Trim21-mediated CCT2 ubiquitination suppresses malignant progression and promotes CD4+T cell activation in breast cancer

Abstract

Breast cancer remains a significant global health challenge, and its mechanisms of progression and metastasis are still not fully understood. In this study, analysis of TCGA and GEO datasets revealed a significant increase in CCT2 expression in breast cancer tissues, which was associated with poor prognosis in breast cancer patients. Functional analysis revealed that CCT2 promoted breast cancer growth and metastasis through activation of the JAK2/STAT3 signaling pathway. Additionally, the E3 ubiquitin ligase Trim21 facilitated CCT2 ubiquitination and degradation, significantly reversing the protumor effects of CCT2. Most interestingly, we discovered that exosomal CCT2 derived from breast cancer cells suppressed the activation and proinflammatory cytokine secretion of CD4⁺ T cell. Mechanistically, exosomal CCT2 constrained Ca²⁺-NFAT1 signaling, thereby reducing CD40L expression on CD4⁺ T cell. These findings highlight CCT2 upregulation as a potential driver of breast cancer progression and immune evasion. Our study provides new insights into the molecular mechanisms underlying breast cancer progression, suggesting that CCT2 is a promising therapeutic target and prognostic predictor for breast cancer.

Fig. 1. Expression of CCT2 in breast cancer tissues with its clinical significance.

A The expression of CCT2 was elevated in breast cancer tissues compared with normal tissues according to Metabric, TCGA and GEO database. B the RNA expression of CCT2 was upregulated in breast cancer tissues compared with normal tissues based on Qilu cohort. C The RNA expression of CCT2 in breast cancer tissues and paired normal mammary tissues (n = 11). D IHC staining of CCT2 in primary breast cancer tissues (n = 26) and non-tumor tissues (n = 26) based on Qilu cohort. Scale bar = 100 μm. E the RNA and protein expression of CCT2 was upregulated in most breast cancer cells compared with that in normal cell (MCF-10A). F, G The correlation between CCT2 expression and overall survival or disease-free survival in breast cancer tissues based on Qilu cohort was calculated with the log-rank test. H CCT2 expression levels at different tumor stages of breast cancer cohorts in the metabric database (Stage1:n = 371, Stage2:n = 571, Stage3:n = 90, Stage4:n = 10). I CCT2 expression levels at different grades of breast cancer cohorts in the metabric database (G1:n = 170, G2:n = 770, G3:n = 592). (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 2. CCT2 promotes breast cancer cells malignant progression in vitro.

MDA-MB-231 and MDA-MB-468 were transfected with siCCT2 or CCT2 plasimd for functional experiments. The effect of CCT2 on cell proliferation was evaluated by MTT A, B and EdU assay C, D. Scale bar = 100 μm. E, F cell cycle distributions in CCT2 knockdown or overexpressing cells were presented by flow cytometry. G, H Flow cytometry apoptosis analysis of CCT2 knockdown or overexpressing cells. I, J cell migration and invasion abilities of CCT2 knockdown or overexpressing cells were evaluated by the transwell assays. Scale bar =200μm. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 3. Mutual binding between CCT2 and Trim21 in breast cancer cell.

A CCT2-Flag plasmid was transfected into MDA-MB-231 cells for CO-IP. The protein pulled down were analyzed by silver staining. The images of silver staining were displayed. Trim21 was identified as a binding partner of CCT2, and the representative spectra were included. B HEK293T cells were transfected with CCT2-Flag and Trim21-HA, after which immunoprecipitation (IP) was performed to assess interaction between CCT2 and Trim21 using anti-Flag or anti-HA antibodies. C validation of biochemical interaction between CCT2 and Trim21 in MDA-MB-231 and MDA-MB-468 cells by coimmunoprecipitation of endogenous CCT2 and Trim21. D Co-localization of CCT2 and Trim21 in MDA-MB-231 and MDA-MB-468 cells were examined by using a fluorescence microscope. Scale bar=20μm. E schematic diagram of human Trim21 domains and strategy to engineer a series of Trim21 deletion mutants. F The interactions between CCT2 and Trim21 fragments were examined by coimmunoprecipitation experiments in HEK293T cells. G Schematic diagram of human CCT2 domains and strategy to engineer a series of CCT2 deletion fragments. H The interactions between Trim21 and CCT2 fragments were examined by coimmunoprecipitation experiments in HEK293T cells. I I-TASSER was used to generate 3D structures for CCT2 and Trim21, the CCT2-Trim21 complex was predicted using ZDOCK.

Fig. 4. Trim21 mediates the ubiquitination and degradation of CCT2.

