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Induction of the p21/CDK6 pathway and alteration of the immune microenvironment by the stem cell marker CBX3 in melanoma

Stem Cell Research & Therapy volume 16, Article number: 63 (2025) Cite this article

Abstract

Background

As one of the stem cell markers, chromobox protein homolog 3 (CBX3) participates in multiple signaling pathways that affect the progression of various tumors. However, the role of CBX3 in melanoma remains unclear, and the mechanisms by which CBX3 may regulate immunotherapy outcome remain largely unknown.

Methods

We used the Cancer Genome Atlas, Genotype-Tissue Expression portal, and Gene Expression Omnibus database to estimate CBX3 expression and its prognostic effect in melanoma. The role of CBX3 in proliferation and migration of melanoma cells were examined using the CCK8, cloning, wound healing, and transwell assays. The effect of CBX3 on melanoma tumorigenesis was assessed using an in vivo animal model. The role of CBX3 in cell cycle was examined using flow cytometry, and expression levels of cell cycle-related genes and proteins in cells with altered CBX3 levels were analyzed using qPCR and western blotting. The function of CBX3 in the immune microenvironment of melanoma was studied using single-cell RNA sequencing and public databases.

Results

We found that CBX3 was highly expressed in melanoma with poor prognosis. CBX3 promoted the proliferation and migration of melanoma cells in vivo and in vitro. Functional analysis revealed that CBX3 regulates cell cycle, as it accelerated the G1 to S transition, decreased p21 expression, and increased CDK6 expression. Finally, single-cell sequencing and immune-related assays showed that CBX3 is immunogenic and can change the immune microenvironment of melanoma.

Conclusions

We conclude that the stem cell marker, CBX3 activates the p21/CDK6 pathway and alters the immune microenvironment in melanoma.

Background

Melanoma derives from melanocytes in the epidermis, mucous membranes, and other tissues [1]. Melanoma grows faster than any other solid tumor, with approximately 230,000 new cases and 20,000 deaths reported each year [2]. It is a malignant tumor with the highest mortality rate among skin cancers [3].

In clinical practice, surgery remains the treatment of choice for most patients [4]. For patients with advanced-stage disease, the prognosis is poor; however, the advent of immunotherapy has dramatically improved this situation, increasing the 5-year survival rate from 5% to approximately 40% [5,6,7]. In other words, about 60% of melanoma patients cannot benefit from immunotherapy. Therefore, preventing immune evasion and overcoming immune resistance are among the main themes of current melanoma immunotherapy research.

Chromebox protein homolog 3 (CBX3), also known as heterochromatin protein 1 gamma (HP1γ), is a member of the heterochromatin protein 1 family [8, 9]. As a major reader of the repressor of the histone marker H3K9me2/3, CBX3 is mainly found in euchromatin in the transcribed regions of active genes, regulating transcriptional elongation and cotranscriptional mRNA processing [10,11,12]. For example, depletion of CBX3 in embryonic stem cell leads to defects in endodermal and neuronal differentiation [13, 14]. CBX3 plays an important role in developmental processes and cell fate decisions and is often regarded as one of the stem cell markers. Recent studies have demonstrated that CBX3 participates in multiple biological processes, including cell cycle, apoptosis, and immune infiltration, thereby affects the progression of various malignancies, such as hepatocellular carcinoma, pancreatic cancer, clear cell renal carcinoma, breast cancer, and others [15,16,17,18]. Targeting epigenetic mechanisms has been shown to be an effective anticancer strategy for stem cell-like tumors types, although whether the role of epigenetic factor CBX3 in melanoma remains unclear, and whether CBX3 affects efficacy of immunotherapy remains quite confusing.

Thus, we analyzed the correlation between CBX3 and the prognosis of melanoma patients and further explored the character of CBX3 in melanoma progression and immune environment. This study contributes to the evidence highlighting the significance of CBX3 as a predictive biomarker and immunotherapy for melanoma.

