DNAJC1 Facilitates Glioblastoma Progression by Promoting Extracellular Matrix Reorganization and Macrophage Infiltration

doi:10.21203/rs.3.rs-4002088/v1

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Research Article

DNAJC1 Facilitates Glioblastoma Progression by Promoting Extracellular Matrix Reorganization and Macrophage Infiltration

https://doi.org/10.21203/rs.3.rs-4002088/v1

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published 21 Jun, 2024

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Background: Glioblastoma (GBM) is a high-grade and heterogeneous subtype of glioma that presents a substantial challenge to human health, characterized by a poor prognosis and low survival rates. Despite its known involvement in regulating leukemia and melanoma, the function and mechanism of DNAJC1 in GBM remain poorly understood.

Methods: Utilizing data from the TCGA, CGGA, and GEO databases, we investigated the expression pattern of DNAJC1 and its correlation with clinical characteristics in GBM specimens. Loss-of-function experiments were conducted to explore the impact of DNAJC1 on GBM cell lines, with co-culture experiments assessing macrophage infiltration and functional marker expression.

Results: Our analysis demonstrated frequent overexpression of DNAJC1 in GBM, significantly associated with various clinical characteristics including WHO grade, IDH status, chromosome 1p/19q codeletion, and histological type. Moreover, Kaplan‒Meier and ROC analyses revealed DNAJC1 as a negative prognostic predictor and a promising diagnostic biomarker for GBM patients. Functional studies indicated that silencing DNAJC1 impeded cell proliferation and migration, induced cell cycle arrest, and enhanced apoptosis. Mechanistically, DNAJC1 was implicated in stimulating extracellular matrix reorganization, triggering the epithelial-mesenchymal transition (EMT) process, and initiating immunosuppressive macrophage infiltration.

Conclusions: Our findings underscore the pivotal role of DNAJC1 in GBM pathogenesis, suggesting its potential as a diagnostic and therapeutic target for this challenging disease.

DNAJC1

glioblastoma

prognostic biomarker

extracellular matrix reorganization

macrophage infiltration

Gliomas constitute over 70% of malignant brain tumors in the central nervous system (CNS) and represent the most prevalent primary brain tumors¹. The prognosis for glioma patients is bleak, with a five-year survival rate of a mere 59%. Glioblastoma (GBM), the most aggressive and heterogeneous glioma subtype, exhibits a particularly dismal two-year survival rate of 26%². GBM is characterized by its rapid proliferation, invasiveness, and capacity to modulate the tumor microenvironment—factors that significantly contribute to its malignancy by impairing immune responses through the release of anti-inflammatory cytokines³. Additionally, the blood-brain barrier (BBB) poses a substantial obstacle to the delivery of therapeutics to GBM lesions⁴. Despite advances in various treatment modalities, the median survival for GBM patients hovers between 14 and 17 months^{5, 6}. Recent research has implicated several genetic and epigenetic regulators in GBM pathogenesis; however, the central mechanisms remain elusive⁷, necessitating the identification of effective targets to elucidate GBM's underlying biology and to develop new treatment strategies.

Heat shock proteins (HSPs), a ubiquitous and evolutionarily conserved superfamily of proteins, are crucial molecular chaperones that safeguard the cell from various stresses and contribute to maintaining homeostasis^8–10. Beyond their protective roles, HSPs also modulate an array of pathological states, including cancer¹¹. Their dysregulated expression in various tumor types implicates a significant role in cellular processes such as apoptosis, proliferation, differentiation, and immune modulation^{12 8}. Nonetheless, the precise function and regulation of HSPs in GBM are not fully understood, although some have been identified as playing roles in tumor promotion or suppression¹³.

The DnaJ Heat Shock Protein Family (HSP40) Member C1, also known as DNAJC1, is a highly conserved protein that assists in protein maturation and various cellular processes¹⁴. Abnormal expression levels of DNAJC1 homologs have been observed in gliomas, affecting tumor development and progression¹⁵. Overexpression of DNAJC1 has been associated with tumor growth and invasiveness in leukemia and melanoma, suggesting a potential role in tumorigenesis^{16, 17}. However, the exact functions and mechanisms of DNAJC1 in GBM remain to be elucidated.

