) CRC ( n = 20) and USP21-downregulated (USP21 down ) mCRC ( n = 7) (Fig. 2 N and Supplementary Table 2). Notably, patients exhibiting higher levels of USP21 expression displayed poorer survival outcomes compared to those with lower expression levels; with survival rates of 45% in USP21 up CRCs ( n = 20) versus 71% in USP21 down CRCs ( n = 7) (Fig. 2 O). These results suggest that USP21 expression plays a pivotal role for the tumorigenicity of colon cancer cells and associated with mCRC patient survival. USP21 and EGFR expression is associated with poor survival in mCRC patients and USP21 promotes CRC progression in response to EGF. Given the above results, our investigation extended to exploring the relationship between USP21 and EGFR in mCRCs (Supplementary Table 2, differential magnitude of EGFR expression in 27 mCRC patients; Supplementary Table 2, differential magnitude of EGFR and USP21 expression in 27 mCRC patients). Intriguingly, the survival rates were lower in patients with EGFR up mCRC or EGFR up USP21 up mCRC patients compared to those with EGFR down mCRC or EGFR down USP21 down mCRC patients, respectively (Fig. 3 A, 40% in EGFR up vs. 59% in EGFR down ; Fig. 3 B, 33% in EGFR up USP21 up vs. 67% in EGFR down USP21 down ). Comparing the survival of EGFR up USP21 up mCRC patients with that of EGFR up mCRC patients or USP21 up mCRC patients, a significantly lower survival rate was evident (Fig. 3 B, vs. Figure 2 N or Fig. 3 A; 33% in EGFR up USP21 up vs. 45% in USP21 up or 40% in EGFR up ), supposing a potential connection of survival rate between EGFR and USP21 expression. It might be associated with the USP21-mediated EGFR stabilization (Fig. 1M), thereby enhancing EGFR-mediated cancer progression, as depicted in Fig. 3 C. To validate the functional role of USP21, we investigated whether USP21 contributes to CRC progression upon EGFR stimulation. We evaluated the colon cancer progression capacity in USP21 -KO colon cancer cells following exposure to EGF. Upon EGF stimulation, the transwell migration and wound healing abilities of USP21 -KO HCT-15, USP21 -KO HT-29, and USP21 -KO SW480 cells were notably reduced compared to Ctrl HCT-15, Ctrl HT-29, and Ctrl SW480 cells (Fig. 3 D-F, transwell migration; Supplementary Fig. 4A-F, wound healing). Similar trends were observed in the cell proliferation assay in USP21 -KO CRC cells upon EGF stimulation (Fig. 3 G, H and Supplementary Fig. S5 , USP21 -KO vs. Ctrl cells). Moreover, anchorage-independent and -dependent colony formation assays revealed a significant decrease in the number of colonies in USP21 -KO HCT-15 or USP21 -KO HT-29 cells treated with either vehicle or EGF compared to Ctrl cells (Fig. 3 I, J, anchorage-independent; Fig. 3 K, L, anchorage-dependent). These findings underscore the role of USP21 in modulating colon cancer proliferation, migration, and colony formation in response to EGF. Next, we delved into the role of USP21 in 3D tumor spheroid formation triggered by EGF. We seeded both Ctrl HT-29 and USP21 -KO HT-29 cells, as well as Ctrl HCT-15 and USP21 -KO HCT-15 cells, in 96-well plates. After allowing two days for spheroid formation optimization, spheroids were treated with either vehicle or EGF for various durations, as depicted (Fig. 4 A, B, Ctrl HT-29 and USP21 -KO HT-29; Fig. 4 A, C, Ctrl HCT-15 and USP21 -KO HCT-15). In Ctrl HT-29 cells, tumor spheroids gradually grew larger with EGF treatment compared to the vehicle-treated cells (Fig. 4 D, E, EGF vs. vehicle in Ctrl HT-29). Conversely, the size of tumor spheroids noticeably decreased in USP21 -KO HT-29 cells treated with vehicle compared to Ctrl HT-29 cells treated with vehicle (Fig. 4 D, E, USP21 -KO HT-29 treated with vehicle vs. Ctrl HT-29 treated with vehicle). Crucially, when comparing the sizes of tumor spheroids between EGF-treated Ctrl HT-29 cells and EGF-treated USP21 -KO HT-29 cells, marked decreases were observed in USP21 -KO HT-29 cells (Fig. 4 D, E, USP21 -KO HT-29 treated with EGF vs. Ctrl HT-29 treated with EGF). At day 8 and day 10 post-incubation, we observed invasive cells derived from tumor spheroids in both Ctrl HT-29 and USP21 -KO HT-29 cells treated with either vehicle or EGF (Fig. 4 F, indicated by red-dashed line). Measurements of the dimensions of invasive cells revealed significantly smaller sizes in USP21 -KO HT-29 cells treated with vehicle or EGF compared to Ctrl HT-29 cells (Fig. 4 F, G, USP21 -KO HT-29 vs. Ctrl HT-29). Similar results were obtained in the 3D tumor spheroid formation assay performed with USP21 -KO HCT-15 cells compared to Ctrl HCT-15 cells (Fig. 4 H-K, USP21 -KO HCT-15 vs. Ctrl HCT-15). Taken together, these findings indicate that USP21 can enhance tumor formation and invasion induced by EGF stimulation. BAY-805, an inhibitor of USP21, markedly attenuates the 3D tumor formation induced by EGF treatment. Having shown that USP21 contributes to tumor formation triggered by EGF stimulation, we examined the impact of inhibiting USP21 activity using the pharmacological inhibitor BAY-805 on EGF-induced tumor formation. To do that, we determined the IC 50 value of BAY-805 in HCT-15- or HT-29-induced spheroid formation (Fig. 5 A, B, HCT-15; Fig. 5 C, D, HT-29). 3D tumor spheroids of HCT-15 or HT-29 cells were treated with either vehicle or varying concentrations of BAY-805 (80 nM ~ 50 µM), as indicated (Fig. 5 A, HCT-15; Fig. 5 C, HT-29). The IC 50 value of BAY-805 was found to be 1.6 µM in HCT-15 or 7.5 µM in HT-29 spheroids, respectively (Fig. 5 B, HCT-15; Fig. 5 D, HT-29). Notably, treatment with BAY-805 significantly reduced EGFR expression in HCT-15 or HT-29 cells compared to the vehicle (Fig. 5 B, western blotting in HCT-15; Fig. 5 D, western blotting in HT-29), indicating that inhibition of USP21 with BAY-805 leads to reduced EGFR expression. To assess the therapeutic effects of BAY-805 on EGF-induced spheroid formation in HCT-15 or HT-29 cells, HCT-15 and HT-29 spheroids were exposed to either vehicle or EGF in the presence or absence of BAY-805 (1.6 µM for HCT-15; 7.5 µM for HT-29) for varying durations, as illustrated in Fig. 6 A. EGF treatment significantly increased the size of tumor spheroids in both HCT-15 and HT-29 cells in the absence of BAY-805 (EGF treatment vs. vehicle: Fig. 6 B, C, HCT-15; Fig. 6 D, E, HT-29). Remarkably, treatment with BAY-805 significantly inhibited the formation of both HCT-15 and HT-29 spheroids in response to EGF (BAY-805 plus EGF treatment vs. EGF treatment: Fig. 6 B, C, HCT-15; Fig. 6 D, E, HT-29). These findings suggest that USP21 promotes EGF-induced cancer progression via stabilizing EGFR, whereas inhibiting USP21 activity attenuates cancer progression induced by EGF stimulation (Fig. 6 F). Discussion It has been reported that EGFR is overexpressed in 60%-80% of colorectal cancers (CRCs), prompting consideration of anti-EGFR targeting as a pivotal strategy in CRC treatment [36-38]. EGFR, a multifunctional receptor, plays a crucial role in various cellular processes such as cell division, differentiation, migration, and organogenesis [38,39]. Dysregulated EGFR signaling significantly impacts multiple pathways including PLC-gamma-1, RAS-RAF-MEK-MAPKs, phosphatidylinositol-3 kinase and Akt, Src, stress-activated protein kinases, PAK-JNKK-JNK, and signal transducers and activators of transcription [37]. Consequently, the development of inhibitors targeting the EGFR signaling pathway has emerged as a promising approach in various cancers, including CRC [40]. Moreover, given the importance of EGFR regulation, cellular regulators influencing its stability and expression are gaining attention as potential therapeutic targets. USPs, which play a role in the recycling and degrading pathway of EGFR, have emerged as potential targets for intervening in EGFR-induced cancer development and progression [8-10, 41-43]. Several USPs have been identified as modulators capable of affecting EGFR stability [13-17]. Thus, there's a current focus in cancer medicine on pharmacologically disrupting USP activity to specifically target cancer-causing protein aberrations, including EGFR [39,40]. Among the USPs, USP21 stands out as it interacts with multiple substrate proteins and is considered a critical oncogene in various human cancers [44-47]. Recent findings also suggest its involvement in CRC metastatic progression by stabilizing Fra-1, a protein linked to tumor formation and metastasis [18]. By increasing the expression of MMP-1 and Fra-1 target genes, USP21 influences CRC progression. Given the biochemical activity of USP21 in regulating the ubiquitin-mediated degradation pathway, there's a hypothesis that USP21 could functionally regulate EGF-mediated CRC progression by modulating EGFR stability. The findings presented in this study shed light on the critical role of USP21 in colorectal cancer (CRC) progression, particularly in the context of metastatic CRC (mCRC). Through a series of in vitro and in vivo experiments, we delineated the molecular mechanisms underlying USP21-mediated regulation of EGFR stability and its impact on CRC progression and patient survival. Our results demonstrate that USP21 enhances EGFR expression by stabilizing EGFR through deubiquitination, thereby promoting EGFR-mediated signaling in colon cancer cells. This mechanism is pivotal for the maintenance of EGFR levels, as evidenced by the reduced EGFR expression and impaired tumorigenic potential observed in USP21 -KO colon cancer cells. Moreover, we show that USP21's enzymatic activity is crucial for its ability to deubiquitinate EGFR and stabilize its expression, highlighting the functional significance of USP21 in CRC progression. Importantly, our study uncovers a significant association between USP21 expression levels and patient survival in mCRC. Patients with higher USP21 expression levels exhibited poorer survival outcomes compared to those with lower expression levels, implicating USP21 as a potential prognostic marker for mCRC. Furthermore, our data suggest a synergistic relationship between USP21 and EGFR expression in mCRC, wherein patients with concurrent upregulation of both proteins displayed the lowest survival rates. This observation underscores the clinical relevance of targeting the USP21-EGFR axis in mCRC therapy. Additionally, we provide evidence for the functional relevance of USP21 in CRC progression in response to EGF stimulation. USP21 -KO cells attenuated the proliferative and migratory capacities of colon cancer cells following EGF exposure, underscoring the importance