Expression patterns and clinical implications of chaperonin subunit 3 mRNA and protein in laryngeal squamous cell carcinoma

Abstract

BACKGROUND

Laryngeal squamous cell carcinoma (LSCC) is a prevalent head and neck malignancy with suboptimal survival rates due to late detection and therapeutic resistance.

AIM

To investigate chaperonin-containing TCP1 subunit 3 (CCT3) expression and its clinical implications, and its effects on LSCC cell growth.

METHODS

Systematic data on CCT3 mRNA expression were collected from biomedical databases, and integrated further based on the standardized mean difference and the summary receiver operating characteristic curve. Single-cell RNA-seq data were mined to validate the expression level of CCT3 mRNA. In-house immunohistochemistry was performed to explore the CCT3 protein levels of clinical LSCC samples and their relationship with clinical parameters. The growth function of LSCC cell was analyzed using CRISPR knockout screening. CCT3-related signaling pathway analyses were conducted using gene set enrichment analysis. Protein-protein interaction network construction was performed to identify hub genes.

RESULTS

CCT3 mRNA was significantly overexpressed in 269 LSCC tissues cases across multiple independent datasets (standardized mean difference = 32, area under the curve = 0.93); At the translational level, the in-house immunohistochemical analysis further demonstrated the consistent upregulation of CCT3 protein in 88 cases of LSCC samples (58 non-LSCC samples vs 30 LSCC samples, P = 1.4e^-14). Analysis of clinical parameters showed no significant differences among subgroup. Functional characterization with clustered regularly interspaced short palindromic repeats--mediated gene knockout revealed that depletion of CCT3 potently suppressed LSCC cell viability in vitro. Gene set enrichment analysis indicated that CCT3 was markedly associated with several key oncogenic pathways, including extracellular matrix receptor interaction and cell cycle regulation pathways.

CONCLUSION

CCT3 upregulation in LSCC may influence cellular growth by regulating related pathways, indicating its potential as a biomarker and therapeutic target for LSCC.

Key Words: Laryngeal squamous cell carcinoma; Chaperonin-containing TCP1 subunit 3; Gene expression; Summary receiver operating characteristic; Area under the curve

Core Tip: In this study, single-cell RNA-seq and functional clustered regularly interspaced short palindromic repeats screening demonstrated the overexpression of chaperonin-containing TCP1 subunit 3 in laryngeal squamous cell carcinoma, thereby showing its role in promoting cell viability. Gene set enrichment analysis indicated that chaperonin-containing TCP1 subunit 3 is involved in extracellular matrix receptor interaction and cell cycle pathways, highlighting its potential as a biomarker and therapeutic target for laryngeal squamous cell carcinoma.

INTRODUCTION

Laryngeal squamous cell carcinoma (LSCC) represents one of the most prevalent malignancies within the head and neck region, accounting for approximately 20% of head and neck cancer diagnoses globally[1,2]. Despite advances in multimodal therapies, including surgery, radiation[3,4], and chemotherapy[5], the five-year survival rate for advanced LSCC remains suboptimal, largely due to late-stage detection, metastasis, and therapeutic resistance[6-9]. This underscores the urgent need to identify novel molecular targets that drive tumor progression and that can serve as potential biomarkers or therapeutic vulnerabilities.

Attention has recently turned to the role of molecular chaperones in oncogenesis, particularly the chaperonin-containing TCP1 (CCT) complex, which facilitates the folding of nascent polypeptides such as actin, tubulin, and cell cycle regulators[10]. Among the subunits of this complex, CCT subunit 3 (CCT3) has emerged as a critical player in tumorigenesis, with studies indicating that its overexpression in malignancies such as hepatocellular carcinoma[11], lung cancer[12], and cervical cancer[13] predicts worse survival rates. CCT3 supports cancer cell survival by stabilizing oncoproteins and modulating signaling pathways (e.g., phosphoinositide 3-kinase/protein kinase B)[14-16]. However, its involvement in LSCC remains underexplored, leading to a gap in the understanding of how proteostatic mechanisms influence LSCC biology.