This study employed bioinformatics techniques to analyze clinical characteristics, molecular markers, and immune cell infiltration in GBM, highlighting the prognostic and diagnostic potential of DNAJC1 as a clinical biomarker. In vitro analyses revealed DNAJC1's role in promoting GBM cell proliferation, cell cycle progression, and migration. Moreover, the study uncovered that DNAJC1 facilitates epithelial-mesenchymal transition (EMT) and attracts immunosuppressive macrophage infiltration into the GBM microenvironment, thus exacerbating GBM oncogenesis. This research offers a comprehensive examination of DNAJC1's influence on immune cell infiltration, particularly its interaction with immunosuppressive cell types, and delves into the molecular mechanisms underpinning these processes in GBM, proposing novel diagnostic and therapeutic strategies.

Datasets

We sourced a comprehensive dataset comprising 10,534 tumor samples across various subtypes, including matched normal controls, from The Cancer Genome Atlas (TCGA) to analyze DNAJC1 expression patterns in pan-cancer contexts. This dataset encompassed RNA-seq data from 689 GBM patients and 1,157 normal controls. Additional data for primary GBM specimens were obtained from the China Glioma Genome Atlas (CGGA), consisting of 156 cases and 5 controls, and the Gene Expression Omnibus (GEO), which included 52 GBM specimens and 20 controls from the GSE29796 series. These datasets facilitated a detailed investigation into the differential expression of DNAJC1 in GBM and its potential association with clinical parameters, including WHO grade, IDH status, 1p/19q co-deletion, and histological subtypes.

Bioinformatics Analysis

The TCGA GBM dataset was bifurcated into high and low DNAJC1 expression groups. We sequenced the transcriptomes of these cohorts to pinpoint differentially expressed genes (DEGs) and their enriched functions or pathways. The R software package (Version 4.2.1) was used for group classification and DEG calculation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses identified enriched cellular components (CC), biological processes (BP), molecular functions (MF), and signaling pathways. Gene Set Enrichment Analysis (GSEA) corroborated these findings by assessing biological functions and pathway enrichments, adhering to the cutoff criteria of P < 0.05, |NES| > 1, and FDR < 0.25. We extracted gene markers for 24 immune cell types from previous research to analyze immune infiltration, employing single-sample GSEA (ssGSEA) with the GSVA R package using TCGA-COADREAD datasets¹⁸. Spearman correlation tests quantified the association between DNAJC1 expression and immune cell infiltration. Visualization was achieved using the ggplot2 R package.

Immunohistochemistry on Human Glioma Specimens

Glioma tissues, including a variety of histological grades and normal brain tissues, were procured from the Pathology Department of Xijing Hospital, Air Force Medical University, Xi'an, China. The collection encompassed tissue chips from 63 patients, treated from 2010 to 2015. Ethical approval was granted by the institution’s ethics committee, and informed consent was obtained from all participants. Tissue preparation entailed meticulous microdissection.

For immunohistochemistry (IHC), sections underwent deparaffinization, rehydration through graded ethanol, and antigen retrieval by boiling. Blocking of endogenous peroxidase was with 3% H2O2 in methanol. Primary antibodies were incubated overnight at 4°C in a humidified chamber. Subsequent incubation with secondary antibodies and streptavidin-conjugated horseradish peroxidase was conducted. Visualization utilized 3,3'-diaminobenzidine (DAB), and hematoxylin counterstaining followed. Sections were dehydrated and mounted in Eukitt medium.

Cell Culture

GBM cell lines U251 and U87, and the mononuclear cell line THP-1, were sourced from the Cell Bank of the Chinese Academy of Sciences. Cultivation occurred in RPMI-1640 medium, enriched with 10% fetal bovine serum and 1% penicillin-streptomycin, in a humidified 37°C incubator with 5% CO2.