of USP21 in mediating EGF-induced CRC progression. Furthermore, our results indicate that pharmacological inhibition of USP21 with BAY-805 significantly abrogates EGF-induced tumor formation in vitro , highlighting the therapeutic potential of targeting USP21 in mCRC treatment. In our proposed scenario, depicted in Figure 7, we suggest a functional association between EGFR and USP21 in CRC progression. mCRC patients exhibiting up-regulated levels of USP21 may experience more aggressive cancer progression, especially in response to a tumor microenvironment enriched with EGF, compared to patients with down-regulated USP21 (Fig. 7A). In tumors with upregulated USP21, the enzyme inhibits the ubiquitin-dependent degradation pathway within multivesicular body (MVB)-lysosome vesicles by deubiquitinating EGFR, consequently increasing EGFR expression in CRCs. Subsequently, aberrant EGFR signaling initiated by EGF engagement is transmitted, thereby enhancing CRC progression (Fig. 7B). Conversely, in tumors with downregulated USP21, EGFR undergoes ubiquitination and subsequent degradation within MVB-lysosome vesicles, leading to attenuated cancer progression through diminished engagement with EGF (Fig. 7C). In conclusion, our study elucidates the multifaceted role of USP21 in CRC progression and patient survival, implicating USP21 as a promising therapeutic target for mCRC. Further investigation into the therapeutic efficacy of USP21 inhibitors, such as BAY-805, in preclinical and clinical settings may offer new avenues for improving the prognosis and treatment outcomes of mCRC patients. Declarations Conflict of interest: the author declare that they have no conflict of interest This work was supported by the National Research Foundation of Korea Grants funded by the Korean Government (2023R1A2C1003762, 2021R1A2C1094478, and RS-2023-00217189). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments We would like to thank Hyehwa Forum members for their helpful discussion. Funding This work was supported by the National Research Foundation of Korea Grants funded by the Korean Government (2023R1A2C1003762, 2021R1A2C1094478, and RS-2023-00217189). Authors’ Contributions JHS, MJK, JYK, YK, SHK and HJL performed the experiments and data analysis; BC, DK, JHS and JYK performed in vivo xenografted NSG experiments; JHS, MJK, JYK, YK, SHK and HJL performed the western blots; JHS, MJK, JYK performed the CRISPR/Cas9-based gene editing; JHS, MJK, JYK, YK, SHK and HJL performed cancer progression assay; EC and KYL performed the data analysis; YBC provided clinical and RNA-Seq data; KKK provided experimental materials; EC and KYL are responsible for the conception, design and supervision of the study. Ethics declarations Tumor and matched normal tissues from 27 patients with primary CRC were obtained in accordance with the ethical principles stated in the Declaration of Helsinki. This study was approved by the institutional review board of Samsung Medical Center (IRB# 2010-04-004). We obtained written informed consent from each patient prior to surgery for using their pathological specimens for research use. 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Additional Declarations (Not answered) Supplementary Files OriginalDataFile.pdf Reproducibilitychecklist.pdf SupplementaryTableS1.pdf SupplementaryTableS2.pdf Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Cell Death Discovery → Version 1 posted Unknown event 17 Oct, 2024 Editorial decision: revise 30 Jul, 2024 Review # 1 received at journal 22 Jul, 2024 Review # 3 received at journal 19 Jul, 2024 Reviewer # 3 agreed at journal 17 Jul, 2024 Reviewer # 2 agreed at journal 14 Jul, 2024 Reviewer # 1 agreed at journal 10 Jul, 2024 Reviewers invited by journal 10 Jul, 2024 Submission checks completed at journal 18 Jun, 2024 Editor assigned by journal 17 Jun, 2024 First submitted to journal 17 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4594251","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":325267027,"identity":"b1932820-b3bb-4f77-9604-74f1b11524eb","order_by":0,"name":"Ki-Young 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relationship between USP21 and Fra-1 or EGFR and Fra-1 in CRC metastasis. \u003cstrong\u003eB\u003c/strong\u003e Myc-EGFR, Flag-USP21, and mock vectors were transfected into HEK-293T cells, as indicated. IP assay was performed with an anti-Myc antibody \u003cstrong\u003eC\u003c/strong\u003e H1299 cells were transfected with mock as control vector or Myc-EGFR, as indicated. IP assay was performed with anti-IgG or anti-Myc antibody. \u003cstrong\u003eD\u003c/strong\u003e Myc-EGFR, HA-Ub, Flag-USP21, and mock vectors were transfected into HEK-293T cells, as indicated. IP assay was performed with an anti-Myc antibody. \u003cstrong\u003eE\u003c/strong\u003eMyc-EGFR, HA-Ub, Flag-USP21 wild type (WT), Flag-USP21 C221A mutant, and mock vectors were transfected into HEK-293T cells, as indicated. IP assay was performed with an anti-Myc antibody. \u003cstrong\u003eF\u003c/strong\u003eThree sgRNA to human USP21 were designed on the CRISPR design website (http://crispr.mit.edu/). To generate \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells, sgRNA vector and Cas9 vector were used. \u003cstrong\u003eG\u003c/strong\u003e-\u003cstrong\u003eI\u003c/strong\u003e sgRNAs and Cas9 vector were transfected into HCT-15 (\u003cstrong\u003eG\u003c/strong\u003e), HT-29 (\u003cstrong\u003eH\u003c/strong\u003e), and SW480 (\u003cstrong\u003eI\u003c/strong\u003e) human colon cancer cells. Expression levels of USP21 were analyzed by western blotting assay with an anti-USP21 antibody. \u003cstrong\u003eJ\u003c/strong\u003e-\u003cstrong\u003eL\u003c/strong\u003e Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eJ\u003c/strong\u003e), Ctrl SW480 and \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 cells (\u003cstrong\u003eK\u003c/strong\u003e), or Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eL\u003c/strong\u003e) were treated with vehicle or cycloheximide (CHX, 20-30 μg/mL) for different time periods, as indicated. Western blotting assay was performed with an anti-EGFR or anti-GAPDH antibody. Error bars represent ± SD of three experiments. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, two-tailed Student’s t-test. \u003cstrong\u003eM\u003c/strong\u003eA schematic model of how USP21 stabilizes EGFR.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/f60a7339b61c0062a94ccb00.png"},{"id":62188590,"identity":"4ff60cd0-0552-40e1-9c43-2d63f20fc463","added_by":"auto","created_at":"2024-08-10 12:17:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18721985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eUSP21\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-KO colon cancer cells exhibit attenuation of tumorigenicity both \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e Anchorage-dependent colony formation assay was performed with Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eA\u003c/strong\u003e) or Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eB\u003c/strong\u003e). Results are presented as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, two-tailed Student’s t-test. \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e Anchorage-independent colony formation assay was performed with Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eC\u003c/strong\u003e) or Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eD\u003c/strong\u003e). Results are presented as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, two-tailed Student’s t-test. \u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e Ctrl HCT-15 or \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells were seeded in 96 well plates, 3D spheroid formation assay was performed. Spheroid formation and growth were evaluated using phase-contrast microscopy (\u003cstrong\u003eE\u003c/strong\u003e, scale bar, 100 μm). The size of the spheroid was assessed using Image J Software. Error bars represent ± SD (\u003cem\u003en\u003c/em\u003e = 7) of three experiments (\u003cstrong\u003eF\u003c/strong\u003e). On day 10 and day 12 post-incubation, spheroid (blue dashed line) and non-spheroid cells (red dashed line) were analyzed (\u003cstrong\u003eG\u003c/strong\u003e). Their sizes were assessed with the ImageJ Software. Non-spherical size was measured and presented as ± SD (\u003cem\u003en\u003c/em\u003e = 7) from three experiments (\u003cstrong\u003eH\u003c/strong\u003e). **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003eI\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e Ctrl HT-29 or \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were seeded in 96 well plates. Plates were incubated at 37 °C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was incubated for different time periods, as indicated. Spheroid formation and growth were evaluated using phase-contrast microscopy (\u003cstrong\u003eI\u003c/strong\u003e, scale bar, 100 μm). The size of the spheroid was assessed using Image J Software. Error bars represent ± SD (n = 5) of three experiments (\u003cstrong\u003eJ\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, two-tailed Student’s t-test. \u003cstrong\u003eK\u003c/strong\u003e-\u003cstrong\u003eM\u003c/strong\u003e Ctrl HCT-15 (5 × 10\u003csup\u003e6\u003c/sup\u003e cells per mouse, \u003cem\u003en\u003c/em\u003e = 10) or \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (5 × 10\u003csup\u003e6\u003c/sup\u003e cells per mouse, \u003cem\u003en\u003c/em\u003e = 10) were injected under the NSG mice skin (back area). At five days after cancer cell injection, tumor volume was measured with a caliper every 4 days until 37 days after injection. Tumor volumes (mm\u003csup\u003e3\u003c/sup\u003e) were calculated as (length x width)\u003csup\u003e2\u003c/sup\u003e × 0.5. Tumor growth curves are presented as average tumor volume ± SEM for each group in this study (\u003cstrong\u003eK\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. Tumor-bearing NSG mice injected with Ctrl HCT-15 (\u003cem\u003en\u003c/em\u003e = 3) or \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 (\u003cem\u003en\u003c/em\u003e = 3) cells were photographed on day 37 post-injection (\u003cstrong\u003eL\u003c/strong\u003e). Tumors were isolated from NSG mice injected with Ctrl HCT-15 (n = 3) or \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cem\u003en \u003c/em\u003e= 3) at day 37 post-injection (\u003cstrong\u003eM\u003c/strong\u003e). \u003cstrong\u003eN\u003c/strong\u003e and \u003cstrong\u003eO\u003c/strong\u003e Based on RNA sequencing data of tumor tissues and adjusted matched normal tissues of mCRC patients (\u003cem\u003en\u003c/em\u003e = 27), △Mag of USP21 expression was analyzed and listed according to the ∆Mag of USP21. mCRC patients were stratified into USP21\u003csup\u003eup\u003c/sup\u003e mCRCs (\u003cem\u003en\u003c/em\u003e=20) and USP21\u003csup\u003edown\u003c/sup\u003e mCRCs (\u003cem\u003en\u003c/em\u003e=7) (\u003cstrong\u003eN\u003c/strong\u003e), and the survival rate was analyzed (\u003cstrong\u003eO\u003c/strong\u003e).