Materials and methods

Tissue microarray and immunohistochemical staining

A tissue microarray containing 204 skin tissue samples was commercially obtained from Alenabio (catalog number: ME2082d, Xian, China). IHC staining was performed on human specimens as described previously [15] using an anti‐CBX3 antibody according to the manufacturer’s instructions (1:400; catalog number: 11650–2-AP, Wuhan Sanying Biotechnology, Wuhan, China). Follow up analysis was conducted on 192 samples.

Collection of public melanoma datasets

TCGA and GTEx portal were accessed via the UCSC Xena website (https://xenabrowser.net/datapages/) for the exploration, visualization, and analysis of cancer genomics data [19]. The melanoma dataset selected for our study contained 102 tumor samples and 811 normal samples.

Cell lines

Three melanoma cell lines, A2058, A375, and Mewo (Cell Bank of the Chinese Academy of Sciences) were used in this study. The cell lines were cultured at 37 °C under a humidified atmosphere of 5% CO2 and 95% air as previously described [20].

Establishment of stable CBX3 overexpression and CBX3 knockout cell lines

The lentiviral CBX3/HP1γ overexpression and knockout vectors were purchased from Hanbio Biotechnology Co., Ltd (Shanghai, China). All the procedures were performed using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Quantitative reverse transcription polymerase chain reaction

qRT-PCR assays were performed using a PCR system with primers according to the manufacturer's instructions. The following primers were used: CBX3, 5′-TAGATCGACGTGTAGTGAATGGG-3′ (forward primer) and 5′-TGT CTGTGGCACCAATTATTCTT-3′ (reverse primer); GAPDH, 5′-GTCTCCTCTGACTTCAACAGCG-3′ (forward primer) and 5′-ACCACCCTGTTGCTGTAGCCAA-3′ (reverse primer).

Western blotting assay

Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) with a blocking buffer (Beyotime Biotechnology, Jiangsu, China) for 1 h at room temperature. The PVDF membranes were incubated with primary and secondary antibodies (Proteintech China) and imaged using FluorChem M (Bio-Rad, Hercules, CA, USA). The primary antibodies including CBX3 (catalog number: 11650–2-AP), CDK6 (catalog number: 14052–1-AP), p21 (catalog number: 10355–1-AP) and GAPDH (catalog number: 10494–1-AP) were offered by Proteintech (Shanghai, China).

Cellular proliferation assays

Transfected cells were seeded into 96-well plates at an initial density of 7,000 cells per well and analyzed in the CCK8 assay at 0, 12, 24, 36, 48, 60, 72, 84, and 96 h according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). A BioTek ELx800 microplate reader (BioTek Instruments, Winooski, VT, USA) was used to measure the absorbance of each well at 450 nm.

Colony formation assays

The cells were cultured at 37 °C for 14 d (600 cells/well in 6-well plates). The colonies were fixed using 4% paraformaldehyde and 0.5% crystal violet, counted, and photographed.

Wound healing assay

Transfected cells were cultured in 6-well plates in a serum-free medium for 12 h. The scratches were produced using sterile pipette tips (KIRGRN, Shanghai, China). The cells were washed with PBS and cultured in a medium supplemented with 2% FBS. Micrographs of the assigned areas were obtained after 0 and 24 h of culture.

Transwell assay

The cells were serum-starved for 12 h, replanted onto Falcon Chambers (Corning Inc., Corning, NY, USA) at a density of 1 × 105 cells for each well. Cells that had moved towards the lower chambers were fixed with 0.5% crystal violet after 24 h. Each well was snapped in nine views under an inverted microscope.

Flow cytometry assay

According to the manufacturer's protocols, the assay was conducted by a Cell Cycle Detection Kit (Invitrogen). Cell cycle flow cytometry data obtained using Accuri C6 (BD Biosciences, Franklin Lakes, NJ, USA) were analyzed with Modfit (Verity Software House, Topsham, ME, USA) and FlowJo (TreeStar, Ashland, OR, USA) software.