Plasmid Construction and Lentivirus Production

Plasmids containing specific shRNA sequences targeting DNAJC1 were constructed as described: shDNAJC1_1: 5’ GCAGCTCAACTTCTACCAGTT 3’; shDNAJC1_2: 5’ GGGTCATTATGCTGTGGTTTG 3’; shDNAJC1_3: 5’ AGGTACAAGTTGCTGGTTGAA 3’. A random control sequence for targeting was also generated: 5’ ACTACCGTTGTTATAGGTG 3’. The DNAJC1 shRNAs and control shRNA were synthesized and inserted downstream of a U6 promoter in the pLVX-U6-EF1α-GFP-Puro lentiviral vector. Validation of all cloning steps was conducted through sequencing. The transfer plasmid containing the shRNA, the viral envelope plasmid, and the packaging plasmid were co-transfected into HEK293T cells using PEI transfection reagent (AC04L091, Shanghai Life-iLab Biotech). After 48 and 72 hours post-transfection, lentivirus-containing supernatants were harvested, filtered through a 0.45 µm filter (Millipore), and subsequently combined with PEG6000 overnight at 4°C. The mixture was concentrated via centrifugation at 4500 × g for 30 minutes at 4°C. U251 and U87 cells were transduced with the lentivirus at a multiplicity of infection (MOI) of 50 accompanied by 10 µg/ml polybrene. Following 72 hours of viral infection, the culture medium was supplemented with 2 µg/ml puromycin, and the cells were maintained for an additional week to establish stable cell lines expressing both GFP and shRNA.

Protein Isolation and Western Blot Analysis

Total protein was isolated from cell lysates using RIPA buffer (Sangon, Shanghai, China) with a protease inhibitor cocktail (Genstar, Shenzhen, China). Protein concentration was determined via bicinchoninic acid assay (BCA kit, Biosharp, Anhui, China). Then, 25 µg of protein per sample was resolved on a 10% SDS-PAGE gel at 110 V and transferred to a PVDF membrane (Millipore, USA) at 300 mA. Blocking was performed using 5% BSA in TBST for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C. Following washes with TBST, membranes were incubated with secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a FluorChem FC2 system (Alpha Innotech, USA) as per the manufacturer's protocol.

Growth Curve Assay

Cell proliferation was assessed using a CCK-8 kit (Beyotime, Shanghai, China). Cells (2 × 10^3 per well) were seeded in 96-well plates with 200 µL of 10% FBS medium. Post attachment, 10 µL of CCK-8 solution was added and incubated at 37°C for 90 minutes. Absorbance at 450 nm was measured with a microplate reader (Bio-Rad, USA). Assays were conducted at 0, 24, 48, and 72 hours post-seeding and replicated thrice.

Colony Formation Assay

Logarithmically growing cells (2 × 10^3) were plated in a 6-cm dish with 5 mL of 10% FBS medium, ensuring even dispersion. After 2–3 weeks of incubation at 37°C and 5% CO2, cells were fixed with methanol, stained with Giemsa, and colonies were counted upon dish inversion.

Cell Apoptosis and Cycle Analyses

Flow cytometry was employed for apoptosis and cell cycle analyses. For apoptosis, cells (5 × 10^5) were cultured for 24, 48, or 72 hours, then exposed to 2% FBS medium for 24 hours. Post-harvesting, cells were stained with 7AAD and Annexin V-FITC for 30 minutes at 4°C in darkness. Analysis was performed using an EPICS XL flow cytometer (Beckman Coulter, USA) with CellQuest software. For cell cycle analysis, cells were fixed in 70% ethanol overnight at 4°C, rinsed, and stained with PI and RNase. A FACScan flow cytometer (BD Bioscience) was used for detection. Each assay was conducted in triplicate.

Wound Healing and Transwell Assay

Cell migration was evaluated through wound healing and transwell assays. For wound healing, cells (5 × 10^5) in a 6-well plate were grown to 90% confluence, scratched with a pipette tip, and imaged at 0 and 24 hours. In transwell assays, cells (1 × 10^4) were placed in the upper chamber of a transwell setup with complete medium as chemoattractant below. After 48 hours, cells were fixed, stained with crystal violet, and counted under a microscope. Each assay was performed thrice.

