Identification of DNAJC24 as a potential therapeutic target in breast cancer

Identification of DNAJC24 as a potential therapeutic target in breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of DNAJC24 as a potential therapeutic target in breast cancer Wenjing Meng, Wei Liu, Xiaorui Wang, Linlin Zhan, Yuchao He, Yi Luo, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3823704/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Purpose On the basis of current markers, a variety of targeted therapies have been proposed for clinical practice, but treatment resistance and recurrence remain the leading 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. Approach and Key Results In this study, we focused on investigating the role of DnaJ heat shock protein family member C24 (DNAJC24) in breast cancer through bioinformatic analysis by using public databases and clinicopathological samples. In addition, we performed in vitro functional assays to explore the biological functions of DNAJC24. In vitro experiments showed that the overexpression of DNAJC24 significantly inhibited the proliferation and invasion of breast cancer cells. Meanwhile, the downregulation of DNAJC24 enhanced cell proliferation, invasion, and migration. The findings provide a theoretical basis for the identification of a novel molecular therapeutic target for breast cancer. Conclusion and Implications Our research revealed that DNAJC24 was weakly expressed in breast cancer tissues, suggesting its involvement in breast carcinogenesis. Furthermore, we observed an inverse correlation between DNAJC24 expression and malignant progression of breast cancer including clinical stage among different pathological subtypes and molecular staging of breast cancer. Therefore, given the significant association between low levels of DNAJC24 expression and poor prognosis in patients with breast cancer, DNAJC24 may serve as a predictive marker. breast cancer DNAJC24 molecular subtype prognosis biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction In terms of incidence, breast cancer has currently risen to the top spot worldwide(Sung et al., 2021). Studies have shown that early diagnosis, radiotherapy, and chemotherapy can significantly improve the prognosis of patients with breast cancer; however, but an extremely high proportion of breast cancer cases progress to distant metastasis and recurrence within a short time after clinical diagnosis (Hou, Peng, & Li, 2022). The identification of key regulatory genes involved in the onset and development of breast cancer at the molecular level, the elucidation of the molecular mechanism of breast cancer pathogenesis and invasion, the discovery of reliable and highly specific diagnostic and prognostic biomarkers, and the continuous search for new targets for the development of new treatments are essential for reducing the incidence and mortality of breast cancer. Eukaryotic elongation factor-2 (eEF2) undergoes diphtheria amidation modification by DnaJ heat shock protein family member C24 (DNAJC24), a member of the dipthamide biosynthesis protein (DPH) family and commonly known as DPH4(Kespohl et al., 2020; Robinson, Henriksen, & Maxwell, 1974). 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. Diphtheria toxin further modifies eEF2 by recognizing diphtheria amide, which inhibits protein synthesis and induces cell death(Xu et al., 2020). The biological role of DNAJC24 (DPH4), which has been found to be associated with diphtheria amide in the current investigation, 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(Wei et al., 2012). Our previous study confirmed for the first time that DNAJC24 plays an important role in the proliferation and migration of hepatocellular carcinoma (HCC) cells by affecting ammonia metabolism(Li et al., 2022). However, the role of DNAJC24 in the occurrence and development of breast cancer or other cancers has not yet been reported. In the current study, we first investigated the expression of DNAJC24 (DPH4) in various breast tumor cell lines, established the knockdown and overexpression cell lines of DNAJC24 in breast tumor cells, and preliminarily evaluated the effect of DNAJC24 (DPH4) protein on breast cancer. The preliminary investigation of DNAJC24 (DPH4) 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 closely related to the development of breast cancer, providing a theoretical basis and foundation for the development of new tumor markers for breast cancer. 2. Materials and methods 2.1 Human breast cancer tissue samples In this study, samples from patients who were undergoing surgical resection of breast cancer 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. 2.2 Cell culture In this study, four human breast cancer cell lines, namely, T47D, MDA-MB-231, SKBR3, and MCF-7, were all derived from the American Type Culture Collection (ATCC) and provided by the Department of Tumor Cell Biology, Cancer Research Institute, Tianjin Cancer Hospital. The cell lines generated in this study were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) with 10% v/v fetal bovine serum (FBS; HyClone Laboratories Inc., Novato, CA, USA) and 1% penicillin–streptomycin solution (PS; Hyclone Laboratories Inc.) at 37°C in an incubator with 5% CO 2 . 2.3 Cell transfection Packaging plasmids (VSVG and ΔR) and expression plasmids (OE and matched Ctrl, KD and matched Ctrl) were transfected into HEK293T cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 h, the medium supernatant was collected to obtain lentiviral particles. T47D and MDA-MB-231 cells were infected with lentivirus that was carrying DNAJC24 short hairpin RNA (shRNA) and their respective scrambled controls to produce DNAJC24 knockdown (KD), DNAJC24 overexpression (OE), and matched control (KD SCR and OE SCR) cell lines. Stably transfected clones were selected using a puromycin (Gibco)-containing medium. Transfection efficiency was validated through Western blot assay. 2.4 Western blot The cells in a 10 cm dish were washed three times with ice-cold phosphate-buffered saline (PBS) (pH 6.8) and then lysed with 1 × sodium dodecyl sulfate (SDS) lysis buffer (Tris-HCl, pH 6.8, 62.5 mM, 2% SDS, 10% glycerol) supplemented with 1 mM of NaF, 1 mM of Na 3 VO 4 , 1 × protease, and phosphatase inhibitor cocktail (Hoffman-la Roche Ltd., Basel, Switzerland) on ice for 30 min. Protein denaturation was performed at 95°C for 10 min. The cell lysates were stored at − 20 ℃. Equal amounts of protein (30–90 µg/cell line) were loaded, separated via SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane (hydrophobic) (Merck KGaA, Darmstadt, Germany). Antibodies against the following proteins were used: anti-DNAJC24 (1:500) from Abcam; anti-glyceraldehyde 3-phosphate dehydrogenase (1:1000), goat anti-rabbit (1:4000), and goat anti-mouse (1:4000) secondary antibody from Santa Cruz Biotechnology. 2.5 Colony formation assay For the colony formation assay, 1000 cells in DMEM supplemented with 10% FBS were plated in six-well plates. After 2 weeks of incubation, the surviving colonies were fixed, stained with 0.5% crystal violet, imaged, and counted. The data are presented as the means ± standard deviation (SD) of triplicate dishes in the same experiment. 2.6 Cell proliferation assay The cells were grown in 96-well plates at a density of 1 × 10 3 cells per well for the initial concentration. Then, 5 duplicate wells for each cell line were set to reduce differences within a group. Then, the cells were incubated with Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan; 10 µL/well) for 2 h at 37°C and 5% CO 2 . Optical density (OD) was measured using an enzyme-linked immunosorbent assay reader (BioTek SynergyH1, Burlington, VT, USA). 2.7 Chemotaxis assay DMEM with 20% FBS was loaded into the lower chambers (600 µL per chamber) and then the cells were suspended and loaded into the upper chambers of a transwell (1×10 5 /200 µL per chamber). After incubating at 37°C in 5% CO 2 (MDA-MB-231 cells for 4 h; T47D cells for 6 h), the membrane was rinsed with PBS, fixed in 4% paraformaldehyde, and stained. Three fields were randomly selected to count the number of migrated cells at ×200 via light microscopy (Olympus BX61). 