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Inhibition of CCT5-mediated asparagine biosynthesis and anti-PD-L1 produce synergistic antitumor effects in colorectal cancer
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Yujie Zhanga, b, c, Weiyi Zhaoa, d, Ling Wua, b, c, Tianjing Aib, Jie Heb, Zetao Chene, Chuangyuan Wange, Hui Wangf, Rui Zhoub, Chaoqun Liua, Liang Zhaoa, b, c, *
Acta Pharmaceutica Sinica B | 2025, 15(5) : 2480 - 2497
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Acta Pharmaceutica Sinica B | 2025, 15(5): 2480-2497
• ORIGINAL ARTICLES •
Inhibition of CCT5-mediated asparagine biosynthesis and anti-PD-L1 produce synergistic antitumor effects in colorectal cancer
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Yujie Zhanga, b, c, Weiyi Zhaoa, d, Ling Wua, b, c, Tianjing Aib, Jie Heb, Zetao Chene, Chuangyuan Wange, Hui Wangf, Rui Zhoub, Chaoqun Liua, Liang Zhaoa, b, c, *
Affiliations
  • aDepartment of Pathology, Nanfang Hospital, Southern Medical University Guangzhou 510515, China
  • bDepartment of Pathology & Guangdong Province Key Laboratory of Molecular Tumor Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
  • cDepartment of Pathology, Shunde Hospital, Southern Medical University (the First People's Hospital of Shunde), Foshan 528399, China
  • dDepartment of Pathology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310009, China
  • eDepartment of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510260, China
  • fDepartment of Medical Oncology, Affiliated Tumour Hospital of Guangzhou Medical University, Guangzhou 510095, China
About Author:

E-mail address: (Liang Zhao).

†

These authors made equal contributions to this work.

Author contributions

Liang Zhao: Conceptualization, Supervision, Project administration, and Funding acquisition. Yujie Zhang: Conceptualization, Methodology, Investigation, Data Curation, Formal analysis, Writing - Original Draft, Writing - Review & Editing, Visualization. Weiyi Zhao: Investigation, Validation, Writing - Original Draft. Ling Wu: Formal analysis, Investigation, Writing - Review & Editing. Tianjing Ai: Validation, Investigation. Jie He: Validation. Zetao Chen: Methodology, Resources. Chuangyuan Wang: Software. Hui Wang: Funding acquisition. Rui Zhou: Funding acquisition. Chaoqun Liu: Funding acquisition.

doi: 10.1016/j.apsb.2025.03.026
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Abnormal amino acid metabolism promotes tumor progression by inducing malignant behaviors in tumor cells and altering the immune landscape within the tumor microenvironment. However, the underlying mechanisms remain unclear. In this study, we constructed colorectal cancer (CRC) organoids and patient-derived tumor xenograft (PDX) models, performing multifaceted validation to confirm that T-complex protein 1 subunit epsilon (CCT5), mediates the biosynthesis of aspartate and enhances sensitivity to anti-PD-L1 immunotherapy. Mechanistically, CCT5 directly binds to asparagine synthetase (ASNS) and promotes the synthesis of aspartate (Asn). The Asn–mTORC1 axis facilitates tumor cell proliferation while upregulating PD-L1 expression, which leads to a reduction in the number of effector CD8+ T cells. Treatment with l-asparaginase (ASNase) combined with anti-PD-L1 therapy effectively reverses the growth of CRC characterized by high CCT5 expression. In summary, we identify CCT5 as a potential biomarker to guide the combined use of ASNase and anti-PD-L1 antibodies in CRC treatment.

Colorectal cancer  /  CCT5  /  Asparagine metabolism  /  ASNase  /  Anti-PD-L1 immunotherapy  /  Combination therapy  /  CD8+ T cell  /  ASNS
Yujie Zhang, Weiyi Zhao, Ling Wu, Tianjing Ai, Jie He, Zetao Chen, Chuangyuan Wang, Hui Wang, Rui Zhou, Chaoqun Liu, Liang Zhao. Inhibition of CCT5-mediated asparagine biosynthesis and anti-PD-L1 produce synergistic antitumor effects in colorectal cancer[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (5) : 2480 -2497 . DOI: 10.1016/j.apsb.2025.03.026
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1.　Introduction
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Colorectal cancer (CRC) ranks third in prevalence and second in mortality among all types of cancer1. Over the past decade, the focus of CRC treatment has shifted from systemic radiotherapy and chemotherapy to precise therapies targeting molecular drivers and immunotherapy aimed at the protective tumor microenvironment (TME)2. However, due to the high molecular heterogeneity of CRC, many patients do not benefit from advanced clinical treatments. Clinical trials have shown that individualized treatment based on tumor molecular and pathological characteristics can improve overall survival rates3. Therefore, in addition to exploring new drugs and therapeutic strategies, it is urgent to develop new molecular biomarkers that can stratify CRC patients for precise treatment based on their unique biological characteristics.
Chaperone proteins were initially identified for their role in folding and preventing protein aggregation under stress conditions. Recent research has revealed their significant association with various aspects of tumorigenesis4, progression5, therapy resistance6, and immune tolerance induction7 across multiple cancer types. This highlights their potential as diagnostic and prognostic biomarkers, as well as novel therapeutic targets for cancer8,9. The T-complex protein 1-ring complex (TRiC, also known as CCT) is a Group II eukaryotic cytosolic chaperonin that consists of two sets of eight subunits (CCT1–8) forming an identical hetero-oligomeric ring10. T-complex protein 1 subunit epsilon (CCT5) is one of the subunits of TRiC. According to existing literature, CCT5 can bind to proteins and participate in multiple biological processes such as embryonic development, cell cycle regulation, and cell migration, thereby promoting the metastasis of gastric and lung cancer11,12.