Cell Coculture

A cell coculture system was established by differentiating THP-1 cells into macrophages using 100 ng/mL phorbol 12-myristate 13-acetate (PMA). After 24 hours, the macrophages were polarized into M2 phenotype by incubating with 20 ng/mL IL-4 for an additional 48 hours. For the coculture, 1 × 10^5 macrophages were suspended in 500 µL of serum-free medium and seeded onto the upper transwell chamber membrane without Matrigel coating (24-well, 8 µm; Millipore). The lower compartment was filled with 500 µL of GBM cell supernatants. Following a 24-hour incubation period, the transwell chamber was washed thrice with PBS, stained with 0.1% crystal violet for 20 minutes, air-dried, photographed, and cell counts were conducted in at least five randomly chosen fields.

Statistical Analysis

Data were analyzed with SPSS 25.0, presented as mean ± SD, and derived from a minimum of three assays. R software (Version 4.2.1) was utilized for bioinformatics. Differences between two groups were evaluated using a two-tailed Student's t-test, while one-way ANOVA with Bonferroni's test compared multiple groups. Kaplan-Meier and log-rank tests assessed survival, Spearman's test examined correlations, and Cox regression analyzed group characteristics. ROC curve and AUC analyses gauged diagnostic accuracy. Significance levels were denoted as *, P < 0.05; **, P < 0.01; ***, P < 0.001.

DNAJC1 Expression is Elevated and May Play a Role in Human GBM Tumorigenesis

To examine the expression of DNAJC1 in tumors, we analyzed data from TCGA database. Our analysis revealed a significant upregulation of DNAJC1 in 20 different tumor types, including GBM and brain lower-grade glioma (LGG), compared to adjacent normal tissues (Fig. 1A). To validate these findings, we also analyzed RNA-seq data from TCGA (Fig. 1B), GSE29796 (Fig. 1C), and CGGA databases (Fig. 1D). Consistently, the expression of DNAJC1 was significantly higher in GBM specimens compared to normal specimens, indicating a positive association between DNAJC1 and GBM.

Furthermore, we investigated the correlation between DNAJC1 and various clinical factors using the TCGA database (Table 1). Our results demonstrated that the expression of DNAJC1 gradually increased with the progression of GBM from WHO grade I to IV (Fig. 1E). Additionally, GBM specimens with IDH mutations exhibited lower levels of DNAJC1 compared to those with wild-type IDH (Fig. 1F). Moreover, DNAJC1 expression was significantly lower in GBM specimens with chromosome 1p/19q co-deletion compared to those without co-deletion (Fig. 1G). Notably, GBM showed higher levels of DNAJC1 expression compared to less malignant subtypes such as astrocytoma, oligoastrocytoma, and oligodendroglioma (Fig. 1H).

To further evaluate the expression pattern of DNAJC1 in different glioma grades, we performed immunohistochemical staining on tissue samples obtained from patients with various tumor grades as well as normal brain tissue samples. Our results demonstrated a gradual increase in DNAJC1 expression from normal cerebral cortex to astrocytoma (WHO grade II) or oligoastrocytoma (WHO grade II), and further to anaplastic astrocytoma (WHO grade III) and anaplastic oligodendroglioma (WHO grade III). Notably, the highest expression level of DNAJC1 was observed in GBM (WHO grade IV) (Fig. 1I). These findings suggest that DNAJC1 is positively associated with clinical progression and may potentially act as a tumor activator in human GBM.

DNAJC1 Serves as an Unfavorable Prognostic Indicator and a Potential Diagnostic Biomarker for Human GBM.

To evaluate the relationship between DNAJC1 expression and patient survival, we analyzed survival data obtained from the TCGA database. GBM patients were divided into two cohorts based on their DNAJC1 expression: high and low. The data, illustrated in Figs. 2A-C, show that patients with elevated DNAJC1 expression have significantly reduced overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in contrast to those with diminished expression. In scenarios involving IDH mutation, 1p/19q codeletion, or primary therapy outcome, the high-expression cohort consistently presented with poorer OS outcomes (Figs. 2D-F). Both univariate and multivariate analyses corroborated the status of DNAJC1 expression as an independent prognostic factor for GBM (Table 2). The CGGA database analysis corroborated these findings, revealing an inverse relationship between DNAJC1 expression and OS in patients with either initial or recurrent GBM (Figs. 2G-H), further supporting DNAJC1's role as a negative prognostic indicator.

