HSPA8 globally modulates transcriptome profile and PI3K/AKT signaling pathway to facilitate malignant progression phenotypes of triple-negative breast cancer
An important pro-oncogenic role of HSPA8 is highlighted in TNBC progression, mediated in part through transcriptomic remodeling and activation of the PI3K/AKT signaling pathway.
Abstract
Objective Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer. HSPA8 is a chaperone protein involved in the proper folding or degradation of many proteins that are implicated in various types of cancers, while its role in TNBC remains unclear. Methods In this study, we extensively explored the cellular function and molecular targets of HSPA8 in TNBC cells by performing small interfering RNA (siRNA)-mediated knockdown, followed by cytological assays and transcriptome sequencing (RNA-seq). Results The results of HSPA8 expression levels across distinct breast cancer subtypes indicated that TNBC exhibits higher HSPA8 expression. Then, cytological experiments demonstrated that knockdown of HSPA8 significantly inhibited TNBC cell proliferation, invasion and migration, and induced apoptosis. Mechanistically, RNA-seq showed that HSPA8 globally regulates gene expression and alternative splicing (AS), particularly modulating expression of genes in PI3K/AKT signaling pathway, and AS of genes in protein phosphorylation. Finally, we experimentally confirmed that knockdown of HSPA8 significantly suppressed the activation of the PI3K/AKT axis and AKT phosphorylation in TNBC cells. Conclusions Our findings highlight an important pro-oncogenic role of HSPA8 in TNBC progression, mediated in part through transcriptomic remodeling and activation of the PI3K/AKT signaling pathway. These results may inform the development of novel targets and strategies for future targeted therapy in TNBC.
BACKGROUND
G protein-coupled receptor 19 (GPR19) is an orphan G protein-coupled receptor with emerging relevance in cancer; however, its role in breast cancer remains poorly understood. Given the high frequency of tumor protein p53 (TP53) alterations in aggressive breast cancer, particularly triple-negative breast cancer (TNBC), we investigated the clinical significance, biological function, and molecular mechanism of GPR19 in TP53-mutant breast cancer.
METHODS
Public datasets (Gene Expression Omnibus and The Cancer Genome Atlas) were analyzed to assess GPR19 expression in relation to TP53 status, molecular subtype, and prognosis. Expression was validated in breast cancer cell lines, paired clinical tissues, and tissue microarrays by quantitative PCR, Western blotting, immunofluorescence, and immunohistochemistry. Stable GPR19 knockdown models in MDA-MB-231 and BT-549 cells were used to evaluate proliferation, cell cycle distribution, and apoptosis. RNA sequencing, rescue experiments with the extracellular signal-regulated kinase (ERK) activator Ro 67-7476, and a nude mouse xenograft model were employed to investigate the underlying mechanism.
RESULTS
GPR19 was significantly upregulated in TP53-mutant breast cancer, primary tumors, and especially TNBC, and its high expression was associated with poor survival. Functionally, GPR19 depletion markedly suppressed cell proliferation, colony formation, and DNA synthesis, while inducing G2/M arrest and apoptosis in TP53-mutant breast cancer cells. Mechanistically, GPR19 knockdown reduced ERK phosphorylation and downregulated forkhead box protein M1 (FOXM1) and its downstream G2/M regulators cyclin B1 (CCNB1) and polo-like kinase 1 (PLK1), whereas total ERK levels remained largely unchanged. Pharmacological activation of ERK partially restored FOXM1 expression, alleviated cell cycle disturbance and apoptosis, and reversed the growth-inhibitory effects of GPR19 depletion. In vivo, GPR19 knockdown suppressed xenograft growth, reduced Ki-67 staining, increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positivity, and inhibited the ERK-FOXM1-CCNB1/PLK1 signaling cascade, all of which were partially rescued by ERK activation.
CONCLUSIONS
GPR19 functions as a novel oncogenic driver and clinically relevant biomarker in breast cancer, particularly in TP53-mutant and TNBC subsets. By activating the ERK-FOXM1 axis, GPR19 sustains cell cycle progression and suppresses apoptosis, highlighting this pathway as a potential therapeutic vulnerability in aggressive breast cancer.