\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/7e33855c9b95590e5d2d3868.png"},{"id":62188587,"identity":"95791c80-0713-46ba-b957-9d5407c5ab75","added_by":"auto","created_at":"2024-08-10 12:17:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18042641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP21 is functionally involved in colon cancer proliferation and colony formation in response to EGF.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Based on RNA sequencing data of tumor tissues and adjusted matched normal tissues of mCRC patients (\u003cem\u003en\u003c/em\u003e = 27), △Mag of EGFR expression was analyzed and listed according to the ∆Mag of EGFR. mCRC patients were stratified into EGFR\u003csup\u003eup\u003c/sup\u003e mCRCs (\u003cem\u003en\u003c/em\u003e=10) and EGFR\u003csup\u003edown\u003c/sup\u003e mCRCs (\u003cem\u003en\u003c/em\u003e=17), and the survival rate was analyzed. \u003cstrong\u003eB\u003c/strong\u003e Based on RNA sequencing data of tumor tissues and adjusted matched normal tissues of mCRC patients (\u003cem\u003en\u003c/em\u003e = 27), differential magnitude (△Mag) of EGFR and USP21 expression was analyzed and listed according to the ∆Mag of EGFR. mCRC patients were stratified into EGFR\u003csup\u003eup\u003c/sup\u003eUSP21\u003csup\u003eup\u003c/sup\u003e mCRCs (\u003cem\u003en\u003c/em\u003e=9) and EGFR\u003csup\u003edown\u003c/sup\u003eUSP21\u003csup\u003edown\u003c/sup\u003e mCRCs (\u003cem\u003en\u003c/em\u003e=6), and the survival rate was analyzed. \u003cstrong\u003eC\u003c/strong\u003e A possible model of how the up-regulated USP21 is implicated in EGFR-mediated cancer progression. \u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eF\u003c/strong\u003e Transwell migration assay was performed with Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eD\u003c/strong\u003e), Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eE\u003c/strong\u003e), or Ctrl SW480 and \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 cells (\u003cstrong\u003eF\u003c/strong\u003e) treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). Results are presented as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e Cell proliferation assay was assay was performed with Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eG\u003c/strong\u003e) or Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eH\u003c/strong\u003e) treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). Results are presented as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003eI\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e Anchorage-independent colony formation assay was performed with Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eI\u003c/strong\u003e) or Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eJ\u003c/strong\u003e) treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). The number of colonies was counted. Results are presented as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003eK\u003c/strong\u003e and \u003cstrong\u003eL\u003c/strong\u003e Anchorage-dependent colony formation assay was performed with Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eK\u003c/strong\u003e) or Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eL\u003c/strong\u003e) treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). The number of colonies was counted. Results are presented as mean ± SD of three independent experiments. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/2ec8d47afd435ed3d706cfec.png"},{"id":62188581,"identity":"e273314e-4f2c-4d60-97b5-a25c60707f93","added_by":"auto","created_at":"2024-08-10 12:17:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12667034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D tumor spheroid formation is decreased in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eUSP21\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-KO colon cancer cells in response to EGF.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e A procedure of 3D tumor spheroid formation. Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e) or Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e) were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37 °C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (0.01% DMSO) and EGF (20 ng/mL) and incubated for different time periods. The culture medium containing vehicle or EGF was exchanged with the time interval of day 3 as indicated (B and C). \u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e Spheroids of Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were treated with vehicle or EGF and incubated for different time periods, as indicated (\u003cstrong\u003eD\u003c/strong\u003e, scale bar, 100 μm). Spheroid formation and growth were evaluated using phase-contrast microscopy. Sizes of spheroids were assessed using the Image J Software. Error bars represent ± SD (\u003cem\u003en\u003c/em\u003e =7) of three experiments (\u003cstrong\u003eE\u003c/strong\u003e). On day 8 and day 10 post-incubation, spheroid (blue dashed line) and non-spheroid cells (red dashed line) were analyzed (\u003cstrong\u003eF\u003c/strong\u003e). Their sizes were assessed with the ImageJ Software. The non-spherical size was measured and presented as ± SD (\u003cem\u003en\u003c/em\u003e = 7) of three experiments (\u003cstrong\u003eG\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 treated EGF versus Ctrl HT-29 treated EGF, two-tailed Student’s t-test. \u003cstrong\u003eH\u003c/strong\u003e-\u003cstrong\u003eK\u003c/strong\u003e Spheroids of Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells were treated with vehicle or EGF and incubated for different time periods, as indicated (\u003cstrong\u003eH\u003c/strong\u003e, scale bar, 100 μm). Spheroid formation and growth were evaluated using phase-contrast microscopy. Sizes of spheroids were assessed using the Image J Software. Error bars represent ± SD (\u003cem\u003en\u003c/em\u003e =7) of three experiments (\u003cstrong\u003eI\u003c/strong\u003e) On day 10 and day 12 post-incubation, spheroid (blue dashed line) and non-spheroid cells (red dashed line) were analyzed (\u003cstrong\u003eJ\u003c/strong\u003e). Their sizes were assessed with the ImageJ Software. The non-spherical size was measured and presented as ± SD (\u003cem\u003en\u003c/em\u003e = 7) of three experiments (\u003cstrong\u003eK\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 treated EGF versus Ctrl HCT-15 treated EGF, two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/eee1298f89925c8fb7771665.png"},{"id":62190163,"identity":"7754f4e1-69b8-4788-80eb-b4e22f565fd1","added_by":"auto","created_at":"2024-08-10 12:25:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18404835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe determination of IC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e value of BAY-805 in wild-type (WT) HCT-15 and WT HT-29 tumor spheroids\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e WT Ctrl HCT-15 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37 °C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (0.01% DMSO) or different concentrations of BAY-805, as indicated. Spheroids were incubated for different time periods (\u003cstrong\u003eA\u003c/strong\u003e, scale bar, 100 μm). IC\u003csub\u003e50\u003c/sub\u003e value of BAY-805 was calculated by GraphPad Prism 8 software (\u003cem\u003en\u003c/em\u003e=7, spheroids). WT HCT-15 cells were treated with vehicle or different concentrations of BAY-805, as indicated (\u003cstrong\u003eB\u003c/strong\u003e, down). The levels of EGFR were evaluated by western blotting analysis with anti-EGFR antibody. \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e. WT HT-29 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37 °C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (0.01% DMSO) or different concentrations of BAY-805, as indicated. Spheroids were incubated for different time periods (\u003cstrong\u003eC\u003c/strong\u003e, scale bar, 100 μm). IC\u003csub\u003e50\u003c/sub\u003e value of BAY-805 was calculated by GraphPad Prism 8 software (\u003cem\u003en\u003c/em\u003e=7, spheroids). WT HT-29 cells were treated with vehicle or different concentrations of BAY-805, as indicated (\u003cstrong\u003eD\u003c/strong\u003e, down). The levels of EGFR were evaluated by western blotting analysis with anti-EGFR antibody.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/25b8a518e14943e486f157c2.png"},{"id":62188585,"identity":"011a7752-4b9d-4209-ae93-f2b7baf60ea9","added_by":"auto","created_at":"2024-08-10 12:17:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15196146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBAY-805, an USP21 inhibitor, effectively attenuates HCT-15- or HT-29- tumor spheroid formation in response to EGF stimulation.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e A procedure for evaluating BAY-805 effects in 3D tumor spheroid formation. Wild-type (WT) HT-29 or WT HCT-15 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37 °C for an additional 48 hours to allow the formation of 3D spheroids in culture, and spheroids were treated with vehicle (0.01% DMSO) or 7.5 mM BAY-805 in HT-29 spheroids and 1.6 mM BAY-805 in HCT-15 spheroids. After 24 hr, spheroids were further treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e. HCT-15-spheroid formation and growth were evaluated using phase-contrast microscopy (\u003cstrong\u003eB\u003c/strong\u003e, scale bar, 100 μm). Sizes of spheroids were assessed using the Image J Software. Error bars represent ± SD (\u003cem\u003en\u003c/em\u003e =5) of three experiments (\u003cstrong\u003eC\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01;\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, HCT-15 treated without BAY-805 versus HCT-15 treated with BAY-805, two-tailed Student’s t-test. \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003e HT-29-spheroid formation and growth were evaluated using phase-contrast microscopy (\u003cstrong\u003eD\u003c/strong\u003e, scale bar, 100 μm). Sizes of spheroids were assessed using the Image J Software. Error bars represent ± SD (\u003cem\u003en\u003c/em\u003e =5) of three experiments (\u003cstrong\u003eE\u003c/strong\u003e). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001;\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, HT-29 treated without BAY-805 versus HT-29 treated with BAY-805, two-tailed Student’s t-test. \u003cstrong\u003eF\u003c/strong\u003e A schematic model of how USP21 inhibitor attenuates EGFR-mediated cancer progression.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/31a47d5dd480ec7c550044de.png"},{"id":62188588,"identity":"abf59446-999d-44f0-9f42-691922e4c190","added_by":"auto","created_at":"2024-08-10 12:17:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7629494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA schematic representation of the proposed model illustrating how USP21 promotes EGFR-mediated progression in CRC patients\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e Based on USP21 expression levels in CRC tumor tissues, tumors are categorized into USP21-upregulated (USP21\u003csup\u003eup\u003c/sup\u003e) CRC or USP21-downregulated (USP21\u003csup\u003edown\u003c/sup\u003e) CRC. \u003cstrong\u003eB\u003c/strong\u003e In tumors with upregulated USP21, the enzyme inhibits the ubiquitin-dependent degradation pathway within MVB-lysosome vesicles by deubiquitinating EGFR, consequently increasing EGFR expression in CRC tumors. Subsequently, aberrant EGFR signaling initiated by EGF engagement is transmitted, thereby enhancing CRC progression. (\u003cstrong\u003eC\u003c/strong\u003e) Conversely, in tumors with downregulated USP21, EGFR undergoes ubiquitination and subsequent degradation within MVB-lysosome vesicles, leading to attenuated cancer progression through diminished engagement with EGF.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/ac806fb00553644da5c0ca69.png"},{"id":62190166,"identity":"639f0d29-719f-4ff0-9c79-9e262a3de958","added_by":"auto","created_at":"2024-08-10 12:25:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":998207,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/d2cd2b56-0e81-4945-8a20-bcaf30ec1c14.pdf"},{"id":62188579,"identity":"12a3bb29-fb40-43ec-bfa6-4f064fd2648a","added_by":"auto","created_at":"2024-08-10 12:17:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":610672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"OriginalDataFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/415191c493a68dc674f7d7f4.pdf"},{"id":62190161,"identity":"2d1d9e0d-ad61-4816-ab7b-cc3aadd68153","added_by":"auto","created_at":"2024-08-10 12:25:36","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1752275,"visible":true,"origin":"","legend":"","description":"","filename":"Reproducibilitychecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/3cbfdbb00b9c280b8ca23d0f.pdf"},{"id":62188583,"identity":"9d3890f5-bdce-4da5-b28f-a949f5c38b20","added_by":"auto","created_at":"2024-08-10 12:17:37","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":133219,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/e49a59e1ad9b2a61da1d9978.pdf"},{"id":62190162,"identity":"366c90dd-2ebb-4282-8941-e4eea5a29455","added_by":"auto","created_at":"2024-08-10 12:25:37","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":204638,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/37cd268dbd56f2c972414b09.pdf"},{"id":62188589,"identity":"01b29c8f-d60d-4fef-a703-5f5331723ce9","added_by":"auto","created_at":"2024-08-10 12:17:37","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1880542,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4594251/v1/240d9b62f0e4f46d7fd97de9.docx"}],"financialInterests":"(Not answered)","formattedTitle":"USP21-EGFR signaling axis is functionally implicated in metastatic colorectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) stands as a pervasive and deadly malignancy worldwide, driven by a myriad of factors encompassing lifestyle choices, environmental exposures, viral infections, and smoking habits [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recent advancements in omics data analysis of CRC patients have unearthed a wealth of genetic information crucial for delineating the pathophysiological landscape of CRC progression and for crafting targeted therapeutic interventions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Dysregulation of epidermal growth factor receptor (EGFR)-mediated signaling pathways emerges as a potent instigator of CRC initiation and advancement [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. EGFR, a pivotal member of the ErbB protein family, exerts profound influence over CRC cellular processes including proliferation, angiogenesis, migration, invasion, and tumorigenicity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite gaps in understanding the precise molecular and cellular mechanisms governing EGFR upregulation in CRC, omics data from CRC patient cohorts consistently highlight elevated EGFR expression levels in tumor tissues, correlating with dismal prognosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, concerted research endeavors have been directed towards unraveling the intricate cellular regulatory mechanisms modulating EGFR expression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe regulation of cellular EGFR expression and activation hinges on intricate EGFR trafficking pathways, exerting significant influence over biological outcomes driven by EGFR signaling in cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. EGFR fate is governed by two principal pathways: the recycling pathway and the degradative multivesicular bodies (MVBs)-lysosome pathway [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Various cellular factors, including Rab4, Rab35, calcium-modulating cyclophilin ligand (CAML), and Eps15S, play crucial roles in the EGFR-recycling pathway, orchestrating prolonged EGFR signaling [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, ubiquitin-specific peptidases (USPs) have emerged as key regulators of EGFR signaling, with several members of the USP family implicated in the modulation of EGFR degradation [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among these, USP21 has garnered attention for its role in stabilizing Fra-1 and PD-L1, promoting CRC metastasis [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Given the interconnectedness between EGFR signaling, Fra-1 activation, and PD-L1 expression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], targeting USP21 holds promise as a strategy for combating metastatic CRC driven by EGFR signaling. However, the functional and clinical relevance of USP21 in metastatic CRC, particularly in association with EGFR expression, remains largely unexplored.\u003c/p\u003e \u003cp\u003eThis study aims to elucidate the relationship between USP21 and EGFR in metastatic CRC, unraveling their clinical significance and functional implications. Through comprehensive biochemical and functional analyses, we demonstrate that USP21 plays a pivotal role in stabilizing EGFR and driving CRC progression triggered by EGF. Conversely, knockout of USP21 in CRC cells impedes CRC progression and attenuates tumor formation in response to EGF stimulation. Importantly, pharmacological inhibition of USP21 with BAY-805 exerts a potent inhibitory effect on CRC tumor spheroid formation, highlighting the therapeutic potential of targeting USP21 in mCRC. Collectively, our findings underscore the promise of USP21 as both a therapeutic target and a predictive biomarker for mitigating mCRC induced by EGFR signaling.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCRC patient specimens\u003c/h2\u003e \u003cp\u003ePrimary tumor tissues and adjusted matched normal tissues of mCRC patients (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) were collected at Samsung Medical Center (SMC, Seoul, Korea). Licensed pathologists confirmed histologic diagnoses and estimated all formalin-fixed paraffin-embedded samples with purity\u0026thinsp;\u0026ge;\u0026thinsp;40% according to H\u0026amp;E staining. Written informed consent was obtained from all participants. All methods, including authorization for utilization of patients\u0026rsquo; specimens, were carried out in accordance with relevant guidelines and regulations. Experiments conducted on patient samples were approved by the Institutional Review Board (IRB) of Samsung Medical Center (IRB# 2010-04-004). RNA sequencing was conducted for all samples, as previously described [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eXenografted NSG mouse model\u003c/h2\u003e \u003cp\u003eNOD/SCID/IL-2Rγ\u003csup\u003enull\u003c/sup\u003e (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained under specific pathogen-free conditions in accordance with ethical guidelines for the care of these mice at the Bioanalysis Center Animal Facility, GenNBio Inc. (Seongnam, Korea). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Bioanalysis Center Animal Facility (IACUC #: 23-10-01). NSG mice (6\u0026ndash;8 weeks old) were used to generate xenografted NSG mice. Control (Ctrl) HCT-15 (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) or \u003cem\u003eUSP21\u003c/em\u003e-knockout (KO) HCT-15 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) were injected under NSG mice skin (back area) within serum-free PBS. The final injection volume was 100 \u0026micro;L/mouse containing a 1:1 v/v mixture of ice-chilled Matrigel (BD Biosciences, La Jolla, CA, USA), which was kept on ice until injection. Five days after cancer cell injection, tumor volume was measured with a caliper every 4 days until 37 days after injection. Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) was calculated as (length x width) \u0026times; 0.5. Tumor growth curves are presented as average tumor volume\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM for each group in this study. All studies involving mice were approved by IACUC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCells\u003c/h2\u003e \u003cp\u003eHCT-15 (human colorectal cancer cell line; CCL-225, American type culture collection (ATCC), Manassas, VA, USA), SW480 (human colon cancer cell line; CCL-228, ATCC), and HT-29 (human colorectal adenocarcinoma cell line; HTB-38, ATCC) were maintained in a medium recommended by ATTC, supplemented with 10% fetal bovine serum (FBS), penicillin (100 \u0026micro;g/mL), and streptomycin (100 \u0026micro;g/mL) in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere at 37\u0026deg;C. Human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM; Welgene, LM-001-05) supplemented with 10% fetal bovine serum (FBS).