This study was conducted to determine the clinical relevance of CCT3 in case of LSCC by investigating its expression patterns, its effects on cell growth, and its putative functional contributions to LSCC occurrence and aggressiveness.

MATERIALS AND METHODS

This study was designed to comprehensively evaluate the role of CCT3 mRNA and protein in LSCC through a combination of in-silico analysis, experimental validation, and functional assessment.

Data collection and standardized mean difference analysis

A systematic search and collection of high throughput data was conducted to identify datasets containing expression levels of CCT3 mRNA in LSCC and non-cancerous laryngeal squamous epithelium tissues. A total of 269 cases were included after screening. The inclusion criteria for the data were as follows: Human subjects, primary LSCC tissues, and tissues containing control groups. The exclusion criterion was the absence of CCT3 expression values. After removing the batch effect using the sva package in R (v4.2.1), the differential CCT3 mRNA expression levels obtained were compared across multiple public repositories, namely The Gene Expression Omnibus, The Cancer Genome Atlas (TCGA), the Sequence Read Archive, and ArrayExpress. The standardized mean difference (SMD) was calculated to compare the comprehensive expression levels of CCT3. Heterogeneity among the studies was assessed using the I² statistic.

Publication bias and sensitivity analysis

Publication bias was assessed using Begg’s test and Egger’s test to ensure the reliability of the SMD results based on State (v12.0) software. To explore the potential sources of heterogeneity, a sensitivity analysis was conducted by excluding individual studies one at a time to evaluate their impact on the overall results and to identify key studies that might affect the outcomes.

Summary receiver operating characteristic analysis

A summary receiver operating characteristic curve was generated using State (v12.0) software to evaluate the comprehensive discriminatory performance of CCT3 mRNA. As a result, the area under the curve (AUC), sensitivity, specificity, positive diagnostic likelihood ratio, and negative diagnostic likelihood ratio were calculated accordingly.

Single-cell expression analysis

Single-cell RNA sequencing data from untreated LSCC tissues (GSM4546858[17]) were analyzed to investigate the expression patterns of CCT3 and LSCC-related marker genes. The cell selection criteria included cells with nCount_RNA > 1000, nFeature_RNA < 5000, percent.mt < 30%, and nFeature_RNA > 600. The seurat package (v5.0.0) was used to process the dataset. The filtered dataset was normalized using the LogNormalize method. Dimensionality reduction was performed via principal component analysis, t-distributed stochastic neighbor embedding, and uniform manifold approximation and projection. Based on CellTypist[18], the distribution and clustering of different cell types were visualized to elucidate the cellular heterogeneity and the role of CCT3 in various cell populations.

In-house immunohistochemical validation

Immunohistochemical staining was performed on in-house LSCC and non-cancerous squamous epithelial tissue samples to validate the protein expression levels of CCT3. The clinical samples were purchased from Guilin Fanpu Biotech (Guangxi Zhuang Autonomous Region, China), a wholly owned subsidiary of Pantomics, Inc HNT961. Other pathological samples were provided by the Pathology Department of Foshan Hospital of Traditional Chinese Medicine, Foshan, Guangdong and the Pathology Department of Wuzhou Red Cross Hospital, Wuzhou, Guangxi Zhuang Autonomous Region. The numbers of the microarrays (HNT961) included 30 cases of LSCC and 58 cases of controls. The study was approved by the Ethics Committee of Guilin Fanpu Biotech in Guangxi Zhuang Autonomous Region, the Pathology Department of Foshan Hospital of Traditional Chinese Medicine, in Foshan, Guangdong, and the Pathology Department of Wuzhou Red Cross Hospital, in Wuzhou, Guangxi Zhuang Autonomous Region. All patients provided signed informed consent. The discriminatory performance of CCT3 protein was assessed by multiplying the staining intensity score by the distribution score, in accordance with a previously described 12-point scoring system. All procedures were conducted as previously described[19-21].

Analysis of clinical variables

SPSS 23.0 software was used to explore the relationship between the expression levels of CCT3 protein and clinical parameters in 30 patients from the immunohistochemistry (IHC) dataset, thereby elaborating on the clinical significance in greater depth.