In addition, receiver operating characteristic (ROC) curve analysis highlighted DNAJC1's capacity to differentiate between primary GBM tumors and normal tissue controls, with an impressive area under the curve (AUC) of 0.931 (Fig. 2I). The discriminative power of DNAJC1 was also consistent across variables such as IDH mutation status, 1p/19q codeletion status, and primary therapy outcomes, yielding AUC values of 0.709, 0.654, and 0.621, respectively (Figs. 2J-L). These findings suggest that DNAJC1 is a promising diagnostic biomarker for GBM in a clinical context.

DNAJC1 Enhances Proliferation and Suppresses Apoptosis in GBM Cells In Vitro

To elucidate DNAJC1's oncogenic function in GBM, we performed in vitro experiments using GBM cell lines. U251 cells were infected with lentiviruses carrying GFP alongside various DNAJC1-targeting shRNAs. GFP-positive cells underwent selection via puromycin (Fig. 3A). Western blot analysis revealed marked downregulation of DNAJC1 in cells treated with shDNAJC1_2 and shDNAJC1_3, and a modest reduction with shDNAJC1_1, relative to controls (Fig. 3B). For validation, we generated U87 GBM cell lines transfected with either a control vector or shDNAJC1_2. Consistent expression patterns were confirmed in both U87 and U251 cells through flow cytometry and western blotting (Figs. 3C-D).

Proliferation assays indicated that both U251 and U87 cells with DNAJC1 knockdown (shDNAJC1_2 and shDNAJC1_3) exhibited reduced growth compared to control vector-transfected cells (Figs. 3E-F). Colony formation assays showed a substantial reduction in colony numbers in DNAJC1-silenced U251 cells (Figs. 3G-H). Cell cycle analysis indicated a decrease in S-phase fraction and an increase in G0/G1 and G2 phases upon DNAJC1 silencing in both cell lines (Figs. 3I-L). Furthermore, apoptotic rates increased in a time-dependent manner following DNAJC1 downregulation in serum-deprived U251 (Figs. 3M-N) and U87 (Figs. 3O-P) cells, as determined by flow cytometry with Annexin V-FITC and 7AAD staining. Collectively, these data imply that DNAJC1 facilitates GBM cell proliferation, expedites cell cycle progression, and inhibits apoptosis in vitro.

DNAJC1 Influences ECM Organization and Immune Response in the GBM Microenvironment

To decipher DNAJC1's role in the evolution and advancement of GBM, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses on GBM samples exhibiting elevated DNAJC1 expression. The analysis presented in Fig. 4A identified significant enrichment in biological functions related to the extracellular matrix structural constituent (GO-MF), the immunoglobulin complex (GO-CC), and complement activation (GO-BP). The KEGG pathway analysis revealed a notable correlation of the DNAJC1-associated gene cluster with pivotal processes and pathways, including the PI3K/AKT signaling pathway, cytokine-cytokine receptor interaction, focal adhesion, ECM-receptor interaction, and the IL-17 signaling pathway (Fig. 4B). These data point to DNAJC1's possible involvement in modulating the ECM and immune response within the GBM microenvironment.

Gene set enrichment analysis (GSEA) further corroborated these observations in the high DNAJC1 expression sample subset. We focused on gene clusters meeting the threshold of a nominal p-value (NOM) < 0.05 and a false discovery rate (FDR) q < 0.25, calculating the normalized enrichment score (NES) to ascertain specific pathway enrichments. The analysis underscored significant enrichment in genes related to MET PROMOTES CELL MOTILITY (Fig. 4C), COLLAGENS (Fig. 4D), and ECM REGULATORS (Fig. 4E), reinforcing DNAJC1's critical role in ECM modulation. Additionally, the enriched gene cluster was associated with pathways pertinent to INITIAL TRIGGERING OF COMPLEMENT (Fig. 4F), PD-1 SIGNALING (Fig. 4G), and INTERLEUKIN 10 SIGNALING (Fig. 4H), illustrating DNAJC1's intimate connection with immune response mechanisms within the GBM microenvironment. Notably, DNAJC1 was found to negatively regulate neuronal system development and was enriched in associated pathways such as NEURONAL SYSTEM (Fig. 4I), DOPAMINE NEUROTRANSMITTER RELEASE (Fig. 4J), and NEUROACTIVE LIGAND-RECEPTOR INTERACTION (Fig. 4K). Collectively, the evidence emphasizes DNAJC1's central role in orchestrating ECM organization and immune dynamics, thereby promoting an environment conducive to GBM progression, invasion, and immune escape.

