Bioinformatics analysis of the clinicopathological and prognostic significance of BAG3 mRNA in gynecological cancers

Abstract BAG3 is a co-chaperone BAG family protein that plays important roles in protein homeostasis, cell survival, cell motility, and tumour metastasis. This study aimed to clarify the clinicopathological and prognostic implications of BAG3 mRNA expression in tumours. We performed bioinformatics analysis on BAG3 mRNA expression using TCGA, XIANTAO, UALCAN, and Kaplan-Meier plotter databases. BAG3 mRNA expression was downregulated in breast and endometrial cancers and positively correlated with favourable PAM50 subtyping in breast cancer，clinical stage and short overall survival in ovarian cancer and negatively correlated with T stage, clinical stage, and histological grade in cervical and endometrial cancers. The top BAG3-related pathways included ligand-receptor interactions and activity, DNA packaging and nucleosomes, hormonal responses, membrane regions, microdomains and rafts, and endosomes in breast cancer; ligand-receptor interactions, transmembrane transporters and channels, cell adhesion, and keratinisation in cervical cancer; ligand-receptor interactions, anion transmembrane transporters, lipoproteins, keratinisation, cell adhesion, and protein processing in endometrial cancer; metabolism of porphyrin, chlorophyll, pentose, uronic acid, ascorbate, and alternate and cell adhesion in ovarian cancer. BAG3 expression could represent a potential marker for carcinogenesis, histogenesis, aggressive behaviours, and prognosis in gynecological cancers. IMPACT STATEMENT What is already known on this subject? BAG3 regulates cell activity, autophagy, and resistance to apoptosis through multiple domains and plays an important role in tumour development. BAG3 positively regulates tumour cell invasion and migration in cervical and ovarian cancers. What do the results of this study add? BAG3 expression is closely associated with histogenesis, clinicopathology, and prognosis in gynecological cancers and is involved in signalling pathways associated with the control of cell proliferation, migration, invasion, and drug resistance in tumours. What are the implications of these findings for clinical practice and/or further research? Abnormal BAG3 expression can be employed as a possible marker of tumour development, invasion, and prognosis, providing new ideas for treating cancer.