Specific metabolic activities contribute to biological processes that promote tumor growth13. A recent study revealed a significant increase in various amino acid metabolites in both mouse and human CRC, suggesting a role for dysregulated amino acid metabolism14. Reprogramming of amino acid metabolism partially fulfills the heightened nutritional demands for tumor proliferation under nutrient-limited conditions15. Amino acid deprivation may be an effective strategy for tumor treatment16. In recent years, asparagine (Asn) has been implicated in tumor progression17,18. Asparagine synthetase (ASNS) catalyzed de novo Asn synthesis, which consumes glutamine and ATP, and plays a critical role in protein synthesis. L-asparaginase (ASNase), which deprives Asn by hydrolyzing it to aspartate, has been used as a first-line treatment for childhood acute lymphoblastic leukemia (ALL)19. Although ASNase has been reported to exhibit therapeutic effects in solid tumors, including ovarian cancer, pancreatic cancer, breast cancer, and CRC17,18, the absence of accurate biomarkers significantly limits its clinical application. The personalized therapeutic effect of ASNase in CRC patients remains unclear.
Reprogramming tumor amino acid metabolism not only affects tumor cell survival and proliferation but also impacts the surrounding TME20,21. Multiple studies have shown that aberrant amino acid metabolism in tumor cells can promote T cell dysfunction and exhaustion, leading to evasion of immunological surveillance and facilitating tumor progression22,23. The therapeutic potential of targeting abnormal amino acid metabolism to enhance anti-tumor immunity has been preliminarily validated in various tumor types, including CRC22,24,25.
In this study, functioning as a molecular chaperone that regulates the homeostasis of intercellular proteins, CCT5 was first found to be involved in the regulation of Asn metabolism and to mediate CD8+ T cell immunosuppression. Most importantly, we propose CCT5 as a specific biomarker and then support the application of ASNase combined therapy with programmed death-ligand 1 (PD-L1) antibody treatment in patients with CRC with high CCT5 expression.
2.　Materials and methods
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2.1.　 Cell lines and cell culture
The NCM460 cell line, derived from normal colon epithelial cells, along with various human colorectal cancer cell lines (SW480, SW620, HCT116, RKO, HCT15, LoVo, and Caco-2) and the human embryonic kidney cell line 293T, were procured from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI 1640 medium (KeyGEN BioTECH, Jiangsu, China) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Invitrogen, Paisley, UK). Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. All cell lines used in this study were negative for mycoplasma and were authenticated by short tandem repeat (STR) profiling within four years.
The final concentrations of the chemicals used for the in vitro treatment of CRC cells or CRC patient-derived tumor organoids (PDTOs) in this study are listed as follows: 10 nmol/L rapamycin (RAPA, S1039, Selleckchem, Houston, TX, USA) for CRC cells and 10 μmol/L for PDTOs; 2 UI/mL L-asparaginase (ASNase, ENZ-287, ProSpec, East Brunswick, NJ, USA); 0.1 mmol/L L-asparagine (A8381, Sigma–Aldrich, St. Louis, MO, USA); 4 mmol/L L-albizziine (Alb, A-230-250, Goldbio, MO, USA); and 50 μg/mL anti-PD-L1 antibody (atezolizumab, αPD-L1, A2004, Selleckchem, Houston, TX, USA). Transfection of cells with plasmid vectors and siRNAs was performed using Lipofectamine 3000 reagent (ThermoFisher Scientific, MA, USA), following the guidelines provided by the manufacturer's instructions.
2.2.　 Clinical tissue samples
Fresh clinical CRC tissues were obtained from the general surgery department at Shunde Hospital, Southern Medical University (Foshan, China) after informed consent was obtained from patients. Formalin-fixed paraffin-embedded tissue samples were collected from 222 cases of primary CRC patients, and the 103 CRC tissues with paired normal mucosa were obtained from the Tumor Tissue Bank of Shunde Hospital, Southern Medical University. The clinical follow-up data for these samples is all available. In each case, a diagnosis of primary CRC had been made before the elective surgery was carried out at Shunde Hospital between 2017 and 2022. None of the patients had received any preoperative chemotherapy or radiotherapy before surgery. The Ethics Committee of Shunde Hospital, Southern Medical University has approved the study (Foshan, China) (Application No: KYLS20230731), and all aspects of the study comply with the Declaration of Helsinki.
2.3.　 CRC patient-derived tumor organoids culture
PDTOs were constructed as previously described26. PDTOs were established using fresh tissue obtained from colon adenocarcinoma patients. Fresh patient tumor tissues were mechanically minced, and digested using Tumor tissue digestion solution (K601003, bioGenous, Hangzhou, China) at 37 °C for 30 min, and filtered through a 100 μm mesh. The filtered samples were then centrifuged at 300×g for 5 min (Centrifuge, 5810R, Eppendorf, Hamburg, Germany). The organoids were mixed with Matrigel (356231, Corning, NY, USA), and the mixture was seeded into 24-well plates. The culture medium (K2103-CR, bioGenous, Hangzhou, China) was added after the Matrigel solidified and was refreshed every 2 days. The organoids were passaged every 7–14 days by dissociation with an organoid dissociation solution (E238001, bioGenous, Hangzhou, China).