DNAJC1 Knockdown Inhibits GBM Cell Migration and EMT

Reflecting on the GO and GSEA results that linked DNAJC1 expression to ECM organization, we explored the gene's effects on GBM cell motility. Wound healing assays demonstrated a marked reduction in migratory capabilities of U251 and U87 cells with silenced DNAJC1 (Figs. 5A-D). Similarly, transwell assays indicated fewer migrating cells on the membranes of the lower chambers when DNAJC1 expression was inhibited in U251 and U87 cells (Figs. 5E-H). These results robustly suggest that DNAJC1 is instrumental in promoting GBM cell migration in vitro.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the metastasis of various solid tumors. We assessed the role of DNAJC1 in EMT using quantitative real-time PCR (qRT-PCR) to measure the impact of DNAJC1 knockdown on EMT marker expression in U251 cells. A significant reduction in the mRNA expression of EMT markers, including Vimentin, FN1, ETS1, SNAI1, and ZEB1, was noted following DNAJC1 suppression (Fig. 5I). Western blot analysis corroborated these findings, showing an upregulation of the epithelial marker E-cadherin and downregulation of the mesenchymal marker Vimentin in U251 cells with reduced DNAJC1 levels (Fig. 5J). These results affirm DNAJC1's role in EMT activation, thereby contributing to the migratory and invasive behavior of GBM cells.

DNAJC1 Augments Immunosuppressive Cell Infiltration in the GBM Microenvironment

Our investigation delved into the role of DNAJC1 in modulating immune cell infiltration within GBM. A distinct link was established between DNAJC1 expression and the presence of immunosuppressive cell types, such as macrophages, neutrophils, and T helper type 2 (Th2) cells, known to facilitate GBM tumorigenesis within the tumor microenvironment (TME) [references 19–21]. Positive correlations were observed between DNAJC1 expression and macrophage (r = 0.485, P < 0.001), neutrophil (r = 0.394, P < 0.001), and Th2 cell (r = 0.485, P < 0.001) infiltration (Figs. 6B-D). Notably, groups with high DNAJC1 expression had significantly greater enrichment of macrophages (Fig. 6G), neutrophils (Fig. 6H), and Th2 cells (Fig. 6I) when compared to low-expression groups. These results point to DNAJC1's involvement in the recruitment of pro-tumorigenic immune cells within the GBM microenvironment. In contrast, there was no discernible association between DNAJC1 expression and the infiltration of anti-tumorigenic immune cells, such as CD8 + T cells and Th1 cells, which were similarly distributed across both high-expression and low-expression DNAJC1 groups (Figs. 6A, E, F, J, K). Thus, our data underscore DNAJC1's role in fostering the infiltration of immune cells that support GBM tumorigenesis.

DNAJC1 Knockdown Reduces Macrophage Infiltration and M2 Macrophage Marker CD163 Expression In Vitro

Further investigating the interplay between DNAJC1 expression and macrophage infiltration, we employed a coculture system consisting of GBM cells and macrophages. When macrophages were cocultured with U251 and U87 cells with DNAJC1 knockdown (shDNAJC1_2 and shDNAJC1_3 for U251, shDNAJC1_2 for U87), we noted fewer labeled macrophages on the lower chamber membranes relative to control cells (Figs. 7A-D), aligning with our bioinformatic predictions (Figs. 6A and 6B). This suggests DNAJC1's potential role in enhancing macrophage infiltration within the GBM microenvironment. Additionally, DNAJC1 suppression led to a decrease in the M2 macrophage marker CD163 (Figs. 7E and 7F), implying that GBM cell-derived DNAJC1 expression may be linked to the immunosuppressive M2 phenotype in macrophages.

