DNAJC24 suppresses breast cancer malignancy and serves as a prognostic biomarker

Abstract

Background

Based on the current markers, numerous targeted therapies have been put forward for clinical application; however, treatment resistance and recurrence still remain the main causes of breast cancer-related mortality. In addition, breast cancer exhibits significant heterogeneity, and patients with apparently similar tumor subtypes exhibit variable responses to identical drug treatments. Therefore, accurate prediction of breast cancer progression and personalized treatment plans will maximize patient benefit by avoiding overtreatment and undertreatment.

Methods

In this study, our emphasis was placed on exploring the function of DnaJ heat shock protein family member C24 (DNAJC24, also known as DPH4) in breast cancer through bioinformatic analysis by using public databases and clinicopathological samples. We performed in vitro functional assays to explore the biological roles of DNAJC24.

Results

Bioinformatics analysis of TCGA data demonstrated significantly lower DNAJC24 expression in breast cancer tissues compared to adjacent paracancer tissues (p < 0.001). Immunohistochemistry showed low DNAJC24 expression in 72.4% (210/290) of breast cancer tissues versus 50.8% (134/264) of adjacent paracancer tissues. DNAJC24 expression decreased progressively with advanced clinical stages (p < 0.05), was lowest in HER2-enriched subtype, and highest in Luminal-A subtype. Overexpression of DNAJC24 in breast cancer cells significantly reduced colony formation (p < 0.05), proliferation rates, chemotaxis (p < 0.05), invasion (p < 0.05), and migration abilities (p < 0.05) compared to controls. Conversely, DNAJC24 knockdown in cancer cells produced opposite effects, significantly enhancing chemotaxis, invasion, and migration (all p < 0.05).

Conclusions

Lower DNAJC24 expression is strongly associated with breast cancer malignancy, advanced clinical stages, aggressive molecular subtypes, and poorer prognosis. Functionally, DNAJC24 inhibits proliferation, invasion, and migration of breast cancer cells, underscoring its potential as a prognostic biomarker in breast cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-03918-4.

Introduction

In terms of cancer incidence among women, breast cancer is the most common worldwide [1]. Studies have shown that early diagnosis, radiotherapy, and chemotherapy can considerably enhance the prognosis of patients suffering from breast carcinoma. Unfortunately, an extremely high proportion of breast cancer cases progress to distant metastasis and recurrence within a short time after clinical diagnosis [2]. The identification of crucial regulatory genes related to the onset and development of breast carcinoma at the molecular level, the elucidation of the molecular mechanism underlying the pathogenesis of breast carcinoma and invasion, the discovery of reliable and highly specific diagnostic and prognostic biomarkers, and the persistent search for new targets for the advancement of new treatments are essential for decreasing the prevalence and death rate of breast cancer.

One such gene that may be contribute to breast cancer onset and progression is DnaJ heat shock protein family member C24 (DNAJC24). Diphthamide is a post-translational modification of eEF2 that is essential for translational accuracy and cell growth. Mutations or inhibition of diphthamide synthesis have been shown to be linked to cancer development. Eukaryotic elongation factor-2 (eEF2) undergoes diphtheria amidation modification by DNAJC24, a prominent member of the dipthamide biosynthesis protein (DPH) family and commonly known as DPH4 [3, 4]. Diphtheria amidation modification occurs at the His715 site of eEF2, a specific modification on the histidine residue of the eEF2 protein that is widely found in archaea and eukaryotes, including humans. The biological role of DNAJC24, which has been found to be associated with diphtheria amide, but has received relatively minimal attention. However, one study identified a link between DNAJC24 and tumor necrosis factor (TNF)-mediated apoptotic sensitivity in breast cancer cells [5]. Our previous research originally affirmed that DNAJC24 has a considerable impact on proliferation and migration of hepatocellular carcinoma(HCC) cells by affecting ammonia metabolism [6]. Regardless, the part played by DNAJC24 in the occurrence and advancement of breast cancer or other cancers has not yet been reported hitherto.

In the current research, we initially investigated the expression of DNAJC24 in various breast tumor cell lines, established the knockdown(KD) and overexpression(OE) cell lines of DNAJC24 in breast tumor cells, and preliminarily evaluated the effect of DNAJC24 protein on breast cancer. The preliminary investigation of DNAJC24 protein on breast cancer cell proliferation, invasion, and migration indicates its anticancer biological function, which is consistent with previous bioinformatics and immunohistochemistry studies. In addition, the downregulation or upregulation of its expression is highly related to the advancement of breast cancer, providing a theoretical basis and foundation for the development of new tumor markers for breast cancer.