Clustered regularly interspaced short palindromic repeats screening for functional analysis

On DepMap[22,23], a clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 (CRISPR-Cas9) knockout screen analysis was conducted with seven LSCC cell lines, including JHU029, JHU022, JHU011, PCI4B, SNU1066, SNU46, and SNU1076. The effect of CCT3 knockout on cell growth was evaluated by measuring the negative effect scores: Which indicated the potential inhibitory role of CCT3 in LSCC cells. The result was plotted using the ggplot2 (v3.5.2).

Gene set enrichment analysis

The clusterProfiler package[24-26] was used to perform a gene set enrichment analysis (GSEA) of genes sorted by log₂FC from a differential analysis between the CCT3 high-expression group and CCT3 low-expression group, to screen out the signaling pathways enriched in CCT3 high expression. This analysis aimed to provide insights into the functional impact of CCT3 on cellular processes and its potential role in tumorigenesis.

Construction of protein-protein network and hub genes screening

To explore the downstream molecular mechanism by which CCT3 exerts its function, this study took CCT3 as the core node and constructed its related protein-protein interaction (PPI) network using the Search Tool for the Retrieval of Interacting Genes (STRING; Version 12.0) database, so as to observe the correlation and interaction between proteins.

Statistical analysis

Statistical analyses were performed using Stata (v12.0) and R (v4.2.1) software, with a P value < 0.05 considered statistically significant. SMD was calculated using R (v4.2.1) and the meta (v4.18-2). A random-effects model was applied for SMD calculation when significant heterogeneity was indicated by a P value < 0.05 and I² value > 50%; otherwise, a fixed-effects model was used.

RESULTS

mRNA expression and discriminatory performance of CCT3 mRNA in LSCC

Following the above inclusion and exclusion processes, a total of 13 datasets were integrated into 12 platforms for inclusion (Figure 1). In most datasets (e.g., E-MTAB-8588, GPL6244, GSE127165, GSE143224, GSE29330, GSE51985, GSE84957, and TCGA-LSCC), the CCT3 mRNA levels in LSCC tissue were consistently higher than in non-cancerous tissue (P < 0.05; Figure 1). CCT3 mRNA exhibited a strong discriminative performance in GPL6244, GSE127165, GSE29330, GSE51985, and TCGA-LSCC (AUC > 0.90; Figure 2). After integration, the comprehensive mRNA level of CCT3 showed a notable upregulation in 269 cases of LSCC tissue compared to non-cancerous tissues (SMD = 1.32, 95%confidence interval: 0.79-1.84) (Figure 3A). Significant heterogeneity was observed among the studies (I² = 75%, τ² = 0.5671, P < 0.01), so the random effects model was employed. The summary receiver operating characteristic curve analysis revealed a strong discriminative performance of CCT3 mRNA [AUC = 0.93, (0.90-0.95), with sensitivity and specificity values of 0.84 and 0.91, respectively (Figure 3B). The positive diagnostic likelihood ratio was 9.48, and the negative diagnostic likelihood ratio was 0.18, indicating a strong diagnostic potential for CCT3 (Figure 4).

Figure 1 Chaperonin-containing TCP1 subunit 3 mRNA levels in laryngeal squamous cell carcinoma are consistently higher compared to non-cancerous tissue. NS: Not significant.

Figure 2 Chaperonin-containing TCP1 subunit 3 mRNA has a marvelous performance in discriminating between laryngeal squamous cell carcinoma and non-cancerous tissue. AUC: Area under the curve.

Figure 3 Chaperonin-containing TCP1 subunit 3 mRNA was significantly increased and has a strong discriminatory performance between laryngeal squamous cell carcinoma and non-cancerous tissue. A: The expression level of chaperonin-containing TCP1 subunit 3 mRNA in the Laryngeal squamous cell carcinoma group was significantly higher than that in the control group overall (the standardized mean difference = 1.32, 95% confidence interval: 0.79-1.84), but there was significant heterogeneity among the studies (I² = 75%); B: The summary receiver operating characteristic curve indicates that the discriminative performance of chaperonin-containing TCP1 subunit 3 mRNA is strong (area under the curve = 0.93), with sensitivity and specificity of 0.84 and 0.91, respectively. SMD: Standardized mean difference; CI: Confidence interval; SROC: Summary receiver operating characteristic; SENS: Sensitivity; SPEC: Specificity; AUC: Area under the curve.