Gene editing of PDTOs was performed according to previously established protocols26. The viral supernatant was concentrated via high-speed centrifugation at 80,000×g for 2 h at 4 °C (Centrifuge, Optima XPN-100, Beckman, Fullerton, CA, USA). The collected viral particles were resuspended in an organoid culture medium containing 8 μg/mL polybrene (107689, Sigma–Aldrich, St. Louis, MO, USA), followed by a 4 h incubation with the organoids. The organoids were then embedded in Matrigel and incubated in an organoid culture medium supplemented with 10 μmol/L Y-27632 (S6390, Selleckchem, Houston, TX, USA). Approximately 48 h after viral infection, organoids were selected in a culture medium containing 2 μg/mL puromycin (s7417, Selleckchem, Houston, TX, USA) for seven consecutive days. The PDTOs were seeded in 96-well plates and incubated. PDTOs growth was evaluated using the CellTiter-Glo 3D cell viability assay (G9683, Promega, Madison, WI, USA) according to the manufacturer's instructions. Luminescence was quantified using a Synergy Neo2 HTS multimode microplate reader (BioTek, Winooski, VT, USA).
2.4.　 Animals, subcutaneous tumor models, PDX models, and treatment
The 3–5-week-old male BALB/c mice weighing 15–20 g were purchased from the Nanfang Medical University Experimental Animal Center (Guangzhou, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China) (Application No: NFYY-2021-0566). The gene-edited CT26 cells were resuspended in PBS and injected subcutaneously into the flanks of the hind legs on both sides of the mice.
We examined the expression of CCT5 in primary tumor tissues of different patients by WB and classified them into high and low-expression groups. The patient-derived tumor xenograft (PDX) model was established by subcutaneous inoculation of fresh CRC patient tissue fragments into 6-week-old male NSG mice, which were purchased from Guangdong Zhiyuan Biopharmaceutical Technology Co., Ltd. (Guangzhou, China). The specific build information is as previously described27. When PDX mice are passed on to the 3rd generation for dosing treatment. Start treatment when tumor volume reaches 100–200 mm3.
The mice were treated with PBS or ASNase (5 IU/g of body weight, ENZ-287, ProSpec, East Brunswick, NJ, USA) intraperitoneally (i.p.) every 3 days, and isotype IgG (A2116, Selleckchem, Houston, TX, USA) or anti-PD-L1 antibody (10 mg/kg, A2115, Selleckchem, Houston, TX, USA) every 6 days. Tumor size was measured using a Vernier caliper, and tumor volume (mm3) was calculated according to Eq. (1):
Tumor volume = Length  (Width)2/2
The tumor growth inhibitory value (TGI) was calculated according to Eq. (2)
TGI = (1 － (TVtreated Day n － TVtreated Day 0) / (TVcontrol Day n－ TVcontrol Day 0))  100%
Here, TV represents the tumor volume, treated represents the drug treatment group, and control represents the control group. n represents the final day of treatment and 0 represents the first day of treatment.
After approximately 4–6 weeks, euthanize the mice using carbon dioxide as an anesthetic. Hematoxylin and eosin (H&E) staining of the lungs shows no evidence of pulmonary hemorrhage in the mice (Supporting Information Fig. S1A). H&E staining of the liver, spleen, and kidneys also shows no signs of abnormal tissue damage (Fig. S1B). This complies with animal ethics. The subcutaneous tumor tissue was collected for subsequent histological examination.
2.5.　 Statistical analyses
Public microarray data were downloaded from The Cancer Genome Atlas (TCGA) datasets (http://cancergenome.nih.gov/) and GEO databases (http://www.ncbi.nlm.nih.gov/geo/). Gene set enrichment analysis (GSEA) was performed using GSEA 4.0.2 software (http://www.broadinstitute.org/gsea/). Figures and graphical elements in this manuscript were created and compiled using BioRender (https://www.biorender.com/) and Adobe Illustrator 2020 (Adobe, San Jose, CA, USA). The results were analyzed using SPSS statistical software (version 24.0, SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8 software (GraphPad Software, Inc., CA, USA). Statistical tests included Student's t-tests (paired or unpaired), one-way ANOVA, Pearson Correlations, Pearson's chi-squared test (χ2), and Kaplan–Meier analysis. P < 0.05 was considered statistically significant (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns not significant).
3.　Result
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3.1.　 CCT5 is upregulated in CRC predicts a poor prognosis and promotes the proliferation of PDTOs
Chaperone proteins play a crucial and irreplaceable role in tumor biology. To identify potential molecular chaperone biomarkers in CRC, we analyzed integrated gene expression profiles from five human CRC datasets obtained from the Gene Expression Omnibus (GEO) database (GSE39582, GSE73360, GSE77955, GSE83889, and GSE87211). By cross-referencing the upregulated genes in these datasets with a chaperone protein set, we identified four differentially expressed chaperone proteins: CCT5, CCT4, HSPD1, and HSP90AA1 (Fig. 1A). Notably, CCT5 was identified as the sole molecular chaperone associated with poor prognosis in CRC (Supporting Information Fig. S2A). CCT5 is a subunit of the molecular chaperone TRiC. We further analyzed the effects of other subunits of this complex on the prognosis of CRC. The results indicated that CCT5 independently contributes to poor prognosis in CRC (Fig. S2B). The expression of CCT5 was significantly upregulated in CRC tissues compared to normal colorectal mucosa (Supporting Information Fig. S3A and S3B) and in paired CRC tissues (Fig. S3C). Clinical sample analysis revealed that CCT5 is highly expressed in CRC tissues (Fig. 1B–D), and elevated CCT5 expression is positively correlated with poor disease-specific survival in the Shunde Hospital cohort (Fig. 1E). Additionally, we observed higher CCT5 expression in CRC cell lines compared to normal colonic epithelial NCM460 cells (Fig. S3D–S3F). These findings strongly suggest that high CCT5 expression is associated with a poor prognosis in CRC.