Materials and methods

Human breast cancer tissue samples

In the research, samples from patients who were undergoing surgical resection of breast carcinoma were collected from the Department of Breast Pathology, Cancer Hospital of Tianjin Medical University from 2010 to 2013. The tumor samples from all the patients were confirmed as breast cancer via pathological identification. Cancer tissues and paired adjacent tissues were collected separately and then sliced after paraffin embedding. The immunohistochemical results were scored by two experienced breast pathologists. This study was reviewed and approved by the Ethics Committee of Tianjin Medical University (NO.bc20240204).

Immunohistochemistry

Immunohistochemistry (IHC) staining of DNAJC24 was meticulously conducted in a comprehensive collection of 290 pairs of cancerous tissue and the corresponding paracancer tissues (collected from histologically confirmed paracancer-appearing breast tissue located at least 1 cm beyond the tumor margin, with confirmation of being tumor-free through pathological examination). The breast cancer samples were infiltrated with xylene and ethanol of a gradient concentration. Subsequently, a crucial step of antigen retrieval was performed in citrate. The endogenous peroxidase activity was effectively inhibited by 3% H₂O₂ for a duration of 10 min at a stable room temperature. The samples reacted with specific antibodies, namely rabbit anti-DNAJC24 (1:50, Abcam, ab246925) for 30 min at room temperature and throughout the night at 4 °C. Subsequently, they were carefully maintained at room temperature for a duration of 30 min. After that, they were stored overnight at a precisely controlled temperature of 4 °C. Next, they were rinsed multiple times using freshly prepared phosphate-buffered saline (PBS) to ensure the removal of any remaining impurities or unbound substances. Subsequently, they were precisely stained with a secondary antibody for a full hour at a consistently maintained room temperature. The reaction products were treated with a 3,3-diaminobenzidine solution (ZSGB-Bio, ZLI-9017) for the purpose of color development. Subsequently, they were counterstained with hematoxylin in a meticulous manner to enhance the visualization and clarity of the samples. The final immunohistochemistry score of DNAJC24 was obtained by multiplying the staining intensity score and the percentage score. A final staining score of equal to or greater than 1 was regarded as an indication of high expression of DNAJC24. On the contrary, a final staining score that was less than 1 was considered as an indication of low expression of DNAJC24. DNAJC24 expression was evaluated using a standardized immunohistochemical scoring system that combines both staining intensity and positive cell percentage. The staining intensity was classified into four categories: 0 (negative, no detectable immune response), 1 (weak positive, indicating minor immune activity), 2 (moderate intensity, representing notable immune reaction), and 3 (strong positive, showing vigorous immune activation). The positive cell percentage was graded as: 1 (1–30% positive cells), 2 (30–60% positive cells), or 3 (> 60% positive cells). The final immunohistochemical score (IHC score) was calculated by multiplying the intensity score (0–3) by the positive rate score (1–3), yielding a range of 0–9 points. For statistical analysis, samples were categorized into low expression (0–6 points) and high expression (7–9 points) groups, with this dichotomization providing optimal discrimination of biological and clinical differences in DNAJC24 expression patterns. Representative photomicrographs of immunostaining were acquired on a light microscope (Olympus BX61). Staining assessment was performed in a blinded manner by two pathologists using a semi-quantitative assay.

Cell culture

In this research, the four human breast cancer cell lines—T47D, MDA-MB-231, SKBR3, and MCF-7—were all sourced from the American Type Culture Collection (ATCC) and supplied by the Department of Tumor Cell Biology at the Cancer Research Institute, Tianjin Cancer Hospital. The cell lines used in this study were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA), supplemented with 10% v/v fetal bovine serum (FBS; HyClone Laboratories Inc., Novato, CA, USA) and 1% penicillin–streptomycin solution (PS; HyClone Laboratories Inc.), under controlled conditions at a stable temperature of 37 °C in an incubator maintained with 5% CO2.

Cell transfection

Plasmids for packaging, specifically VSVG and ΔR, as well as expression plasmids such as OE, KD, and their respective controls, were carefully introduced into HEK293T cells using Lipofectamine 2000, a reagent obtained from Invitrogen based in Carlsbad, CA, USA. After a period of exactly 48 h, the supernatant of the medium was carefully collected through a meticulous process in order to successfully acquire the lentiviral particles. Lentivirus containing short hairpin RNA (shRNA) targeting DNAJC24 was used to infect T47D and MDA-MB-231 cells, resulting in the establishment of cell lines for DNAJC24 KD, OE, and their respective scrambled controls (SCR). Stably transfected cells were chosen with specific medium containing puromycin (Gibco). The efficiency of transfection was verified by the widely recognized Western blotting.