Bcl-2-associated athanogene 3 (BAG3, BIS, CAIR-1, or MFM6) is a member of the Hsc70 binding co-chaperone BAG-family of proteins and plays important roles in apoptosis and autophagy, protein homeostasis, cell adhesion, cell migration, and tumour metastasis.Human BAG3 is located on chromosome 10q25.2-q26.2,and its 2608-nucleotide cDNA encodes a 575-amino-acid product of 61 kDa (Lee et al. 1999).BAG3 is a cytoplasmic protein located in the rough endoplasmic reticulum that can exhibit a binuclear state or nuclear localisation in certain cell types or when certain cells are exposed to external stimuli.The BAG3 protein interacts with the ATPase domain of Hsc70 in a Hip-modulated manner through the BAG domain (110-124 amino acids) and with other molecules through its WW domain, IPV motifs, PXXP repeats, and a proline-rich region containing SH3-binding motifs (Liu et al. 2022).BAG3 binds to the SH3 domain of PLC-c via its PXXP region, forming a ternary complex regulated by epidermal growth factor.Through an indirect effect on focal adhesion kinase, its proline-rich repeat controls cell adhesion and migration (Rosati et al. 2011).The proline-rich structural domain of BAG3 can be removed to re-establish migration and the stickiness to matrix molecules in MDA435 human breast cancer cells (Kassis et al. 2006).In contrast, the PPDY motif at the C-terminus of guanine nucleotide exchange factor 2 (PDZGEF2) binds to the WW domain of BAG3, the deletion of which can lead to a loss of cell adhesion and motility activity (Iwasaki et al. 2007).BAG3 overexpression promotes cancer cell survival by modifying the interaction between IKKgamma and HSP70, boosting IKKgamma and shielding it from proteasome-dependent degradation in the NF-jB pathway (Ammirante et al. 2011).
BAG3 silencing causes abnormal cell cycle progression, most notably in the S and G 2 phases, confirming a decrease in cyclin B1 expression levels.(Gentilella et al. 2010).Upon activation of the eIF2alpha signalling pathway, BAG3 interacts with heat shock protein HSPB8 to initiate macroautophagy and protein quality control (Carra et al. 2010, Fuchs et al. 2009).BAG3 overexpression is cytoprotective against heat shock and protects ubiquitinated proteins, such as Akt, from being degraded by the proteasome (Virador et al. 2009).Zhang et al. (2022) found that miR-135a-5p reduced breast cancer cell proliferation, transplantation, and invasion by regulating BAG3.The effects of miR-135a-5p on the malignant phenotype of breast cancer cells are reversed by BAG3 overexpression, which triggers cell cycle, mTOR, and TGF-signalling pathways.
BAG3 is a multifunctional protein induced by intracellular stress and overexpressed in skeletal muscle, cardiomyocytes, and various malignancies (Qu et al. 2022).BAG3 knockout mice have retarded growth and fulminant myopathy after birth, characterised by non-inflammatory myofibrillar degeneration with apoptotic characteristics (Homma et al. 2006).BAG3 expression in prostate cancer progressively decreases as the degree of differentiation decreases, along with the loss of polarisation of the signal in metastatic cancer (Staibano et al. 2010).Numerous human epithelial cancer cell lines express high BAG3 protein levels, which are strongly correlated with tumour invasion and metastasis (Iwasaki et al. 2007).The function of BAG3 expression in colorectal carcinogenesis and progression was discovered by measuring BAG3 mRNA and protein expression in colorectal cancer tumours and comparing it with clinicopathological indicators and tumour survival time.
Women are particularly susceptible to breast, vulvar, vaginal, cervical, endometrial, and ovarian cancer (Paepke et al. 2020, Aquil et al. 2021, Segev et al. 2021, Seland et al. 2022).Vargiu et al. (2022) reported that assessing a woman's BMI is fundamental to developing treatment strategies and reducing complications in gynecological oncology.Various pieces of evidence indicate that obesity and metabolic diseases may increase cancer risk and regulate pivotal cross-talk pathways for cell proliferation and differentiation (Giannini et al. 2022, Vargiu et al. 2022).In the present study, we aimed to illustrate the clinicopathological characteristics and prognostic significance of BAG3 mRNA expression and BAG3-related signalling pathways in breast, cervical, endometrial, and ovarian cancers using XIANTAO, The Cancer Genome Atlas (TCGA), Kaplan-Meier plotter, and UALCAN datasets as summarised in a previous report (Zheng et al. 2023).

KM plotter analysis
The predictive significance of BAG3 mRNA in cervical, endometrial, breast, and ovarian cancer was examined using the Kaplan-Meier plotter (http://kmplot.com).

UALCAN analysis
BAG3 expression and methylation were examined using the UALCAN database (http://ualcan.path.uab.edu).These data were contrasted with the predictive parameters and clinicopathological characteristics of ovarian, breast, cervical, and endometrial cancer.

XIANTAO analysis
The XIANTAO platform (https://www.XIANTAO.love/)was used to examine BAG3 expression and methylation.Utilising the XIANTAO platform, we discovered the differential and relevant genes between low and high BAG3 expression groups.The differentially expressed genes were used to construct a PPI network (Protein-Protein Interaction Network), and the crucial hub genes were identified.KEGG (Kyoto Encyclopaedia of Genes and Genomes analysis on these genes was used to determine the related signal pathways of differential and relevant genes.

Statistical analysis
The means were compared using the Student's t-test.Chisquare test was employed to compare the positive rates.Kaplan-Meier survival curves were generated, and the curves were compared using the log-rank test.Multivariate analysis was conducted using Cox's proportional hazards model.Statistical differences (two-sided) were considered significant when p < 0.05.Statistical analysis was performed using SPSS 17.0 software.

Ethical approval statement
The Ethics Committee of The First Affiliated Hospital of Jinzhou Medical University authorised the research plan (Project Ethics number: 202334).