To more effectively preserve the heterogeneity of CRC, we established patient-derived tumor organoids (PDTOs) from fresh human CRC samples (Fig. 1F). The three PDTOs showed a high degree of consistency with the original tumor tissues in terms of morphological characteristics and Ki67 expression levels (Fig. 1G). The PDTOs were circular or oval with a hollow lumen, and the expression of EpCAM and Ki67 in the PDTOs indicated their epithelial characteristics and proliferative capacity (Fig. 1H). Subsequently, we performed overexpression and knockdown treatments in CRC 1# PDTOs to manipulate the expression of CCT5 (Fig. 1I). The Ki67 expression and growth of PDTOs were significantly affected by changes in CCT5 expression (Fig. 1J–L). We similarly constructed CRC cell lines with CCT5 overexpression and knockdown (Supporting Information Fig. S4A and S4B, and Supporting Information Fig. S5A). The results showed that CCT5 promoted CRC cell proliferation both in vivo and in vitro (Figs. S4C–S4E, S5B and S5C). Furthermore, the expression levels of other TRiC complex subunits did not significantly change with alterations in CCT5 expression, indicating that the changes in CRC proliferation capacity are primarily regulated independently by CCT5 (Supporting Information Fig. S6).
3.2.　 Direct interaction between CCT5 and ASNS in CRC
To elucidate the potential mechanisms underlying CCT5-mediated CRC proliferation, we conducted an immunoprecipitation (IP) experiment using a CCT5-specific antibody to enrich CCT5-interacting proteins. We identified three distinct bands at positions of 50–70 kDa, which were absent in the IgG control group (Fig. 2A). Subsequent qualitative mass spectrometry (MS) assays and GO function enrichment analysis of the identified proteins revealed significant enrichment in small molecule metabolism, enzyme binding, and biosynthetic processes, suggesting a role for CCT5 in these biological processes in CRC (Fig. 2B). ASNS was identified based on its ranking (Supporting Information Fig. S7A and S7B). Molecular docking results indicated potential binding between ASNS (amino acids 321, 317, 491, 150, 503, 523, and 200) and CCT5 (amino acids 260, 259, 264, 256, 414, 303, 252, and 526) (Fig. 2C and D, Fig. S7C). Our study confirmed the interaction and spatial co-localization between CCT5 and ASNS (Fig. 2E and F). Additionally, truncated plasmids with fusion tags were constructed (Fig. S7D), demonstrating that amino acids 181–360 of CCT5 can bind to both truncated variants of ASNS (Fig. S7E, Fig. 2G and H). GST pull-down experiments further confirmed the direct binding of CCT5 to ASNS (Fig. 2I and Fig. S7F). To determine whether the binding of CCT5 to ASNS is dependent on the integrity of the TRiC complex, we knocked down the adjacent subunits CCT2 and CCT7 to disrupt its function28. Our results showed that the knockdown of CCT2 or CCT7 alone or both simultaneously did not affect CCT5 expression, and CCT5 binding to ASNS was reduced but still present (Fig. 2J and K). This finding suggests that CCT5 can specifically bind to ASNS independently of the integrity of the TRiC complex.
3.3.　 CCT5 stimulates the activity of ASNS to increase Asn biosynthesis
In biological organisms, aspartate and glutamine serve as substrates for the biosynthesis of Asn catalyzed by ASNS in the presence of ATP. Remarkably, ASNase was capable of reversing this process29 (Fig. 3A). Pathways related to the regulation of amino acid metabolism and activation were enriched in CRC tissues with high CCT5 expression (Fig. 3B and C, Supporting Information Fig. S8A). This finding is consistent with the GO enrichment results for CCT5-interacting proteins (Fig. 2B). Liquid chromatography–mass spectrometry (LC–MS) was employed to detect Asn levels. As predicted, CCT5 expression positively correlated with Asn levels in CRC cells, PDTOs, and the corresponding culture medium (CM) (Fig. 3D–G). L-Albizziine (Alb), a competitive inhibitor of ASNS, reversed the increased Asn levels induced by CCT5 overexpression (Fig. 3H and I). Additionally, it was observed that changes in CCT5 expression did not affect ASNS transcription or protein levels (Fig. 3J–M). Surprisingly, CCT5 enhanced ASNS activity to generate more Asn in vitro (Fig. 3N and O, Fig. S8B). The increase in Asn levels was reversed after treatment with Alb (Fig. 3P).
3.4.　 The Asn/mTORC1 axis plays a critical role in CCT5-mediated CRC proliferation
To investigate the mechanism by which CCT5 mediates CRC proliferation via Asn, we performed Gene Set Enrichment Analysis (GSEA) on four CRC datasets based on CCT5 expression profiles and identified their intersection (Fig. 4A). The results indicated that the mTORC1 signaling pathway is significantly and positively associated with high CCT5 expression (Fig. 4B). It has been demonstrated that Asn can function as an amino acid exchange molecule to activate the mTORC1 signaling pathway in tumor cells29. Western blot assays showed that after adding the mTORC1-specific inhibitor rapamycin (RAPA), the mTORC1 activated by overexpressed CCT5 was restored, indicating that CCT5 specifically activated the mTORC1 pathway (Fig. 4C). Similarly, ASNase treatment reversed the activation of mTORC1 resulting from CCT5 overexpression (Fig. 4D). Moreover, Asn supplementation restored mTORC1 inhibition due to CCT5 knockdown (Fig. 4E). Administration of RAPA or ASNase resulted in significant growth inhibition of PDTOs overexpressing CCT5 (Fig. 4F, G, I, and J). Furthermore, the growth inhibition induced by CCT5 knockdown was reversed upon the addition of Asn (Fig. 4H and K). CRC cells also confirmed the mediating role of the Asn/mTORC1 axis in CCT5-stimulated proliferation (Fig. 4L–T). Notably, ASNase significantly inhibited the growth of PDTOs and CRC cells overexpressing CCT5, while no obvious inhibitory effect was observed in the control group (Fig. 4G). This finding suggests that CCT5 may serve as a specific biological marker for ASNase application.