Western blot analysis

The cells cultivated in a 10 cm dish were meticulously rinsed 3 times by employing ice-cold phosphate-buffered saline (PBS) with a pH value of 6.8. Subsequently, the cells were lysed with 1 × sodium dodecyl sulfate (SDS) lysis buffer, which consisted of 2% SDS, Tris–HCl with a pH of 6.8 and a concentration of 62.5 mM, as well as 10% glycerol. To this lysis buffer, 1 mM of Na3VO4, 1 mM of NaF, 1 × protease, and a comprehensive cocktail of phosphatase inhibitor sourced from Hoffman-la Roche Ltd. based in Basel, Switzerland, were added. The lysis was conducted on ice for a duration of 30 min, allowing for the efficient disruption of the cells and the release of intracellular components. The protein was denatured by a metal bath at 95 ℃ for ten minutes. Cell lysates were kept at − 20 ℃. Equal amounts of protein, between 30 and 90 μg per cell line, were meticulously loaded and separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Afterward, the samples were carefully transferred to the polyvinylidene difluoride membrane (hydrophobic) (Merck KGaA, Darmstadt, Germany) to maintain the integrity and quality of the protein for subsequent analysis and experimentation. The following antibodies against specific proteins were employed in the experiment: anti-DNAJC24 (with a dilution ratio of 1:500), which was sourced from Abcam; anti-glyceraldehyde 3-phosphate dehydrogenase (1:1000), the secondary antibody of goat anti-rabbit (1:4000), and goat anti-mouse (1:4000) from Santa Cruz Biotechnology.

Colony formation assay

In this experiment, 1 × 10³ cells and DMEM containing 10% FBS were added to 6-well plates. Following a 2-week incubation period, the remaining clonal clusters were carefully fixed and then subjected to staining with 0.5% crystal violet. Subsequently, they were precisely photographed and counted. Results of counting were displayed in the form of the means ± standard deviation (SD) of triplicate dishes with the same treatment.

Cell proliferation assay

Cells were seeded in 96-well plates, ensuring 1 × 10³ cells/well initially. Then, 5 duplicate wells were established for each cell line. This approach was adopted to minimize any potential differences that might occur in a group. Subsequently, the 96-well plates were subjected to incubation with Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan; 10 μL/well) for a period of 2 h at a controlled temperature of 37 °C and a stable environment with 5% CO₂. On day 0, the CCK8 solution should be added approximately 4 h after seeding (just before proliferation). The absorbance was determined by enzyme-linked immunosorbent assay reader (BioTek SynergyH1, Burlington, VT, USA). CCK-8 absorbance (450 nm) was background-corrected by subtracting the blank control and normalized to Day 0 (Fold = 1). Cell growth (Fold) was calculated as: (Corrected OD_{Day n})/(Corrected OD_{Day 0}).

Chemotaxis assay

DMEM supplemented with 20% FBS (600 μL per chamber) was carefully added to the lower chamber. Subsequently, the freshly prepared cell suspension was placed in the upper chamber at a density of 1 × 10⁵ cells in a volume of 200 μL per chamber. Following incubation at 37 °C in 5% CO₂, the incubation period was set at 4 h for MDA-MB-231 and 6 h for T47D. The bottom of the upper chamber was carefully washed with PBS multiple times to ensure thorough cleaning. Subsequently, it was fixed with 4% paraformaldehyde and stained with crystal violet. Three randomly chosen fields were photographed under a high-resolution light microscope (Olympus BX61). The counting of the migrated cells was carried out at a magnification of × 200.

Matrigel invasion assay

In this assay, we added matrigel to the bottom of upper transwell chamber and inserted it into a 24-well plate, which was then left at 37 °C to solidify over one hour. DMEM containing 20% FBS (200 μL) was added to the lower chamber, 50 μL of cell suspension (2 × 10⁵ cells) in serum-free DMEM were placed in the upper chamber. After being incubated for 24 h under suitable conditions, the 24-well plate was fixed, stained, and finally counted by light microscopy (Olympus BX61).

Scratch assay

One day before treatment, cells were seeded in 6-well plates(1 × 10⁶ cells/well) to achieve confluent monolayers. Note that the medium used was DMEM without FBS. A small pipette tip was utilized to draw a uniform wound. Three sites were selected to measure the wound width, and the time points of measurement were after 0, 3, 6, 9, 12, and 24 h. The data are presented in the form of means ± SD, while representative images were captured at the initial and final time points.

Statistical analysis

Statistical analysis was performed using SPSS 26.0. Survival curves compared by log-rank test. Continuous variables analyzed via Student’s t-test or ANOVA. Categorical variables assessed by χ² test. Multivariate Cox regression adjusted for age, stage, and receptor status. Statistical significance was defined as p < 0.05.

