RNA helicase SKIV2L impedes tumor immunity by reprogramming arginine metabolism of hepatocellular carcinomas.

RNA helicase SKIV2L impedes tumor immunity by reprogramming arginine metabolism of hepatocellular carcinomas - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice J Immunother Cancer . 2026 Apr 9;14(4):e014192. doi: 10.1136/jitc-2025-014192 Search in PMC Search in PubMed View in NLM Catalog Add to search RNA helicase SKIV2L impedes tumor immunity by reprogramming arginine metabolism of hepatocellular carcinomas Lisheng Zhu Lisheng Zhu 1 Department of Medical Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Cancer Center, Zhejiang University, Hangzhou, China 3 Binjiang Institute of Zhejiang University, Hangzhou, China Find articles by Lisheng Zhu 1, 2, 3, 0 , Meng Wu Meng Wu 1 Department of Medical Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Cancer Center, Zhejiang University, Hangzhou, China 3 Binjiang Institute of Zhejiang University, Hangzhou, China Find articles by Meng Wu 1, 2, 3, 0 , Bingbing Feng Bingbing Feng 4 School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Find articles by Bingbing Feng 4 , Yu Zhang Yu Zhang 5 Department of Cardiology, Heart Regeneration and Repair Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 6 State Key Laboratory of Transvascular Implantation Devices, Zhejiang University, Hangzhou, China Find articles by Yu Zhang 5, 6 , Yang Xu Yang Xu 2 Cancer Center, Zhejiang University, Hangzhou, China 3 Binjiang Institute of Zhejiang University, Hangzhou, China 5 Department of Cardiology, Heart Regeneration and Repair Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 6 State Key Laboratory of Transvascular Implantation Devices, Zhejiang University, Hangzhou, China Find articles by Yang Xu 2, 3, 5, 6, ✉ , Lili Yu Lili Yu 1 Department of Medical Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Cancer Center, Zhejiang University, Hangzhou, China Find articles by Lili Yu 1, 2, * Author information Article notes Copyright and License information 1 Department of Medical Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Cancer Center, Zhejiang University, Hangzhou, China 3 Binjiang Institute of Zhejiang University, Hangzhou, China 4 School of Basic Medical Sciences, Southern Medical University, Guangzhou, China 5 Department of Cardiology, Heart Regeneration and Repair Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 6 State Key Laboratory of Transvascular Implantation Devices, Zhejiang University, Hangzhou, China Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Additional supplemental material is published online only. To view, please visit the journal online ( https://doi.org/10.1136/jitc-2025-014192 ). None declared. ✉ Dr Yang Xu; [email protected] * Dr Lili Yu; [email protected] 0 LZ and MW contributed equally. Received 2025 Nov 5; Accepted 2026 Mar 26; Collection date 2026. Copyright © Author(s) (or their employer(s)) 2026. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See https://creativecommons.org/licenses/by-nc/4.0/ . PMC Copyright notice PMCID: PMC13084862  PMID: 41956538 Abstract Background and aims Human solid tumors such as hepatocellular carcinomas (HCC) establish a complex immunosuppressive tumor microenvironment (TME) that undermines the efficacy of existing immunotherapies such as chimeric antigen receptor T (CAR T) cell therapy. To advance immunotherapy for HCC, it is crucial to delineate the molecular mechanisms that drive TME formation and immune evasion. Methods We integrated bulk and single-cell RNA sequencing analysis to assess immune regulation and conduct pathway enrichment analyses. The oncogenic roles of SKIV2L were revealed by comparing the proliferation and tumorigenesis of HCC with or without SKIV2L knockdown. SKIV2L-driven mechanisms were investigated using RNA sequencing, RNA immunoprecipitation sequencing, immunoprecipitation-mass spectrometry, and proximity ligation assay. Functional studies of SKIV2L in arginine metabolism and anti-tumor immunity were performed using flow cytometry and tumor-T cell co-culture assays. Results SKIV2L was overexpressed in HCC, and its expression levels negatively correlated with patient prognosis and tumor immune cell infiltration. Depletion of SKIV2L disrupted the immunosuppressive landscape of HCC, enhanced systemic antitumor immunity, and significantly augmented the efficacy of CAR T cell therapy. Mechanistically, SKIV2L promoted arginine metabolism in HCC by recruiting GNL3 to stabilize mRNAs encoding key regulators of arginine uptake and metabolism, including the arginine transporter SLC7A1 and the arginine-catabolizing enzyme ARG2. This SKIV2L–arginine axis fostered an immunosuppressive TME and impaired T-cell function. Furthermore, SKIV2L was identified as a direct transcriptional target of c-Myc, positioning SKIV2L as a druggable mediator of c-Myc-driven oncogenesis. Conclusions Our findings identify a novel c-Myc–SKIV2L–arginine metabolism axis that drives HCC progression and immune evasion. Targeting SKIV2L reprograms TME and reinvigorates antitumor immunity, providing a promising therapeutic strategy to overcome resistance to immunotherapy such as CAR T cell therapy in HCC. Keywords: Immunotherapy, Hepatocellular Carcinoma, Tumor microenvironment - TME, Adoptive cell therapy - ACT WHAT IS ALREADY KNOWN ON THIS TOPIC Arginine availability critically influences the antitumor efficacy of CAR T cells. SKIV2L, a core component of the SKI complex in RNA decay, is known to modulate RNA metabolism. WHAT THIS STUDY ADDS SKIV2L enhances arginine metabolism in hepatocellular carcinoma (HCC) independently of its helicase activity by recruiting GNL3 to stabilize mRNAs encoding key regulators of arginine uptake and degradation, thereby promoting an immunosuppressive tumor microenvironment (TME) and impairing T-cell function. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Depletion of SKIV2L remodels the immunosuppressive TME and enhances anti-tumor immunity, providing a novel therapeutic strategy to overcome immunotherapy resistance in HCC. Introduction Cancer immunotherapies have achieved substantial clinical success in the treatment of malignant tumors. For example, chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable efficacy against hematologic malignancies such as leukemia and lymphoma. 1 2 However, CAR T cells exhibit limited activity in solid tumors, largely due to the complex immunosuppressive tumor microenvironment (TME). The immune checkpoint blockades such as anti-PD1 and anti-CTLA4 antibody therapies are only effective for a small percentage of solid tumors due to the highly heterogeneous TME. 3 4 Thus, to improve the efficacy of immunotherapy against solid tumors, it is critical to elucidate the mechanisms that drive TME development and to identify strategies that can remodel the TME to favor antitumor immunity. 