Figure 4 Chaperonin-containing TCP1 subunit 3 mRNA has a strong discriminatory performance in laryngeal squamous cell carcinoma. A: The positive diagnostic likelihood ratio is 9.48, and the negative diagnostic likelihood ratio is 0.18; B: The overall sensitivity of the test is 0.84, and the specificity is 0.91.

Publication bias and sensitivity analysis

Both Begg’s test and Egger’s test indicated the absence of publication bias (Supplementary Figure 1). The sensitivity analysis demonstrated that the overall results were robust, with no single study significantly affecting the overall estimates (Supplementary Figure 2).

Single-cell expression analysis

Single-cell RNA sequencing analysis revealed distinct expression patterns of CCT3 and LSCC-related marker genes (P < 0.05; Figure 5). The plots illustrated the distribution and clustering of different cell types, highlighting the cellular heterogeneity in LSCC tissues and the potential role of CCT3 in specific cell populations.

Figure 5 Single cell expression analysis of chaperonin-containing TCP1 subunit 3 in laryngeal squamous cell carcinoma. These plots illustrate the expression patterns of Chaperonin-containing TCP1 subunit 3 and laryngeal squamous cell carcinoma-related marker genes, as well as the distribution and clustering of different cell types. tSNE: T-distributed stochastic neighbor embedding.

In-house IHC

The positive signals of the CCT3 protein were mainly localized in the cell membrane and the cytoplasm of non-cancerous epithelial cells and LSCC cells (LSCC samples = 30 vs non-LSCC samples = 58). A small number of positive signals appeared in the stroma, in accordance with the findings from scRNA-seq (Figure 5). Hence, the in-house immunohistochemical staining confirmed that CCT3 protein expression was significantly higher in LSCC tissues compared to non-cancerous squamous epithelium (median of non-LSCC samples = 9.8 vs median of LSCC samples = 0.8, P < 0.05, Figure 6).

Figure 6 Immunohistochemical verification of chaperonin-containing TCP1 subunit 3 protein in laryngeal squamous cell carcinoma tissue. A and B: Chaperonin-containing TCP1 subunit 3 (CCT3) protein show negative intensity in non-cancerous squamous epithelium; C and D: CCT3 protein show positive intensity in the cytoplasm of laryngeal squamous cell carcinoma cell; E: The expression of CCT3 protein is significantly higher in laryngeal squamous cell carcinoma than in non-cancerous squamous epithelium; F: CCT3 protein has perfect diagnostic performance, accurately distinguishing between laryngeal squamous cell carcinoma and non-cancerous squamous epithelium. CCT3: Chaperonin-containing TCP1 subunit 3; LSCC: Laryngeal squamous cell carcinoma; AUC: Area under the curve.

Clinical parameter analysis

Thirty patients from the IHC dataset were enrolled as subjects in this study. The results showed that there was no statistical significance between the protein levels and general clinical parameters such as age, gender, pathological grade, and tumor node metastasis stage (Table 1). This indicates that CCT3 was overexpressed in LSCC tissue, with no differences observed among subgroups.

Table 1 The relationship between chaperonin-containing TCP1 subunit 3 protein expression and clinical characteristics in patients with laryngeal squamous cell carcinoma from the in-house immunohistochemistry dataset.

Clinical features	CCT3 protein expression			t or F (ANOVA test)	P value
	Number	Mean	SD
Age				0.538	0.5951
> 55	12	9.500	1.698
≤ 55	18	9.822	1.548
Sex				0.448	0.6573
Male	28	9.729	1.608
Female	2	9.200	1.697
Clinical stage				0.537	0.7097
Stage I	12	9.533	1.533
Stage II	8	9.900	1.720
Stage III	4	8.900	1.800
Stage I-II	2	9.800	0.0283
Stage II-III	4	10.050	1.915
TNM-N
N1	24	9.683	1.565	-0.678	0.9465
N2	6	9.733	1.836
TNM-T				2.533	0.0982
T1	2	9.800	1.979
T2	26	9.508	1.519
T3	2	12.000	0.000

CCT3: Chaperonin-containing TCP1 subunit 3; ANOVA: Analysis of variance; TNM-N: Tumor node metastasis-node; TNM-T: Tumor node metastasis-tumor.