Results

Expression of DNAJC24 in cancer tissues is different from that in paracancer tissues

Analysis of The Cancer Genome Atlas (TCGA) database indicated that the mRNA level of DNAJC24 in tissues of carcinoma, including breast carcinoma, pancreatic carcinoma, prostate cancer, thymus cancer, and endometrial cancer was lower than that of adjacent paracancer tissues (Fig. 1A). In accordance with the screening conditions, the raw data of 841 cases of female breast cancer were downloaded from the TCGA database. These samples included 104 pairs of paracancer tissues and paired adjacent tissues, and 737 cases of malignant tumor samples. After data analysis, 7 patients had a paracancerous sample that corresponded to multiple cancer tissue samples. By querying the original data description, multipoint sampling and sequencing were considered. In addition, the DNAJC24 expression of 1 case was more than 3 times higher than the average level of the remaining 96 patients, and thus, this outlier was discarded. Finally, 96 patients were included in the calculation of follow-up data. The expression level of DNAJC24 in the relevant cancer tissues was notably lower than that in the adjacent tissues (p < 0.001) (Fig. 1B, left). Similar results were obtained when comparing paracancer tissue samples with malignant tumor samples in which the expression level of DNAJC24 in cancerous tissues was markedly lower than that in paracancer ones, p < 0.001 (Fig. 1B, right). Patient age, primary site (left breast or right breast), pathological type (intraductal or lobular breast cancer), ER, PR, HER2, and TNM stage were downloaded from the database that corresponded to the transcriptome data. The expression of DNAJC24 is significantly different in PAM50 molecular subtypes of breast cancer. The expression level of DNAJC24 in HER2-enriched breast cancer is the lowest, while it is higher in Luminal-A and Luminal-B than that of basal-like and HER2E subtypes, and highest in Luminal-A. (Fig. 1C).

Fig. 1.

Expression of DNAJC24 in cancer tissues is different from paracancer tissues. A The expression level of DNAJC24 in cancer and paracancer tissues of 5 malignant tumors including breast cancer by TCGA database analysis. B DNAJC24 mRNA expression level: breast cancer tissues versus paired paracancer tissues (left), breast cancer tissues versus Paracancer tissues (right). C Expression of DNAJC24 in different PAM50 molecular subtypes of breast cancer. D Expression level of DNAJC24 in different clinical stages of breast cancer. E Correlation analysis of DNAJC24 expression with PR, ER and HER2 expression. F Analysis of DNAJC24 expression and disease-free survival of breast cancer patients (the right figure shows disease-free survival of more than 7 years).The values were shown as mean ± SEM, p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***, p ≤ 0.0001 ****, p > 0.05 ns(not significant)

At present, the TNM staging system is the most commonly used staging system for malignant tumors. Through analysis, the expression of DNAJC24 was found to decrease gradually with tumor progression, irrespective of the T stage, the N stage, or the TNM stage, showing a statistical difference (p < 0.05) (Fig. 1D). In addition, PR, ER, and HER2 are among the most important immunohistochemical indicators of breast carcinoma. In this research, patients were separated into negative and positive groups according to the IHC expression levels of the three aforementioned indicators (all data were derived from the TCGA database). Whether differences in DNAJC24 expression levels exist between the two groups was analyzed via t-test. The results revealed that the level of DNAJC24 was higher in the ER-positive and PR-positive groups compared to the negative group. However, the trend was the opposite in the HER2-positive group. In contrast to the negative group, DNAJC24 was downregulated in the HER2-positive group, p < 0.05 (Fig. 1E). Finally, Kaplan–Meier survival analysis showed that DNAJC24 generally exerted no significant effect on relapse-free survival (DFS). Interestingly, in patients with long DFS (i.e., more than 7 years), DNAJC24 was significant in predicting the risk of relapse, and the higher the expression level, the lower the risk of relapse (p = 0.032) (Fig. 1F). Generally, the results of the database analysis indicated that the mRNA level of DNAJC24 was notably higher in adjacent tissues as opposed to cancerous tissues. Moreover, the expression of DNAJC24 was diverse in different pathological and molecular types. The DNAJC24 expression level of luminal breast cancer was higher than that of HER2-positive and basal-like breast cancer. Moreover, minimal variation of DNAJC24 expression was observed between cancer and paracancer in the two tumor types. In addition, DNAJC24 levels gradually decreased with tumor progression, regardless of T stage, N stage, or TNM stage. Among breast cancer patients who have been free of recurrence for a long time (i.e., more than 7 years), a higher expression of DNAJC24 is frequently related to a lower risk of recurrence.