2.8 Matrigel invasion assay For the invasion assay, Matrigel-coated transwell chambers were incubated in 24-well plates for more than 1 h at 37 ℃. DMEM supplemented with 20% FBS was loaded in the lower chamber, and 2 × 10 5 cells in serum-free DMEM were added to the upper chamber. After 24 h incubation at 37 ℃ in a humidified incubator with 5% CO 2 , the invaded cells on the bottom were fixed with 4% paraformaldehyde for 20 min, stained, and counted via light microscopy (Olympus BX61). 2.9 Scratch assay The cells were seeded in six-well plates 1 day prior to the assay, with each well containing a density of 1 × 10 6 cells/well to achieve a confluent monolayer. The culture medium used was DMEM without FBS. A 10 µL pipette tip was used to create a uniform wound. Subsequently, the cells were incubated in a humidified atmosphere at 37°C and 5% CO 2 . The distance between the wounds was measured at three random sites within specific time intervals (0, 3, 6, 9, 12, and 24 h). The data are presented as means ± SD, while representative images were captured at the initial and final time points. 2.10 Immunohistochemical assays Immunohistochemistry (IHC) staining for DNAJC24 was performed in 290 pairs of cancerous and matched adjacent nonmalignant tissue. The tissues were sequentially immersed in xylene and gradient ethanol for dewaxing and rehydration. Antigen retrieval was performed using a citrate target retrieval solution at pH 6.0 (ZSGB-BIO, ZLI-9065) in a pressure cooker for 2.5 min and then cooled naturally to room temperature. Subsequently, 3% hydrogen peroxide was used to inhibit endogenous hydrogen peroxidase activity. Subsequently, the samples were incubated with rabbit anti-DNAJC24 (1:50, Abcam, ab246925) at room temperature for 30 min and overnight at 4°C. After washing with PBS, the samples were stained with secondary antibody for 1 h at room temperature. The cells were visualized with a 3,3-diaminobenzidine solution (ZSGB-Bio, ZLI-9017) and counterstained with hematoxylin. Finally, the sections were photographed using a light microscope (Olympus BX61). The IHC score of DNAJC24 was the product of the staining intensity and percentage scores. DNAJC24 positive expression intensity: Grade 0 is negative, Grade 1 is low, Grade 2 is moderate, and Grade 3 is high. 3. RESULTS 3.1 Expression of DNAJC24 in cancer tissues is different from that in normal tissues Analysis of The Cancer Genome Atlas (TCGA) database indicated that the mRNA level of DNAJC24 in cancer tissues, such as breast cancer, pancreatic carcinoma, prostate cancer, thymus cancer, and endometrial cancer was lower than that in adjacent normal tissues (Fig. 1 .A). In accordance with the screening conditions, 841 cases of female breast cancer raw data were downloaded from the TCGA database, including 104 pairs of normal 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 corresponding cancer tissues was significantly decreased compared with that in the adjacent tissues (p < 0.001) (Fig. 1 .B, left). Meanwhile, similar results were obtained when comparing normal tissue samples with malignant tumor samples, and the expression level of DNAJC24 in cancer tissues was significantly lower than that in normal tissues, p < 0.001 (Fig. 1 .B, 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. 1 .C). 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, regardless of T stage, N stage, or TNM stage, with statistical difference (p < 0.05) (Fig. 1 .D). In addition, PR, ER, and HER2 are among the most important immunohistochemical indicators of breast cancer. In the current study, patients were divided into negative and positive groups based on 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 positive and negative groups was analyzed via t -test. The results showed that the expression level of DNAJC24 was higher in the ER-positive and PR-positive groups than in the negative group. However, the trend was the opposite in the HER2-positive group. Compared with the negative group, DNAJC24 was downregulated in the HER2-positive group, p < 0.05 (Fig. 1 .E). Finally, Kaplan–Meier survival analysis showed that DNAJC24 generally exerted no significant effect on relapse-free survival (DFS). However, 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. 1 .F). In general, the results of the database analysis showed that the mRNA level of DNAJC24 was significantly higher in adjacent tissues than in 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 associated with a lower risk of recurrence. 3.2 Higher expression of DNAJC24 is associated with better prognosis in breast cancer tissues In the current study, samples from patients who underwent surgical resection for breast cancer 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. 2 .A). For comparison, three-negative breast cancer has the lowest expression (Fig. 2 .B). 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. 2 .C). That is, the expression level of DNAJC24 in normal tissues was higher than that in cancer tissues. This result was consistent with the analysis of the TCGA database. Among breast cancer patients who did not express ER (Fig. 2 .D and E) and breast cancer patients who did not express PR (Fig. 2 .F and G), DNAJC24 was positively correlated with overall survival (OS) and disease-free survival. Similar results were obtained in the total samples, where DNAJC24 was positively associated with OS and disease-free survival of the patients. 3.3 DNAJC24 inhibits proliferation of breast cancer cells The database analysis and immunohistochemical analysis of cancer and adjacent tissues from 290 breast cancer patients showed that patients with decreased expression of DNAJC24 had poor prognosis. Does DNAJC24 inhibit the malignant behavior of breast cancer? Western blot was used to analyze the protein expression of DNAJC24 in 4 wild-type human breast cancer cell lines, namely, T47D, MDA-MB-231, SKBR3, and MCF-7. The results showed that the expression of DNAJC24 protein was highest in T47D and lowest in MDA-MB-231 among the 4 cell lines (Fig. 3 .A). In accordance with the expression of DNAJC24 protein in the 4 cell lines, MDA-MB-231 was selected for OE (Fig. 3 .B), while T47D cells were selected for KD (Fig. 3 .C) to investigate the effect of DNAJC24 on the biological function of the two cell lines. The number of colony formation was proportional to the cell proliferation ability. Therefore, a plate cloning experiment was used to detect the changes in cell proliferation ability after the overexpression of DNAJC24 in MDA-MB-231 and the down-expression of DNAJC24 in T47D. The results were statistically analyzed via two independent sample t -tests. After the overexpression of DNAJC24, the number of colony formed in the MDA-MB-231 OE/DNAJC24 group was significantly lower than that in the MDA-MB-231 OE/SCR group (p < 0.05) (Fig. 3 .D). CCK-8 is also an important tool for detecting cell proliferation ability. Therefore, CCK-8 was used to detect the proliferation of MDA-MB-231 OE cell lines at 24, 48, 72, 96, and 120 h. The cell growth curve 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. 3 .E). 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. 3 .F). The proliferation rate of the T47D KD/SCR cell line was lower than that of the T47D KD/DNAJC24 cell line (Fig. 3 .G). These results indicate that the overexpression of DNAJC24 inhibits tumor cell proliferation, while the down-expression of DNAJC24 may promote tumor cell proliferation. 3.4 DNAJC24 inhibits invasion and migration of breast cancer cells Transwell migration assay was used to detect the ability of cell chemotaxis and invasion in vitro. A medium with 20% FBS was added to the lower chamber, and cells with the medium were added to the upper chamber. Differences in nutrient composition promoted the chemotaxis of cells. In the invasion experiment, Matrigel was added to the bottom of the upper chamber. Compared with the control group, DNAJC24-overexpressed MDA-MB-231 that passed through the transwell chamber was less (Fig. 4 .A), suggesting that DNAJC24 could significantly reduce the chemotactic ability of MDA-MB-231 breast cancer cells in vitro. The Matrigel-coated transwell mimics the ability of 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. 