Xenograft studies

Mice were anesthetized with 15–40 mg/kg of sodium pentobarbital and injected intraperitoneally, depending on their body weight. After completion of anesthesia, A375 cells and A2058 cells (2 × 106 cells ml−1) were injected subcutaneously into BALB/c nude mice (Hunan SJA Laboratory Animal Co., Ltd., Changsha, China). Monitored every 4 days, tumor volume was determined using the formula (width2 × length)/2 with a slide caliper. Four weeks after implantation, the mice were euthanized by overdose anesthesia (sodium pentobarbital, 100–200 mg/kg) and the tumors were surgically excised. This assay obtained ethical approval from the Shantou University Medical College Ethics Committee.

Differential gene enrichment analysis

The data from TCGA were classified into two groups (high-expression and low-expression) for CBX3, with the median values serving as boundaries between these groups. GSEA was conducted on the mRNA expression data of the selected genes using R package "clusterProfiler" [21]. Gene sets with a false discovery rate of < 0.25 and p < 0.05 were regarded as significantly enriched.

Interactive prediction model of CBX3 and p21

UniProt database (http://www.uniprot.com) was used for searching against protein, while Protein Data Bank database (https://www.rcsb.org) was used for developing structures. The interaction between CBX3 and p21 was predicted from H-DOCK (http://hdock.phys.hust.edu.cn). Visualize the amino acids involved in the interactions using Pymol software.

Single-cell sequencing analysis

Melanoma scRNA-seq data were obtained from GEO dataset GSE72056. The data were analyzed by R package "Seurat". The cell subpopulations were annotated based on previously published studies [22]. R package "monocle2" was used to estimate the variation in gene expression levels over time in different cell subpopulations and for the construction of cell lineage development.

Exploration of CBX3 role in the immune microenvironment of melanoma

We downloaded 22 infiltrating immune cell components using the LM22 characteristic matrix from CIBERSORT [23]. R software was used to calculate the correlation coefficients of various immune cells. TIDE is used to evaluate the potential of tumor immune escape from the gene expression profiles of cancer samples [24].

Statistical analysis

Statistical differences between samples were analyzed using the Student’s t-test for independent samples. Differences were considered statistically significant if p < 0.05.

Results

High CBX3 expression is associated with poorer prognosis in melanoma

Using the GEIPA database, we showed high expression of CBX3 in most cancer tissues, especially skin cutaneous melanoma (Fig. 1A). To investigate CBX3 mRNA expression levels in melanoma, RNA sequencing data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression portal (GTEx) were analyzed. CBX3 expression was significantly higher in melanoma than in normal tissues (Fig. 1B), which was consistent with data from the Gene Expression Profiling Interactive Analysis resource (http://gepia.cancer-pku.cn/). The survival analysis comparing samples with low and high CBX3 expression levels in the TCGA and Gene Expression Omnibus database revealed that high expression of CBX3 was closely linked to poor prognosis in patients with melanoma, as measured by overall survival (Fig. 1C–E). We performed IHC staining of tissue microarrays containing samples from 192 patients with melanoma to evaluate the relationship between CBX3 expression and clinicopathological characteristics (Table 1). CBX3 staining in melanoma tissues was mainly nuclear, as evidenced by the presence of brownish-yellow granules in the nucleus with varying signal intensity (Fig. 1F). The chi-squared test indicated that CBX3 expression was strongly associated with age, tumor grade, clinical stage, lymph node metastasis, and tissue type (Table 2).

Fig. 1
figure 1

CBX3 expression in melanoma and its correlation with disease prognosis. (A) Upregulation of CBX3 in most cancers was confirmed in the GEPIA database. (B) CBX3 expression was significantly increased in melanoma compared to its level in normal tissues (102 primary tumor samples vs. 811 normal samples, p < 0.001). (C–E) Survival curves of patient groups differentiated by CBX3 expression level from TCGA, GSE133713, and GSE19234 datasets. (F) Representative image of CBX3/HP1γ staining (high/low expression) in tumor cell nuclei (left × 40, right × 400)

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Table 1 Clinical and pathological characteristics of 192 melanoma patients
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Table 2 Correlation analysis between the expression of CBX3 and clinical case factors of melanoma
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CBX3 promotes proliferation and migration of melanoma cells

There was some heterogeneity in the expression of CBX3 in different skin cancer cell lines, as well as inconsistencies between the mRNA and protein levels (Fig. 2A, B). To explore the function of CBX3 in the development and progression of melanoma, we established melanoma cell lines in which we stably overexpressed or knocked out CBX3 using lentiviruses for subsequent examination by western blotting and qPCR (Fig. 2C–E).