Higher expression of DNAJC24 is related to better prognosis in breast carcinoma tissues

For this research, samples from patients who underwent surgical resection for breast carcinoma were collected from the Department of Breast Pathology, Cancer Hospital of Tianjin Medical University from 2010 to 2013. Tumor samples from all the patients were confirmed as breast cancer through pathological identification. Cancer tissues and adjacent tissues were collected separately, and then sectioned after paraffin embedding. The immunohistochemical staining of 290 female breast cancer patients showed that DNAJC24-specific immune response particles were mostly localized in the cytoplasm. The expression level of DNAJC24 in breast cancer was classified into four grades: negative, low, medium, and high (Fig. 2A). For comparison, triple-negative breast cancer has the lowest expression (Fig. 2B). Furthermore, the proportion of low DNAJC24 expression was 72.4% (210/290) in breast cancer tissues, corresponding to 50.8% (134/264) in paracancer tissues (Fig. 2C). That is, the level of DNAJC24 in paracancer tissues was consistently observed to be higher compared to corresponding cancer tissues. The finding was in accordance with the analysis of TCGA database. In breast carcinoma patients lacking ER expression (Fig. 2D, E) and those without PR expression (Fig. 2F, G), a positive correlation was observed between the expression of DNAJC24 and both overall survival (OS) and disease-free survival (DFS). Consistent results were found across the overall cohort, where DNAJC24 expression was similarly associated with favorable OS and DFS outcomes for the patients.

Fig. 2.

DNAJC24 is associated with the better prognosis. A The expression of DNAJC24 in breast cancer tissues was divided into four categories: negative, low, moderate and high. B Expression level of DNAJC24 in different types of breast cancer. C The proportion of high expression of DNAJC24 in tumor tissues was lower than that in paracancer tissues. D Relationship between DNAJC24 expression and OS in ER- patients. E Relationship between DNAJC24 expression and DFS in ER- patients. F Relationship between DNAJC24 expression and OS in PR-patients. G Relationship between DNAJC24 expression and DFS in PR-patients. H The expression levels of DNAJC24 were correlated with breast cancer patients’ DFS. I The expression levels of DNAJC24 were correlated with breast cancer patients’ OS

DNAJC24 inhibits proliferation of breast carcinoma cells

The database analysis and immunohistochemical analysis of cancer and adjacent tissues from 290 breast carcinoma patients showed that patients with low DNAJC24 expression indicated poor prognosis. We were curious if DNAJC24 expression restricted the malignant behavior of breast carcinoma. The protein levels of DNAJC24 in 4 wild-type human breast cancer cell lines(T47D, MDA-MB-231, SKBR3, MCF-7) were analyzed through Western blot. The findings indicated that the expression of DNAJC24 protein was highest in T47D and lowest in MDA-MB-231 among the 4 cell lines (Fig. 3A). In accordance with the expression of DNAJC24 protein in the 4 cell lines, MDA-MB-231 was selected for overexpression(OE) (Fig. 3B), while T47D cells were selected for knockdown(KD) (Fig. 3C) to conduct an investigation into the effect of DNAJC24 on various biological processes of the two cell lines.

Fig. 3.

DNAJC24 inhibits proliferation of breast cancer cells. A DNAJC24 level in four wild-type breast cancer cell lines. B Lentivirus infected breast cancer cells MDA-MB-231 to construct stable DNAJC24 overexpression cell line. C Lentivirus infected breast cancer cell T47D to construct stable DNAJC24 knockdown cell line. D Upregulation of DNAJC24 suppresses the number of colonies per well in MDA-MB-231 cells. E Upregulation of DNAJC24 suppresses proliferation of MDA-MB-231 cells. *Y-axis shows cell growth (Fold) relative to Day 0 (set as baseline = 1).* F Downregulation of DNAJC24 promotes the number of colonies per well in T47D cells. G Downregulation of DNAJC24 promotes proliferation of T47D cells. *Y-axis shows cell growth (Fold) relative to Day 0 (set as baseline = 1).* Results were normalized to viability at day 0 and represented as fold change. The values were shown as mean ± SEM, p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***, p ≤ 0.0001 ****, p > 0.05 ns(not significant)