Jihan Qiu, Cheng Tian, Hanzhi Li et al.· Frontiers in Bioscience· 0 citations
Tumor protein p53-regulated apoptosis-inducing protein 1 (TP53AIP1) has been implicated in tumor suppression, but its role in breast cancer remains unclear. This study evaluated the expression pattern, prognostic value, immune infiltration association, methylation status, and biological function of TP53AIP1 in breast cancer using TCGA transcriptomic data, public methylation datasets, immunohistochemistry, and in vitro experiments. TP53AIP1 was significantly downregulated in breast cancer tissues and was identified as an independent prognostic factor for poor survival. TP53AIP1 expression was positively associated with transcriptome-estimated infiltration of natural killer cells, mast cells, and plasmacytoid dendritic cells. Methylation analysis showed that TP53AIP1 promoter hypermethylation was associated with reduced TP53AIP1 expression, suggesting a potential epigenetic silencing mechanism. Functionally, TP53AIP1 overexpression suppressed breast cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition and, promoted apoptosis and cell-cycle arrest. Mechanistically, TP53AIP1 overexpression reduced MEK/ERK phosphorylation, whereas MAPK pathway reactivation partially reversed its inhibitory effects on malignant phenotypes. These findings suggest that TP53AIP1 may serve as a potential prognostic biomarker and tumor-suppressive candidate in breast cancer, with its effects at least partly associated with MAPK pathway attenuation.
Meihai Deng, Xueting Wu, J. Bai et al.· Discover Oncology· 0 citations
The role of T-box transcription factor 6 (TBX6), a developmental transcription factor, in tumor initiation and progression in colorectal cancer (CRC) remains unclear. The present study investigated the expression pattern, biological functions, and downstream transcriptional regulatory networks of TBX6 in CRC. Public databases, clinical cohorts, functional assays, xenograft models, and RNA sequencing coupled with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction analyses were used to define TBX6-associated molecular alterations. TBX6 was markedly upregulated in CRC tissues and cell lines, and its high expression was associated with advanced stage, lymph node metastasis, and poor survival. Functionally, TBX6 promoted proliferation, migration, invasion, and epithelial-mesenchymal transition in CRC cells in vitro, whereas TBX6 knockdown suppressed these malignant phenotypes and inhibited tumor growth with reduced proliferation and increased apoptosis in vivo. Transcriptomic analyses showed that TBX6 knockdown induced coordinated gene expression reprogramming and altered multiple tumor-related pathways. Protein-protein interaction analysis identified C-X-C motif chemokine ligand 14 (CXCL14), NOTUM, adrenoceptor α2A (ADRA2A), and melanin-concentrating hormone receptor 1 (MCHR1) as hub nodes within the TBX6-regulated network. Mechanistically, TBX6 knockdown regulated CXCL14 expression and attenuated Wnt/β-catenin signaling, findings that were further validated in xenograft tissues. These results indicate that TBX6 is aberrantly reactivated in CRC and promotes tumor progression through transcriptional network remodeling, suggesting that TBX6 may represent a potential prognostic biomarker and therapeutic target.
Nianjie Zhang, Leiting Wan, N. He et al.· Gene· 0 citations
As the most aggressive subtype of breast cancer, triple-negative breast cancer (TNBC) is frequently treated with radiotherapy. Downregulation of syntaxin binding protein 1 (STXBP1) gene expression is notably associated with poor prognosis in patients with breast cancer and its protein expression level is associated with tumor radioresistance. However, the precise molecular mechanisms by which STXBP1 regulates breast cancer pathogenesis and radiotherapy resistance remain to be elucidated. In the present study, STXBP1 was overexpressed in MDA-MB-231 cells to investigate its effects on cell proliferation and apoptosis. Transcriptomic sequencing (RNA-sequencing; RNA-seq) was performed to identify differentially expressed genes and alternative splicing events regulated by STXBP1. Additionally, a publicly available RNA-seq dataset (GSE189495) associated with breast cancer radiotherapy, including three fractionally irradiated 20 Gy and three age-matched control MCF-7 cell samples were analyzed to identify radiotherapy-associated alternative splicing alterations. Integrated analysis of these two datasets was conducted to explore the potential mechanisms underlying STXBP1-mediated radiotherapy response in breast cancer. Key gene expression changes and alternative splicing events were validated using reverse transcription-quantitative (RT-q) PCR. In MDA-MB-231 cells, the overexpression of STXBP1 notably inhibited cell proliferation and enhanced levels of apoptosis. Through an analysis of RNA-seq data, the present study discovered that STXBP1 regulates global gene expression and alternative splicing profiles in MDA-MB-231 cells by modulating 111 differentially expressed genes and 1,161 regulated alternative splicing events. STXBP1 plays a key role in regulating the splicing patterns of numerous DNA repair-related genes, including USP48 and PLEC. In addition, the present study conducted an overlap analysis on the transcriptome data from radiotherapy-treated MCF-7 cells alongside the dataset generated in the present study, which revealed 21 alternative splicing events associated with radiotherapy. Notably, these include the DNA repair-related gene UBE2I, and its expression pattern has been confirmed through RT-qPCR. The present study systematically delineated the downstream targets and functional mechanisms of STXBP1 in breast cancer cells, revealing its antitumor molecular role. Moreover, STXBP1 may be associated with radiation sensitivity by regulating the alternative splicing of genes associated with DNA repair. These molecular targets, such as UBE2I, hold potential as novel therapeutic avenues for breast cancer treatment, particularly for TNBC.