4 .C), suggesting that DNAJC24 could significantly reduce the cell invasion ability of MDA-MB-231 breast cancer cells. Scratch experiments were performed in a serum-free conditioned medium. Therefore, the effect of cell proliferation was excluded and the rate of cell migration could be measured. In the scratch experiments, the migration rate of MDA-MB-231 cells overexpressed DNAJC24 was lower than that of the control group (Fig. 4 .E). Meanwhile, invasion chemotaxis and migration experiments were also performed in T47D cells with KD (Fig. 4 .B, D, and F). The results were consistent with those of the MDA-MB-231 cell line, i.e., DNAJC24 not only inhibited the proliferation of breast cancer cells, but also inhibited the chemotactic, invasion, and migration abilities of breast cancer cells. 4. DISCUSSION Biomarkers are quantifiable indicators of normal biological processes, pathogenic, or responses to exposures or interventions(Cserni et al., 2011). Advances in the field of pathology and in-depth research into the molecular mechanisms of breast cancer progression have brought an increasing number of breast cancer biomarkers to the fore. These include immunohistochemical markers (e.g., ER, PR, HER2[ERBB2], and the proliferation marker protein Ki-67[MKI67]), genomic markers (e.g., BRCA1, BRCA2, and PIK3CA), and immunological markers (e.g., tumor-infiltrating lymphocytes and PD-L1)(Veronesi, Boyle, Goldhirsch, Orecchia, & Viale, 2005). 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 causes of breast cancer-related mortality(Crimini et al., 2021). 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 group of proteins whose function is to reverse or inhibit the denaturation or unfolding of proteins in cells under pressure or high temperature. Traditionally, HSPs have also been referred to as molecular chaperones because of their role in maintaining the structural stability 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)(Ciocca & Calderwood, 2005). Many studies have been conducted on the relationship between the HSP family and cancer. For example, the inhibition of HSP27 has been demonstrated to reverse epithelial-to-mesenchymal transition (EMT), reduce matrix metalloproteinase (MMP) activity, proliferation, migration, and invasion of cancer cells(Gibert, Simon, Dimitrova, Diaz-Latoud, & Arrigo, 2013). HSP60 promotes cancer cell survival by binding to and inhibiting intracellular protein aggregins in neuroblastoma cells(Chaiwatanasirikul & Sala, 2011). Jaattela et al. reported that HSP70 protected cancer cells from TNF-induced cytotoxicity, and they suggested that HSP70 may enhance the carcinogenic potential of certain cancer cells through immune escape mechanisms(Rérole, Jego, & Garrido, 2011). The HSP40/DNAJ family of proteins comprises homologues of the bacterial DnaJ HSP. Most members contain a “J” domain through which they can interact with HSP70, and thus, HSP40 is also known as an HSP70 co-partner (Alderson, Kim, & Markley, 2016; Faust & Rosenzweig, 2020). Among all the major human HSP families, the HSP40 family has the largest number of members. The proteins of HSP40 are classified into three DNAJ subclasses: DNAJA, DNAJB, and DNAJC. Our previous studies have shown that an increased expression of DNAJC24 in HCC cells can promote the proliferation and migration of HCC cells. Targeting DNAJC24 can interfere with ammonia metabolism, affecting the proliferation and autophagy of HCC cells and ultimately inhibiting the malignant progression of HCC. However, Diane L N Trinh et al. found that Tid1 (also known as DNAJA3) interacts with HSP70 protein, which has been identified as a regulator of p53-mediated apoptosis, and interacts directly with p53, leading to the mitochondrial translocation of the complex and the induction of apoptosis in MCF-7 breast cancer cells(Trinh, Elwi, & Kim, 2010). 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 contrast behavior compared with its role in HCC(Li et al., 2022), because it significantly inhibited tumor cell proliferation and the invasion of tumor cells in breast cancer. In the current study, the analysis of TCGA data showed differences in the expression of DNAJC24 in the cancer and paracancerous 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 cancer tissues compared with in adjacent tissues or normal tissues. Therefore, we further investigated the expression 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 than in cancer tissues. This finding was consistent with the results of database analysis. In addition, DNAJC24 expression was positively correlated with DFS and OS in ER-negative or PR-negative patients. This result suggests that DNAJC24 may have a function in inhibiting tumor malignant progression in breast cancer. 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 acts as a tumor suppressor in breast cancer and that the loss of DNAJC24 expression may be associated with the progression of breast cancer, 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 effect of DNAJC24 on breast cancer progression, 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(Chen & Behringer, 2004; Liu et al., 2006; Webb et al., 2008), but no such lethal phenotype occurs when similar knockout is performed in cell lines. This 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 DPH4: the conserved J domain and the CSL domain, which are helixally connected by a flexible linker. The adaptor helix regulates the conformational flexibility between the two domains, and thus, the function of the protein(Liu, Milne, Kuremsky, Fink, & Leppla, 2004). DPH4 exhibits the unique ability to bind iron in a tetrahedral coordination geometry through the cysteine of its CSL domain. Iron-bound DPH4 (Fe-DPH4) also undergoes oligomerization, and thus, may function as a transient “iron storage protein” to regulate intracellular iron homeostasis(Thakur et al., 2012). 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(Cinà et al., 2019). 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 DPH4 is to participate in the diacetylamine modification of human eEF2, which is involved in protein synthesis(Liu et al., 2004; Stahl et al., 2015). 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, DPH4 can protect brain endothelial cells from stress conditions(Sun et al., 2015), but the consequence of the lack of this protective effect on tumor cells is unknown. At present, research on DNAJC24 remains scarce. Our study first shows that DNAJC24 can inhibit the malignant behavior of breast cancer, exhibiting potential as a diagnostic and therapeutic target for breast cancer. Our study also demonstrates the diversity of HSP family members on cancer and lays the foundation for further research. Declarations Statements & Declarations Funding This research was supported by The Science & Technology Development Fund of Tianjin Education Commission for Higher Education(2021KJ191). Conflict of interest statement The authors declare they have no conflict of interest. Author contributions All authors contributed to the study conception and design. Wenjing Meng: Data curation; investigation(equal); methodology(equal); funding acquisition(equal). Wei Liu: Writing original draft(equal); visualization(equal). Xiaorui Wang: Software(equal). Linlin Zhan: Investigation(equal); validation(equal). Yuchao He: Methodology(equal); formal analysis(equal). Yi Luo: Writing-Review & Editing(equal); supervision(equal). Liwei Chen: Writing-Review & Editing(equal); resources(equal). Yu Wang: Investigation(equal). Guangtao Li ： Investigation(equal); validation(equal). Yehui Shi: Resources(equal). Zhongsheng Tong: Resources(equal). Hua Guo: Project administration(equal); methodology(equal); conceptualization(equal). Data availability statement All data have been included within this manuscript. References Alderson, T. R., Kim, J. H., & Markley, J. L. (2016). Dynamical Structures of Hsp70 and Hsp70-Hsp40 Complexes. Structure (London, England : 1993), 24 (7), 1014-1030. doi:10.1016/j.str.2016.05.011 Chaiwatanasirikul, K. A., & Sala, A. (2011). The tumour-suppressive function of CLU is explained by its localisation and interaction with HSP60. Cell death & disease, 2 (10), e219. doi:10.1038/cddis.2011.99 Chen, C. M., & Behringer, R. R. (2004). Ovca1 regulates cell proliferation, embryonic development, and tumorigenesis. 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Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming. Science advances, 6 (11), eaay1109. doi:10.1126/sciadv.aay1109 Li, G., He, Y., Liu, H., Liu, D., Chen, L., Luo, Y., . . . Guo, H. (2022). DNAJC24 is a potential therapeutic target in hepatocellular carcinoma through affecting ammonia metabolism. Cell death & disease, 13 (5), 490. doi:10.1038/s41419-022-04953-z Liu, S., Milne, G. T., Kuremsky, J. G., Fink, G. R., & Leppla, S. H. (2004). Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Molecular and cellular biology, 24 (21), 9487-9497. doi:10.1128/mcb.24.21.9487-9497.2004 Liu, S., Wiggins, J. F., Sreenath, T., Kulkarni, A. B., Ward, J. M., & Leppla, S. H. (2006). Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development. Molecular and cellular biology, 26 (10), 3835-3841. doi:10.1128/mcb.26.10.3835-3841.2006 Rérole, A. L., Jego, G., & Garrido, C. (2011). Hsp70: anti-apoptotic and tumorigenic protein. Methods in molecular biology (Clifton, N.J.), 787 , 205-230. doi:10.1007/978-1-61779-295-3_16 Robinson, E. A., Henriksen, O., & Maxwell, E. S. (1974). Elongation factor 2. Amino acid sequence at the site of adenosine diphosphate ribosylation. The Journal of biological chemistry, 249 (16), 5088-5093. Stahl, S., da Silva Mateus Seidl, A. R., Ducret, A., Kux van Geijtenbeek, S., Michel, S., Racek, T., . . . Brinkmann, U. (2015). Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor. Proceedings of the National Academy of Sciences of the United States o f America, 112 (34), 10732-10737. doi:10.1073/pnas.1512863112 Sun, P., Esteban, G., Inokuchi, T., Marco-Contelles, J., Weksler, B. B., Romero, I. A., . . . Solé, M. (2015). Protective effect of the multitarget compound DPH-4 on human SSAO/VAP-1-expressing hCMEC/D3 cells under oxygen-glucose deprivation conditions: an in vitro experimental model of cerebral ischaemia. British journal of pharmacology, 172 (22), 5390-5402. doi:10.1111/bph.13328 Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians, 71 (3), 209-249. doi:10.3322/caac.21660 Thakur, A., Chitoor, B., Goswami, A. V., Pareek, G., Atreya, H. S., & D'Silva, P. (2012). Structure and mechanistic insights into novel iron-mediated moonlighting functions of human J-protein cochaperone, Dph4. The Journal of biological chemistry, 287 (16), 13194-13205. doi:10.1074/jbc.M112.339655 Trinh, D. L., Elwi, A. N., & Kim, S. W. (2010). Direct interaction between p53 and Tid1 proteins affects p53 mitochondrial localization and apoptosis. Oncotarget, 1 (6), 396-404. doi:10.18632/oncotarget.100902 Veronesi, U., Boyle, P., Goldhirsch, A., Orecchia, R., & Viale, G. (2005). Breast cancer. Lancet (London, England), 365 (9472), 1727-1741. doi:10.1016/s0140-6736(05)66546-4 Webb, T. R., Cross, S. H., McKie, L., Edgar, R., Vizor, L., Harrison, J., . . . Jackson, I. J. (2008). Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development. J Cell Sci, 121 (Pt 19), 3140-3145. doi:10.1242/jcs.035550 Wei, H., Xiang, L., Wayne, A. S., Chertov, O., FitzGerald, D. J., Bera, T. K., & Pastan, I. (2012). Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy. Proceedings of the National Academy of Sciences of the United States o f America, 109 (18), 6898-6903. doi:10.1073/pnas.1204523109 Xu, L., Raabe, M., Zegota, M. M., Nogueira, J. C. F., Chudasama, V., Kuan, S. L., & Weil, T. (2020). Site-selective protein modification via disulfide rebridging for fast tetrazine/trans-cyclooctene bioconjugation. Organic & biomolecular chemistry, 18 (6), 1140-1147. doi:10.1039/c9ob02687h Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3823704","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264808373,"identity":"58f466a7-a00d-414a-98a7-79b43ed71b0f","order_by":0,"name":"Wenjing Meng","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Meng","suffix":""},{"id":264808374,"identity":"69318724-1598-4b3f-94b0-e8948da4bf07","order_by":1,"name":"Wei Liu","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Liu","suffix":""},{"id":264808375,"identity":"3ffc9f6b-33ce-4627-a713-922f018b0fbf","order_by":2,"name":"Xiaorui Wang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaorui","middleName":"","lastName":"Wang","suffix":""},{"id":264808376,"identity":"5fce0f68-eb02-4531-bbf4-0fbaa8263949","order_by":3,"name":"Linlin Zhan","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linlin","middleName":"","lastName":"Zhan","suffix":""},{"id":264808377,"identity":"c1925237-087f-474a-8c87-c69270076671","order_by":4,"name":"Yuchao He","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuchao","middleName":"","lastName":"He","suffix":""},{"id":264808378,"identity":"42f5f493-83d6-4c08-aecb-1b5728506cf4","order_by":5,"name":"Yi Luo","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Luo","suffix":""},{"id":264808379,"identity":"1932ad59-76fd-420d-944e-0ecc62266185","order_by":6,"name":"Liwei Chen","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liwei","middleName":"","lastName":"Chen","suffix":""},{"id":264808380,"identity":"1d503558-441d-4271-9560-49d9fd55491e","order_by":7,"name":"Yu Wang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Wang","suffix":""},{"id":264808381,"identity":"68e6c876-f7bb-47a4-a00c-a99a3ee9d4b5","order_by":8,"name":"Guangtao Li","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guangtao","middleName":"","lastName":"Li","suffix":""},{"id":264808382,"identity":"fea3df37-623f-483d-86e2-cba7096d6f9d","order_by":9,"name":"Yehui Shi","email":"","orcid":"","institution":"National Clinical Research Center for Cancer, Tianjin’s Clinical Research Center for Cancer","correspondingAuthor":false,"prefix":"","firstName":"Yehui","middleName":"","lastName":"Shi","suffix":""},{"id":264808383,"identity":"1e8704f7-7988-4bf7-8232-47355902c661","order_by":10,"name":"Zhongsheng Tong","email":"","orcid":"","institution":"National Clinical Research Center for Cancer, Tianjin’s Clinical Research Center for Cancer","correspondingAuthor":false,"prefix":"","firstName":"Zhongsheng","middleName":"","lastName":"Tong","suffix":""},{"id":264808384,"identity":"12fd7ee9-00b2-4eb5-a024-2a7b2c04b118","order_by":11,"name":"Hua Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYHACNiC2YWBsYGBgJkVLmgTJWg5LgFjEaZGPSD72mKfmfB3ztDOGnwsY7OR0GwhoMbyRlm7Mc+y2BOPsHGPpGQzJxmYHCGmZkWMmzcMG1mLGzMNwIHEbcVr+nSNBi7wEUAtv2wEStBjwPEuTnNuXLNk4O61YmseACL/Itycfk3jzzY7fcHbyxs88FXZyBLUYwBQYNoC5BJSDbWmAMYhQPApGwSgYBSMUAAC71zjP7aLrgAAAAABJRU5ErkJggg==","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hua","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2023-12-30 09:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3823704/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3823704/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49237300,"identity":"8594a92a-8e05-43ca-a2bc-f7fd71488f35","added_by":"auto","created_at":"2024-01-05 18:00:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1152581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of DNAJC24 in cancer tissues is different from normal tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e \u003cstrong\u003eThe expression level of DNAJC24 in cancer and normal tissues of 5 malignant tumors including breast cancer by TCGA database analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e \u003cstrong\u003eDNAJC24 mRNA expression level: breast cancer tissues versus paired paracancer tissues (left), breast cancer tissues versus Normal tissues (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e \u003cstrong\u003eExpression of DNAJC24 in different PAM50 molecular subtypes of breast cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e \u003cstrong\u003eExpression level of DNAJC24 in different clinical stages of breast cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e \u003cstrong\u003eCorrelation analysis of DNAJC24 expression with PR, ER and HER2 expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e \u003cstrong\u003eAnalysis 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\u0026lt; 0.05, *p\u0026lt; 0.01*** p\u0026lt; 0.001. ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3823704/v1/68e35ab5905ce614e71b4826.png"},{"id":49236147,"identity":"84ad4975-0c56-4c86-a561-ed15b9bab666","added_by":"auto","created_at":"2024-01-05 17:52:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2798725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining of 290 breast cancer patients shows that the higher expression of DNAJC24 is associated with the better prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e \u003cstrong\u003eThe expression of DNAJC24 in breast cancer tissues was divided into four categories: negative, low, moderate and high.