3.5.　 CCT5 as a biological marker to guide ASNase application for CRC growth inhibition
As ASNase has been used in the clinical treatment of ALL, we further investigated its therapeutic potential in the CRC treatment by targeting the CCT5/Asn axis. We observed that ASNase reduced the size and weight of subcutaneous tumors formed by both control (LV-Ctrl) and Cct5-overexpressing (LV-Cct5) CRC cells compared to those treated with PBS. Subcutaneous tumors formed by LV-Cct5 cells were more sensitive to ASNase treatment than those formed by LV-Ctrl cells (Fig. 5A and B). Consistently, the expression of Cct5 was positively correlated with the expression of Ki67 and mTORC1 signaling pathway-related markers in subcutaneous tumors (Supporting Information Fig. S9A and S9B). Furthermore, we observed that ASNase treatment dramatically decreased the weight and size of subcutaneous tumors formed by LV-Cct5 cells compared to those formed by LV-Ctrl cells, whereas it did not affect subcutaneous tumors formed by shCct5 cells compared to those formed by shNC cells (Fig. 5C and D). There was no significant difference in the expression of Ki67 and mTORC1 signaling pathway-related markers in subcutaneous tumors formed by shCct5 cells after treatment with ASNase (Fig. S9C and S9D). We further validated these findings using PDTOs models, and consistent results were obtained (Fig. 5E). To better preserve the genetic characteristics and heterogeneity of primary tumors, as well as to better predict the potential clinical efficacy of ASNase, we collected CRC tissues with high CCT5 expression and low CCT5 expression and constructed PDX models (Fig. 5F and G). We obtained the same results as before in the PDX models (Fig. 5H). These results strongly suggest a potential therapeutic role for ASNase in CCT5-overexpressing CRC tissues.
3.6.　 The Asn/mTORC1 axis mediates the effect of CCT5 on CD8+ T cell function by upregulating the expression of PD-L1 in CRC cells
Several recent reports have demonstrated the regulatory relationship between amino acids and TME25,30. We observed a negative correlation between elevated expression of CCT5 and immune pathways (Fig. 6A) as well as a negative correlation with the infiltration of CD8+ T lymphocytes (Fig. 6B and Supporting Information Fig. S10A). The clinical analysis of CRC tissues showed that tumors with low CCT5 expression had 56% (10/18) of the CRC samples being CD3-positive and 67% (12/18) were CD8-positive (Fig. 6C). Subcutaneous tumors revealed greater infiltration of CD3- and CD8-positive cells in the control group compared to the group with Cct5 overexpression (Fig. 6D).
We speculate whether CCT5 regulates CD8+ T cells in the TME through Asn. CCT5-overexpressing CRC cells were co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. As expected, a decrease in the proportion of CD8+ and CD69+CD8+ T cells in PBMCs and a decrease in cytokine levels. Additionally, the effect of CCT5 on CD8+ T cells was reversed by the addition of ASNase (Fig. 6E and Fig. S10B). However, the direct addition of Asn to PBMCs increased the quantity and activity of CD8+ T cells, while the administration of ASNase could remove the effects caused by Asn (Fig. S10C and S10D). These findings suggest that CCT5-regulated effector CD8+ T cell reduction is not directly triggered by Asn and may be driven by Asn-mediated changes in tumor cells.
GSEA was performed on the top 50 genes that were highly correlated with CCT5 and revealed enrichment of PD-L1 expression and the PD-1 checkpoint pathway (Fig. 6F and Supporting Information Fig. S11A). Initially, co-culture experiments were performed using CCT5-overexpressing HCT116 cells and PBMCs. The results show that the proportion of CD8+PD-1+ cells did not change significantly (results not shown). However, CCT5 overexpression was found to increase the expression of PD-L1, which was reversed by the administration of ASNase (Fig. 6G, I, and Fig. S11B). Moreover, the knockdown of CCT5 reduced PD-L1 expression, while Asn supplementation restored the expression of PD-L1 (Fig. 6H and J, Fig. S11C). This result suggests that CCT5 regulates the expression of PD-L1 through Asn. Furthermore, to clarify whether the reduced CD8+ T cell function caused by CCT5 overexpression is specifically triggered by the upregulation of PD-L1 expression, we added anti-PD-L1 antibody (atezolizumab, αPD-L1) treatment to the co-culture system. Results show that the suppression of CD8+ T cell numbers and activity due to CCT5 overexpression can be reversed after αPD-L1 treatment (Fig. 6K).
Previous research results have confirmed that Asn activates mTORC1 in CRC cells (Fig. 4A–E), and literature has reported that mTORC1 activation can upregulate the expression of PD-L131—34. Based on this, we applied the mTORC1 inhibitor and found that administration of RAPA could reduce the upregulation of PD-L1 expression caused by CCT5 (Fig. 6L and M, Fig. S11D) and restore effector CD8+ T cell numbers in PBMCs cocultured with CCT5-overexpressing HCT116 cells (Fig. 6N and Fig. S11E). Therefore, we propose that CCT5 upregulates the expression of PD-L1 in CRC cells through the Asn/mTORC1 axis, thereby reducing the function of CD8+ T cells. Drug blockade at key links can effectively restore this negative effect (Fig. 6O). This provides us with a new idea for the treatment of CRC.