Fig. 2
figure 2

CBX3 expression in melanoma cell lines. (A) CBX3 expression in skin cancer cell lines (n = 62). (B, C) CBX3 mRNA expression in A375, Mewo, and A2058 cell lines was explored by qPCR and western blot. (D) Overexpression of CBX3 (oe) in a transfected A2058 cell line and knockout of CBX3 (sh) in transfected A375 and Mewo cell lines in comparison with CBX3 levels in the cells transfected with the control vector (nc) were verified by western blotting. All full-length blots are presented in Supplementary file 1. (n = 3, *p < 0.05, **p < 0.01)

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Next, we used the CCK8 and colony formation assays to investigate the role of CBX3 in melanoma proliferation. The proliferative ability of A375 and Mewo cells with downregulated CBX3 expression was lower than that of the cells transfected with an empty vector. In contrast, the upregulation of CBX3 expression increased the proliferation ability of A2058 cells (Fig. 3A, B). In wound healing assays, the knockdown of CBX3 expression leading to a lower wound healing rate, whereas the increase in CBX3 expression was associated with a higher wound healing rate, indicating an increase in cell migration ability (Fig. 3C). The number of cells invading the chamber decreased when CBX3 was knocked out in the transwell assays, explicitly indicating a reduction in cell migration ability. In contrast, when CBX3 was upregulated using a CBX3-harboring plasmid, cell migration was increased (Fig. 3D).

Fig. 3
figure 3

CBX3 promotes proliferation and migration of melanoma cells. (A) Growth rates of melanoma cells with altered CBX3 expression were determined using the CCK8 assay. (B) Tumorigenicity of melanoma cells with altered CBX3 expression was measured using the colony assay. (C, D) Migration ability of melanoma cells with altered CBX3 expression was assessed using the wound healing and transwell assays. (n = 3, *p < 0.05, **p < 0.01)

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CBX3 promotes tumor formation in xenograft mice models

To investigate the effects of modulating CBX3 expression in vivo models, we established melanoma xenograft tumor models by implanting A375 cells and A2058 cells with attenuated and increased CBX3 expression level, respectively, as well as corresponding negative control (nc) cells, in nu/nu mice. The rate of melanoma growth and tumor weight in animals implanted with A375 cells expressing short hairpin RNA against CBX3 (sh-CBX3) were significantly lower than those in the nc group (Fig. 4A, C). In contrast, the upregulation of CBX3 expression in A2058 cells enhanced tumor growth as the volume and weight of the tumors were larger than those in the control group (Fig. 4B, D). These findings were consistent with those of in vitro assays that showed a stimulatory effect of CBX3 overexpression on the proliferation and migration of melanoma cells.

Fig. 4
figure 4

CBX3 promotes tumor formation in xenograft mice. (A, B) Growth of melanoma tumors in xenograft nude mice depending on CBX3 expression levels in implanted cells. (C) Relationship between CBX3 expression and tumor volume in vivo. (D) Relationship between CBX3 expression and tumor weight in vivo. (n = 3, *p < 0.05, **p < 0.01)

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CBX3 affects melanoma proliferation by regulating cell cycle

To further explore the oncologic mechanism of CBX3, we performed the analysis of differentially expressed genes based on CBX3 expression and showed that 62 genes were upregulated and 1,110 genes were downregulated (Fig. 5A). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that these differentially expressed genes were enriched in those encoding proteins relevant to cell cycle (Fig. 5B, C). Flow cytometry showed that CBX3 knockdown impeded the transition from the G1 to S phase, indicating suppression of DNA replication. In contrast, CBX3 overexpression accelerated the G1 to S phase transition and increased cell proliferation (Fig. 5D).