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e \u003cstrong\u003eExpression level of DNAJC24 in different types of breast cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e \u003cstrong\u003eThe proportion of high expression of DNAJC24 in tumor tissues was lower than that in normal tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e \u003cstrong\u003eRelationship between DNAJC24 expression and OS in ER- patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e \u003cstrong\u003eRelationship between DNAJC24 expression and DFS in ER- patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e \u003cstrong\u003eRelationship between DNAJC24 expression and OS in PR-patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e \u003cstrong\u003eRelationship between DNAJC24 expression and DFS in PR-patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e \u003cstrong\u003eThe expression levels of DNAJC24 were correlated with breast cancer patients’ DFS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u003c/strong\u003e \u003cstrong\u003eThe expression levels of DNAJC24 were correlated with breast cancer patients’ OS.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3823704/v1/5d8ae35e466fad366ef80bbd.png"},{"id":49236145,"identity":"96ad9e73-f424-41ae-bfc2-1cd1f1535bb6","added_by":"auto","created_at":"2024-01-05 17:52:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1045542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNAJC24 inhibits proliferation of breast cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. DNAJC24 level in four wild-type breast cancer cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Lentivirus infected breast cancer cells MDA-MB-231 to construct stable DNAJC24 overexpression cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Lentivirus infected breast cancer cell T47D to construct stable DNAJC24 knockdown cell line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. Upregulation of DNAJC24 suppresses clonogenesis of MDA-MB-231 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. Upregulation of DNAJC24 suppresses proliferation of MDA-MB-231 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. Downregulation of DNAJC24 promotes clonogenesis of T47D cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. Downregulation of DNAJC24 promotes proliferation of T47D cells. The values were shown as mean ± SEM, p\u0026lt; 0.05, *p\u0026lt; 0.01*** p\u0026lt; 0.001. ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3823704/v1/4f229fb0ae51e99d4a516bf2.png"},{"id":49236148,"identity":"6ffdae63-bab6-44d3-9df4-acea92e2354c","added_by":"auto","created_at":"2024-01-05 17:52:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3241707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNAJC24 inhibits chemotaxis, invasion and migration of breast cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Upregulation of DNAJC24 inhibits chemotactic ability of MDA-MB-231 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Downregulation of DNAJC24 promotes chemotactic ability of T47D cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Upregulation of DNAJC24 restrains the invasion of MDA-MB-231.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. Downregulation of DNAJC24 enhances invasion ability of T47D cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. Upregulation of DNAJC24 decreases migration ability of MDA-MB-231 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. Downregulation of DNAJC24 increases migration ability of T47D cells. The values were shown as mean ± SEM, p\u0026lt; 0.05, *p\u0026lt; 0.01*** p\u0026lt; 0.001. ns, not significant.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3823704/v1/5dfde7a46c75ffdfa9512382.png"},{"id":49590701,"identity":"278f8142-6b74-4a29-bc3c-856bdce3c8a0","added_by":"auto","created_at":"2024-01-14 21:07:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3414198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3823704/v1/2864ab36-9b41-4bb2-aec5-bfbc2222448c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of DNAJC24 as a potential therapeutic target in breast cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn terms of incidence, breast cancer has currently risen to the top spot worldwide(Sung et al., 2021). Studies have shown that early diagnosis, radiotherapy, and chemotherapy can significantly improve the prognosis of patients with breast cancer; however, but an extremely high proportion of breast cancer cases progress to distant metastasis and recurrence within a short time after clinical diagnosis (Hou, Peng, \u0026amp; Li, 2022). The identification of key regulatory genes involved in the onset and development of breast cancer at the molecular level, the elucidation of the molecular mechanism of breast cancer pathogenesis and invasion, the discovery of reliable and highly specific diagnostic and prognostic biomarkers, and the continuous search for new targets for the development of new treatments are essential for reducing the incidence and mortality of breast cancer.\u003c/p\u003e \u003cp\u003eEukaryotic elongation factor-2 (eEF2) undergoes diphtheria amidation modification by DnaJ heat shock protein family member C24 (DNAJC24), a member of the dipthamide biosynthesis protein (DPH) family and commonly known as DPH4(Kespohl et al., 2020; Robinson, Henriksen, \u0026amp; Maxwell, 1974). 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. Diphtheria toxin further modifies eEF2 by recognizing diphtheria amide, which inhibits protein synthesis and induces cell death(Xu et al., 2020). The biological role of DNAJC24 (DPH4), which has been found to be associated with diphtheria amide in the current investigation, 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(Wei et al., 2012). Our previous study confirmed for the first time that DNAJC24 plays an important role in the proliferation and migration of hepatocellular carcinoma (HCC) cells by affecting ammonia metabolism(Li et al., 2022). However, the role of DNAJC24 in the occurrence and development of breast cancer or other cancers has not yet been reported.\u003c/p\u003e \u003cp\u003eIn the current study, we first investigated the expression of DNAJC24 (DPH4) in various breast tumor cell lines, established the knockdown and overexpression cell lines of DNAJC24 in breast tumor cells, and preliminarily evaluated the effect of DNAJC24 (DPH4) protein on breast cancer. The preliminary investigation of DNAJC24 (DPH4) 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 closely related to the development of breast cancer, providing a theoretical basis and foundation for the development of new tumor markers for breast cancer.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Human breast cancer tissue samples\u003c/h2\u003e \u003cp\u003eIn this study, samples from patients who were undergoing surgical resection of breast cancer 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003e In this study, four human breast cancer cell lines, namely, T47D, MDA-MB-231, SKBR3, and MCF-7, were all derived from the American Type Culture Collection (ATCC) and provided by the Department of Tumor Cell Biology, Cancer Research Institute, Tianjin Cancer Hospital. The cell lines generated in this study were grown in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, Carlsbad, CA, USA) with 10% v/v fetal bovine serum (FBS; HyClone Laboratories Inc., Novato, CA, USA) and 1% penicillin\u0026ndash;streptomycin solution (PS; Hyclone Laboratories Inc.) at 37\u0026deg;C in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell transfection\u003c/h2\u003e \u003cp\u003ePackaging plasmids (VSVG and ΔR) and expression plasmids (OE and matched Ctrl, KD and matched Ctrl) were transfected into HEK293T cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 h, the medium supernatant was collected to obtain lentiviral particles. T47D and MDA-MB-231 cells were infected with lentivirus that was carrying DNAJC24 short hairpin RNA (shRNA) and their respective scrambled controls to produce DNAJC24 knockdown (KD), DNAJC24 overexpression (OE), and matched control (KD SCR and OE SCR) cell lines. Stably transfected clones were selected using a puromycin (Gibco)-containing medium. Transfection efficiency was validated through Western blot assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot\u003c/h2\u003e \u003cp\u003eThe cells in a 10 cm dish were