5 Accumulating data have shown that metabolic reprogramming plays a key role in shaping the TME. 6 7 For instance, the metabolic switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis increases lactate production, which impairs T-cell function both directly—via engagement of its receptors on immune cells—and indirectly, by acidifying the local microenvironment. 8 Metabolic reprogramming also contributes to the differentiation and maintenance of immunosuppressive macrophages and cancer-associated fibroblasts. 9 10 Tumor cells can further suppress T-cell responses in the TME by competing for key nutrients that are essential for T-cell survival and function. Among these metabolites, arginine is particularly notable as an amino acid indispensable for the survival and proliferation of both T cells and tumor cells. 11 12 Arginine has been shown to activate multiple proliferative signaling pathways, including PI3K–AKT–mTOR, 13 14 MEK–ERK, 15 and OXPHOS. 16 When T lymphocytes fail to outcompete tumor cells for arginine within TME, their survival and antitumor effector functions are compromised. 17 18 Arginine metabolic reprogramming thus represents a promising therapeutic avenue to both inhibit tumor growth and disrupt the immunosuppressive microenvironment. However, the molecular mechanisms that orchestrate arginine metabolic reprogramming in tumor cells remain incompletely understood. To elucidate the mechanisms driving arginine reprogramming in hepatocellular carcinoma (HCC), we performed a comprehensive analysis of HCC datasets and identified the SKI2-like (SKI2L) family RNA helicase SKIV2L as a candidate regulator. The SKI2L family of RNA helicases constitutes a distinct subset of helicases with robust RNA unwinding and ribonucleoprotein (RNP) complex–remodeling activity, thereby playing central roles in mRNA turnover and surveillance. 19 We found that SKIV2L is required to maintain the immunosuppressive TME in HCC by enhancing the ability of HCC cells to compete for arginine within TME and thereby suppress T-cell activity. Mechanistically, SKIV2L binds to and stabilizes the mRNAs of SLC7A1 and ARG2, whose gene products are critical for arginine transport and metabolism, leading to increased arginine uptake and utilization by tumor cells. Thus, SKIV2L emerges as a promising therapeutic candidate for disrupting the TME of solid tumors and improving the efficacy of CAR-T cell–based immunotherapy. Methods and materials Patient samples Human HCC and adjacent normal tissue samples (ANT, exceeding the edge of the tumor by at least 2 cm) were obtained from 62 patients with HCC informed consent. The approval by the Institutional Review Board of Nanfang Hospital and the detailed clinical information were previously published. Human HCC and adjacent normal tissue samples (ANT, exceeding the edge of the tumor by at least 2 cm). 20 21 Total RNA was extracted from samples using TRIzol reagent (Invitrogen, Cat# 15596018CN), reversely transcribed, and analyzed by quantitative real-time PCR assay. The PCR primers used are listed in online supplemental table 1 . Animal experiments 6-week-old male specific pathogen-free C57BL/6, BALB/c, BALB/c nude mice, or NSG mice were used in these experiments. The mice were housed in a standard experimental environment at the Animal Experimental Center. For subcutaneous tumor models, mice received flank injections of tumor cells. For the Tet-inducible SKIV2L knockdown experiment, BALB/c mice bearing H22 tumors with Tet-inducible SKIV2L knockdown were used. When tumor volume reached approximately 100 mm³, all tumor-bearing mice were randomly assigned to different groups using computer-generated randomization. Specifically, animals were ranked by unique IDs, and an independent researcher generated random numbers via the RAND() function in Excel. Group divided: (1) a control group injected intraperitoneally with Doxycycline (Doxy) (20 mg/kg body weight) daily and provided Doxy (2 mg/L) in their drinking water; (2) a Tet-inducible SKIV2L knockdown group treated similarly. Tumor tissues from HCC patients were cut into small pieces and transplanted into NSG mice. After 3–5 generations of propagation in recipient mice, stable tumor growth was achieved, and the tumors were used for experiments, including the injection of shSKIV2L lentivirus or scramble virus to establish an inducible SKIV2L knockdown system in solid tumors. For orthotopic HCC models, H22 cells were injected into the hepatic portal vein of 6-week-old male BALB/c mice. 5 days after implantation, the mice were injected intraperitoneally with Doxy (20 mg/kg body weight) daily and provided Doxy (2 mg/L) in their drinking water. Mice were humanely euthanized on meeting predefined endpoints: >20% body weight loss, hunched posture, immobility, piloerection, anorexia, or hypothermia (<34°C core temperature). 22 For in vivo arginine deprivation experiments, SKIV2L-overexpressing and control H22 cell lines were used. Mice were fed either an arginine-free or normal diet. In arginine rescue experiments, H22 cells were implanted subcutaneously into BALB/c mice. When the tumor volume reached approximately 200 mm³, the tumor-bearing mice were randomly assigned to different groups and fed with or without 7 mg/mL L-arginine. 23 3 days after treatment, the tumors were harvested and analyzed. Tumor growth was monitored and evaluated using the formula: (length×width²)/2. Mice were euthanized when the tumor volume reached approximately 1000 mm³ or when the maximum tumor diameter measured approximately 15 mm. Tumor tissues and vital organs were harvested for further analysis. Results SKIV2L promotes tumorigenesis of HCC To identify potential RNA helicases involved in the tumorigenesis of HCC, we conducted a targeted analysis of the HCC datasets from TCGA and the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). Two RNA helicases, SKIV2L and NAV2, were found to be associated with oncogenic events in HCC, including MYC , KRAS , CTNNB1 , and TP53 alterations ( figure 1A,B ). 24 , 26 In bulk RNA-seq data from patients treated with anti-PD-1 plus lenvatinib ( GSE235863 ), 27 the expression levels of both SKIV2L and NAV2 were associated with the treatment response ( figure 1C ). However, integrative analysis of transcriptomic profiles from GSE25097 and GSE105130 revealed that only SKIV2L was markedly upregulated in HCC tissues ( figure 1D,E ). Consistently, single-cell RNA sequencing (scRNA-seq) analysis of HCC showed that the proportion of tumor cells expressing SKIV2L was higher than that of nonmalignant cells, and normalized SKIV2L expression levels were significantly elevated in tumor cells compared with normal cells ( figure 1F,G ). 