DNAJC1 facilitates glioblastoma progression by promoting extracellular matrix reorganization and macrophage infiltration

Abstract

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.

Keywords: DNAJC1; Extracellular matrix reorganization; Glioblastoma; Macrophage infiltration; Prognostic biomarker.

Fig. 1

Correlation between DNAJC1 expression and clinicopathological characteristics of GBM. A DNAJC1 expression in different cancers from the TCGA database. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, low grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma. The expression of DNAJC1 in GBM samples and normal tissues from B TCGA, C GSE29796 or D CGGA. The expression of DNAJC1 in different E glioma grades, F IDH mutant status, G chromosome 1p/19q codeletion or H histological types. I Representative immunohistochemical staining of DNAJC1 in different histological gliomas using a tissue chip. * P < 0.05; ** P < 0.01; *** P < 0.001

Fig. 2

Correlation between DNAJC1 expression and GBM survival prognosis. Correlation between DNAJC1 expression and A overall survival, B disease-specific survival or C progression-free interval in GBM patients. Correlation between DNAJC1 expression and the overall survival of D IDH mutation status, E chromosome 1p/19q noncodeletion or F primary therapy outcome in GBM patients. Correlation between DNAJC1 expression and the overall survival of GBM patients with G primary tumors or H recurrent tumors. Receiver operating characteristic (ROC) curve analyses were used to distinguish DNAJC1 expression in I primary GBM tumors, J IDH mutation, K 1p/19q codeletion or L primary therapy outcome in GBM patients

Fig. 3

DNAJC1 silencing inhibits proliferation and promotes apoptosis in GBM cells. A–D Establishment of GMB cell lines stably expressing control shRNA or DNAJC1-targeting shRNA. U251 and U87 cells were transduced with lentivirus encoding GFP and shRNA. Puromycin (2 μg/mL) was added to the culture medium 72 h post-virus infection, and the cells were cultured for another week to obtain stable GFP- and shRNA-expressing cell lines. Flow cytometry analysis of GFP-positive A U251 cells and C U87 cells was performed. Western blot analysis was used to detect DNAJC1 expression in DNAJC1- silencing B U251 cells and D U87 cells. E–H DNAJC1 silencing inhibits GBM cell growth. A growth curve assay was performed with DNAJC1- silencing E U251 cells and F U87 cells. G Colony formation assay and H statistical analysis were performed with DNAJC1- silencing U251 cells. Cell cycle experiments and statistical analyses were performed in DNAJC1- silencing I,J U251 cells and K,L U87 cells. M–P U251 or U87 cells were cultured in medium containing 2% FBS for 24 h, 48 h, or 72 h before harvesting. Flow cytometry analysis was performed with Annexin V-FITC and 7AAD staining to detect apoptosis at 72 h (M, O) or over a period of time (N, P) after serum deprivation

Fig. 4

Functional enrichment analysis of DNAJC1 in GBM from the TCGA database. A Enriched Gene Ontology terms of DNAJC1 coexpression genes in GBM based on biological processes, cellular components, and molecular function. B KEGG analysis of GBM patients with high DNAJC1 expression. Enrichment plots from GSEA, including C MET promotes the cell motility pathway, D collagens pathway, E ECM regulators pathway, F initial triggering of the complement pathway, G PD-1 signaling pathway, H interleukin 10 signaling pathway, I neuronal system pathway, J dopamine neurotransmitter release pathway, and K neuroactive ligand receptor interaction pathway

Fig. 5

DNAJC1 promotes the migration of GBM cells and induces the EMT process. Wound healing experiments were conducted along with statistical analysis to assess the migratory capacity of DNAJC1- silencing A,B U251 cells and C,D U87 cells. The migration ability of E,F U251 cells and G,H U87 cells was examined using the transwell assay. I qRT-PCR analysis was performed to assess the expression of EMT markers in U251 cells with DNAJC1 knocked down and control cells. J Western blot analysis was conducted to examine the expression of the epithelial marker E-cadherin and the mesenchymal marker Vimentin in U251 cells with DNAJC1 knocked down and control cells

Fig. 6

Correlation analysis between DNAJC1 expression and macrophage infiltration in GBM. A Correlation between DNAJC1 expression and 24 immune cells. Relationship between DNAJC1 expression level and GBM infiltrating B macrophages, C neutrophils, D Eosinophils, E Th2 cells, F CD8⁺ T cells, and G Th1 cells. H–K Coculture and recruitment experiments were performed using GBM cells and macrophages. Macrophages, differentiated from THP-1 cells by PMA, were seeded in the upper transwell chambers, while DNAJC1- silencing H,I U251 cells or J,K U87 cells were placed in the lower transwell chambers. Following 24 h of incubation, macrophages that migrated through the chamber membrane were stained with 0.1% crystal violet and counted for statistical analysis. L,M Flow cytometry was employed to analyze the expression of the M2 marker CD163 on macrophages after coculture with DNAJC1-silenced U251 cells or control cells