washed three times with ice-cold phosphate-buffered saline (PBS) (pH 6.8) and then lysed with 1 \u0026times; sodium dodecyl sulfate (SDS) lysis buffer (Tris-HCl, pH 6.8, 62.5 mM, 2% SDS, 10% glycerol) supplemented with 1 mM of NaF, 1 mM of Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1 \u0026times; protease, and phosphatase inhibitor cocktail (Hoffman-la Roche Ltd., Basel, Switzerland) on ice for 30 min. Protein denaturation was performed at 95\u0026deg;C for 10 min. The cell lysates were stored at \u0026minus;\u0026thinsp;20 ℃. Equal amounts of protein (30\u0026ndash;90 \u0026micro;g/cell line) were loaded, separated via SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane (hydrophobic) (Merck KGaA, Darmstadt, Germany). Antibodies against the following proteins were used: anti-DNAJC24 (1:500) from Abcam; anti-glyceraldehyde 3-phosphate dehydrogenase (1:1000), goat anti-rabbit (1:4000), and goat anti-mouse (1:4000) secondary antibody from Santa Cruz Biotechnology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Colony formation assay\u003c/h2\u003e \u003cp\u003eFor the colony formation assay, 1000 cells in DMEM supplemented with 10% FBS were plated in six-well plates. After 2 weeks of incubation, the surviving colonies were fixed, stained with 0.5% crystal violet, imaged, and counted. The data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of triplicate dishes in the same experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eThe cells were grown in 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well for the initial concentration. Then, 5 duplicate wells for each cell line were set to reduce differences within a group. Then, the cells were incubated with Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan; 10 \u0026micro;L/well) for 2 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Optical density (OD) was measured using an enzyme-linked immunosorbent assay reader (BioTek SynergyH1, Burlington, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Chemotaxis assay\u003c/h2\u003e \u003cp\u003eDMEM with 20% FBS was loaded into the lower chambers (600 \u0026micro;L per chamber) and then the cells were suspended and loaded into the upper chambers of a transwell (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/200 \u0026micro;L per chamber). After incubating at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e (MDA-MB-231 cells for 4 h; T47D cells for 6 h), the membrane was rinsed with PBS, fixed in 4% paraformaldehyde, and stained. Three fields were randomly selected to count the number of migrated cells at \u0026times;200 via light microscopy (Olympus BX61).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Matrigel invasion assay\u003c/h2\u003e \u003cp\u003eFor the invasion assay, Matrigel-coated transwell chambers were incubated in 24-well plates for more than 1 h at 37 ℃. DMEM supplemented with 20% FBS was loaded in the lower chamber, and 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in serum-free DMEM were added to the upper chamber. After 24 h incubation at 37 ℃ in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e, the invaded cells on the bottom were fixed with 4% paraformaldehyde for 20 min, stained, and counted via light microscopy (Olympus BX61).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Scratch assay\u003c/h2\u003e \u003cp\u003eThe cells were seeded in six-well plates 1 day prior to the assay, with each well containing a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well to achieve a confluent monolayer. The culture medium used was DMEM without FBS. A 10 \u0026micro;L pipette tip was used to create a uniform wound. Subsequently, the cells were incubated in a humidified atmosphere at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The distance between the wounds was measured at three random sites within specific time intervals (0, 3, 6, 9, 12, and 24 h). The data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, while representative images were captured at the initial and final time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Immunohistochemical assays\u003c/h2\u003e \u003cp\u003eImmunohistochemistry (IHC) staining for DNAJC24 was performed in 290 pairs of cancerous and matched adjacent nonmalignant tissue. The tissues were sequentially immersed in xylene and gradient ethanol for dewaxing and rehydration. Antigen retrieval was performed using a citrate target retrieval solution at pH 6.0 (ZSGB-BIO, ZLI-9065) in a pressure cooker for 2.5 min and then cooled naturally to room temperature. Subsequently, 3% hydrogen peroxide was used to inhibit endogenous hydrogen peroxidase activity. Subsequently, the samples were incubated with rabbit anti-DNAJC24 (1:50, Abcam, ab246925) at room temperature for 30 min and overnight at 4\u0026deg;C. After washing with PBS, the samples were stained with secondary antibody for 1 h at room temperature. The cells were visualized with a 3,3-diaminobenzidine solution (ZSGB-Bio, ZLI-9017) and counterstained with hematoxylin. Finally, the sections were photographed using a light microscope (Olympus BX61). The IHC score of DNAJC24 was the product of the staining intensity and percentage scores. DNAJC24 positive expression intensity: Grade 0 is negative, Grade 1 is low, Grade 2 is moderate, and Grade 3 is high.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Expression of DNAJC24 in cancer tissues is different from that in normal tissues\u003c/h2\u003e \u003cp\u003eAnalysis of The Cancer Genome Atlas (TCGA) database indicated that the mRNA level of DNAJC24 in cancer tissues, such as breast cancer, pancreatic carcinoma, prostate cancer, thymus cancer, and endometrial cancer was lower than that in adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A). In accordance with the screening conditions, 841 cases of female breast cancer raw data were downloaded from the TCGA database, including 104 pairs of normal 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 corresponding cancer tissues was significantly decreased compared with that in the adjacent tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B, left). Meanwhile, similar results were obtained when comparing normal tissue samples with malignant tumor samples, and the expression level of DNAJC24 in cancer tissues was significantly lower than that in normal tissues, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B, 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.C).\u003c/p\u003e \u003cp\u003eAt 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, regardless of T stage, N stage, or TNM stage, with statistical difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.D). In addition, PR, ER, and HER2 are among the most important immunohistochemical indicators of breast cancer. In the current study, patients were divided into negative and positive groups based on 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 positive and negative groups was analyzed via \u003cem\u003et\u003c/em\u003e-test. The results showed that the expression level of DNAJC24 was higher in the ER-positive and PR-positive groups than in the negative group. However, the trend was the opposite in the HER2-positive group. Compared with the negative group, DNAJC24 was downregulated in the HER2-positive group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.E). Finally, Kaplan\u0026ndash;Meier survival analysis showed that DNAJC24 generally exerted no significant effect on relapse-free survival (DFS). However, 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\u0026thinsp;=\u0026thinsp;0.032) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.F). In general, the results of the database analysis showed that the mRNA level of DNAJC24 was significantly higher in adjacent tissues than in 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 associated with a lower risk of recurrence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Higher expression of DNAJC24 is associated with better prognosis in breast cancer tissues\u003c/h2\u003e \u003cp\u003eIn the current study, samples from patients who underwent surgical resection for breast cancer 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A). For comparison, three-negative breast cancer has the lowest expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.C). That is, the expression level of DNAJC24 in normal tissues was