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eUSP21\u003c/b\u003e\u003cb\u003e-knockout (KO) colon cancer cell lines with CRISPR/Cas9 two vector system\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo generate \u003cem\u003eUSP21-\u003c/em\u003eKO colon cancer cells with CRISPR/Cas9 gene editing method, we used two vector systems, single guide RNA (sgRNA) and CRISPR-associated protein 9 (Cas9) vectors. sgRNA and Cas9 vectors were kindly provided by Dr. Daesik Kim (Sungkyunkwan University School of Medicine, Suwon, Korea). Guide RNA sequences for CRISPR/Cas9 were designed on the CRISPR design website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.mit.edu/\u003c/span\u003e\u003cspan address=\"http://crispr.mit.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) provided by the Feng Zhang Lab. Insert oligonucleotides for human USP21 gRNA were 5\u0026rsquo;-ATGACCGAGCCAACCTAATG-3\u0026rsquo; (gRNA-1) / 5\u0026rsquo;- GTTTCCACATTAGGTTGGCT-3\u0026rsquo; (gRNA-2) / 5\u0026rsquo;-CTTCTCTGGATACAGCCAGC-3\u0026rsquo; (gRNA-3). Complementary oligonucleotides to guide RNAs (gRNAs) were annealed and cloned into a sgRNA vector. The sgRNA vector expressing gRNA of USP21 and Cas9 vector expressing Cas9 were transfected into HCT-15, SW480, and HT-29 colon cancer cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. At two weeks after transfection, colonies were isolated and single-cell selection was performed. The expression of USP21 in \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells was analyzed by western blotting assay with an anti-USP21 antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and reagents\u003c/h2\u003e \u003cp\u003eAnti-Myc (sc40), anti-USP21 (sc-515911), Anti-HA (sc-7392), and anti-GAPDH (sc-47724) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Flag (F3165) antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-EGFR antibody (2232S) was purchased from Cell Signaling Technology (Danvers, MA, USA). TrueBlot\u0026reg; secondary antibody (18-8817-33) were purchased from Rockland Immunochemicals (Pottstown, PA, USA). Goat anti-rabbit IgG (HRP) (GTX213110-01) antibody was purchased from GeneTex Inc. (Irvine, CA, USA). Rabbit anti-mouse IgG H\u0026amp;L (HRP) (ab6728) antibody was purchased from Abcam (Cambridge, MA, USA). Dimethyl sulfoxide (DMSO; D4540), Phosphate-buffered saline (PBS; CBP007A), glutaraldehyde (G6257-100ml), crystal violet (C6158-50g), cycloheximide (CHX; C1988), EGF (SRP3027), and thiazolyl blue tetrazolium bromide (MTT; M5655) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000 (11668019) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). BAY-805 (HY-153045) was purchased form MedChemExpress (Monmouth Junction, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid constructs\u003c/h2\u003e \u003cp\u003eEGFR-GFP (32751) and Flag-HA-USP21 (22574) vectors were purchased from Addgene (Watertown, MA, USA). pCMV-3Tag-7 (240202) and pCMV-3Tag-6 (240200) vectors were purchased from Agilent technologies (Santa Clara, CA, USA). Using Flag-HA-USP21 plasmid, full-length USP21 was cloned into the pCMV-3Tag-6 vector to generate a Flag-USP21 vector. Using the EGFR-GFP plasmid, full-length EGFR was cloned into the pCMV-3Tag-7 vector to generate Myc-EGFR vector. Flag-USP21 C221A mutant was generated by site-directed mutagenesis using Flag-USP21 wild-type (WT) plasmid as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting assay\u003c/h2\u003e \u003cp\u003eWestern blotting and immunoprecipitation (IP) assays were performed as previously described [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, cell lysates were prepared from control (Ctrl) HCT-15, Ctrl SW480, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells. They were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8\u0026ndash;12%) and immune-probed with an anti-USP21 or anti-GAPDH antibody. HEK-293T cells were transfected with a mock control vector, Myc-EGFR, or Flag-USP21 vector. Cells were then incubated at 37\u0026deg;C for 24 h. After collecting cells, cell lysates were prepared and immunoprecipitated with an anti-Myc antibody. IP complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8\u0026ndash;12%) and immune-probed with an anti-Myc or anti-Flag antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eUbiquitination and deubiquitination assay\u003c/h2\u003e \u003cp\u003eHEK-293T cells were transiently transfected with mock, Myc-EGFR, HA-Ub, Flag-USP21 wild type (WT), or Flag-USP21 C221A mutant vector. Cells were then incubated at 37\u0026deg;C for 24 h. After collecting cells, cell lysates were prepared and immunoprecipitated with an anti-Myc antibody. IP complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8\u0026ndash;12%) and immune-probed with an anti-Myc, anti-Flag, or anti-HA antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCycloheximide (CHX) chase assay\u003c/h2\u003e \u003cp\u003eCycloheximide (CHX) chase assay was performed to determine the half-life of EGFR following previous protocols [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, Control (Ctrl) HCT-15, Ctrl SW480, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were treated with CHX (20\u0026ndash;30 \u0026micro;g/mL; Sigma-Aldrich, St. Louis, MO, USA) for different time periods. EGFR was then detected using western blotting assay with an anti-EGFR antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWound-healing migration assay\u003c/h2\u003e \u003cp\u003eA wound-healing migration assay was performed following previous protocols [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, Control (Ctrl) HCT-15, Ctrl SW480, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were seeded into 12-well plates and cultured to reach confluence. Cell monolayers were gently scratched and washed with a culture medium. After floating cells and debris were removed, cells attached to culture plates were treated with vehicle (DMSO, 0.01% v/v concentration) or EGF (20 ng/mL) for different time periods. Cell images were captured after culturing for different time periods as indicated in each experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration assay\u003c/h2\u003e \u003cp\u003eCtrl HCT-15, Ctrl SW480, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were suspended in a culture medium (250 \u0026micro;L) and added to the upper compartment of a 24-well Transwell\u0026reg; chamber (8 \u0026micro;m pore; Corning, 3422). Ctrl HCT-15, Ctrl SW480, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells and culture medium (250 \u0026micro;L) were mixed with vehicle (DMSO, 0.1% v/v concentration) or EGF (20 ng/mL) and incubated at 37\u0026deg;C for 24 h. Migratory cells would pass through polycarbonate membrane and cling to the bottom side. Non-migratory cells would stay in the upper chamber. After removing non-migratory cells, migratory cells were fixed using 2.5% glutaraldehyde (Sigma-Aldrich, G6257-100 mL) and then stained with 0.1% crystal violet (Sigma-Aldrich, C6158-50g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnchorage-independent soft agar colony formation assay\u003c/h2\u003e \u003cp\u003eAnchorage-independent soft agar colony formation assay was performed following previous protocols [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, Ctrl HCT-15, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells /well) mixed with 0.3% Agarose (BioShop Canada, AGA001.500) in complete medium were plated onto the bottom of a 0.5% agar layer in a 6-well plate with a complete medium. Growth medium (2 mL) with vehicle (DMSO, 0.01% v/v concentration) or EGF (20 ng/mL) was added to the top of the layer and cells were incubated at 37\u0026deg;C for 28 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnchorage-dependent colony formation assay\u003c/h2\u003e \u003cp\u003eThe ability of a single cell to grow into a colony was assessed by the colony formation assay as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, Ctrl HCT-15, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were harvested with trypsin-EDTA and resuspended as single cells. Cells (1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well) were plated into 6-well plates and treated with vehicle (DMSO, 0.01% v/v concentration) or EGF (20 ng/mL). Cells were incubated for ~\u0026thinsp;12 days. Colonies were stained with 0.5% crystal violet (Sigma-Aldrich, C6158-50g) for 30 min at room temperature. The number of colonies was counted using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThree-dimensional (3D) spheroids formation assay using agarose-coated plates\u003c/h2\u003e \u003cp\u003e3D spheroids formation assay was performed following previous protocols [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Briefly, 1.5% agarose hydrogel was added to each well of a 96-well culture plate. The plate was then incubated at room temperature (RT) for 30 min. Ctrl HCT-15, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were seeded in 100 \u0026micro;l growth medium at a concentration of 500 cells per well. Plates were incubated at 37\u0026deg;C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (DMSO, 0.1% v/v concentration) or EGF (20 ng/mL) and incubated for additional time periods. Spheroid formation and growth were evaluated using phase-contrast microscopy. Sizes of spheroids and non-spherical cells were assessed using ImageJ Software (National Institutes of Health, Bethesda, MD, USA). For the determination of IC\u003csub\u003e50\u003c/sub\u003e value of BAY-805 in wild-type HCT-15 or HT-29 spheroids, WT HCT-15 or WT HT-29 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37\u0026deg;C for an additional 48 hours to allow the formation of 3D spheroids in culture. The spheroid was added with vehicle (0.01% DMSO) or different concentrations of BAY-805. Spheroids were incubated for different time periods. IC\u003csub\u003e50\u003c/sub\u003e value of BAY-805 was calculated by GraphPad Prism 8 software. To evaluate the inhibitory effect of BAY-805 on HCT-15 or HT-29 spheroids induced by EGF, WT HT-29 or WT HCT-15 cells were seeded into 96-well plates at a concentration of 500 cells per well. These 96-well plates were then incubated at 37\u0026deg;C for an additional 48 hours to