The number of colony formation was proportional to the cell proliferation ability. Therefore, a colony formation assay was employed to examine the alterations in the cell proliferation ability after overexpressing DNAJC24 in MDA-MB-231 and downregulating DNAJC24 in T47D. The results were statistically analyzed via two independent sample t-tests. After the overexpression of DNAJC24, the colony formation of MDA-MB-231 OE/DNAJC24 cell line was considerably lower compared to MDA-MB-231 OE/SCR cell line (p < 0.05) (Fig. 3D). CCK-8 is also an important tool for detecting cell proliferation ability. Therefore, the proliferation of MDA-MB-231 OE cell lines was detected by using Cell Counting Kit-8(CCK-8) at 24, 48, 72, 96, and 120 h. The curve of cell growth was drawn on the basis of the absorption values at OD 450 nm, proving that the proliferation of the MDA-MB-231 OE/DNAJC24 cell line was slower than that of MDA-MB-231 OE/SCR (Fig. 3E). In addition, the cloning and proliferation ability of T47D downregulated DNAJC24 was measured. Compared with that in T47D KD/SCR, the number of colony formed in T47D KD/DNAJC24 was significantly higher (Fig. 3F). The proliferation rate of the T47D KD/SCR cell line was lower than that of the T47D KD/DNAJC24 cell line (Fig. 3G). These results prove that the overexpressing DNAJC24 inhibits tumor cell proliferation, while downregulating DNAJC24 may promote tumor cell proliferation.

DNAJC24 inhibits invasion and migration of breast cancer cells

Transwell was used to detect cell chemotaxis and invasion ability in vitro. Medium containing 20% fetal bovine serum was carefully added to the lower chamber. Meanwhile, the cells, which had been resuspended by serum-free medium, were gently placed in the upper chamber. Differences in nutrient composition promoted the chemotaxis of cells. In the invasion experiment, Matrigel was placed at the bottom of the upper chamber. In contrast to the control group, DNAJC24-overexpressed MDA-MB-231 that passed through the transwell chamber was less (Fig. 4A), suggesting that DNAJC24 could significantly restrain the chemotaxis of MDA-MB-231 in vitro. The matrigel-coated transwell was used to simulate the cells to penetrate the basement membrane and enter blood vessels. The MDA-MB-231 cells overexpressed DNAJC24 passing through the transwell chamber was significantly reduced (Fig. 4C), suggesting that DNAJC24 could significantly suppress the invasion of MDA-MB-231.

Fig. 4.

DNAJC24 inhibits chemotaxis, invasion and migration of breast cancer cells. A Upregulation of DNAJC24 inhibits chemotactic ability of MDA-MB-231 cells. B Downregulation of DNAJC24 promotes chemotactic ability of T47D cells. C Upregulation of DNAJC24 restrains the invasion of MDA-MB-231. D Downregulation of DNAJC24 enhances invasion ability of T47D cells. E Upregulation of DNAJC24 decreases migration ability of MDA-MB-231 cells. F Downregulation of DNAJC24 increases migration ability of T47D cells. The values were shown as mean ± SEM, p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***, p ≤ 0.0001 ****, p > 0.05 ns(not significant)

Scratch experiments were performed in a serum-free conditioned medium. Therefore, the effect of cell proliferation was excluded, while being able to measure cell mobility. In the scratch experiments, the rate of migration of MDA-MB-231 overexpressed DNAJC24 was slower compared to control group (Fig. 4E). Meanwhile, invasion, chemotaxis and migration experiments were also performed in T47D cells with KD (Fig. 4B, D, and F). The outcome was consistent with MDA-MB-231 cell line, i.e., DNAJC24 not only restrained the proliferation of breast carcinoma cells, but also decreased the chemotactic, invasion, and migration abilities of breast carcinoma cells.

Discussion

Biomarkers are measurable indicators of physiological processes, pathogenicity, or responses to exposures and interferences [7]. Advances in pathology and molecular mechanisms of breast cancer progression have brought a rising number of breast cancer biomarkers to the forefront. These consist of immunohistochemical indicators (for instance, ER, PR, HER2[ERBB2], and the proliferation protein Ki-67[MKI67]), genomic indicators (such as BRCA1, BRCA2, and PIK3CA), along with immunological indicators (including tumor-infiltrating lymphocytes and PD-L1) [8]. On the basis of these markers, a variety of targeted therapies have been proposed for clinical practice. Although approximately 87% of breast cancer patients benefit from currently available therapies, treatment resistance and recurrence remain the leading factors contributing to breast cancer-related mortality [9]. In addition, breast cancer exhibits significant heterogeneity, and patients with apparently similar tumor subtypes exhibit variable responses to identical drug treatments. Therefore, the need for novel biomarkers, particularly in triple-negative breast tumors and ER and HER2-positive tumors that develop resistance, is ongoing. Accurate prediction of breast cancer progression and personalized treatment plans will maximize patient benefit by avoiding overtreatment and undertreatment.