Fig. 5
figure 5

Regulation of cell cycle by CBX3. (A) Differential gene expression analysis based on the CBX3 expression level. (B, C) Functional enrichment of differentially expressed genes. (D) Analysis of cell cycle in cells with different levels of CBX3 expression was performed using flow cytometry

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Through the above bioinformatics analyses and cell function experiments, it is indicated that CBX3 plays a crucial role in the cell cycle pathway. Changes in cell cycle proteins were verified using western blotting. In cells with downregulated CBX3 expression, the p21 expression level was higher and CDK6 expression level was lower, whereas CBX3 overexpression changed expression levels of these cell cycle-related proteins in the opposite direction (Fig. 6A). CBX3 also regulated p21 and CDK6 expression levels in vivo, confirming its role in the modulation of cell cycle and melanoma progression (Fig. 6B). Furthermore, the molecular binding sites of CBX3 and p21 were predicted by H-DOCK (Fig. S1). The residues within CBX3 formed strong hydrogen bonds with P21, thus demonstrating the potential for strong interactions (Fig. 6C-E). After visualization using Pymol software, it was found that the predicted amino acid residues of p21 binding to the B chain in the CBX3 structure remained basically unchanged, further clarifying the significant interaction between CBX3 and p21.

Fig. 6
figure 6

Regulation of cell cycle by CBX3. (A) Expression levels of hallmark cell cycle proteins were determined using western blotting. (B) In vivo expression of cell cycle hallmark proteins. All full-length blots are presented in Supplementary file 1. (C–E) The 3D protein–protein interaction models created by HDOCK and pymol. Model 1 (C), model 2 (D), and model 4 (E) were selected based on the Docking Score, Confidence Score, and Ligand rmsd. All full-length blots are presented in Supplementary file 1. (n = 3, *p < 0.05, **p < 0.01)

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CBX3 influences the immune microenvironment in melanoma

Given that the advent of immunotherapy has revolutionized the treatment of melanoma, we sought to investigate whether CBX3 regulates melanoma immune microenvironment as this may provide new perspectives for treatment. Cell heterogeneity was further examined by analyzing single-cell sequencing data from dataset GSE72056 (Fig. 7A, Fig. S2A–D). CBX3 mRNA was present in almost all cells but particularly enriched in immune T cells (Fig. 7B, Fig. S2E). The pseudotime analysis showed that single cells in the dataset had two directions of differentiation (Fig. 7C, Fig. S2F) and CBX3 was mainly enriched in differentiated T cells (Fig. S2G). To further establish whether CBX3 expression affected immune microenvironment, the immune cell content of each sample in the TCGA was calculated. The “CIBERSORT” package was used to estimate the coefficient of CBX3 expression and immune cell infiltration in groups of samples segregated by the level of CBX3 expression (Fig. 7D). Levels of regulatory T cells (Spearman’s R =  − 0.47, p < 0.01) and activated natural killer cells (Spearman’s R =  − 0.22, p < 0.05) negatively correlated with CBX3 expression level. In contrast, abundances of M2 macrophages (Spearman’s R = 0.24, p < 0.05) and follicular helper T cells (Spearman’s R = 0.2, p < 0.05) correlated positively with CBX3 expression (Fig. S2H-K).

Fig. 7
figure 7

Relationships between CBX3 expression and abundances of different immune cell populations in melanoma. (A) Single-cell sequencing (sc-seq) data from dataset GSE72056. (B) CBX3 expression in sc-seq data. (C) Directions of differentiation in sc-seq data determined by the pseudotime analysis. (D) Relationship between CBX3 expression coefficient and immune cell infiltration determined by the “CIBERSORT” package. (E) CBX3 expression levels in the cancer-immunity cycle

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Relationship between expression levels of CBX3 and different immune checkpoint proteins and CBX3 role in the cancer-immunity cycle.