allow the formation of 3D spheroids in culture, and spheroids were treated with vehicle (0.01% DMSO) or 7.5 \u0026micro;M BAY-805 in HT-29 spheroids and 1.6 \u0026micro;M BAY-805 in HCT-15 spheroids. After 24 hr, spheroids were further treated with vehicle (0.01% DMSO) or EGF (20 ng/mL). Tumor spheroid formation and growth were evaluated using phase-contrast microscopy. Sizes of spheroids were assessed using the Image J Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMTT Assay\u003c/h2\u003e \u003cp\u003eCtrl HCT-15, Ctrl SW480, Ctrl HT-29, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480, and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells were seeded into 96-well culture plates at a density of 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well, treated with vehicle (DMSO, 0.01% v/v concentration) or EGF (2.5 ng/mL), and grown in a culture medium supplemented with 10% FBS for different time periods. Cell viability was measured using an MTT reagent (Sigma-Aldrich, M5655) dissolved in PBS (1 mg/mL). On the day when measurements were taken, the medium was carefully replaced with fresh RPMI\u0026thinsp;+\u0026thinsp;10% FBS, added with diluted MTT (1:10, 10% MTT), and incubated at 37\u0026deg;C for 3 h. After removing the incubation medium, formazan crystals were dissolved in a 100 \u0026micro;L solution of DMSO. MTT reduction was quantified by measuring light absorbance at 595 nm using a Bio-Rad Model 680 microplate reader (Bio-Rad, CA, USA). Each test was repeated at least four times in quadruplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003ePrimary tumor tissues and adjusted matched normal tissues were obtained from CRC patients (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) and RNA sequencing was done using the Illumina TruSeq RNA Sample Preparation Kit v2, as described in the previous report [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The experiments conducted on patient samples were approved by the institutional review board of Samsung Medical Center (IRB# 2010-04-004). Written informed consents were obtained from all participating patients. All experiments and analysis procedures were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation) or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was determined by Student\u0026rsquo;s t-test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). \u003cem\u003eP\u003c/em\u003e-values are marked by asterisks (*, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eUSP21 stabilizes EGFR by deubiquitinating EGFR in colon cancer cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the context of colorectal cancer (CRC) metastasis, USP21 plays a pivotal role by stabilizing Fra-1 through deubiquitination, thereby facilitating the expression of metastasis-related genes like MMPs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, the activation of Fra-1 is intertwined with EGFR signaling in an MMP-dependent manner [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Despite the recognized interconnectedness of USP21-Fra-1 and Fra-1-EGFR, the regulatory axis of USP21-EGFR in metastatic CRC remains largely unexplored, as depicted in Fig.\u0026nbsp;1A. Our study began by elucidating the biochemical relationship between USP21 and EGFR. We observed a direct interaction between USP21 and EGFR (Fig.\u0026nbsp;1B, lane 3), a finding further validated through semi-endogenous immunoprecipitation (Fig.\u0026nbsp;1C). Remarkably, we found that USP21 induced the deubiquitination of EGFR in a dose-dependent manner (Fig.\u0026nbsp;1D, lane 1 vs. lanes 2\u0026ndash;4). To probe whether this deubiquitination activity hinged on the enzymatic function of USP21, we generated a catalytically inactive mutant, USP21 C221A, and conducted deubiquitination assays comparing USP21 wild type (WT) and the USP21 C221A mutant. The deubiquitination of EGFR consistently occurred in the presence of USP21 WT, but not in the USP21 C221A mutant (Fig.\u0026nbsp;1E, lane 3 vs. lane 2), suggesting that the deubiquitination of EGFR might indeed be reliant on the catalytic activity of USP21. Subsequently, we investigated whether USP21-mediated deubiquitination of EGFR affected EGFR stability. To do so, we utilized CRISPR-Cas9 gene editing to generate three distinct \u003cem\u003eUSP21\u003c/em\u003e-knockout (KO) colon cancer cell lines: \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29, and \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 (Fig.\u0026nbsp;1F, CRISPR-Cas9 gene editing; Fig.\u0026nbsp;1G, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15; Fig.\u0026nbsp;1H, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29; Fig.\u0026nbsp;1I, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480). Assessing EGFR's half-life via cycloheximide (CHX) chase assay in control (Ctrl) cells and \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells revealed significantly reduced EGFR levels in \u003cem\u003eUSP21\u003c/em\u003e-KO cells compared to their respective Ctrl counterparts (Fig.\u0026nbsp;1J, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 vs. Ctrl HCT-15; Fig.\u0026nbsp;1K, \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 vs. Ctrl SW480; Fig.\u0026nbsp;1L, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 vs. Ctrl HT-29). These findings suggest that USP21 interacts with and stabilizes EGFR, potentially by preventing its ubiquitin-mediated degradation within multivesicular body (MVB)-lysosome vesicles, thus leading to elevated EGFR expression levels (Fig.\u0026nbsp;1M).\u003c/p\u003e \u003cp\u003e \u003cb\u003eUSP21 plays a crucial role in the progression of colon cancer and impacts survival rates in patients with metastatic CRC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the above biochemical results, we investigated the involvement of USP21 in CRC progression through both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assays using \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells. Notably, we observed a significant reduction in both transwell migration and wound healing in \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29, and \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 cells as compared to Ctrl HCT-15, Ctrl HT-29, and Ctrl SW480 cells, respectively (Supplementary Fig.\u0026nbsp;1A-C, transwell migration; Supplementary Fig.\u0026nbsp;2A-C, wound healing). Moreover, cell proliferation was significantly reduced in \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29, and \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 cells as compared to Ctrl HCT-15, Ctrl HT-29, and Ctrl SW480 cells, respectively (Supplementary Fig.\u0026nbsp;3A-C). Additionally, anchorage-dependent or -independent colony formation was markedly attenuated in \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells as compared to Ctrl HCT-15 and Ctrl HT-29 cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, anchorage-dependent; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D, anchorage-independent). To assess the tumorigenic potential of USP21, we performed \u003cem\u003ein vitro\u003c/em\u003e three-dimensional (3D) tumor spheroid assays and \u003cem\u003ein vivo\u003c/em\u003e xenograft assays using NSG mice. Tumor spheroids derived from \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells exhibited significantly smaller sizes compared to those from Ctrl HCT-15 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F, Ctrl HCT-15 vs. \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15). Additionally, the number of invasive cells originating from spheroids increased in Ctrl HCT-15 cells after day 10, whereas it significantly decreased in \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H, Ctrl HCT-15 vs. \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15). While the size of tumor spheroids progressively increased in Ctrl HT-29 cells, it was notably diminished in \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J, Ctrl HT-29 vs. \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29). Importantly, xenografting NSG mice with either Ctrl HCT-15 or \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells revealed a notable attenuation in tumor growth and size in mice harboring \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells compared to those with Ctrl HCT-15 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-M, Ctrl HCT-15 vs. \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15), strongly indicating the critical role of USP21 expression in the tumorigenicity of colon cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the insights gained from our biochemical and cellular studies into the functional role of USP21, we sought to examine its correlation with the survival outcomes of CRC patients. To achieve this, we analyzed a cohort of CRC patients with metastasis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27, Supplementary Table\u0026nbsp;1). Utilizing RNA sequencing data, we compared the differential magnitude of USP21 expression in tumor tissues (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) versus matched normal tissues (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) and categorized the 27 CRC patients into two groups: USP21-upregulated (USP21\u003csup\u003eup\u003c/sup\u003e) CRC (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) and USP21-downregulated (USP21\u003csup\u003edown\u003c/sup\u003e) mCRC (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eN and Supplementary Table\u0026nbsp;2). Notably, patients exhibiting higher levels of USP21 expression displayed poorer survival outcomes compared to those with lower expression levels; with survival rates of 45% in USP21\u003csup\u003eup\u003c/sup\u003e CRCs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) versus 71% in USP21\u003csup\u003edown\u003c/sup\u003e CRCs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). These results suggest that USP21 expression plays a pivotal role for the tumorigenicity of colon cancer cells and associated with mCRC patient survival.\u003c/p\u003e \u003cp\u003e \u003cb\u003eUSP21 and EGFR expression is associated with poor survival in mCRC patients and USP21 promotes CRC progression in response to EGF.