28 , 31 In line with these findings, SKIV2L expression was predominantly detected in tumor cells among all major cell types in the TME ( online supplemental figure 1A ). A pan-cancer survey further demonstrated SKIV2L overexpression across multiple malignancies—including bladder, breast, colon adenocarcinoma, and lung cancers ( online supplemental figure 1B ). Survival analyses revealed that higher SKIV2L expression was associated with poorer prognosis in patients with HCC ( online supplemental figure 1C , online supplemental table 2 ). Together, these results suggest that SKIV2L contributes to HCC tumorigenesis. Figure 1. SKIV2L promotes tumorigenesis of human HCC. ( A ) The flow chart for identifying RNA helicase SKIV2L as the potential gene driving HCC tumorigenesis. ( B ) Venn diagrams illustrate the differential genes associated with HCC driver genes ( MYC , KRAS , CTNNB1 , and TP53 ). The lists of differential genes were analyzed from GSE156744 ( MYC and KRAS ), GSE213517 ( CTNNB1 ), GSE34760 ( TP53 ), and TCGA database. ( C ) Bulk RNA-seq analysis of SKIV2L and NAV2 expression in HCC patients exhibiting either response or non-response to anti-PD-1 plus Lenvatinib combination therapy. The differential expression levels of SKIV2L ( D ) and NAV2 ( E ) between tumor and adjacent normal tissues in GSE25097 and GSE105130 . For the GSE25097 dataset: n=243 for normal sample, n=268 for tumor sample, unpaired t-test; For the GSE105130 dataset: n=25, paired t-test. ( F ) Single-cell RNA-seq analysis showing SKIV2L expression in normal versus tumor cells visualized using t-SNE plots ( GSE229772 ). ( G ) Comparative SKIV2L expression between normal and tumor cells across three single-cell RNA-seq datasets: GSE229772 , GSE151530 , and GSE189903 . HCC, hepatocellular carcinoma; tSNE, t-distributed stochastic neighbor embedding. Open in a new tab In support of its roles in HCC tumorigenesis, SKIV2L knockdown in human HCC cells reduced their proliferative capacity and colony-forming ability in vitro ( online supplemental figure 1D–F ). Moreover, inducible knockdown of SKIV2L in established human HCC tumors in NSG mice significantly suppressed tumor growth ( online supplemental figure 1G–I ). The clinical relevance of SKIV2L in promoting HCC tumorigenesis was further validated using patient-derived HCC samples, in which intratumoral injection of lentivirus expressing SKIV2L shRNA markedly impaired tumor growth ( online supplemental figure 1J–L ). SKIV2L is required for developing immunosuppressive TME of HCC To examine the potential roles of SKIV2L in developing TME of HCC, we tested the impact of SKIV2L knockdown on the tumorigenesis of mouse HCC cells in both immunodeficient and immunocompetent syngeneic mice. SKIV2L knockdown impaired the proliferation of the murine HCC H22 cell line in vitro ( online supplemental figure 2A–C ). While SKIV2L knockdown in H22 cells did not significantly affect tumor growth in immunodeficient mice ( online supplemental figure 2D,E ), it markedly suppressed tumor growth in immunocompetent syngeneic mice in a subcutaneous tumor model. These findings suggest that SKIV2L expression in HCC cells is required to establish an immunosuppressive TME that enables tumor cells to evade anti-tumor immunity in immunocompetent hosts ( online supplemental figure 2F,G ). Consistent with this interpretation, SKIV2L knockdown in HCC cells disrupted the TME of tumors formed in syngeneic immunocompetent mice in the subcutaneous model, as evidenced by a reduction in intratumoral regulatory T (Treg) cells, an increase in CD8 + IFN-γ + T cells, and a shift in tumor-associated macrophages from an M2-like to an M1-like phenotype ( online supplemental figure 2H–L ). To further examine the roles of SKIV2L in maintaining TME of HCC, we used the inducible knockdown system to determine the impact of SKIV2L depletion on the growth and TME of already established H22 tumors in syngeneic orthotopic HCC mouse model. Inducible SKIV2L knockdown significantly suppressed HCC growth ( figure 2A–D ) and remodeled the TME, including a reduction of intratumoral Tregs, exhausted T cells, and M2-type macrophages, along with an increase in intratumoral CD8 + T cells and M1-type macrophages ( figure 2E–I ). In line with the conclusion that SKIV2L drives an immunosuppressive TME, inducible SKIV2L knockdown in the subcutaneous tumor model similarly led to decreased intratumoral Tregs and exhausted T cells and increased CD8 + IFN-γ + T cells and MHC-II + dendritic cells (DCs) ( online supplemental figure 3A–H ). Conversely, SKIV2L overexpression in HCC cells enhanced the immunosuppressive TME in syngeneic mice by increasing Treg and M2 macrophage frequencies while reducing CD8 + IFN-γ + T cells and activated DCs ( online supplemental figure 2M–R ). Imaging mass cytometry analysis further demonstrated that SKIV2L depletion promoted CD8 + T-cell infiltration into the tumor core ( figure 2J–L ), accompanied by a locally elevated concentration of GZMB. This region is characterized by high CD44 expression, and the extent of immune cell infiltration in this area is a key prognostic indicator for immunotherapy response, suggesting a potential mechanistic link. 32 33 Figure 2. SKIV2L is required to maintain immune suppressive TME of HCC in H22 orthotopic HCC model. ( A–H ) Doxycycline (Doxy)-inducible knockdown of SKIV2L in an orthotopic HCC model established with H22 cells in immunocompetent BALB/c mice significantly suppressed tumor growth. ( A ) Representative images of livers harvested from each group. ( B ) Liver weight to body weight ratio. n=5; data are presented as mean±SD; unpaired t-test. ( C ) Body weight was monitored every other day following daily intraperitoneal administration of Doxy. n=5; two-way ANOVA with Tukey’s multiple comparisons test. ( D ) Representative liver H&E images. Scale bar, 2 mm. ( E ) Inducible SKIV2L knockdown altered the TME. Shown are the percentages of: ( E ) CD4 + CD25 + FOXP3 + T cells, ( F ) CD8 + T cells, ( G ) CD8 + PD-1 + exhausted T cells, ( H ) M1-type macrophages, and ( I ) M2-type macrophages. n=5; data are presented as mean±SD; unpaired t-test. ( J ) Representative imaging mass cytometry (IMC) images from the H22 HCC model with inducible SKIV2L knockdown or scramble control. Quantification of intratumoral CD8 + ( K ) and GZMB + ( L ) cells in tumor tissue. Arrowheads indicate the GZMB + cells. n=5 for iSC+Doxy group, n=4 for iKD+Doxy group; data are presented as mean±SD; unpaired t-test. ( M–P ) Single-cell RNA-seq analysis ( GSE151530 ) of immune cell composition in SKIV2L high-expression versus low-expression groups. ( P ) Pathway signatures of T-cell differential gene expression between SKIV2L-high and SKIV2L-low groups ( GSE151530 ). ANOVA, analysis of variance; CAFs, cancer-associated fibroblasts; HCC, hepatocellular carcinoma; iKD, inducible knockdown; iSC, inducible scramble control; TAMs, tumor-associated macrophages; TECs, tumor-associated endothelial cells; TME, tumor microenvironment. Open in a new tab To confirm the critical role of SKIV2L in shaping the TME of HCC, we repeated our experiments using another murine HCC cell line Hepa1–6 in syngeneic C57BL/6 mice. SKIV2L knockdown markedly inhibited HCC (Hepa1–6) proliferation in vitro and suppressed their tumor‐forming capacity in immunodeficient hosts ( online supplemental figure 4A–F ). In immunocompetent mice, SKIV2L depletion likewise remodeled the TME of Hepa1–6–derived tumors ( online supplemental figure 4G–N ). Global transcriptome profiling further demonstrated that SKIV2L silencing activates