The diagnostic paradigm for GBM has evolved to integrate molecular profiling alongside traditional histopathological examination. Clinically, molecular markers such as IDH mutations and 1p/19q codeletion are now routinely utilized for GBM diagnosis, prognosis assessment, and therapeutic decision-making¹⁹. IDH, a critical enzyme in the Krebs cycle, catalyzes the conversion of isocitric acid into α-ketoglutarate (α-KG) and CO2, essential for energy production and biosynthetic precursor synthesis²⁰. Typically occurring in the early phases of glioma development, IDH mutations are prevalent in oligodendroglioma, astrocytoma, and secondary GBM, but rare in primary GBM²¹. Patients with IDH-mutant GBM generally have a protracted disease course and improved outcomes compared to those with wild-type IDH²². The codeletion of the short arm of chromosome 1 and the long arm of chromosome 19 is often found in younger glioma patients²³. The WHO's classification system identifies the 1p/19q codeletion as a distinctive marker for oligodendroglioma²⁴. As these chromosomal regions harbor essential genes for cellular growth and differentiation, this codeletion can inhibit GBM cell proliferation and vasculature formation, enhancing response to chemotherapy and ultimately leading to better patient prognoses²⁵.

Nonetheless, despite the employment of molecular markers such as IDH mutations and 1p/19q codeletion in the clinical management of GBM, patient outcomes remain dismal, with median survival rates of 14–17 months⁵. Bioinformatic analysis of public databases has uncovered numerous novel differentially expressed genes that hold significant potential for improving GBM diagnostics and treatment strategies^26–28.

This study commenced with an analysis of the TCGA database to ascertain the expression pattern of DNAJC1. The gene was significantly upregulated in at least 20 tumor subtypes, with notably high levels in GBM or LGG, suggesting a potential oncogenic role for DNAJC1. Further examination of TCGA, CGGA, and GEO databases corroborated the elevated expression of DNAJC1 in GBM tissues, which increased concomitantly with higher WHO tumor grades. Additionally, DNAJC1 expression was inversely correlated with both IDH mutation and 1p/19q codeletion. Kaplan–Meier survival analysis revealed that higher DNAJC1 expression was associated with poorer OS, DSS, and PFI in GBM patients. Conversely, patients with lower DNAJC1 expression displayed enhanced responses to chemotherapy, with higher complete response (CR), partial response (PR), and stable disease (SD) rates. These findings underscore DNAJC1's potential as a prognostic biomarker and its diagnostic relevance in GBM.

The TME is instrumental in GBM growth and progression and consists of immune and non-immune elements that interact with tumor cells^{19, 29}. Components such as collagen, laminin, and fibronectin are integral to the TME structure, alongside extracellular matrix elements, cancer-associated fibroblasts (CAFs), and immunosuppressive cytokines like IL-10 and IL-17^30–32. Our analyses via GO, KEGG, and GSEA identified DNAJC1's involvement in extracellular matrix organization and collagen binding within the GBM TME. DNAJC1 also regulates complement and cytokine signaling, including IL-10, IL-17, and PD-1 pathways, and is associated with EMT. The suppression of DNAJC1 results in decreased EMT marker expression, suggesting its role in promoting EMT and thereby enhancing GBM cell invasion and migration.

Moreover, the infiltration of immune cells into the TME is critical in gliomagenesis³³. Macrophages, particularly those with the M2 phenotype, dominate the inflammatory cell population within tumors and facilitate tumor progression by promoting growth, angiogenesis, migration, and resistance to various therapies^34–36. Th2 cells and immature neutrophils in the TME contribute to an immunosuppressive milieu that aids tumor immune evasion^{37, 38}. In our research, we found that elevated DNAJC1 expression is positively associated with the infiltration of macrophages, neutrophils, and Th2 cells into the glioma TME. Inhibition of DNAJC1 led to reduced expression of the M2 macrophage marker CD163. Collectively, these findings support DNAJC1's role in gliomagenesis through the recruitment of immunosuppressive cells into the TME.