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the above results, our investigation extended to exploring the relationship between USP21 and EGFR in mCRCs (Supplementary Table\u0026nbsp;2, differential magnitude of EGFR expression in 27 mCRC patients; Supplementary Table\u0026nbsp;2, differential magnitude of EGFR and USP21 expression in 27 mCRC patients). Intriguingly, the survival rates were lower in patients with EGFR\u003csup\u003eup\u003c/sup\u003e mCRC or EGFR\u003csup\u003eup\u003c/sup\u003eUSP21\u003csup\u003eup\u003c/sup\u003e mCRC patients compared to those with EGFR\u003csup\u003edown\u003c/sup\u003e mCRC or EGFR\u003csup\u003edown\u003c/sup\u003eUSP21\u003csup\u003edown\u003c/sup\u003e mCRC patients, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, 40% in EGFR\u003csup\u003eup\u003c/sup\u003e vs. 59% in EGFR\u003csup\u003edown\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, 33% in EGFR\u003csup\u003eup\u003c/sup\u003eUSP21\u003csup\u003eup\u003c/sup\u003e vs. 67% in EGFR\u003csup\u003edown\u003c/sup\u003eUSP21\u003csup\u003edown\u003c/sup\u003e). Comparing the survival of EGFR\u003csup\u003eup\u003c/sup\u003eUSP21\u003csup\u003eup\u003c/sup\u003e mCRC patients with that of EGFR\u003csup\u003eup\u003c/sup\u003e mCRC patients or USP21\u003csup\u003eup\u003c/sup\u003e mCRC patients, a significantly lower survival rate was evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, vs. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eN or Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; 33% in EGFR\u003csup\u003eup\u003c/sup\u003eUSP21\u003csup\u003eup\u003c/sup\u003e vs. 45% in USP21\u003csup\u003eup\u003c/sup\u003e or 40% in EGFR\u003csup\u003eup\u003c/sup\u003e), supposing a potential connection of survival rate between EGFR and USP21 expression. It might be associated with the USP21-mediated EGFR stabilization (Fig.\u0026nbsp;1M), thereby enhancing EGFR-mediated cancer progression, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. To validate the functional role of USP21, we investigated whether USP21 contributes to CRC progression upon EGFR stimulation. We evaluated the colon cancer progression capacity in \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells following exposure to EGF. Upon EGF stimulation, the transwell migration and wound healing abilities of \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29, and \u003cem\u003eUSP21\u003c/em\u003e-KO SW480 cells were notably reduced compared to Ctrl HCT-15, Ctrl HT-29, and Ctrl SW480 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F, transwell migration; Supplementary Fig.\u0026nbsp;4A-F, wound healing). Similar trends were observed in the cell proliferation assay in \u003cem\u003eUSP21\u003c/em\u003e-KO CRC cells upon EGF stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H and Supplementary Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e, \u003cem\u003eUSP21\u003c/em\u003e-KO vs. Ctrl cells). Moreover, anchorage-independent and -dependent colony formation assays revealed a significant decrease in the number of colonies in \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 or \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells treated with either vehicle or EGF compared to Ctrl cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J, anchorage-independent; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, L, anchorage-dependent). These findings underscore the role of USP21 in modulating colon cancer proliferation, migration, and colony formation in response to EGF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we delved into the role of USP21 in 3D tumor spheroid formation triggered by EGF. We seeded both Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells, as well as Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells, in 96-well plates. After allowing two days for spheroid formation optimization, spheroids were treated with either vehicle or EGF for various durations, as depicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C, Ctrl HCT-15 and \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15). In Ctrl HT-29 cells, tumor spheroids gradually grew larger with EGF treatment compared to the vehicle-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E, EGF vs. vehicle in Ctrl HT-29). Conversely, the size of tumor spheroids noticeably decreased in \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells treated with vehicle compared to Ctrl HT-29 cells treated with vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 treated with vehicle vs. Ctrl HT-29 treated with vehicle). Crucially, when comparing the sizes of tumor spheroids between EGF-treated Ctrl HT-29 cells and EGF-treated \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells, marked decreases were observed in \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 treated with EGF vs. Ctrl HT-29 treated with EGF). At day 8 and day 10 post-incubation, we observed invasive cells derived from tumor spheroids in both Ctrl HT-29 and \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells treated with either vehicle or EGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, indicated by red-dashed line). Measurements of the dimensions of invasive cells revealed significantly smaller sizes in \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 cells treated with vehicle or EGF compared to Ctrl HT-29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G, \u003cem\u003eUSP21\u003c/em\u003e-KO HT-29 vs. Ctrl HT-29). Similar results were obtained in the 3D tumor spheroid formation assay performed with \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 cells compared to Ctrl HCT-15 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-K, \u003cem\u003eUSP21\u003c/em\u003e-KO HCT-15 vs. Ctrl HCT-15). Taken together, these findings indicate that USP21 can enhance tumor formation and invasion induced by EGF stimulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBAY-805, an inhibitor of USP21, markedly attenuates the 3D tumor formation induced by EGF treatment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHaving shown that USP21 contributes to tumor formation triggered by EGF stimulation, we examined the impact of inhibiting USP21 activity using the pharmacological inhibitor BAY-805 on EGF-induced tumor formation. To do that, we determined the IC\u003csub\u003e50\u003c/sub\u003e value of BAY-805 in HCT-15- or HT-29-induced spheroid formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, HCT-15; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D, HT-29). 3D tumor spheroids of HCT-15 or HT-29 cells were treated with either vehicle or varying concentrations of BAY-805 (80 nM\u0026thinsp;~\u0026thinsp;50 \u0026micro;M), as indicated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, HCT-15; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, HT-29). The IC\u003csub\u003e50\u003c/sub\u003e value of BAY-805 was found to be 1.6 \u0026micro;M in HCT-15 or 7.5 \u0026micro;M in HT-29 spheroids, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, HCT-15; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, HT-29). Notably, treatment with BAY-805 significantly reduced EGFR expression in HCT-15 or HT-29 cells compared to the vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, western blotting in HCT-15; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, western blotting in HT-29), indicating that inhibition of USP21 with BAY-805 leads to reduced EGFR expression. To assess the therapeutic effects of BAY-805 on EGF-induced spheroid formation in HCT-15 or HT-29 cells, HCT-15 and HT-29 spheroids were exposed to either vehicle or EGF in the presence or absence of BAY-805 (1.6 \u0026micro;M for HCT-15; 7.5 \u0026micro;M for HT-29) for varying durations, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. EGF treatment significantly increased the size of tumor spheroids in both HCT-15 and HT-29 cells in the absence of BAY-805 (EGF treatment vs. vehicle: Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C, HCT-15; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E, HT-29). Remarkably, treatment with BAY-805 significantly inhibited the formation of both HCT-15 and HT-29 spheroids in response to EGF (BAY-805 plus EGF treatment vs. EGF treatment: Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C, HCT-15; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E, HT-29). These findings suggest that USP21 promotes EGF-induced cancer progression via stabilizing EGFR, whereas inhibiting USP21 activity attenuates cancer progression induced by EGF stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt has been reported that EGFR is overexpressed in 60%-80% of colorectal cancers (CRCs), prompting consideration of anti-EGFR targeting as a pivotal strategy in CRC treatment [36-38]. EGFR, a multifunctional receptor, plays a crucial role in various cellular processes such as cell division, differentiation, migration, and organogenesis [38,39]. Dysregulated EGFR signaling significantly impacts multiple pathways including PLC-gamma-1, RAS-RAF-MEK-MAPKs, phosphatidylinositol-3 kinase and Akt, Src, stress-activated protein kinases, PAK-JNKK-JNK, and signal transducers and activators of transcription [37]. Consequently, the development of inhibitors targeting the EGFR signaling pathway has emerged as a promising approach in various cancers, including CRC [40].\u003c/p\u003e\n\u003cp\u003eMoreover, given the importance of EGFR regulation, cellular regulators influencing its stability and expression are gaining attention as potential therapeutic targets. USPs, which play a role in the recycling and degrading pathway of EGFR, have emerged as potential targets for intervening in EGFR-induced cancer development and progression [8-10, 41-43]. Several USPs have been identified as modulators capable of affecting EGFR stability [13-17]. Thus, there's a current focus in cancer medicine on pharmacologically disrupting USP activity to specifically target cancer-causing protein aberrations, including EGFR [39,40]. Among the USPs, USP21 stands out as it interacts with multiple substrate proteins and is considered a critical oncogene in various human cancers [44-47]. Recent findings also suggest its involvement in CRC metastatic progression by stabilizing Fra-1, a protein linked to tumor formation and metastasis [18]. By increasing the expression of MMP-1 and Fra-1 target genes, USP21 influences CRC progression. Given the biochemical activity of USP21 in regulating the ubiquitin-mediated degradation pathway, there's a hypothesis that USP21 could functionally regulate EGF-mediated CRC progression by modulating EGFR stability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe findings presented in this study shed light on the critical role of USP21 in colorectal cancer (CRC) progression, particularly in the context of metastatic CRC (mCRC). Through a series of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, we delineated the molecular mechanisms underlying USP21-mediated regulation of EGFR stability and its impact on CRC progression and patient survival. Our results demonstrate that USP21 enhances EGFR expression by stabilizing EGFR through deubiquitination, thereby promoting EGFR-mediated signaling in colon cancer cells. This mechanism is pivotal for the maintenance of EGFR levels, as evidenced by the reduced EGFR expression and impaired tumorigenic potential observed in \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells. Moreover, we show that USP21's enzymatic activity is crucial for its ability to deubiquitinate EGFR and stabilize its expression, highlighting the functional significance of USP21 in CRC progression.