3.7.　 Pharmacological values of targeting the CCT5/Asn/mTORC1/PD-L1 axis in CRC immunotherapy
Based on the positive regulation of PD-L1 expression by CCT5, we aimed to investigate whether CCT5 could serve as a molecular marker for predicting the efficacy of clinical anti-PD-L1 antibodies. We collected tumor tissues from six CRC patients who underwent αPD-L1 treatment. Among these patients, three exhibited a favorable response to the treatment, while the other three did not respond. IHC results indicated that, compared to the non-responsive group, the expression of CCT5 in the tumor epithelium of patients who responded to treatment was significantly elevated (Fig. 7A). These findings suggest that CCT5 expression in CRC patients is positively correlated with sensitivity to clinical αPD-L1 treatment. Further, we targeted the CCT5/Asn/mTORC1/PD-L1 axis to explore the potential of using ASNase and immune checkpoint inhibitors (ICIs) in combination therapy for CRC. In the co-culture system of CRC cells and PBMCs, we found that the combined application of ASNase and αPD-L1 seemed to be more effective compared to the individual treatment with either ASNase or αPD-L1, as indicated by the highest level of tumor cell apoptosis (Fig. 7B). Moreover, in the combination treatment group, the killing function of CD8+ T cells was significantly restored, accompanied by a decrease in the expression of PD-L1 on tumor cells (Fig. 7C and D). The comparison revealed that the therapeutic effect of ASNase combined with αPD-L1 was the best in tumor cells of the CCT5 overexpression group.
We further explored the value of in vivo combination therapy (Fig. 7E). ASNase and αPD-L1 combined therapy reduced the subcutaneous tumors formed by LV-Cct5 and LV-Ctrl cells compared to those treated with PBS, ASNase, or αPD-L1 antibodies separately (Fig. 7F–H). Moreover, subcutaneous tumors formed by LV-Cct5 cells were more sensitive to both ASNase monotherapy and combined ASNase and αPD-L1 therapy. However, no significant difference was observed in the subcutaneous tumors formed by shCct5 cells among the various treatment groups. TGI analyses indicated that, among all the treatments conducted, the combined ASNase and αPD-L1 therapy exhibited the strongest tumor inhibition effect in subcutaneous tumors formed by LV-Cct5 cells, with a TGI of up to 94% (Fig. 7I). The expression of proliferation index Ki67, mTORC1 signaling pathway-related markers, and PD-L1 was the lowest in subcutaneous tumors formed by LV-Cct5 cells after ASNase and αPD-L1 combined therapy (Fig. 7J and Supporting Information Fig. S12A). Moreover, we collected mouse liver, lung, and spleen tissues after the combination treatment, and H&E staining showed that the combination therapy was not toxic (Fig. S12B). These results confirmed that combined treatment with ASNase and αPD-L1 antibodies can effectively inhibit CCT5-stimulated CRC proliferation.
4.　Discussion
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The molecular complexity of CRC contributes to its aberrant proliferation and suboptimal clinical response to therapies. In this study, we have provided the first evidence that CCT5 is associated with CRC progression. We found that CCT5 increases Asn biosynthesis by directly surrounding the unfolded asparagine synthetase via the apical domain and enhancing its enzymatic activity. On one hand, the augmented CCT5-mediated Asn synthesis activates the mTORC1 pathway in CRC cells, promoting CRC proliferation. On the other hand, mTORC1 activation upregulates the expression of PD-L1 in tumor cells, leading to a decrease in effector CD8+ T cells in the TME, promoting immune escape of tumor cells, and further enhancing CRC proliferation. Importantly, we have demonstrated the efficacy of ASNase in CRC treatment, and the combination therapy of αPD-L1 antibodies effectively suppresses the proliferation of CCT5-high-expressing CRC (Fig. 8). These findings reveal CCT5 as a biological marker for assessing the response to targeted Asn metabolism combined with anti-PD-L1 immunotherapy in CRC.
The primary function of the chaperonin protein TRiC is to assist protein folding and contribute to the maintenance of cellular protein homeostasis. It is currently estimated that TRiC can fold approximately 10% of the newly synthesized proteins35. Elevated TRiC expression has also been demonstrated to be closely associated with abnormal proliferation of various tumor cells36. Here, we established for the first time the stimulatory role of one of its subunits, CCT5, in CRC proliferation. The ATP-binding equatorial domain (1–154 and 418–541 aa), the apical domain (227–380 aa) that binds to unfolded proteins, and the intermediate domain (155–226 and 381–417 aa) that undergoes nucleotide-induced conformational changes can be found in all the subunits of TRiC37, and individual TRiC subunits also possess the ability to assist protein folding38. A major difference among various TRiC subunits exists in the apical domain, which might contribute to the unique recognition specificity of their interacting proteins39. In this study, ASNS was identified as a protein that specifically interacts with CCT5. Consistent with its binding properties to unfolded proteins, we confirmed direct binding between the CCT5 apical domain and the entire ASNS protein. Moreover, the interaction between CCT5 and ASNS did not alter the abundance of ASNS but induced a pronounced elevation in enzymatic activity, thereby facilitating the biosynthesis of Asn. Molecular chaperones maintain the active conformation of interacting proteins, and the CCT complex has been reported to stabilize the conformation of wild-type p53 to promote its activity40. Therefore, we speculated that the aberrantly high expression of CCT5 in conjunction with ASNS could stabilize the enzymatically active conformation of ASNS and stimulate the synthesis of Asn in CRC. This finding expands our understanding of TRiC's function as a molecular chaperone for protein homeostasis and provides the first evidence for its regulatory role in cellular amino acid metabolism. However, this finding was investigated only in CRC, and whether it is a universal phenomenon in normal colorectal mucosa or in other tumor cells remains to be explored.