To ascertain whether CBX3 could modulate the ability of the human immune system to respond to tumor antigens and kill tumor cells, we assessed CBX3 expression level throughout the cancer-immunity cycle. High CBX3 expression promoted step 1 (release of cancer cell antigens) and step 4 (Th17 cell recruitment), whereas low expression of CBX3 was linked to step 2 (cancer antigen presentation), step 4 (T cell recruitment), step 4 (dendritic cell recruitment), step 4 (Treg cell recruitment), and step 5 (infiltration of immune cells into malignancies) (Fig. 7E). The Tumor Immune Dysfunction and Exclusion (TIDE) score was used to evaluate the relationship between CBX3 expression and tumor immune escape. The high-CBX3 group presented insignificant indices in comparison with those in the low-CBX3 group (Fig. S3A). Current immunotherapies most commonly target immune checkpoint proteins such as PD1, PD-L1, and CTLA4. The immune positive scores calculated using The Cancer Imaging Archive did not significantly differ in samples with high and low expression of CBX3 (Fig. S3B–E). In addition to examining the relationship of CBX3 expression level with that of the common immune checkpoint proteins mentioned above, we also studied whether CBX3 expression correlates with other immune checkpoint molecules. We showed that CBX3 expression significantly positively correlated with that of immune checkpoints CD200 and TNFSF4, and significantly negatively correlated with expression of immune checkpoints CD40LG, RNFRSF8, LAIR1, TNFRSF4, and TNFRSF18 (Fig. S3F).

Discussion

In the present study, we investigated a possible involvement of CBX3 in melanoma, showed positive association of CBX3 expression with melanoma progression, and assessed potential role of CBX3 in immunotherapy. There is growing evidence that CBX3 is involved in tumorigenesis and cancer development [25, 26]. However, the roles of CBX3 in cancer remain controversial as this protein is involved in multiple regulatory networks in tumors. For example, CBX3 is often supposed to play a pro-carcinogenic role in tumor development [15,16,17]; however, it has also been shown to inhibit tumor progression in gliomas [27, 28]. Our results indicate that CBX3 is highly expressed in melanoma and its high expression correlates with poor prognosis in patients with melanoma, as confirmed by in vivo and in vitro experiments. Data from in vitro experiments demonstrated that CBX3 regulates the proliferation and migration of melanoma stem cells; therefore, CBX3 plays a pro-tumorigenic role in melanoma progression. In clinical practice, CBX3 may have a great potential as a biomarker to assess the prognosis of patients with melanoma.

Although the molecular mechanism by which CBX3 promotes melanoma cell proliferation is still unclear, the Gene Set Enrichment Analysis (GSEA) showed that cells with highly expressed CBX3 were enriched in genes encoding cell cycle signaling pathway components. Furthermore, flow cytometry experiments demonstrated that high levels of CBX3 promoted the G1/S transition in melanoma cells, which is accompanied by massive DNA replication. Similar results have been observed in other cancers [29, 30]; for example, CBX3 was found to promote the G1/S cell cycle transition in prostate cancer cells [31]. In squamous cell carcinoma of the tongue, CBX3 overexpression suppressed p21 expression and also promoted the G1/S cell cycle transition [32]. We further explored the mechanism by which CBX3 promotes melanoma development and by using in vitro and in vivo experiments demonstrated that high levels of CBX3 inhibited the activation of AKT, which phosphorylates p21. This, in turn, disrupted the inhibitory effect of p21 on the formation of the cell cycle protein kinase complex (cyclin D1–CDK4–CDK6) and promoted the G1/S phase transition [33, 34]. Induced by mitogenic signals, cyclin D binds to CDK6 and promotes the phosphorylation of the retinoblastoma protein (RB) [35, 36], which separates the transcription factor E2F from the RB1–E2F1 complex, leading to the entry of the cell into the S phase and initiation of DNA replication [37, 38]. CBX3 binds to the pocket structural domain of RB1 to release E2F1 in cancer cells, promoting cell cycle progression [31]. In addition, RB1 inhibits E2F activity through chromatin remodeling enzymes such as histone deacetylases [39, 40]. The removal of acetyl groups from the tails of histone octamers facilitates the condensation of nucleosomes into chromatin, thereby suppressing gene expression by inhibiting the binding of transcription factors to their promoters [41, 42]. E2F acetylation enhances its ability to bind to DNA; therefore, RB1 can inhibit the transcriptional activity of CBX3 by deacetylating E2F through histone deacetylases (Fig. 8).