higher than that in cancer tissues. This result was consistent with the analysis of the TCGA database. Among breast cancer patients who did not express ER (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.D and E) and breast cancer patients who did not express PR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.F and G), DNAJC24 was positively correlated with overall survival (OS) and disease-free survival. Similar results were obtained in the total samples, where DNAJC24 was positively associated with OS and disease-free survival of the patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 DNAJC24 inhibits proliferation of breast cancer cells\u003c/h2\u003e \u003cp\u003eThe database analysis and immunohistochemical analysis of cancer and adjacent tissues from 290 breast cancer patients showed that patients with decreased expression of DNAJC24 had poor prognosis. Does DNAJC24 inhibit the malignant behavior of breast cancer? Western blot was used to analyze the protein expression of DNAJC24 in 4 wild-type human breast cancer cell lines, namely, T47D, MDA-MB-231, SKBR3, and MCF-7. The results showed that the expression of DNAJC24 protein was highest in T47D and lowest in MDA-MB-231 among the 4 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A). In accordance with the expression of DNAJC24 protein in the 4 cell lines, MDA-MB-231 was selected for OE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B), while T47D cells were selected for KD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.C) to investigate the effect of DNAJC24 on the biological function of the two cell lines.\u003c/p\u003e \u003cp\u003eThe number of colony formation was proportional to the cell proliferation ability. Therefore, a plate cloning experiment was used to detect the changes in cell proliferation ability after the overexpression of DNAJC24 in MDA-MB-231 and the down-expression of DNAJC24 in T47D. The results were statistically analyzed via two independent sample \u003cem\u003et\u003c/em\u003e-tests. After the overexpression of DNAJC24, the number of colony formed in the MDA-MB-231 OE/DNAJC24 group was significantly lower than that in the MDA-MB-231 OE/SCR group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.D). CCK-8 is also an important tool for detecting cell proliferation ability. Therefore, CCK-8 was used to detect the proliferation of MDA-MB-231 OE cell lines at 24, 48, 72, 96, and 120 h. The cell growth curve 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.E). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.F). The proliferation rate of the T47D KD/SCR cell line was lower than that of the T47D KD/DNAJC24 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.G). These results indicate that the overexpression of DNAJC24 inhibits tumor cell proliferation, while the down-expression of DNAJC24 may promote tumor cell proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 DNAJC24 inhibits invasion and migration of breast cancer cells\u003c/h2\u003e \u003cp\u003eTranswell migration assay was used to detect the ability of cell chemotaxis and invasion in vitro. A medium with 20% FBS was added to the lower chamber, and cells with the medium were added to the upper chamber. Differences in nutrient composition promoted the chemotaxis of cells. In the invasion experiment, Matrigel was added to the bottom of the upper chamber. Compared with the control group, DNAJC24-overexpressed MDA-MB-231 that passed through the transwell chamber was less (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A), suggesting that DNAJC24 could significantly reduce the chemotactic ability of MDA-MB-231 breast cancer cells in vitro. The Matrigel-coated transwell mimics the ability of 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.C), suggesting that DNAJC24 could significantly reduce the cell invasion ability of MDA-MB-231 breast cancer cells.\u003c/p\u003e \u003cp\u003eScratch experiments were performed in a serum-free conditioned medium. Therefore, the effect of cell proliferation was excluded and the rate of cell migration could be measured. In the scratch experiments, the migration rate of MDA-MB-231 cells overexpressed DNAJC24 was lower than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.E). Meanwhile, invasion chemotaxis and migration experiments were also performed in T47D cells with KD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.B, D, and F). The results were consistent with those of the MDA-MB-231 cell line, i.e., DNAJC24 not only inhibited the proliferation of breast cancer cells, but also inhibited the chemotactic, invasion, and migration abilities of breast cancer cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eBiomarkers are quantifiable indicators of normal biological processes, pathogenic, or responses to exposures or interventions(Cserni et al., 2011). Advances in the field of pathology and in-depth research into the molecular mechanisms of breast cancer progression have brought an increasing number of breast cancer biomarkers to the fore. These include immunohistochemical markers (e.g., ER, PR, HER2[ERBB2], and the proliferation marker protein Ki-67[MKI67]), genomic markers (e.g., BRCA1, BRCA2, and PIK3CA), and immunological markers (e.g., tumor-infiltrating lymphocytes and PD-L1)(Veronesi, Boyle, Goldhirsch, Orecchia, \u0026amp; Viale, 2005). 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 causes of breast cancer-related mortality(Crimini et al., 2021). 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.\u003c/p\u003e\n\u003cp\u003eHeat shock proteins (HSPs) are a group of proteins whose function is to reverse or inhibit the denaturation or unfolding of proteins in cells under pressure or high temperature. Traditionally, HSPs have also been referred to as molecular chaperones because of their role in maintaining the structural stability 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)(Ciocca \u0026amp; Calderwood, 2005). Many studies have been conducted on the relationship between the HSP family and cancer. For example, the inhibition of HSP27 has been demonstrated to reverse epithelial-to-mesenchymal transition (EMT), reduce matrix metalloproteinase (MMP) activity, proliferation, migration, and invasion of cancer cells(Gibert, Simon, Dimitrova, Diaz-Latoud, \u0026amp; Arrigo, 2013). HSP60 promotes cancer cell survival by binding to and inhibiting intracellular protein aggregins in neuroblastoma cells(Chaiwatanasirikul \u0026amp; Sala, 2011). Jaattela et al. reported that HSP70 protected cancer cells from TNF-induced cytotoxicity, and they suggested that HSP70 may enhance the carcinogenic potential of certain cancer cells through immune escape mechanisms(R\u0026eacute;role, Jego, \u0026amp; Garrido, 2011). The HSP40/DNAJ family of proteins comprises homologues of the bacterial DnaJ HSP. Most members contain a \u0026ldquo;J\u0026rdquo; domain through which they can interact with HSP70, and thus, HSP40 is also known as an HSP70 co-partner (Alderson, Kim, \u0026amp; Markley, 2016; Faust \u0026amp; Rosenzweig, 2020). Among all the major human HSP families, the HSP40 family has the largest number of members. The proteins of HSP40 are classified into three DNAJ subclasses: DNAJA, DNAJB, and DNAJC. Our previous studies have shown that an increased expression of DNAJC24 in HCC cells can promote the proliferation and migration of HCC cells. Targeting DNAJC24 can interfere with ammonia metabolism, affecting the proliferation and autophagy of HCC cells and ultimately inhibiting the malignant progression of HCC. However, Diane L N Trinh et al. found that Tid1 (also known as DNAJA3) interacts with HSP70 protein, which has been identified as a regulator of p53-mediated apoptosis, and interacts directly with p53, leading to the mitochondrial translocation of the complex and the induction of apoptosis in MCF-7 breast cancer cells(Trinh, Elwi, \u0026amp; Kim, 2010). 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 contrast behavior compared with its role in HCC(Li et al., 2022), because it significantly inhibited tumor cell proliferation and the invasion of tumor cells in breast cancer.