Guanine nucleotide-binding protein subunit gamma-12 (GNG12), a G protein γ-subunit, has been identified as a potential regulator of tumor biology. However, its functional relevance in triple-negative breast cancer (TNBC) remains unclear. In this study, we integrated pan-cancer transcriptomic analyses with experimental validation in TNBC models to investigate the expression patterns, clinicopathological significance, and biological roles of GNG12. GNG12 was found to be broadly downregulated across diverse cancer types, and its reduced expression was significantly associated with unfavorable patient survival outcomes. Low GNG12 expression correlated with adverse clinicopathological features in breast cancer (BRCA). Moreover, it was associated with poorer outcomes in TNBC patient cohorts. Functional assays demonstrated that GNG12 overexpression inhibited TNBC cell proliferation, migration, and invasion, whereas GNG12 knockdown exerted the opposite effects. Pathway analysis and western blotting indicated that GNG12 expression was associated with altered PI3K/AKT pathway activity. Additionally, mutations at S2 and S33—both located within predicted phosphorylation sites—were identified as candidate regulatory alterations whose functional relevance requires further validation. Moreover, GNG12 expression was positively correlated with RNA methylation-related markers and inversely associated with promoter methylation. Taken together, GNG12 was downregulated in TNBC and associated with suppression of malignant cellular phenotypes; however, its clinical significance and mechanistic relationship with PI3K/AKT signaling require further validation in larger cohorts and additional experimental models.
Biao-Feng Shan, Le Zhao, Tao Hu et al.· Scientific Reports· 0 citations
Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by significant intratumoral heterogeneity and poor prognosis. Our study identified Establishment Factor-Like Protein 2 (EFO2) as a key oncogenic driver of TNBC progression. Using GEO datasets, we identified differentially expressed genes in TNBC. Functional roles of EFO2 were assessed via knockdown in human (MDA-MB-231, HCC1937) and mouse (4 T1, EMT6) TNBC cells, examining proliferation, glycolysis, and co-culture with CD8⁺ T cells. In vivo tumor growth was evaluated. Molecular mechanisms were investigated through co-immunoprecipitation, mutagenesis, luciferase reporter, and ChIP-qPCR assays. We observed that EFO2 was highly expressed in TNBC tissues, and this high expression correlated with shorter patient survival. Functional experiments showed that EFO2 knockdown suppressed tumor growth and proliferation both in vitro and in vivo. Furthermore, EFO2 knockdown inhibited glycolysis, as evidenced by decreased glucose uptake, ATP production, and lactate production. Moreover, EFO2 deficiency enhanced CD8⁺ T cell-mediated cytotoxicity against TNBC cells. Mechanistically, we demonstrated that EFO2 promoted the acetylation of Upstream Transcription Factor 1 (USF1), thereby enhancing transcriptional upregulation of the SLC2A1 promoter, a key glucose transporter. This EFO2/USF1/SLC2A1 signaling axis accelerated glycolysis in TNBC cells, which concurrently sustained tumor proliferation and impaired CD8⁺ T cell effector function, reducing TNBC cell susceptibility to T cell-mediated killing. Our findings identify a novel EFO2/USF1/SLC2A1 signaling axis that modulates glycolytic metabolism and CD8⁺T cell cytotoxicity, positioning EFO2 as a promising therapeutic target for TNBC treatment.
Lin Jia, Liru Li, Junning Peng et al.· Biochemical Pharmacology· 0 citations