antitumor immune pathways within HCC ( online supplemental figure 4O ). To rule out the potential confounding effects of tumor burden, we analyzed TME when the volume of SKIV2L-silenced and control tumors was comparable. Our findings confirm that SKIV2L knockdown specifically reduced the frequencies of Tregs and exhausted T cells while increasing the proportion of CD8 + IFN-γ + T cells ( online supplemental figure 5A–F ). Collectively, these data establish that SKIV2L is indispensable for the development and maintenance of an immunosuppressive TME in HCC. To validate these findings in human HCC, we interrogated the scRNA-seq data from HCC patients ( GSE229772 ) by selecting the top 10 and bottom 10 samples based on SKIV2L expression. The SKIV2L-low group exhibited a higher proportion of intratumoral CD8 + T cells and a lower proportion of M2 macrophages compared with the SKIV2L-high group ( online supplemental figure 5G ). Using AddModuleScore, we found that SKIV2L-low tumors had a significantly higher effector-to-exhaustion score ratio ( online supplemental figure 5H ). Analysis of an independent scRNA-seq dataset ( GSE151530 ) similarly revealed enhanced T-cell infiltration and heightened immune activation in SKIV2L-low tumors ( figure 2M,N ). Expression of immune activation markers (such as CD69, 4–1BB, IFN-γ, and TNF-α) was significantly increased in the SKIV2L low-expression group, whereas inhibitory markers were markedly reduced ( figure 2O ). Moreover, immune activation pathways were significantly upregulated in SKIV2L-low tumors ( figure 2P ). These findings further support a central role for SKIV2L in promoting an immunosuppressive TME in HCC. Depletion of SKIV2L in established tumors activates systemic tumor immunity and CAR T cells To evaluate whether knockdown of SKIV2L in established tumors can elicit systemic antitumor immune responses, we implanted wild-type (WT) mouse HCC (H22) cells on one flank of BALB/c mice and inducible scramble control or SKIV2L-inducible knockdown H22 cells on the contralateral flank. Following doxycycline administration in the SKIV2L-inducible knockdown group, we observed significant growth inhibition not only of the primary SKIV2L-depleted tumors but also of the distant WT tumors ( online supplemental figure 6A,B ). Importantly, the distal WT tumors exhibited a reversed TME, characterized by reduced intratumoral Tregs, exhausted T cells, and M2 macrophages, together with increased frequencies of intratumoral IFN-γ + T cells, CD11c + MHCII + DCs, and M1 macrophages ( online supplemental figure 6C–H ). Consistent immune alterations were detected in the peripheral blood 3 days after inducible SKIV2L knockdown via lentiviral injection, including a reduction in Tregs and CD8 + PD-1 + T cells, accompanied by an expansion of effector T cells ( online supplemental figure 6I–K ). Collectively, these data demonstrate that silencing SKIV2L expression in HCC reverses TME and activates systemic antitumor immune responses. We hypothesized that SKIV2L knockdown in HCC could enhance the therapeutic efficacy of CAR T cells. Supporting this hypothesis, anti-EphA2 CAR-T cells (structure shown in online supplemental figure 7A ) co-cultured with EphA2 + scramble control or SKIV2L-knockdown HCC cells revealed that SKIV2L depletion in HCC cells markedly enhanced CAR-T cell activation, as indicated by increased frequencies of CD8 + CD44 + T cells, CD8 + IFN-γ + T cells, and CD8 + TNF-α + T cells ( figure 3A–C ). The expression level of EphA2 in H22 cells was confirmed by flow cytometry ( figure 3D ). Furthermore, the depletion of SKIV2L in HCC tumors established in syngeneic mice significantly improved the in vivo antitumor efficacy of CAR T cells ( figure 3E–G ). SKIV2L knockdown remodeled the tumor immune landscape, resulting in increased infiltration of CD3 + IFN-γ + and CD3 + TNF-α + T cells and a decreased population of CD3 + TIM-3 + exhausted T cells ( figure 3H–K ). To further characterize the activation status of intratumoral CAR T cells, we analyzed G4S-positive CAR T cells and found elevated levels of CD3 + G4S + IFN-γ + and CD3 + G4S + TNF-α + CAR-T cells in SKIV2L-knockdown tumors, accompanied by reduced proportions of CD3 + G4S + PD-1 + and CD3 + G4S + TIM-3 + CAR T cells ( figure 3L–P ). The clinical relevance of these findings was supported by the observation that tumor samples from patients resistant to immunotherapy exhibited higher SKIV2L expression ( figure 1C ), enrichment of mRNA modification and helicase activity signatures ( figure 3Q ), and a more pronounced immunosuppressive TME, including negative regulation of cytokine-mediated signaling, reduced T-cell and natural killer (NK) cell-mediated cytotoxicity, and downregulation of antigen-processing and antigen-presentation pathways ( figure 3Q ). Figure 3. Knockdown of SKIV2L enhanced the anti-tumor efficacy of CAR-T cells in established HCC tumor. ( A–C ) EphA2 CAR-T cells were cocultured with H22 with or without SKIV2L knockdown. The percentage of CD8 + CD44 + T cells ( A ), CD8 + IFNγ + T cells ( B ), CD8 + TNFα + T cells ( C ) was measured by flow cytometry analysis. n=3. Data are presented as mean value±SD. Unpaired t-test. ( D ) EphA2 expression was detected on the surface of H22 cells by flow cytometry. ( E, F ) Knockdown of SKIV2L in established H22 tumors was performed by intratumoral injection of SKIV2L shRNA lentivirus, followed by the transfusion of CAR-T cells. ( E ) Tumor weight at endpoint (representative tumor images shown on the left). n=8. Data are presented as mean±SD. Unpaired t-test. ( F ) Tumor volume was measured once every 2 days. n=8. Data are presented as mean value±SD. Statistical analysis was conducted using two-way ANOVA, followed by Tukey’s multiple comparisons test. ( G ) The SKIV2L mRNA levels in tumors after various treatments were analyzed with qPCR assay. n=4. Data are presented as mean value ±SD. ( H–K ) Flow-cytometric quantification of CD3 + IFN-γ + ( H ), CD3 + TNF-α + T cells ( I ), CD3 + PD1 + T cells ( J ), and CD3 + TIM3 + T cells ( K ), infiltrating H22 tumors with or without SKIV2L knockdown, assessed 2 days after administration of EphA2-targeted CAR-T cells. ( L ) CD3 + G4S + CAR-T cells were quantified in tumor tissues at 2 days postinfusion. n=3. One-way ANOVA with Tukey’s multiple comparisons test. ( M–P ) Flow cytometry was employed to determine the percentages of CD3 + G4S + IFN-γ + T ( M ), CD3 + G4S + TNF-α + T ( N ), CD3 + G4S + PD1 + T ( O ), and CD3 + G4S + TIM-3 + T ( P ) cells in tumor tissues at 2 days post-infusion. n=3. Data are presented as mean value±SD. Unpaired t-test. ( Q ) Gene pathway enrichment of differentially expressed genes from HCC bulk RNA-seq in anti-PD-1 plus Lenvatinib responders vs non-responders ( GSE235863 ). ANOVA, analysis of variance; HCC, hepatocellular carcinoma; KD, knockdown; PBS, phosphate buffered saline; qPCR, quantitative PCR; SC, scramble control. Open in a new tab Consistent with the observation that SKIV2L depletion in HCC enhances CAR T cell efficacy, SKIV2L knockdown in murine lung tumors established in syngeneic mice also markedly improved CAR T cell-mediated tumor control ( online supplemental figure 7B–D ). SKIV2L depletion reduced expression of exhaustion markers TIM-3 and PD-1 while increasing IFN-γ expression ( online supplemental figure 7E–H ). Therefore, SKIV2L depletion could be a common strategy to treat solid tumors by augmenting the antitumor efficacy of CAR T cells by alleviating the immunosuppressive TME. SKIV2L reprograms arginine metabolism of HCC Given the crucial role of tumor metabolic reprogramming in shaping the TME, we employed global gene expression profiling and metabolomics to elucidate the mechanisms by which SKIV2L promotes an immunosuppressive TME in HCC. Differentially expressed genes on SKIV2L knockdown were significantly enriched in metabolic pathways ( figure 4A ). Metabolomics analysis further revealed that amino acid metabolism, particularly arginine metabolism, was profoundly altered in HCC samples following SKIV2L knockdown ( figure 4B,C ). 