Clinicopathological and prognostic significance of BAG3 mRNA expression in breast cancer
According to the UALCAN analysis, BAG3 is expressed at a lower level in breast cancer than in normal tissue (p < 0.05; Figure 1(A)).It was higher in luminal breast cancer than in triple-negative and Her2-positive breast cancer (p < 0.05; Figure 1(B)).BAG3 expression was positively associated with the Asian race, infiltrating lobular carcinoma, ER (Estrogen Receptor) and PR (Progesterone Receptor) expression, and favourable PAM50 subtyping (p < 0.05; Table 1).

Clinicopathological and prognostic significance of BAG3 mRNA expression in ovarian cancer
TCGA analysis showed that BAG3 expression was higher in young than in old [95% CI ¼ 465: 205 À 734] patients and in stages 3 and 4 than in stage 2 (p < 0.05; Supplementary Figure 3(A)).Kaplan-Meier analysis demonstrated that high BAG3 expression in ovarian cancer had poorer overall survival than those with low expression.There was also a positive correlation between BAG3 expression and the recurrence-free survival rate of White cancer patients (Supplementary Figure 3(B), p < 0.05).

Relationship between BAG3 mRNA expression and infiltrating immune cells in gynecological cancer
Based on the XIANTAO analysis, BAG3 expression is favourably associated with the infiltration of mast cells, eosinophils, NK CD56 bright cells, and NK cells but negatively correlated with CD8 T cells, TFH, Tem, macrophages, NK CD56dim cells, Treg, cytotoxic cells, aDC, T cells, Th1 cells, DC and B cells in breast cancer (p < 0.05; Figure 2).It was also favourably correlated with the infiltration of Tcm, Tgd, T helper cells, iDC, DC, Th1 cells, NK CD56dim cells, Th2 cells, and aDC but unfavourable with CD8 T cells, Th17 cells, TFH, Tem, NK cells, pDC, and NK CD56bright cells in cervical cancer (p < 0.05; Figure 2).BAG3 expression was also favourably correlated with the infiltration of Th2 cells, Tgd, and Tcm but negatively related to cytotoxic cells, B cells, and pDC in endometrial cancer (p < 0.05; Figure 2).Moreover, it was favourably correlated with the infiltration of Tem, eosinophils, Tcm, NK cells, NK CD56dim cells, neutrophils, Th2 cells, macrophages, mast cells, iDC, TFH, CD8 T cells, Th1 cells, Tgd, DC, and pDC in ovarian cancer (p < 0.05; Figure 2).

BAG3-related genes and pathways in gynecological cancer
Using the XIANTAO platform, we found the differentially expressed genes between the low and high BAG3 mRNA expression groups in gynecological malignancies.According to the KEGG analysis, the major signalling pathways related to BAG3 included: ligand-receptor interactions and activity, DNA packaging and nucleosomes, and hormonal response in breast cancer; ligand-receptor interactions, transmembrane transporters and channels, apical part, and keratinisation in cervical cancer; ligand-receptor interactions, anion transmembrane transporter, lipoproteins, and keratinisation in endometrial cancer; metabolism of porphyrin, chlorophyll, pentose, uronic acid, ascorbate, and aldarate, extracellular matrix, and glucuronidation in ovarian cancer (Figure 3(A)).Additionally, PPI pairs were identified using STRING, and Cytoscape was used to identify the top ten nodes ranked according to their level of connectivity  (Figure 3(B)).The top hub genes mainly included: chemokines, histones, and clusters of differentiation in breast cancer; late cornified envelope proteins and small proline-rich proteins in cervical and endometrial cancers; mucins, synovial sarcoma X, and melanoma-associated antigens in ovarian cancer.
The genes correlated with BAG3 in the gynecological cancers in the XIANTAO database were subjected to KEGG analysis (Supplementary Figure 4).The BAG3-correlated genes were involved in the following processes: endocytosis, membrane regions, microdomains and rafts, and endosomes in breast cancer; cell adhesion, skin development, and desmosomes in cervical cancer; protein processing, cell adhesion, and Golgi vesicles in endometrial cancer; cell adhesion in ovarian cancer.Zhao et al. (2021) found that BAG3 controlled the epigenetic expression of GALNT10 through WDR5 and ZBTB2 and promotes the stem cell-like characteristics of platin-resistant ovarian cancer cells.Zhang et al. (2022) reported that CHIP-HSP70-BAG3 complex formation enabled the M2 isoform of pyruvate kinase to promote the aggregation of ubiquitinated misfolded proteins, thereby protecting cells from proteasome stress.An et al. (2017) reported that BAG3 directly stabilised hexokinase 2 mRNA and promoted aerobic glycolysis in pancreatic cancer cells.BAG3-mediated Mcl-1 stability promotes treatment resistance with USP9X in ovarian cancer (Habata et al. 2016) and resistance to Bcl-2 antagonists in triple-negative breast cancer and androgen receptor-negative prostate cancer cells (Boiani et al. 2013).BAG3 overexpression was observed in endometrioid endometrial adenocarcinomas (Esposito et al. 2017), medulloblastoma (Yang et al. 2016), seminoma (Bartsch et al. 2016), colorectal cancer (Yang et al. 2013), pancreatic cancer (Rosati et al. 2012), and astrocytic tumours (Festa et al. 2011).Raffone et al. (2020) found that BAG3 expression gradually increased maldevelopment in cervical squamous intraepithelial lesions.However, we found that BAG3 mRNA expression was downregulated in breast and endometrial cancers, suggesting that its low expression might be involved in their tumorigenesis.