A, B Immunoblotting was performed to analyze lysates prepared from HEK293T cells A, MDA-MB-231 and MDA-MB-468 B following transfection with Flag-CCT2 and different doses of the HA-Trim21 expression plasmid. C, D Immunoblotting were performed to evaluate the expression of CCT2 in Trim21-silenced C or Trim21- overexpressing D breast cancer cells. E MDA-MB-231 and MDA-MB-468 cells were transfected with indicated plasmid, followed by cycloheximide (CHX) treatment for indicated duration. Immunoblotting was then performed using lysates prepared from these cells, with the ImageJ software being used to quantify CCT2 expression and β-actin being used for normalization. F Following transfection with the CCT2 and Trim21 plasmids and treatment with CQ, and MG132 for 4 h, breast cancer cell lysates were subjected to western blotting. G Anti-flag was used to immunoprecipitated lysates prepared from 293 T cells following transient HA-Ub, Flag-CCT2 and His-Trim21 co-transfection, after which anti-HA was used for immunoblotting. H immunoprecipitation analyses were performed for lysates from 293 T cells following the transient co-transfection of Flag-CCT2, His-Trim21, and total-Ub or K48-Ub or K63-Ub mutant. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 5. Trim21 suppresses oncogenic properties of breast cancer cells through a CCT2 dependent manner.

MTT A and EdU B assays indicated the increased proliferative ability in breast cancer cells transfected with Trim21 siRNAs. Scale bar = 100 μm.The migration C and invasion D capabilities of breast cancer cells were promoted by Trim21 knockdown. Scale bar = 200 μm. Breast cancer cells were transfected with CCT2 and Trim21 alone or simultaneously. Then the ability of cell proliferation, apoptosis, migration, and invasion was respectively assessed by MTT E, EdU F, colony assay G, flow cytometry assay H, and transwell assays I, J. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 6. Loss of CCT2 impedes breast cancer growth and promotes the CD4⁺T cells activation.

A Tumors were obtained from 4T1-shCCT2 and 4T1-shNC group mice sacrificed on day 17(n = 5, Scale bar =1 cm). B The tumor volumes were measured every two days. C Tumor weights were recorded after sacrifice of the mice. Flow cytometry analysis of CD3⁺CD4⁺T cells D, CD3⁺CD4⁺CD40L⁺T cells E, CD3⁺CD4⁺IFNγ⁺T cells and CD3⁺CD4⁺IL-4⁺T cells F from 4T1 tumors harvested from mice in three different groups (n = 5). G H&E staining showed the tissue morphology of transplanted tumors. Representative pictures of IHC staining of CCT2, Ki67, and CD4 in the tumor tissues. Scale bar =100μm. qPCR (H) and flow cytometry I analysis were used to detect CD40L expression on CD4⁺T cells under different conditions: normal media, shCCT2 or shNC breast cancer cell CM, and pCMV6 or CCT2 overexpressing breast cancer cell CM. J the levels of mRNA expression of IFNγ and TNFα in CD4⁺T cells from each group were determined by qPCR. K Immunohistochemical staining depicted the correlation between the expression of CCT2 and CD40L in breast cancer tissues (N = 93) based on Qilu cohort (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 7. Exosomal CCT2 suppresses CD4⁺T cells activation and inflammatory cytokine production.

Characterization of harvested breast cancer exos. A Transmission electron microscope was used to detect the spheroid morphology and size of breast cancer exos. Scale bar = 500 nm. B Nanoparticle tracking analysis was performed to detect the diameter quantitation of breast cancer exos. C Western blot analysis of exosomal markers in the exos isolated from breast cancer cells. D A representative fluorescence image of the internalization of fluorescently labeled breast cancer exos in CD4⁺T cells. Scale bar = 5 μm. E CCT2 expression of CD4⁺T cells treated with exos released by indicated cells were detected by qPCR and western blot. qPCR F and flow cytometry G analysis were used to detect CD40L expression on CD4⁺T cells under different conditioned breast cancer exos: PBS, shCCT2 or shNC breast cancer cell exos, and pCMV6 or CCT2 overexpressing breast cancer cell exos. H the levels of mRNA expression of IFNγ and TNFα in CD4⁺T cells from each group were determined by qPCR. I The expression levels of NFAT1 in the cytosolic and nuclear fractions of CD4⁺T cells from each group were detected by western blot. J The location of NFAT1 (fluorescent green) in CD4⁺T cells from each group were visualized using a fluorescent microscope. The nucleus was stained with DAPI (fluorescent blue). Scale bar =5μm. K Representative curves of Ca²⁺ influx dynamics in CD4⁺T cells from each group were depicted using Fluo-4AM fluorescence. L The fluorescence of Fluo-4AM (fluorescent green) was investigated to measure the level of Ca²⁺ influx in CD4⁺T cells from each group. Scale bar = 40 μm. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 8. Schematic model on the proposed role of CCT2 in breast cancer progression and immune evasion.

CCT2 promotes breast cancer tumorigenesis through JAK2/STAT3 signaling pathway. Trim21, functioning as an E3 ligase, interacts with CCT2 and promotes its ubiquitination and degradation, partially counteracting the pro-tumor effect of CCT2. Furthermore, exosomal CCT2 derived from breast cancer cells suppresses the activation and pro-inflammatory cytokine secretion of CD4⁺T cell through modulating Ca²⁺-NFAT1 signaling.