Amino acids are essential building blocks of cells, and aberrant amino acid metabolism plays a crucial role in tumor progression, whereas depriving them can impede the aberrant growth of tumor cells41. The bioavailability of asparagine drives breast cancer metastasis and restores tumor cell death induced by glutamine depletion42,43. In this study, we demonstrated that CCT5 could stimulate the biosynthesis of Asn and promote the subsequent release of Asn into the extracellular space. The secreted Asn could potentially activate mTORC1 signaling to promote CRC cell proliferation by functioning as an amino acid exchange factor to facilitate the uptake of extracellular amino acids, such as serine, arginine, and histidine29. The impact of tumor amino acid metabolism on TME should not be overlooked. Here, we investigated the interaction between tumor and immune cells in the TME from the perspective of amino acid metabolism. We observed a significant decrease in effector CD8+ T cells in CRC tissues with high CCT5 expression. Considering that high CCT5 expression is correlated with increased synthesis of Asn in tumors, we initially hypothesized that tumor-secreted Asn could directly inhibit the function of CD8+ T cells in the TME. However, the administration of Asn directly to PBMCs in vitro could increase the quantity of CD8+ T cells while simultaneously stimulating their function. This is consistent with an existing report that Asn can activate LCK signaling to enhance the anti-tumor response of CD8+ T cells44. These phenomena contradicted our observations. As tumor cells have a greater demand for nutrients than T cells23, they may compete with T cells for Asn uptake in the TME to accomplish cell proliferation and survival. Considering the positive regulatory role of Asn in breast cancer cell metastasis42, we speculate that tumor cells could prioritize the utilization of Asn to alter Asn levels in the TME, thereby manipulating T cells to evade immune surveillance44.
PD-L1/PD-1 signaling is a key mechanism by which tumor cells inhibit T cell-mediated anti-tumor immune responses. Activation of this signaling axis inhibits T cell activation, proliferation, and cytotoxic secretion45. Specifically, upregulation of PD-L1 in tumor cells can bind to PD-1 on T cells, leading to dephosphorylation of CD3ζ and ZAP70 associated with the T cell receptor (TCR), and T cell activation is inhibited. This process inhibits the transmission of downstream signaling pathways (including PI3K/AKT and RAS/MEK/ERK pathways, etc.), thereby impairing T cell survival and proliferation. In addition, inhibition of PKCδ activation leads to reduced secretion of cytotoxic cytokines (such as IFN-γ) by T cells46. The expression of PD-L1 is regulated by multiple mechanisms. In addition to classical IFN-γ/JAK/STAT1 signaling, activation of the PI3K/AKT/mTOR oncogenic pathway promotes translation and expression of PD-L1 in various tumors such as gastric cancer, CRC, non-small cell lung cancer, and glioma31—34. It has been reported that mTORC1 activates downstream proteins such as 4E-BP1, STAT3, NF-κB, and c-MYC, which can upregulate the post-transcriptional translation of PD-L147. Rapamycin inhibits mTORC1 and promotes autophagic degradation of PD-L1 protein48. In this study, we found that high expression of CCT5 in CRC upregulates the expression of PD-L1 in tumor cells and inhibits the cytotoxic function of CD8+ T cells. The upregulation of PD-L1 may be mediated by Asn-activated mTORC1 signaling, as the expression of PD-L1 was restored upon supplementation with Asn in CCT5-silencing CRC cells. Moreover, either the addition of ASNase or treatment with the mTORC1 inhibitor RAPA resulted in decreased expression of PD-L1 and restoration of CD8+ T cell function in CCT5-overexpressing CRC cells. Consequently, the overexpression of CCT5 in tumor cells results in enhanced synthesis of Asn, which may be preferentially utilized by tumor cells upon its secretion into the extracellular space.
Targeting PD-L1/PD-1 signaling has become the first-line therapy for multiple tumors. Similarly, targeting amino acid metabolism has been proven to have therapeutic benefits in various preclinical models49. Hence, combination therapy targeting both amino acid metabolism and ICIs may achieve durable antitumor effects and improve patient outcomes. Epacadostat, the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor, which affects tryptophan metabolism, was used in combination with PD-1 inhibitor Pembrolizumab in a further study on melanoma. In the initial non-randomized trials, the combined treatment showed hopeful efficacy without significant toxicity50. However, the therapeutic potential of targeting Asn to reverse the immune suppression in CRC needs to be further evaluated.
Therefore, we explored the therapeutic potential of ASNase-targeted Asn metabolism combined with immunotherapy in CRC. In this study, we demonstrated that ASNase alone has an inhibitory effect on CRC proliferation in BALB/c mice, with a TGI of approximately 40%–66%. Interestingly, in high CCT5-expressing CRC mice or PDX models, the application of ASNase resulted in a TGI of 76%–93%. We propose that this phenomenon occurs because ASNase reduces the elevated extracellular Asn levels resulting from CCT5 overexpression, thereby inhibiting the cancer-promoting Asn/mTORC1 signaling pathway. Simultaneously, it decreases the expression of PD-L1, which is upregulated by mTORC1 activation, thereby partially restoring the tumoricidal function of effector T cells.
In contrast, there was no significant difference in the inhibitory effect of ASNase on CRC when CCT5 was lowly expressed, indicating the potential guiding value of CCT5 expression in the application of ASNase treatment. Amino acid metabolism is a complex process, and CCT5 may not be the only regulator of Asn. After shCCT5, the level of Asn may decrease in a short period, but during the long-term growth of tumor cells, there may be a compensatory pathway to re-promote Asn synthesis, offsetting the Asn level downregulated by shCCT5, so that there was no significant difference in tumor volume compared with the Ctrl group treated with ASNase. Based on existing literature reports we speculate that, on the one hand, CCT5 as a molecular chaperone may cause misfolding or accumulation of proteins synthesized by cells after knockdown; on the other hand, the Asn level downregulated by shCCT5 may cause nutritional deficiency. These two effects may cause endoplasmic reticulum stress51. Under endoplasmic reticulum stress, the transcription factor ATF4 is upregulated, which can promote the transcriptional expression of ASNS and promote the biosynthesis of Asn52. This part of the mechanism needs to be further explored.