Heat shock proteins (HSPs) are a category of proteins that function to prevent or reverse the unfolding and denaturation of cellular proteins when subjected to stress or high temperatures. Traditionally, these proteins have been known as molecular chaperones due to their crucial role in maintaining the structural integrity of proteins. HSPs are typically classified in accordance with their molecular weight, and most of them belong to HSP27, HSP40, HSP60, HSP70, HSP90, and large HSP (HSP110 and glucose-regulated protein 170, GRP170) [10]. Numerous investigations have explored the connection between the HSP family and cancer. For instance, research has shown that the inhibition of HSP27 can reverse the epithelial-to-mesenchymal transition (EMT), while also decreasing the activity of matrix metalloproteinases (MMPs), as well as the proliferation, migration, and invasion capabilities of cancer cells [11]. HSP60 plays a crucial role in the survival of cancer cells by binding to and inhibiting intracellular protein aggregates in neuroblastoma cells [12]. Jaattela et al. revealed that HSP70 protects cancer cells from cytotoxic effects caused by TNF and proposed that HSP70 might augment the carcinogenic potential of specific cancer cells via immune evasion mechanisms [13]. The HSP40/DNAJ protein family includes homologues of the bacterial DnaJ heat shock protein. Most members carry a “J” domain, allowing them to associate with HSP70, which is why HSP40 is frequently termed an HSP70 co-partner [14, 15]. Notably, the HSP40 family stands out among all major human HSP families due to having the highest number of members. HSP40 proteins consist of three subclasses of DNAJ: DNAJA, DNAJB, and DNAJC. Prior research conducted by our team has indicated that the overexpression of DNAJC24 can enhance both proliferation and migration in hepatocellular carcinoma. Targeting DNAJC24 can interfere with ammonia metabolism, affecting the autophagy and proliferation and ultimately restraining the malignant development in HCC. However, Diane L N Trinh et al. found that Tid1 (also known as DNAJA3) interacts with HSP70 protein, which has been recognized as a regulator of p53-mediated apoptosis, and has a direct interaction with p53, resulting in the mitochondrial translocation of the complex and apoptosis in MCF-7 [16]. Consequently, a conclusion can be drawn that different members of the HSP family exert diverse effects on cancer. In the current study, DNAJC24, which belongs to the HSP40 family, exhibited contrasting behavior compared with its role in HCC [6], because it significantly inhibited tumor cell proliferation and the invasion in breast carcinoma. Notably, our data revealed lower DNAJC24 expression in MCF7 (ER + /PR + /HER2 +) compared to T47D, despite both being luminal breast cancer cell lines. This discrepancy may reflect intrinsic phenotypic differences, as MCF7 is reported to exhibit more mesenchymal traits and heightened aggressiveness relative to the epithelial-dominated T47D [17–19]. We speculate that DNAJC24’s tumor-suppressive function is more pronounced in epithelial contexts (e.g., T47D), where it may counteract pro-EMT signals. In MCF7, constitutive EMT activation might downregulate DNAJC24, further reinforcing its invasive phenotype. This aligns with reports of tamoxifen-resistant MCF7 (MCF7-TR) showing EMT upregulation and ERα loss—a phenotype reminiscent of our low-DNAJC24 MCF7 observations [3]. While this study focused on T47D to delineate DNAJC24’s role in luminal cancers without EMT confounders, future comparative studies in MCF7 could clarify how DNAJC24’s function diverges across luminal subtypes with varying epithelial-mesenchymal plasticity.

In this study, the analysis of TCGA data showed differences in the expression of DNAJC24 in the cancerous and adjacent non-cancerous tissues of different cancer types, i.e., a higher expression in paracancerous tissues than in tumor tissues. In breast cancer, the mRNA and protein levels of DNAJC24 were downregulated in comparison to paracancer tissues. As a result, we further detected the level of DNAJC24 in clinical breast cancer samples. The analysis of immunohistochemical results from 290 breast cancer tissues and paired adjacent tissues showed that DNAJC24 was significantly higher in adjacent tissues compared to cancer tissues. The discovery was consistent with database analysis. In addition, DNAJC24 expression was positively correlated with DFS and OS in ER-negative or PR-negative patients. This result indicates that DNAJC24 have potential to inhibit malignant progression in breast carcinoma. We performed in vitro experiments to verify that the downregulation of DNAJC24 not only promoted the proliferation of breast cancer cells, but also their migration and invasion. The overexpression of DNAJC24 inhibited tumor proliferation and invasion. This finding supports the hypothesis that DNAJC24 plays the role of tumor suppressor in breast carcinoma and that downregulation of DNAJC24 may be related to the advancement of breast carcinoma which is in contrast with its role in HCC. This revelation makes us more deeply aware that HSP family members are rich and diverse, and their effects on cancer are also complex and diverse.