CRISPR screening for cell growth analysis

The CRISPR-Cas9 knockout screen in the LSCC cell lines showed a highly negative effect score, indicating that CCT3 knockout induced a strongly inhibitory effect on LSCC cell growth (gene effect score < -1) (Figure 7).

Figure 7 Clustered regularly interspaced short palindromic repeats screen is used to identify the function of chaperonin-containing TCP1 subunit 3 by knocking them out and observing the effect on laryngeal squamous cell carcinoma. Laryngeal squamous cell carcinoma cell lines show a highly negative effect score, indicating that chaperonin-containing TCP1 subunit 3 knocking out may induce strongly inhibitory effect on laryngeal squamous cell carcinoma.

GSEA

With a statistical threshold of normalized enrichment score ≥ 1.39 and false discovery rate ≤ 0.048, GSEA identified several biological pathways enriched in the CCT3 overexpression group of LSCC tissues. The results provide insights into the potential functional impact of CCT3 on cellular processes, such as ECM receptor interaction, the cell cycle, pathways in cancer, DNA replication, the Wnt signaling pathway, basal transcription factors, biosynthesis of unsaturated fatty acids, epidermal growth factor receptor family, transforming growth factor-beta, adipocytokine, and mitogen-activated protein kinase signaling pathway (Figure 8).

Figure 8 Gene set enrichment analysis of chaperonin-containing TCP1 subunit 3 in laryngeal squamous cell carcinoma. This gene set enrichment analysis plot for chaperonin-containing TCP1 subunit 3 provides a visual representation of which biological pathways are significantly enriched in the activated or suppressed conditions, helping to understand the functional impact of chaperonin-containing TCP1 subunit 3 on cellular processes.

Protein-protein network analysis and hub gene screening

The STRING database was used to perform PPI network analysis of the CCT3 gene and its pathway-related genes. The core objective was to leverage the database’s interaction prediction and visualization functionalities to identify the key interacting genes of CCT3. Ultimately, six genes-CDC20, MCM7, PPP2CA, PPP2CB, CSNK2A1, RUVBL1, and CACYBP were screened out, and these genes exhibited stable associations with CCT3 (Figure 9).

Figure 9 Analysis of chaperonin-containing TCP1 subunit 3 protein interaction network. A: Kyoto Encyclopedia of Genes and Genomes (KEGG) cell cycle; B: KEGG DNA replication; C: KEGG Wnt signaling pathway.

DISCUSSION

This study investigated into the multifaceted role of CCT3 in LSCC - a malignancy that remains a formidable challenge despite therapeutic advancements. The findings highlight the discriminatory and functional significance of CCT3 in LSCC progression, thereby underscoring its potential as a biomarker and therapeutic target. However, existing studies have not yet fully explored the mechanism underlying the role of CCT3 in LSCC.

The consistent overexpression of CCT3 mRNA in LSCC tissues across various datasets suggested a fundamental role in tumor biology. The robust discriminatory performance of CCT3 mRNA, as evidenced by the high AUC value and favorable sensitivity and specificity metrics, indicated its potential as a reliable biomarker for distinguishing LSCC tissues from non-cancerous tissues. This was further corroborated by in-house immunohistochemical validation, which confirmed elevated CCT3 protein expression in LSCC tissues. Meanwhile, the results of clinical parameter analysis further confirmed this point. Despite the small sample size and certain limitations of this study, the protein’s discriminatory performance showed promising accuracy, indicating that CCT3 may hold potential as a useful tool in clinical settings for the early detection and diagnosis of LSCC.

The functional analysis via CRISPR-Cas9 knockout screening revealed a critical role for CCT3 in sustaining LSCC cell viability and proliferation. The significant negative effect scores observed in the LSCC cell lines upon CCT3 knockout implied that this chaperone subunit might be essential for tumor cell survival. This finding aligned with the broader understanding of molecular chaperones in cancer, where they often stabilize oncoproteins and modulate the signaling pathways crucial for tumor progression[27-30]. Targeting CCT3 could potentially disrupt these processes, offering a novel therapeutic strategy for cancers[31,32], especially LSCC.