34 Figure 4. SKIV2L promotes the arginine metabolism of HCC. ( A ) KEGG analysis of differentially expressed genes (DEGs) in human HCCs (Huh7) before and after SKIV2L knockdown using hallmark gene set collection. ( B, C ) Metabolomics analysis of human HCCs (Huh7) before and after SKIV2L knockdown revealed significantly altered arginine metabolism. ( D ) Venn diagram showing the overlap between genes identified in the SKIV2L RIP-seq dataset, DEGs on SKIV2L knockdown, and genes involved in amino acid metabolism and transport. ( E ) Motif analysis of SKIV2L RIP-seq data with MEME-chip predicts the potential SKIV2L binding sites, the potential binding motif GATGG was enriched in the target mRNAs downregulated after SKIV2L knockdown, while the potential motif T(A)T(A)T(G)T(A)G(C) was enriched in the target mRNAs upregulated after SKIV2L knockdown. ( F ) SKIV2L mRNA targets important for arginine metabolism were confirmed by RIP assay. ( G, H ) The relative mRNA levels of SKIV2L targets involved in arginine metabolism in human HCC cells Huh7 ( G ) and mouse HCC cells H22 ( H ) with or without SKIV2L knockdown. n=3. Values represent means±SD. Unpaired t-test. ( I ) The protein levels of SLC7A1 and SLC7A2 in human HCC cells Huh7 (left panel) and mouse HCC cells H22 (right panel) with or without SKIV2L knockdown. ( J ) Correlation analysis of SKIV2L mRNA levels with SLC7A1 mRNA levels and immune cell infiltration in various types of human tumors was conducted using TCGA data. CTRL, control; HCC, hepatocellular carcinoma; KD, knockdown; OE, overexpression; RIP-seq, RNA immunoprecipitation sequencing; SC, scramble control. Open in a new tab As an RNA helicase, SKIV2L is expected to regulate the expression of its target RNAs. We therefore performed RNA immunoprecipitation sequencing (RIP-seq) in HCC cells to identify potential SKIV2L-bound transcripts. Integrating these datasets with the transcriptomic changes on SKIV2L knockdown revealed several putative RNA targets linked to amino acid metabolism and transport. Among them, SLC7A1 and SLC7A2 were downregulated after SKIV2L depletion ( figure 4D,E ). Consistent with this, RIP assays further confirmed that SKIV2L binds the mRNAs of several key genes involved in the arginine metabolic pathway, including SLC7A1 and SLC7A2, which mediate arginine transport into cells, as well as ARG2 and SLC25A29, which participate in intracellular arginine metabolism ( figure 4F–I ). The clinical relevance of these findings was supported by analysis of TCGA HCC gene expression data, which showed positive correlations between SKIV2L expression and SLC7A1, ARG2, and SLC25A29 expression ( online supplemental figure 6L ). 35 36 Moreover, SLC7A1 expression was negatively associated with the infiltration of B cells, CD8 + T cells, and DCs in most cancer types ( figure 4J ). Together, these results indicate that SKIV2L plays a central role in reprogramming arginine metabolism in HCC by enhancing arginine uptake and metabolism. To elucidate how SKIV2L regulates the mRNA levels of its target genes, we examined whether SKIV2L affects the stability of SLC7A1 and SLC7A2 mRNAs. Measurement of mRNA half-lives in HCC cells before and after SKIV2L knockdown revealed that SKIV2L depletion reduced the half-lives of SLC7A1 and SLC7A2 mRNAs ( figure 5A,B ). Thus, SKIV2L stabilizes SLC7A1 and SLC7A2 mRNAs to maintain their expression in HCC cells. This function contrasts with the canonical role of SKIV2L in mRNA decay as a component of the super-killer (SKI) complex, which targets specific mRNAs for degradation. 19 Supporting this distinction, depletion of SKI3 (TTC37) or SKI8 (WDR61), key components of the SKI complex, had minimal impact on SLC7A1 and SLC7A2 mRNA levels in HCC cells ( online supplemental figure 8 ). To explore SKI-complex-independent mechanisms of SKIV2L-mediated mRNA stabilization, we tested whether its RNA helicase activity is required by generating a catalytically inactive SKIV2L-E424Q mutant. 37 Remarkably, SKIV2L-E424Q retained the ability to stabilize SLC7A1 and SLC7A2 mRNAs and to promote tumor cell proliferation ( figure 5C,D ). To evaluate the functional consequences in vivo, we established the helicase-dead mutant of SKIV2L, SKIV2L-E421Q. 37 Expression of SKIV2L-E421Q in H22 cells increased SLC7A1 levels, promoted tumor growth, and markedly enhanced the immunosuppression levels of TME ( online supplemental figure 9 ). These findings further support the hypothesis that SKIV2L promotes tumorigenesis by stabilizing a subset of mRNA targets via mechanisms independent of the SKI complex. Consistent with this model, motif analysis of SKIV2L mRNA targets revealed distinct common binding motifs between transcripts downregulated and upregulated after SKIV2L knockdown: mRNAs downregulated on SKIV2L knockdown harbored two putative SKIV2L-binding motifs (TGCTGCTGAGAA and GATGG), whereas mRNAs upregulated after SKIV2L knockdown were enriched for T(A)T(A)T(G)T(A)G(C), likely part of the poly(A) tail ( figure 4E ). Figure 5. SKIV2L stabilizes SLC7A1 and SLC7A2 mRNA through interaction with GNL3. ( A, B ) SKIV2L depletion destabilized SLC7A1 and SLC7A2 mRNA in HCC cells. Huh7 cells with or without SKIV2L knockdown were treated with actinomycin D (5 µM). Total RNA was collected at indicated time points, and levels of SLC7A1 ( A ) and SLC7A2 ( B ) mRNA were quantified by RT-qPCR n=3. mRNA ( C ) and protein ( D ) levels of SKIV2L targets as well as colony-forming ability (right panel, D ) of Huh7 cells expressing empty vector, wild-type SKIV2L, or the helicase-dead mutant SKIV2L E424Q . n=3. One-way ANOVA, followed by Tukey’s multiple comparisons test. ( E ) The interaction between SKIV2L and GNL3 was validated with co-immunoprecipitation. Huh7 cells were harvested and immunoprecipitated with isotype IgG and SKIV2L or GNL3 primary antibodies. ( F ) SLC7A1 and SLC7A2 mRNA levels in Huh7 cells ectopically expressing empty vector or SKIV2L, with or without GNL3 knockdown. n=3. ( G ) GNL3 depletion destabilized SLC7A1 