Fig. 8
figure 8

Hypothetical mechanism of cell cycle regulation by CBX3 in melanoma. CBX3 suppressed p21 expression, disrupted the inhibitory effect of p21 on the formation of the cell cycle protein kinase complex (cyclin D1–CDK6) and promoted the G1/S phase transition. Induced by mitogenic signals, cyclin D binds to CDK6 and promotes the phosphorylation of the retinoblastoma protein (RB), which separates the transcription factor E2F from the RB1–E2F1 complex, leading to the entry of the cell into the S phase and initiation of DNA replication

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Besides the p21/CDK6 signaling pathway, the cell cycle was also regulated by other classic pathways, commonly the PI3K/AKT signaling pathway. CBX3 knockout in gliomas promotes AKT/mTOR phosphorylation [27]. In most tumors, CBX3 regulates EGFR expression, thereby promoting the PI3K/AKT/mTOR pathway [43,44,45,46]. Activated AKT induces nuclear translocation of MDM2 [47, 48]. After entering the nucleus, MDM2 binds to the tumor suppressor p53, inhibits its transcription, and induces its degradation, thereby inhibiting cell cycle arrest and apoptosis [49, 50]. On the other hand, AKT negatively regulates p27 by phosphorylating and thereby promotes cell cycle [51]. Our previous findings also indicate that CBX3 affects the AKT/mTOR signaling pathway; however, further exploration of their phosphorylation level is required.

As a therapeutic strategy for melanoma, immunotherapy efficacy is often diminished by the developing immune resistance and immune escape [52]. To overcome these problems, immunotherapy is now increasingly used in combination with targeted therapies [53]. Different immunotherapies engage innate and acquired immunity, and our study also shows that CBX3 plays distinct roles in the two types of immune responses. During immune cell infiltration, high levels of CBX3 positively correlated with M2 macrophages and negatively correlated with natural killer cells, indicating that high CBX3 expression disrupts the innate immunity in melanoma. In acquired immunity, high CBX3 levels positively correlated with T follicular helper cells and negatively correlated with regulatory T cells, revealing that high levels of CBX3 increase the strength of acquired immunity.

CBX3 plays different roles in innate and acquired immunity, which may be related to its role as an epigenetic factor. CBX3 is a highly conserved protein module that normally inhibits spontaneous embryonic stem cells differentiation and supports embryonic stem cells self-renewal by binding to chromatin regions of genes [25]. Thus, CBX3 sustains a balance between differentiation and pluripotency programs [14]. For example, CBX3 promotes smooth muscle cell differentiation from precursor cells by interacting with serum response factors [54]. CBX3 has been found to be critical in neuronal maturation as well as axonal and dendritic development [55]. Meanwhile, the level of H4K20me3 in cardiomyocytes was closely associated with the maintenance of CBX3 expression [56]. During mouse somatic cell reprogramming, CBX3 activity is necessary for the early stages of induced pluripotent stem cell formation, but CBX3 is inhibitory to the later stages of pluripotency induction [25].

Post-translationally modified by citrulline, the chromodomain of CBX3 was promoted to bind with H3K9me3 [57]. However, the citrullination of CBX3 is reduced with the differentiation process of stem cells, so the chromatin function of CBX3 during lineage commitment can be regulated by this way [57]. The cis-regulatory elements formed by the epigenetic factor CBX3 remodel chromatin structure, conferring special phenotypes and unique functions to innate immune cells by establishing cell-specific gene expression patterns [58]. Innate immune cells have a certain degree of plasticity in innate cell development and innate immune response, which results in the differential expression of CBX3 in innate and acquired immune responses. However, the function of CBX3 in the immune system is not fully understood and lacks support in the literature. Existing evidence only suggests that CBX3 promotes the ability of tumor cells to evade the intrinsic immune system, but does not help tumor cells evade T cell surveillance. However, this justifies the importance of CBX3 in immunotherapy.