In conclusion, bioinformatic analysis reveals that DNAJC1 exhibits high expression levels in human GBM specimens with a strong correlation to clinical prognostic outcomes. DNAJC1 is implicated in enhancing proliferation, migration, and the recruitment of immunosuppressive macrophages in glioblastoma, and its association with the expression of immunosuppressive molecules, such as CTLA-4, PD-1, and PD-L1, suggests a role in immune cell inhibition and tumor immune evasion. It appears that DNAJC1 may facilitate GBM growth and progression within the TME by supporting immune escape mechanisms and drug resistance, potentially through promoting the epithelial-mesenchymal transition (EMT) process and upregulating immune inhibitory signaling.

Clinically, DNAJC1 holds promise as a prognostic marker and diagnostic biomarker for GBM. However, the complexity and heterogeneity of GBM necessitate further clinical validation and translational research. Personalized therapy, tailored to individual patient subtypes and clinical features, represents an important avenue for future GBM treatment strategies. The combined use of DNAJC1 with other molecular markers and clinical indicators may lead to more customized treatment approaches.

Our study, through a blend of bioinformatic inquiry and clinical data, contributes to a nuanced understanding of DNAJC1's function in gliomas. Notwithstanding, reliance on public databases could introduce bias, and in vitro loss-of-function experiments may not fully capture the complexity of human gliomas. Consequently, additional research employing animal models is imperative. Overall, our research underscores DNAJC1's pivotal role in glioma pathogenesis and underscores its potential as a significant prognostic biomarker and a candidate for targeted therapy, opening new pathways for advancing glioma diagnostics and treatment.

Ethics approval and consent to participate: The research was approved by the Institutional Animal Care and Use Committee of the School of Medicine, Fourth Military Medical University.

Consent for publication：All subjects provided written informed consent.

Data Availability Statement: The original contributions presented in the study are included in the article file, and further inquiries can be directed to the corresponding author.

Competing interests: The authors declare no conflict of interest

Funding: This research was funded by grants from the National Natural Science Foundation of China, grant number 82073361 and 82203274; the State Key Laboratory of Cancer Biology Project, grant number CBSKL2022ZZ21; the Key R&D Plan of Shaanxi Province, grant number 2023-YBSF-667; the Xi'an Municipal Health Commission grant, grant number 2022ms06.

Author Contributions: Conceptualization, T.W., L.S. and X.C.; methodology, H.Z., W.Z. and L.S.; validation, H.Z., Y.H. and H.L.; formal analysis, W.Z.; investigation, T.W and L.S.; data curation, H.Z. and Y.H.; writing—original draft preparation, H.Z. and X.C.; writing—review and editing, T.W.; supervision, T.W and L.S.; project administration, T.W and L.S.; funding acquisition, T.W., L.S. and X.C. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: We would like to thank American Journal Experts (www.aje.cn) for its linguistic assistance during the preparation of this manuscript. We also would like to thank Dr. Jing Ye from Department of Pathology, Fourth Military Medical University for his help in immunohistochemical staining.

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Tables 1 to 3 are available in the Supplementary Files section.

No competing interests reported.

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DNAJC1 Facilitates Glioblastoma Progression by Promoting Extracellular Matrix Reorganization and Macrophage Infiltration

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Datasets

Bioinformatics Analysis

Immunohistochemistry on Human Glioma Specimens

Cell Culture

Plasmid Construction and Lentivirus Production

Protein Isolation and Western Blot Analysis

Growth Curve Assay

Colony Formation Assay

Cell Apoptosis and Cycle Analyses

Wound Healing and Transwell Assay

Cell Coculture

Statistical Analysis

Results

DNAJC1 Expression is Elevated and May Play a Role in Human GBM Tumorigenesis

DNAJC1 Enhances Proliferation and Suppresses Apoptosis in GBM Cells In Vitro

DNAJC1 Influences ECM Organization and Immune Response in the GBM Microenvironment

DNAJC1 Knockdown Inhibits GBM Cell Migration and EMT

DNAJC1 Augments Immunosuppressive Cell Infiltration in the GBM Microenvironment

DNAJC1 Knockdown Reduces Macrophage Infiltration and M2 Macrophage Marker CD163 Expression In Vitro

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1