mRNA in Huh7 cells. ( H ) RIP assay confirming the binding of GNL3 to SLC7A1 mRNA. n=3. ( I ) Immunofluorescence analysis showing subcellular co-localization of SKIV2L and GNL3. ( J ) Proximity ligation assay detecting the direct interaction between SKIV2L and GNL3. ( K, L ) Subcellular distribution of GNL3 in Huh7 cells with or without SKIV2L knockdown, detected by immunofluorescence ( K ) and nuclear-cytoplasmic fractionation analysis ( L ). ANOVA, analysis of variance; EV, empty vector; HCC, hepatocellular carcinoma; KD, knockdown; OE, overexpression; qPCR, quantitative polymerase chain reaction; RIP-seq, RNA immunoprecipitation sequencing; SC, scramble control; WT, wild type. Open in a new tab SKIV2L promoted the stability of mRNA by recruiting GNL3 To further elucidate the molecular mechanism underlying SKIV2L-mediated mRNA stabilization, we performed immunoprecipitation followed by mass spectrometry and identified GNL3, known for its role in mRNA metabolism, 38 , 40 as a novel SKIV2L-interacting protein. This interaction was validated by reciprocal co-immunoprecipitation (co-IP) assays ( figure 5E ). Functionally, similar to SKIV2L knockdown, GNL3 knockdown significantly reduced SLC7A1 and SLC7A2 mRNA levels ( figure 5F ). RIP analysis demonstrated that, like SKIV2L, GNL3 interacts with SLC7A1 mRNA and stabilizes it ( figure 5G,H ). Immunofluorescence staining revealed prominent co-localization of GNL3 and SKIV2L in cells ( figure 5I ), which was further confirmed by proximity ligation assay ( figure 5I,J ). While SKIV2L did not alter GNL3 protein levels, SKIV2L knockdown significantly increased GNL3 accumulation in the nucleolus, indicating that SKIV2L regulates the subcellular distribution of GNL3 ( figure 5K,L ). To examine the involvement of GNL3 subcellular localization in regulating the stability of SLC7A1 mRNA, we expressed a nucleolar localization signal-deficient GNL3 mutant (G261V), 41 which showed increased cytoplasmic retention when compared with WT-GNL3 ( online supplemental figure 10A,B ). GNL3 G261V induced higher levels of SLC7A1 mRNA and significantly enhanced SLC7A1 mRNA stability ( online supplemental figure 10C,D ). Together, these findings support a model in which SKIV2L retains GNL3 in the cytoplasm through protein–protein interaction, thereby enabling GNL3-dependent stabilization of SKIV2L target mRNAs. SKIV2L drives tumorigenesis of HCC by increasing its arginine uptake Accumulating evidence indicates that arginine is essential for both tumor growth and T-cell activation, and that tumor cells can suppress T-cell function by competing for and depleting arginine within the TME. 17 18 Because SKIV2L increases the mRNA levels of proteins required for arginine transport and metabolism, we hypothesized that SKIV2L drives HCC tumorigenesis by promoting arginine uptake and metabolism. In support of this notion, when human HCC cells were co-cultured with human T cells, SKIV2L knockdown in HCC cells resulted in a significant reduction of arginine levels in HCC cells ( online supplemental figure 11A ), while concurrently increasing the arginine levels in co-cultured T cells ( online supplemental figure 11B ). In addition, the overexpression of SKIV2L in human HCC cells increased the arginine concentration in HCC cells and decreased the arginine levels in co-cultured T cells ( online supplemental figure 11C,D ). Consistent findings were obtained when EphA2 + HCC cells with or without SKIV2L knockdown were co-cultured with CAR-T cells ( online supplemental figure 11E,F ). Additionally, SKIV2L overexpression in HCC cells reduced arginine concentration in the co-culture supernatant ( online supplemental figure 11G ). The role of the SKIV2L–SLC7A1–arginine axis in enabling HCC cells to outcompete immune cells for arginine was further corroborated in mouse HCC cells ( online supplemental figure 11H–K ). To determine the importance of SKIV2L in reprogramming arginine metabolism of HCC, we restored the expression of SLC7A1 in human HCC cells after SKIV2L knockdown and showed that this manipulation rescued their proliferation defects in vitro and tumor growth in immunodeficient mice ( figure 6A–D ). Figure 6. SKIV2L drives tumorigenesis of HCC by increasing arginine uptake. ( A ) Restoration of the expression levels of SLC7A1 mRNA in HCC cells after SKIV2L knockdown was confirmed by qPCR. n=3. Values represent the mean±SD. One-way ANOVA, followed by Tukey’s multiple comparisons test. ( B ) The restoration of SLC7A1 expression in HCC cells after SKIV2L knockdown rescued their proliferation defects. n=4. Values represent means±SD, two-way ANOVA, followed by Tukey’s multiple comparisons test. The restoration of SLC7A1 expression in HCC cells after SKIV2L knockdown rescued their tumor weight ( C ) and growth curve ( D ) in immunodeficient NSG mice. The tumors were recovered and weighed ( C ). n=8. One-way ANOVA with Tukey’s multiple comparisons test. ( E ) The percentage of Annexin V + apoptotic H22 cells with or without SKIV2L knockdown when co-cultured with CD3/CD28 beads-activated CD3 + T cells was analyzed by flow cytometry. n=3. Values represent the mean±SD. Unpaired t-test. ( F, G ) The percentage of CD8 + IFN-γ + T cells in CD8 + T cells when co-cultured with SKIV2L knockdown and control H22 cells ( F ), and with or without SKIV2L overexpression ( G ). n=3. Values represent the mean±SD. Unpaired t-test. ( H ) The percentage of CD8 + IFN-γ + T cells in CD8 + T cells when co-cultured with SKIV2L knockdown and control H22 cells with or without SLC7A1 overexpression. n=3. Values represent the mean±SD. One-way ANOVA with Tukey’s multiple comparisons test. ( I, J ) The restoration of SLC7A1 expression in HCC formed by SKIV2L knockdown H22 cells in syngeneic mice rescued tumor growth. The tumor volume was measured every other day ( I ). n=10. Two-way ANOVA, followed by Tukey’s multiple comparisons test. The tumors were recovered and weighed ( J ). n=10. One-way ANOVA with Tukey’s multiple comparisons test. Values represent the mean±SD. ( K ) The percentage of CD8 + IFN-γ + T cells in CD8 + T cells in tumor tissues of various groups was measured by flow cytometry. n=5. ( L ) Relative arginine levels in interstitial fluid of tumors from the indicated groups. n=3. ( M ) The percentage of CD8 + IFN-γ + T cells in CD8 + T cells co-cultured with or without SKIV2L overexpression in the presence of high arginine concentration. n=3. Values represent the mean±SD. One-way ANOVA with Tukey’s multiple comparisons test. ANOVA, analysis of variance; E:T, Effector cell to Target cell ratio; EV, empty vector; HCC, hepatocellular carcinoma; OE, overexpression; qPCR, quantitative polymerase chain reaction; SC, scramble control. Open in a new tab While SKIV2L knockdown impaired the proliferation of most HCC cell lines analyzed, including mouse Hepa1–6 cells, it had no apparent effect on the growth of H22-derived tumors in immunodeficient mice. To explain this discrepancy, we examined expression of ASS1, a key enzyme in arginine biosynthesis. 