Discussion
BAG3 negatively regulates ciliogenesis, ciliary function, and the epithelium-mesenchymal transition (EMT) in spongioblastoma and triple-negative breast cancer cells (Linder et al. 2022), but causes the opposite in cervical cancer (Song et al. 2017).Wang et al. (2018) found that miR-206 targets BAG3 to prevent migration and invasion in cervical cancer.Xiao et al. (2014) found that BAG3 promotes EMT and angiogenesis in human hepatocellular carcinoma.Suzuki et al. (2011) found that BAG3 positively regulates ovarian cancer cell invasion and motility via interactions with MMP-2.Liu et al. (2017) found that BAG3 promoted stem cell-like phenotype in breast cancer by upregulating the expression of CXCR4 through an interaction with its transcript.BAG3 levels were significantly correlated with tumour size, clinical stage and gender of colorectal cancer (Yang et al. 2013).Xiao et al. (2014) demonstrated a significant association between BAG3 staining and tumour TNM stage in hepatocellular carcinoma after liver transplant.Staibano et al. (2010) found that BAG3 expression positively correlated with differentiation and polarisation in prostate carcinoma.Shields et al. (2018) found that lower recurrence-free survival in triple-negative breast cancer patients correlated with high BAG3 mRNA expression.Rosati et al. (2012) found that BAG3 expression positively correlated with short survival time in pancreatic cancer patients who underwent radical resection (R0).Yang et al. (2016) found that BAG3 overexpression was an independent predictive marker significantly correlated with poor prognosis in medulloblastoma.In the present study, BAG3 expression positively correlated with favourable PAM50 subtyping in breast cancer and negatively correlated with histological grade in cervical and endometrial cancer, suggesting that it is possible to use BAG3 expression as a latent indicator for aggressive tendencies in these three cancer types.However, BAG3 expression was positively associated with clinical stage and short overall survival in ovarian cancer in a favourable way, indicating the specificity of the clinicopathological and prognostic significance of BAG3 in ovarian cancer.Additionally, BAG3 expression was higher in squamous cell carcinoma than in endocervical, endometrioid, and mucinous carcinoma, in adenosquamous than in endocervical carcinoma, and in mucinous than in endocervical carcinoma in cervical cancer, and in endometrioid carcinoma than in serous adenocarcinoma in endometrial cancer, demonstrating that BAG3 might be involved in the histogenesis of cervical and endometrial cancers.Dufrusine et al. (2022) found that cytokines, such as interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor, were released by fibroblasts following the induction of BAG3, which can support tumour progression and pancreatic cancer cell migration.De Marco et al. (2018) discovered that a secreted form of BAG3 could bind to the macrophageexpressed IFITM2 receptor and induce the release of substances that promote the growth and spread of tumours.In cervical and endometrial cancers, BAG3 mRNA expression was favourably associated with the infiltration of Tcm, Tgd, and Th2 cells but negatively associated with pDC.In breast cancer, it was negatively linked to the infiltration of Tem, macrophages, Th1 cells, CD8 T cells, NK CD56dim cells, TFH, and DC; the opposite was observed in ovarian cancer.These results demonstrated a potential role for BAG3 in immune surveillance and therapy, and tumour-associated immune responses depended on the gynecological cancer type.Li et al. (2018) found that BAG3 regulates signalling pathways involved in CRC cell proliferation, migration, invasion, and chemoresistance.In our study, the top BAG3-related pathways included ligand-receptor interactions and