The single-cell RNA sequencing analysis provided a granular view of CCT3’s expression patterns within the heterogeneous cellular landscape of LSCC tissue. The distinct clustering and distribution of CCT3 highlighted its potential involvement in specific cell populations that contribute to tumor aggressiveness. This cellular heterogeneity is a hallmark of many cancers and underscores the complexity of targeting LSCC. Understanding the specific cell types and pathways influenced by CCT3 could pave the way for more precise therapeutic interventions.

GSEA further elucidated the biological pathways enriched in LSCC samples with high CCT3 expression. These CCT3-participating pathways, including cell cycle regulation[33], DNA replication[34], the WNT signaling pathway[35], and various signaling cascades, are integral to cancer development and progression. The enrichment of these pathways suggested that CCT3 might orchestrate a complex interplay of cellular processes that drive LSCC. This mechanistic insight is crucial for developing targeted therapies that can disrupt these pathways and inhibit tumor growth. Furthermore, by constructing a PPI network, this study identified and screened CDC20, MCM7, PPP2CA, PPP2CB, CSNK2A1, RUVBL1, and CACYBP as potential key proteins that interact with CCT3. CDC20 can activate the activity of the anaphase-promoting complex ubiquitin ligase[36], MCM7 is one of the key DNA replication licensing factors for DNA synthesis and cell entry into the S phase[37], and PPP2CA is the main catalytic subunit of protein phosphatase 2A. Notably, protein phosphatase 2A is well recognized as being involved in mitosis and cell cycle control[38]. These three proteins play important roles in regulating the cell cycle and promoting cancer progression. PPP2CB is associated with M2 macrophage infiltration and can affect the tumor immune microenvironment[39]. RUVBL1, a protein that exhibits both DNA-dependent ATPase and DNA helicase activities, is involved in a variety of cellular processes, including gene regulation, DNA damage repair, and chromatin remodeling[40]. As a serine/threonine kinase, CSNK2A1 participates in the process of cell apoptosis[41]. CACYBP, a highly conserved human calcyclin-binding protein, is involved in regulating multiple cellular processes such as calcium signal transduction, cell proliferation, and cell death[42]. This study hypothesizes that CCT3 may interact with the aforementioned genes to affect biological behaviors such as proliferation and metastasis of LSCC cells, thereby providing new clues for further clarifying the mechanism of action of CCT3 in LSCC. Future research should focus on experimental verification using animal models to clarify the interactions between CCT3 and these proteins as well as to delineate the more explicit mechanism underlying the role of CCT3 in the initiation and progression of LSCC.

Overall, this study integrated multiple lines of evidence to establish CCT3 as a key player in LSCC. The findings highlighted the potential of CCT3 as a discriminatory biomarker and therapeutic target, offering new avenues for improving the management of this challenging malignancy. Future research should focus on validating these findings in larger cohorts and exploring the detailed molecular mechanisms by which CCT3 influences LSCC biology. Additionally, the development of CCT3-targeted therapies and their evaluation in preclinical and clinical settings could significantly advance the treatment landscape for LSCC.

CONCLUSION

In summary, this study verified the overexpression of CCT3 at both the mRNA and protein levels in LSCC tissues. Additionally, the AUC value indicated high diagnostic performance, suggesting CCT3’s potential as a diagnostic biomarker for LSCC. After CRISPR-Cas9 knockout screening, the viability of LSCC cells was impaired, which confirmed that CCT3 is functionally involved in the growth of LSCC cells. GSEA revealed that CCT3 may promote the progression of LSCC by regulating multiple oncogenic pathways, such as extracellular matrix receptor interaction and cell cycle regulation, further demonstrating its potential as a therapeutic target.

ACKNOWLEDGEMENTS

The authors of this paper gratefully acknowledge the Clinical Pathology and Computational Pathology techniques provided by Chen G Lab at the First Affiliated Hospital of Guangxi Medical University. Additionally, we express our sincere appreciation for the public high throughput databases involved in this study.