42 ASS1 levels were substantially higher in H22 cells than in Hepa1–6 cells ( online supplemental figure 12A ), suggesting that H22 cells may compensate for reduced arginine uptake on SKIV2L knockdown by increasing endogenous arginine synthesis. In support of this, whereas intracellular arginine levels were reduced in both H22 and Hepa1–6 cells after SKIV2L knockdown, arginine levels in SKIV2L-depleted H22 cells remained comparable to those in Hepa1–6 cells before knockdown ( online supplemental figure 12B ), indicating a compensatory biosynthetic pathway. SKIV2L knockdown did not affect ASS1 mRNA levels in H22 cells ( online supplemental figure 12C ). Analysis of scRNA-seq data from human HCC tissues showed a mutually exclusive expression pattern of ASS1 and SLC7A1 ( online supplemental figure 12D ). Importantly, dual knockdown of SKIV2L and ASS1 in H22 cells abolished this compensatory mechanism and significantly suppressed tumor growth in immunodeficient mice ( online supplemental figure 12E–G ). These findings indicate that the dependence of HCC cells on SKIV2L for proliferation is determined by their capacity for endogenous arginine synthesis to compensate for reduced arginine uptake. SKIV2L expressed in tumor cells suppresses tumor immunity To investigate the role of SKIV2L in establishing the immunosuppressive TME of HCC, we examined the impact of SKIV2L knockdown in HCC cells on co-cultured T cells. SKIV2L knockdown in mouse HCC cells increased the cytotoxic activity of syngeneic T cells, leading to a marked reduction in viable HCC cells in co-culture ( figure 6E,F and online supplemental figure 13A,B ). Similarly, SKIV2L knockdown in human HCC cells enhanced activation of allogeneic human T cells, as evidenced by increased frequencies of IFN-γ + T cells ( online supplemental figure 13F,G ). In contrast, SKIV2L overexpression in HCC cells suppressed T-cell activation in co-culture ( figure 6G , online supplemental figures 11H and 13C ). Considering the importance of arginine in T cell activation, these data suggest that SKIV2L could suppress T cell function by increasing the uptake of arginine by HCC and depleting arginine in the environment required for T cell activation. Supporting this model, restoration of SLC7A1 expression in HCC cells after SKIV2L knockdown reversed T-cell activation in co-culture ( figure 6H , online supplemental figure 13D,I ). These findings indicate that the SKIV2L–SLC7A1–arginine pathway in HCC cells plays a central role in suppressing T-cell function. To further delineate the physiological roles of SKIV2L in driving an immunosuppressive TME in vivo, we restored SLC7A1 expression in mouse HCC cells after SKIV2L knockdown and implanted these cells into syngeneic mice ( figure 6I,J ). The restoration of SLC7A1 expression in SKIV2L knockdown tumors re-established the immunosuppressive TME, as evidenced by reduced infiltration of CD8 + IFN-γ + T cells and diminished interstitial arginine accumulation ( figure 6K,L ). To directly test the impact of SKIV2L modulation on T-cell–mediated anti-tumor immunity, we administered anti-EphA2 CAR-T cells into nude mice bearing EphA2 + tumors formed by H22 cells with mocked treatment, SKIV2L knockdown, or SKIV2L knockdown with SLC7A1 restoration. When compared with control H22 tumors, SKIV2L knockdown markedly enhanced antitumor activity of CAR T cells, whereas SLC7A1 reconstitution abrogated this beneficial anti-tumor effect ( online supplemental figure 14A,B ). Arginine administration to H22 tumor-bearing mice increased arginine concentration in the tumor interstitial fluid and boosted T-cell immunity ( online supplemental figure 14C–G ). While the overexpression of SKIV2L in HCC cells suppressed the co-cultured T cells, the increased concentrations of arginine in the co-culture medium rescued the defective T cell activation ( figure 6M , online supplemental figure 10J and 13E ). Together, these results establish that SKIV2L expression in HCC cells is critical for establishing an immunosuppressive TME by driving arginine metabolism. We further performed tumor cell–T-cell co-culture experiments to validate the importance of the SKIV2L–GNL3 axis in modulating arginine metabolism and antitumor immunity. Overexpression of GNL3 in Huh7 cells suppressed co-cultured T cells and reduced intracellular arginine levels in these T cells ( online supplemental figure 15A–C ). Importantly, this inhibitory effect was dependent on SKIV2L, as it was abolished by SKIV2L knockdown ( online supplemental figure 15D ). These data indicate that the SKIV2L-GNL3 axis drives immunosuppression of TME. SKIV2L-arginine pathway is a common mechanism to develop TME We subsequently examined the SKIV2L-arginine pathway across various cancer types. No significant differences in tumor growth were observed in Mc38 and 4T1 xenografts following SKIV2L knockdown in nude mice ( online supplemental figure 16A–C and F–H ). However, SKIV2L knockdown in tumor cells impaired the growth of tumors formed by Mc38 and 4T1 tumors in syngeneic mouse models ( online supplemental figure 16D,E,I,J ), accompanied by a reduced arginine uptake and activation of anti-tumor immune responses ( online supplemental figure 16K,L ). Analysis of the TCGA database further revealed a correlation between SKIV2L expression with increased SLC7A1 levels and reduced immune infiltration, a pattern consistent across a broad spectrum of cancer types ( figure 4J ). c-Myc activates the transcription of SKIV2L To identify the oncogenic pathway responsible for SKIV2L overexpression in HCC, we screened transcription factors predicted to bind the SKIV2L promoter across multiple tumor cell types using the ENCODE database ( online supplemental figure 17A ). This analysis revealed enrichment of c-Myc, TAF1, and GABPA binding within CpG islands of the SKIV2L promoter ( figure 7A , online supplemental figure 17A ). JASPAR transcription factor motif analysis further supported potential c-Myc binding to the SKIV2L promoter region ( online supplemental figure 17B ). By silencing c-Myc, TAF1, or GABPA expression in HCC cells, we found that c-Myc is responsible for activating SKIV2L expression ( figure 7B,C and online supplemental figure 17C,D ). Consistently, dual-luciferase reporter assays confirmed that c-Myc directly regulates the transcription of SKIV2L mRNA ( figure 7D ). The clinical relevance of this pathway was supported by positive correlations among c-Myc, SKIV2L, and SLC7A1 mRNA levels in HCC patient samples ( figure 7E–G ). The increase of SLC7A1 mRNA and arginine uptake induced by c-Myc overexpression was reversed by SKIV2L knockdown, indicating that c-Myc regulates arginine metabolism through SKIV2L ( figure 7H,I ). Consistently, the decreased arginine uptake by co-cultured T cells induced by c-Myc overexpression in Huh7 cells was reversed by SKIV2L knockdown ( figure 7J,K , and online supplemental figure 17E ). These findings identify a novel oncogenic c-Myc/SKIV2L/arginine metabolism axis that plays a key role in developing TME ( figure 7L ). Figure 7. SKIV2L is a