activity, DNA packaging and nucleosomes, hormonal response, membrane regions, microdomains and rafts, and endosomes in breast cancer; ligand-receptor interactions, transmembrane transporters and channels, cell adhesion, and keratinisation in cervical cancer; ligand-receptor interactions, anion transmembrane transporters, lipoproteins, keratinisation, cell adhesion, and protein processing in endometrial cancer; metabolism of porphyrin, chlorophyll, pentose, uronic acid, ascorbate and alternate, and cell adhesion in ovarian cancer.These findings provide novel clues about the roles and molecular mechanisms by which BAG3 plays an important role in the tumorigenesis and progression of gynecological cancers, which should be investigated in future studies.
In 2013, TCGA classified endometrial cancer into four main genetic clusters: polymerase epsilon (POLE) ultramutated, MSI hypermutated, low copy-number (CN), and high CN (Hussein et al. 2015), and resolved the numerous limitations of tumour grade and histotype, depth of myometrial invasion and cervical and adnexal involvement.Their prognosis parallels the sequence of molecular subtyping: 1 st > 2 nd > 3 rd > 4 th .Among the four main genetic clusters, patients characterised by ultramutated (POLE) and hyper-mutated (MSI-H) profiles are likely to respond to monoclonal antibody therapy against immune checkpoint-associated proteins (Oaknin et al. 2022).Additionally, p53-mutant endometrial cancer has the worst prognosis and a high risk of recurrence.Endometrial cancer patients carrying CTNNB1 exon 3 mutations have an increased risk of distant recurrence.The molecular characteristics of endometrial cancer with a worse prognosis are PI3K/Akt mutations, the positivity of estrogen and progesterone receptors, and L1CAM positivity (Di Donato et al., 2023, Golia D'Aug� e et al. 2023).In the future, we will investigate the relationship between BAG3 expression and the molecular subtyping of endometrial cancers to guide clinical treatment.
In summary, downregulated BAG3 mRNA expression is strongly associated with the carcinogenic effect of breast and endometrial cancers and the histogenesis of cervical and endometrial cancer.It can be used as a potential marker of the aggressiveness or prognosis of gynecological cancers.However, one limitation of this study is that the bioinformatic data could not be validated using real-time RT-PCR, even with laser capture dissection.In breast, cervical, endometrial and ovarian cancer, the differential genes of BAG3 were subjected to the signal pathway analysis using KEGG (A).STRING was used to identify the protein-protein interaction network of differential genes about BAG3 in cancers, and cytoscape was employed to find out the top 10 hub nodes ranked by degree (B).

Figure 2 .
Figure 2. The relationship between BAG3 mRNA expression and immune infiltration in gynecological cancers.The enrichment of immune cells was explored between low and high expression of BAG3 in breast, cervical, endometrial and ovarian cancers using xiantao.

Figure 3 .
Figure3.The differential genes and related signal pathways about BAG3 in gynecological cancers.In breast, cervical, endometrial and ovarian cancer, the differential genes of BAG3 were subjected to the signal pathway analysis using KEGG (A).STRING was used to identify the protein-protein interaction network of differential genes about BAG3 in cancers, and cytoscape was employed to find out the top 10 hub nodes ranked by degree (B).

Table 1 .
The relationship between BAG3 mRNA expression and clinicopathological features of breast cancer.

Table 2 .
The relationship between BAG3 mRNA expression and clinicopathological features of cervical cancer.

Table 3 .
The relationship between BAG3 mRNA expression and clinicopathological features of endometrial cancer.