However, as shown in Fig. 6G and I, the use of ASNase could only restore the upregulation of PD-L1 expression mediated by CCT5 overexpression, but could not completely block the expression of PD-L1. In the TME, PD-L1 is expressed not only in tumor cells, but also in macrophages, dendritic cells, and B cells46. Therefore, some T cells are still suppressed, preventing them from fully restoring their ability to kill tumors. However, the use of anti-PD-L1 antibodies can completely block the activation of the PD-L1/PD-1 signaling pathway. Furthermore, we attempted to explore the therapeutic effect of anti-PD-L1 antibodies alone and in combination with ASNase in CRC with high expression of CCT5.
Through IHC experiments, we found that the expression of CCT5 in the epithelial tissue of CRC patients who clinically responded well to PD-L1 treatment was significantly higher than that of patients who did not respond to PD-L1 treatment. This suggests that the high expression of CCT5 may be positively correlated with PD-L1 treatment sensitivity in clinical CRC patients. However, there was no significant difference in the TGI between tumor-bearing mice injected with Cct5-overexpressing cells (50%) and those injected with control cells (49%) after anti-PD-L1 antibody treatment. But, tumor-bearing mice injected with Cct5-silencing cells showed a decrease in TGI (13%), indicating that high CCT5 expression is a sufficient but not necessary condition as a biomarker for anti-PD-L1 antibody treatment in CRC. Surprisingly, in mice under the combined treatment of ASNase and anti-PD-L1 antibodies, there was an improvement in TGI in the control, Cct5-overexpressing, and Cct5-knocked-down groups. More importantly, the inhibition rate in the subcutaneous tumors of mice with high Cct5 expression reached 94%. Simultaneously, no significant organ damage was observed in mice after combination treatment.
Overall, the combination of ASNase and anti-PD-L1 therapy showed significant anti-tumor effects in CRC with high CCT5 expression. We hypothesize that, on one hand, ASNase suppresses aberrant tumor proliferation signals by inhibiting the CCT5/Asn/mTORC1 axis, while blocking CCT5-mediated sustained upregulation of PD-L1 expression in tumor cells. On the other hand, anti-PD-L1 antibodies block the activation of the PD-L1/PD-1 signaling axis between tumor cells, other cells in the TME, and T cells. This blockade reverses the immunosuppressive effects on T cells, enhances their tumor-killing capacity, and suppresses abnormal tumor growth.
Rapamycin has been approved by the FDA as an mTOR inhibitor, but we did not explore its effect in combination with anti-PD-L1 antibodies in this study. Because this study reported that Asn is located upstream of mTORC1 and has a positive activation effect on it, the removal of Asn by ANSase can also restore over-activated mTORC1 compared with the use of rapamycin. We believe that blocking the activation of the cancer signaling axis from a more upstream target may be more effective. Besides, the strategy of combining rapamycin or rapamycin analogs targeting mTOR with PD-L1 antibody therapy has been reported in many tumor studies48,53. It has also been reported that there are multiple feedback loops and compensatory pathways that promote cell survival and growth after rapamycin administration, making it impossible to completely block mTORC1-mediated signaling events54. However, as a first-line drug for childhood ALL, the therapeutic effect of ASNase in solid tumors still needs to be further explored, and its combination therapy with ICIs has not been reported, which is more valuable for research and provides new ideas for us to develop new and effective treatment strategies.
In existing clinical treatment guidelines, medication is often guided by the expression of certain biomarkers. For example, high expression of HER-2 promotes tumor progression in breast cancer. Trastuzumab (Herceptin) is effective in patients with HER-2-positive breast cancer but has no effect in patients with low HER-2 expression55. In summary, we have demonstrated the therapeutic potential of the combined treatment of ASNase and anti-PD-L1 antibodies in CRC and demonstrated that CCT5 could be used as a biomarker for combination treatment.
A limitation of the study is that the in vivo experiments were only done with subcutaneous tumor models, dismissing the role of the local tumor microenvironment of CRC, in particular the microbiome. We will further evaluate the effectiveness of ASNase combined with anti-PD-L1 antibody therapy in the orthotopic setting of CRC in follow-up studies.
5.　Conclusions
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This study discovered a new role for the chaperone protein CCT5 in amino acid metabolism and immune regulation. Mechanistically, CCT5 directly binds to ASNS to increase its activity and promote Asn biosynthesis. Tumor cells preferentially utilize the increased Asn synthesis, activate the mTORC1 signaling pathway, and promote tumor proliferation. At the same time, activated mTORC1 upregulates tumor cell PD-L1 expression, induces a decrease in the killing function of CD8+ T cells in the TME, and ultimately leads to the malignant growth of CRC. In summary, we believe that CCT5 is a potential biomarker, which provides evidence for the application of combined therapy of ASNase and PD-L1 antibodies in CRC patients with high CCT5 expression.
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Year 2025 volume 15 Issue 5
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doi: 10.1016/j.apsb.2025.03.026
  • Receive Date：2024-12-15
  • Online Date：2026-09-17
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  • Received：2024-12-15
  • Revised：2025-02-16
  • Accepted：2025-02-18
Affiliations
    aDepartment of Pathology, Nanfang Hospital, Southern Medical University Guangzhou 510515, China
    bDepartment of Pathology & Guangdong Province Key Laboratory of Molecular Tumor Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
    cDepartment of Pathology, Shunde Hospital, Southern Medical University (the First People's Hospital of Shunde), Foshan 528399, China
    dDepartment of Pathology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310009, China
    eDepartment of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510260, China
    fDepartment of Medical Oncology, Affiliated Tumour Hospital of Guangzhou Medical University, Guangzhou 510095, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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