direct transcriptional target of c-Myc. ( A ) The Integrated Genome Viewer (IGV) browser displayed the c-Myc binding site within the SKIV2L promoter region by analyzing ChIP-seq data of c-Myc in human cancer cell lines of the ENCODE database. ( B ) The protein levels of c-Myc, SKIV2L, and SLC7A1 before and after c-Myc knockdown in HCC cells. ( C ) The relative mRNA levels of c-Myc and SKIV2L in HCC cells before and after c-Myc knockdown. n=3. Data are presented as mean value±SD. Unpaired t-test. ( D ) Luciferase reporter assay confirms c-Myc as a transcriptional regulator of SKIV2L. n=3. Values represent the mean±SD. Unpaired t-test. ( E–G ) Correlation analysis of mRNA levels of c-Myc and SKIV2L ( E ), c-Myc and SLC7A1 ( F ), and SKIV2L and SLC7A1 ( G ) using data from the TCGA database. Two-tailed Pearson correlation test. P value and Pearson’s correlation coefficient R are indicated. ( H ) Western blot analysis of c-Myc, SKIV2L, and SLC7A1 expression in Huh7 cells expressing empty vector or c-Myc, with or without SKIV2L knockdown. ( I, J ) Huh7 cells expressing empty vector or c-Myc, with or without SKIV2L knockdown were co-cultured with human T cells. Relative intracellular arginine concentrations in Huh7 cells ( I ) and in co-cultured human T cells ( J ) are shown. n=3. ( K ) Percentage of CD8 + IFN-γ + cells in intratumoral CD8 + T cells of indicated groups. n=3. One-way ANOVA with Tukey’s multiple comparisons test. Values represent the mean±SD. ( L ) Schematic diagram of SKIV2L target genes in arginine metabolic pathway was created with BioRender tool ( https://biorender.com/wuqm1w9 ). ANOVA, analysis of variance; EV, empty vector; HCC, hepatocellular carcinoma; KD, knockdown; OE, overexpression; SC, scramble control. Open in a new tab Discussion Accumulating evidence indicates that tumor cells compete with T cells for arginine uptake and suppress antitumor T-cell activation and effector functions by depleting arginine within the TME. 17 43 In this study, we identify the RNA helicase SKIV2L as a key regulator of arginine metabolism and an important driver of TME in solid tumors. In HCC, SKIV2L increases the expression of the arginine transporters SLC7A1 and SLC7A2 by stabilizing their mRNAs, thereby enabling HCC cells to more effectively compete for arginine in the TME and suppress intratumoral T-cell activation. Inducible downregulation of SKIV2L suppresses HCC cell proliferation, activates systemic antitumor immune responses, and markedly enhances CAR T cell function. In addition, the mRNA targets of SKIV2L include other critical components of the arginine metabolic pathway, such as ARG2 and SLC25A29. Together, these findings define SKIV2L as a master regulator orchestrating arginine metabolic reprogramming in solid tumors. Previous studies have established SKIV2L as a core component of the SKI2–Exo complex, which functions in the cytoplasm to mediate RNA degradation. 19 44 45 Our work reveals a previously unrecognized role of SKIV2L in RNA metabolism by binding to specific mRNAs and protecting them from degradation. Supporting this notion, the putative SKIV2L-binding motifs of mRNA targets whose stability is increased or decreased on SKIV2L knockdown are distinct, suggesting that SKIV2L participates in multiple protein complexes with opposing effects on mRNA metabolism. Moreover, we identified GNL3, a known RNA-binding protein, as a novel SKIV2L-interacting partner. GNL3 knockdown significantly attenuated the upregulation of SLC7A1 and SLC7A2 induced by SKIV2L overexpression. Importantly, a cytoplasmically enriched GNL3 mutant potently stabilized SLC7A1 mRNA, highlighting a key cytoplasmic role of GNL3 in regulating mRNA stability. Collectively, these results reveal the mechanism that SKIV2L stabilizes target mRNA by sequestering GNL3 in the cytoplasm. Using in vitro and in vivo models, we demonstrate that SKIV2L-dependent arginine metabolic reprogramming fuels robust immune suppression within the TME. In this context, depletion of SKIV2L in solid tumors disrupts TME, activates systemic antitumor immunity, and significantly improves the capacity of CAR T cells to eliminate solid tumors. Given the differences between subcutaneous and orthotopic models, 46 we employed both subcutaneous and orthotopic HCC models to confirm the roles of SKIV2L in TME of HCC. Our study also demonstrates that c-Myc/SKIV2L axis mediates the c-Myc-dependent reprogramming of tumor metabolism and establishment of TME. In summary, our findings demonstrate that SKIV2L is a promising therapeutic target for treating solid tumors by disrupting multiple oncogenic pathways important for tumor growth and TME. Supplementary material online supplemental table 1 jitc-14-4-s001.pdf (190.2KB, pdf) DOI: 10.1136/jitc-2025-014192 online supplemental table 2 jitc-14-4-s002.pdf (237.4KB, pdf) DOI: 10.1136/jitc-2025-014192 online supplemental table 3 jitc-14-4-s003.pdf (132.8KB, pdf) DOI: 10.1136/jitc-2025-014192 online supplemental file 1 jitc-14-4-s004.pdf (6.4MB, pdf) DOI: 10.1136/jitc-2025-014192 Footnotes Funding: This work was supported by National Natural Science Foundation of China (No. 82230102, 82472638), the National Key Research and Development Program of China (2022YFC3401600), Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2022R01002), Key Research and Development Program of Zhejiang Province (2022C03006), and the Fundamental Research Funds for the Central Universities (No. 2021FZZX001-42). Provenance and peer review: Not commissioned; externally peer reviewed. Patient consent for publication: Not applicable. Ethics approval: All animal experiments were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (2024-329). Human HCC specimens and matched adjacent non-tumor tissues were obtained from Nanfang Hospital with approval from its Institutional Review Board (NFEC-2018-004). Comprehensive clinical characteristics of these cohorts were previously reported. Data availability free text: All figures and tables including figures1 7 ; online supplemental figures S1–S17 ; onlinesupplemental tables 1 3 . Data availability statement Data are available in a public, open access repository. Data are available on reasonable request. References 1. Zhang J, Hu Y, Yang J, et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature New Biol. 2022;609:369–74. doi: 10.1038/s41586-022-05140-y. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. Hu Y, Feng J, Gu T, et al. 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Supplementary Materials online supplemental table 1 jitc-14-4-s001.pdf (190.2KB, pdf) DOI: 10.1136/jitc-2025-014192 online supplemental table 2 jitc-14-4-s002.pdf (237.4KB, pdf) DOI: 10.1136/jitc-2025-014192 online supplemental table 3 jitc-14-4-s003.pdf (132.8KB, pdf) DOI: 10.1136/jitc-2025-014192 online supplemental file 1 jitc-14-4-s004.pdf (6.4MB, pdf) DOI: 10.1136/jitc-2025-014192 Data Availability Statement Data are available in a public, open access repository. Data are available on reasonable request. 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