\u003c/p\u003e\n\u003cp\u003eIn the current study, the analysis of TCGA data showed differences in the expression of DNAJC24 in the cancer and paracancerous 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 cancer tissues compared with in adjacent tissues or normal tissues. Therefore, we further investigated the expression 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 than in cancer tissues. This finding was consistent with the results of database analysis. In addition, DNAJC24 expression was positively correlated with DFS and OS in ER-negative or PR-negative patients. This result suggests that DNAJC24 may have a function in inhibiting tumor malignant progression in breast cancer. 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 acts as a tumor suppressor in breast cancer and that the loss of DNAJC24 expression may be associated with the progression of breast cancer, 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.\u003c/p\u003e\n\u003cp\u003eWe preliminarily investigated the effect of DNAJC24 on breast cancer progression, 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(Chen \u0026amp; Behringer, 2004; Liu et al., 2006; Webb et al., 2008), but no such lethal phenotype occurs when similar knockout is performed in cell lines. This 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 DPH4: the conserved J domain and the CSL domain, which are helixally connected by a flexible linker. The adaptor helix regulates the conformational flexibility between the two domains, and thus, the function of the protein(Liu, Milne, Kuremsky, Fink, \u0026amp; Leppla, 2004). DPH4 exhibits the unique ability to bind iron in a tetrahedral coordination geometry through the cysteine of its CSL domain. Iron-bound DPH4 (Fe-DPH4) also undergoes oligomerization, and thus, may function as a transient \u0026ldquo;iron storage protein\u0026rdquo; to regulate intracellular iron homeostasis(Thakur et al., 2012). Metabolic disorders are also a hallmark of cancer cells. In addition, shRNA targeting diproylamine biosynthesis proteins 1\u0026ndash;4 (DPH1\u0026ndash;DPH4) can increase the adhesion of human podocytes, and immortalized human podocytes stably expressing these shRNA exhibit increased adhesion(Cin\u0026agrave; et al., 2019). 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 DPH4 is to participate in the diacetylamine modification of human eEF2, which is involved in protein synthesis(Liu et al., 2004; Stahl et al., 2015). 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, DPH4 can protect brain endothelial cells from stress conditions(Sun et al., 2015), but the consequence of the lack of this protective effect on tumor cells is unknown. At present, research on DNAJC24 remains scarce. Our study first shows that DNAJC24 can inhibit the malignant behavior of breast cancer, exhibiting potential as a diagnostic and therapeutic target for breast cancer. Our study also demonstrates the diversity of HSP family members on cancer and lays the foundation for further research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatements \u0026amp; Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003eThis research was supported by The\u0026nbsp;Science \u0026amp; Technology\u0026nbsp;Development Fund\u0026nbsp;of Tianjin\u0026nbsp;Education\u0026nbsp;Commission for\u0026nbsp;Higher\u0026nbsp;Education(2021KJ191).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u0026nbsp;\u003c/strong\u003eThe authors declare they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003eAll authors contributed to the study conception and design. \u0026nbsp;\u003cstrong\u003eWenjing Meng:\u0026nbsp;\u003c/strong\u003eData curation; investigation(equal); methodology(equal); funding acquisition(equal).\u003cstrong\u003e\u0026nbsp;Wei Liu:\u0026nbsp;\u003c/strong\u003eWriting original draft(equal); visualization(equal).\u003cstrong\u003e\u0026nbsp;Xiaorui Wang:\u0026nbsp;\u003c/strong\u003eSoftware(equal). \u003cstrong\u003eLinlin Zhan:\u0026nbsp;\u003c/strong\u003eInvestigation(equal); validation(equal). \u003cstrong\u003eYuchao He:\u0026nbsp;\u003c/strong\u003eMethodology(equal); formal analysis(equal). \u003cstrong\u003eYi Luo:\u0026nbsp;\u003c/strong\u003eWriting-Review \u0026amp; Editing(equal); supervision(equal). \u003cstrong\u003eLiwei Chen:\u0026nbsp;\u003c/strong\u003eWriting-Review \u0026amp; Editing(equal); resources(equal).\u003cstrong\u003e\u0026nbsp;Yu Wang:\u0026nbsp;\u003c/strong\u003eInvestigation(equal).\u003cstrong\u003e\u0026nbsp;Guangtao Li\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eInvestigation(equal); validation(equal). \u003cstrong\u003eYehui Shi:\u0026nbsp;\u003c/strong\u003eResources(equal).\u003cstrong\u003e\u0026nbsp;Zhongsheng Tong:\u0026nbsp;\u003c/strong\u003eResources(equal). \u003cstrong\u003eHua Guo:\u0026nbsp;\u003c/strong\u003eProject administration(equal); methodology(equal); conceptualization(equal).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003eAll data have been included within this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlderson, T. 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Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development. \u003cem\u003eJ Cell Sci, 121\u003c/em\u003e(Pt 19), 3140-3145. doi:10.1242/jcs.035550\u003c/li\u003e\n \u003cli\u003eWei, H., Xiang, L., Wayne, A. S., Chertov, O., FitzGerald, D. J., Bera, T. K., \u0026amp; Pastan, I. (2012). Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy. \u003cem\u003eProceedings of the National Academy of Sciences of the United States o f America, 109\u003c/em\u003e(18), 6898-6903. doi:10.1073/pnas.1204523109\u003c/li\u003e\n \u003cli\u003eXu, L., Raabe, M., Zegota, M. M., Nogueira, J. C. F., Chudasama, V., Kuan, S. L., \u0026amp; Weil, T. (2020). Site-selective protein modification via disulfide rebridging for fast tetrazine/trans-cyclooctene bioconjugation. \u003cem\u003eOrganic \u0026amp; biomolecular chemistry, 18\u003c/em\u003e(6), 1140-1147. doi:10.1039/c9ob02687h\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"breast cancer, DNAJC24, molecular subtype, prognosis biomarker","lastPublishedDoi":"10.21203/rs.3.rs-3823704/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3823704/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Purpose\u003c/h2\u003e \u003cp\u003eOn the basis of current markers, a variety of targeted therapies have been proposed for clinical practice, but treatment resistance and recurrence remain the leading 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.\u003c/p\u003e\u003ch2\u003eApproach and Key Results\u003c/h2\u003e \u003cp\u003eIn this study, we focused on investigating the role of DnaJ heat shock protein family member C24 (DNAJC24) in breast cancer through bioinformatic analysis by using public databases and clinicopathological samples. In addition, we performed \u003cem\u003ein vitro\u003c/em\u003e functional assays to explore the biological functions of DNAJC24. \u003cem\u003eIn vitro\u003c/em\u003e experiments showed that the overexpression of DNAJC24 significantly inhibited the proliferation and invasion of breast cancer cells. Meanwhile, the downregulation of DNAJC24 enhanced cell proliferation, invasion, and migration. The findings provide a theoretical basis for the identification of a novel molecular therapeutic target for breast cancer.\u003c/p\u003e\u003ch2\u003eConclusion and Implications\u003c/h2\u003e \u003cp\u003eOur research revealed that DNAJC24 was weakly expressed in breast cancer tissues, suggesting its involvement in breast carcinogenesis. Furthermore, we observed an inverse correlation between DNAJC24 expression and malignant progression of breast cancer including clinical stage among different pathological subtypes and molecular staging of breast cancer. Therefore, given the significant association between low levels of DNAJC24 expression and poor prognosis in patients with breast cancer, DNAJC24 may serve as a predictive marker.\u003c/p\u003e","manuscriptTitle":"Identification of DNAJC24 as a potential therapeutic target in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-05 17:52:28","doi":"10.21203/rs.3.rs-3823704/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ca87982-4847-4a2b-ad56-564a7758e348","owner":[],"postedDate":"January 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-14T20:59:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-05 17:52:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3823704","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3823704","identity":"rs-3823704","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}