\u003c/p\u003e\n\u003cp\u003eImportantly, our study uncovers a significant association between USP21 expression levels and patient survival in mCRC. Patients with higher USP21 expression levels exhibited poorer survival outcomes compared to those with lower expression levels, implicating USP21 as a potential prognostic marker for mCRC. Furthermore, our data suggest a synergistic relationship between USP21 and EGFR expression in mCRC, wherein patients with concurrent upregulation of both proteins displayed the lowest survival rates. This observation underscores the clinical relevance of targeting the USP21-EGFR axis in mCRC therapy. Additionally, we provide evidence for the functional relevance of USP21 in CRC progression in response to EGF stimulation. \u003cem\u003eUSP21\u003c/em\u003e-KO cells attenuated the proliferative and migratory capacities of colon cancer cells following EGF exposure, underscoring the importance of USP21 in mediating EGF-induced CRC progression. Furthermore, our results indicate that pharmacological inhibition of USP21 with BAY-805 significantly abrogates EGF-induced tumor formation \u003cem\u003ein vitro\u003c/em\u003e, highlighting the therapeutic potential of targeting USP21 in mCRC treatment.\u003c/p\u003e\n\u003cp\u003eIn our proposed scenario, depicted in Figure 7, we suggest a functional association between EGFR and USP21 in CRC progression. mCRC patients exhibiting up-regulated levels of USP21 may experience more aggressive cancer progression, especially in response to a tumor microenvironment enriched with EGF, compared to patients with down-regulated USP21 (Fig. 7A). In tumors with upregulated USP21, the enzyme inhibits the ubiquitin-dependent degradation pathway within multivesicular body (MVB)-lysosome vesicles by deubiquitinating EGFR, consequently increasing EGFR expression in CRCs. Subsequently, aberrant EGFR signaling initiated by EGF engagement is transmitted, thereby enhancing CRC progression (Fig. 7B). Conversely, in tumors with downregulated USP21, EGFR undergoes ubiquitination and subsequent degradation within MVB-lysosome vesicles, leading to attenuated cancer progression through diminished engagement with EGF (Fig. 7C).\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study elucidates the multifaceted role of USP21 in CRC progression and patient survival, implicating USP21 as a promising therapeutic target for mCRC. Further investigation into the therapeutic efficacy of USP21 inhibitors, such as BAY-805, in preclinical and clinical settings may offer new avenues for improving the prognosis and treatment outcomes of mCRC patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003ethe author declare that they have no conflict of interest\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea Grants funded by the Korean Government (2023R1A2C1003762, 2021R1A2C1094478, and RS-2023-00217189).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Hyehwa Forum members for their helpful discussion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea Grants funded by the Korean Government (2023R1A2C1003762, 2021R1A2C1094478, and RS-2023-00217189).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJHS, MJK, JYK, YK, SHK and HJL performed the experiments and data analysis; BC, DK, JHS and JYK performed \u003cem\u003ein vivo\u003c/em\u003e xenografted NSG experiments; JHS, MJK, JYK, YK, SHK and HJL performed the western blots; JHS, MJK, JYK performed the CRISPR/Cas9-based gene editing; JHS, MJK, JYK, YK, SHK and HJL performed cancer progression assay; EC and KYL performed the data analysis; YBC provided clinical and RNA-Seq data; KKK provided experimental materials; EC and KYL are responsible for the conception, design and supervision of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor and matched normal tissues from 27 patients with primary CRC were obtained in accordance with the ethical principles stated in the Declaration of Helsinki. This study was approved by the institutional review board of Samsung Medical Center (IRB# 2010-04-004). We obtained written informed consent from each patient prior to surgery for using their pathological specimens for research use. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Bioanalysis Center Animal Facility (IACUC #: 23-10-01), GenNBio Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanani B, Vijayakumar M, Priya K, et al. 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Cancer Res. 2010;70:3843\u0026ndash;3850.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JC, Vivanco I, Beroukhim R, et al. Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain. PLoS Med. 2006;3:e485.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin J, Liu J, Chen C, Liu Z, et al. The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog. Nat Commun. 2016;7:13594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakagawa T, Kajitani T, Togo S, et al. Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation. Genes Dev. 2008;22:37\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Chen C, Hou X, et al. Identification of the E3 deubiquitinase ubiquitin-specific peptidase 21 (USP21) as a positive regulator of the transcription factor GATA3. J Biol Chem. 2013;288:9373\u0026ndash;9382.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan A, Giri S, Wang Y, et al. BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase. Proc Natl Acad Sci U S A. 2015;112:8338\u0026ndash;8343.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4594251/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4594251/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe emerging significance of ubiquitin-specific peptidase 21 (USP21) in stabilizing Fra-1 (FOSL1) has shed light on their involvement in promoting colorectal cancer (CRC) metastasis. Additionally, EGFR signaling has been linked reciprocally with Fra-1 activation in an MMP-dependent manner. However, the functional implications of the USP21-EGFR signaling axis in metastatic CRC (mCRC) remain incompletely understood. RNA-Seq data from tumor tissues (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) and matched normal tissues (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27) from 27 mCRC patients were analyzed to investigate the clinical correlation between USP21 and EGFR expression. Functional studies including CRISPR/Cas9 gene editing method to generate \u003cem\u003eUSP21\u003c/em\u003e-knockout (\u003cem\u003eUSP21\u003c/em\u003e-KO) CRC cells, \u003cem\u003ein vitro\u003c/em\u003e cancer progression and tumor formation assays, \u003cem\u003ein vivo\u003c/em\u003e xenograft assays in NSG mice, and therapeutic assays with the USP21 inhibitor, BAY-805, were conducted. Elevated levels of USP21 and EGFR expression in mCRC patients correlated with poorer survival outcomes. Mechanistically, USP21 was found to enhance EGFR stability by deubiquitinating EGFR, resulting in reduced EGFR levels in \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells. \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells exhibited significantly attenuated cell proliferation, migration, colony formation, and 3D tumor spheroid formation in response to EGF. Furthermore, tumorigenic activity \u003cem\u003ein vivo\u003c/em\u003e was notably diminished in NSG mice xenografted with \u003cem\u003eUSP21\u003c/em\u003e-KO colon cancer cells. Notably, the USP21 inhibitor, BAY-805, demonstrated a remarkable inhibitory effect on the formation of 3D tumor spheroids in colorectal cancer cells stimulated with EGF. These findings provide valuable insights into the potential of USP21 as both a therapeutic target and a predictive biomarker for intervening in mCRC induced by EGF.\u003c/p\u003e","manuscriptTitle":"USP21-EGFR signaling axis is functionally implicated in metastatic colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-10 12:17:31","doi":"10.21203/rs.3.rs-4594251/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Cell Death Discovery","date":"2024-10-18T00:15:42+00:00","index":"","fulltext":""},{"type":"decision","content":"revise","date":"2024-07-30T14:34:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-22T23:43:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-19T08:55:45+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-17T08:27:10+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-14T13:06:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-10T23:29:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-07-10T15:13:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-18T10:04:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-17T12:48:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-06-17T12:48:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba5571b2-36b8-4ec0-a878-140acb8e28dc","owner":[],"postedDate":"August 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34409066,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer"},{"id":34409067,"name":"Health sciences/Biomarkers/Prognostic markers"}],"tags":[],"updatedAt":"2024-12-19T08:06:46+00:00","versionOfRecord":{"articleIdentity":"rs-4594251","link":"https://doi.org/10.1038/s41420-024-02255-1","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2024-12-18 05:00:00","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-08-10 12:17:31","video":"","vorDoi":"10.1038/s41420-024-02255-1","vorDoiUrl":"https://doi.org/10.1038/s41420-024-02255-1","workflowStages":[]},"version":"v1","identity":"rs-4594251","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4594251","identity":"rs-4594251","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}