Biomarkers associated with poor prognosis are often considered driver genes and potential therapeutic targets and are predominantly found in the widely expressed cell cycle and housekeeping genes [59]. Cell proliferation has a deep impact on the gene expression of the entire genome, so many genes with different functions may still be predicted prognosis by indirectly catching cell cycle activity [60, 61]. Our research indicated that the correlation between CBX3 expression and poor prognosis in cutaneous melanoma was closely linked to the cell cycle signaling pathway. Accordingly, the potential of CBX3 as a biomarker for melanoma prognosis is enormous. Besides, personalized cancer vaccines have achieved promising therapeutic results in melanoma and pancreatic cancer, but are ineffective at high tumor loads [62,63,64]. The relationship between CBX3 expression and poor prognosis in cutaneous melanoma is not linked to a severe mutational load in this cancer. It can be seen that understanding the steps in the cancer-immunity cycle that limit vaccine efficacy is important to improve the chances of cure. Our present data may help construct a more detailed framework of the cancer-immunity cycle and contribute to understanding the complex immune microenvironment of melanoma.

Conclusion

In conclusion, we showed that high CBX3 expression is associated with poor prognosis in patients with melanoma and that CBX3 promotes melanoma stem cell proliferation and migration by regulating the cell cycle through the p21/CDK6 signaling axis. Additionally, CBX3 suppresses innate immunity in melanoma and promotes T cell-induced adaptive immunity, which may provide a new direction for tackling resistance to immunotherapy. Our study highlights the potential of CBX3 as a biomarker for melanoma prognosis and a powerful target for personalized melanoma therapy.

Availability of data and materials

The original contributions presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding authors.

Abbreviations

CBX3:

Chromobox protein homolog 3

HP1γ:

Heterochromatin protein 1 gamma

TCGA:

The cancer genome atlas

GTEx:

Genotype-tissue expression portal

TIDE:

Tumor immune dysfunction and exclusion

GSEA:

Gene set enrichment analysis

RB:

Retinoblastoma protein

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Acknowledgements

The authors declare that they have not use AI-generated work in this manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82071101, 82002068 and 82272281); Natural Science Foundation of Guangdong Province (No. 2021A1515010949 and 2021A1515011142); Shantou Science and Technology Project (No. 200624095260243 and 230503176494983).

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  1. Wanxian Chen, Jingjing Jiang and Jiasheng Chen have contributed equally to this work.

Authors and Affiliations

  1. Department of Plastic and Burns Surgery, The Second Affiliated Hospital of Shantou University Medical College, Shantou, 515000, People’s Republic of China

    Wanxian Chen, Linsa Zhou, Jiasheng Chen, Deyi Geng, Yaokun Chen, Xiaosha Han, Qihu Xie, Genghong Guo, Xuefen Chen, Shijie Tang & Xiaoping Zhong

  2. Plastic Surgery Research Institute, Ear Deformities Treatment Center and Cleft Lip and Palate Treatment Center of Shantou University Medical College, Shantou, China

    Wanxian Chen, Linsa Zhou, Jiasheng Chen, Deyi Geng, Yaokun Chen, Xiaosha Han, Qihu Xie, Genghong Guo, Xuefen Chen, Shijie Tang & Xiaoping Zhong

  3. Joint Shantou International Eye Center of Shantou University and the Chinese University of Hong Kong, Shantou, China

    Jingjing Jiang

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Contributions

XPZ performed study concept and design; WXC, JSC, JJJ and LSZ performed development of methodology and writing, review and revision of the paper; DYG, YKC, XSH, QHX, GHG and XFC provided acquisition, analysis and interpretation of data, and statistical analysis; SJT provided technical and material support. All authors read and approved the final paper.

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Correspondence to Xiaoping Zhong.

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The original sources (Alenabio and Cell Bank of the Chinese Academy of Sciences) have confirmed that there was initial ethical approval for collection of these human cells/ tissues, and that the donors had signed informed consent. The research project was conducted according to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). The animal study was compliant with the protocol of Ethics Committee of Shantou University Medical College (SUMC2020-257). Date of approval is Jan 1, 2021. Title of the approved project: Role of HP1γ in melanoma development and its molecular mechanism.

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Chen, W., Zhou, L., Jiang, J. et al. Induction of the p21/CDK6 pathway and alteration of the immune microenvironment by the stem cell marker CBX3 in melanoma. Stem Cell Res Ther 16, 63 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04179-8

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512