We preliminarily investigated the impact of DNAJC24 on breast carcinoma development, and performed in vitro functional experiments based on the database and immunohistochemical analyses of clinical samples. Notably, the knockout of DPH1, DPH3, or DPH4 is embryologically lethal in mice [20–22], but no such lethal phenotype occurs when similar knockout is performed in cell lines. The result suggests that the function of DNAJC24 is also dependent on the in vivo environment. Therefore, the establishment of stable cell lines with OE or KD is crucial for further studies. To elucidate the underlying mechanism through which DNAJC24 influences breast tumor progression in patients, this cell line can be transplanted into mouse models to assess tumor growth and evaluate its effect on the OS of mice. In terms of mechanistic aspects, the nuclear magnetic resonance (NMR) structure revealed two domains of DNAJC24: the highly conservative J and CSL domains, which are interconnected by a flexible linker helix, play crucial roles in the overall structure and function of the protein [23]. DNAJC24 possesses the distinctive capability of binding iron in a tetrahedral coordination geometry via the cysteine of its CSL domain. Iron-bound DNAJC24 also undergoes oligomerization. Therefore, it may act as a temporary "iron storage protein" for regulating intracellular iron homeostasis [24]. Metabolic disorders are also a hallmark of cancer cells. In addition, shRNA targeting diproylamine biosynthesis proteins 1–4 (DPH1–DPH4) can increase the adhesion of human podocytes, and immortalized human podocytes stably expressing these shRNA exhibit increased adhesion [25]. Can DNAJC24 alter the adhesion of breast cancer cells? Whether DNAJC24 deletion in vivo can promote breast cancer metastasis is also worth investigating. Another important function of DNAJC24 is to participate in the diacetylamine modification of human eEF2, which is involved in protein synthesis [23, 26]. On the basis of this function, DNAJC24 is likely to participate in the protein synthesis of cancer cells, affecting their life activities. Under stress, the HSP family maintains the structure of intracellular proteins to ensure their functions. For example, under the conditions of oxygen and glucose deprivation, DNAJC24 can protect brain endothelial cells from stress conditions [27], but the consequence of the lack of this protective effect on tumor cells is unknown. At present, research on DNAJC24 remains scarce.

Conclusions

Our study initially reveals that DNAJC24 is capable of suppressing the malignant behavior of breast carcinoma cells, demonstrating the potential to serve as a diagnostic and therapeutic target for breast carcinoma. Our study also demonstrates the diversity of HSP family members on cancer and lays the foundation for further research.

Abbreviations

ATCC

American Type Culture Collection

CCK-8

Cell Counting Kit8

CSL

C-Terminal Src Kinase-Like

DFS

Disease-Free Survival

DMEM

Dulbecco’s Modified Eagle’s Medium

DPH

Diphthamide Biosynthesis Protein

DNAJ

DNAJ Subclass

DNAJC24

DnaJ Heat Shock Protein Family Member C24

eEF2

Eukaryotic Elongation Factor2

EMT

Epithelial-To-Mesenchymal Transition

ER

Estrogen Receptor

FBS

Fetal Bovine Serum

GRP170

Glucose-Regulated Protein 170

HER2

Human Epidermal Growth Factor Receptor 2

HCC

Hepatocellular Carcinoma

HSP

Heat Shock Protein

IHC

Immunohistochemistry

KD

Knockdown

NMR

Nuclear Magnetic Resonance

OS

Overall Survival

OE

Overexpression

PS

Penicillin–Streptomycin Solution

PR

Progesterone Receptor

SCR

Scrambled Control

SDS

Sodium Dodecyl Sulfate

Tid1

Tumour Protein p53-Inducible Protein 1

TNM

Tumor-Node-Metastasis

TNF

Tumor Necrosis Factor

TCGA

The Cancer Genome Atlas

VSVG

Vesicular Stomatitis Virus G Protein

Author contributions

WJM: performed data curation, investigation, methodology, and funding acquisition. WL: wrote the original draft and created visualizations. YLY: reviewed and edited the manuscript. XRW: developed the software. LLZ: conducted investigations and validations. YL: reviewed and edited the manuscript and provided supervision. LWC: reviewed and edited the manuscript and provided resources. YW: conducted investigations. GTL: conducted investigations and validations. YHS: provided resources. YCH: administered the project, developed methodology, and conceptualized the study. ZST: provided resources. HG: administered the project, developed methodology, and conceptualized the study.

Funding

This study was supported by the Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2021KJ191) and Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-009A).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Research Ethics Committees of the Tianjin Medical University Cancer Institute and Hospital(approval number: bc20240204).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.