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Deep analysis of neuroblastoma core regulatory circuitries using online databases and integrated bioinformatics shows their pan-cancer roles as prognostic predictors

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Abstract

Aim: Neuroblastoma is a heterogeneous childhood cancer derived from the neural crest. The dual cell identities of neuroblastoma include Mesenchymal (MES) and Adrenergic (ADRN). These identities are conferred by a small set of tightly-regulated transcription factors (TFs) binding super enhancers, collectively forming core regulatory circuitries (CRCs). The purpose of this study was to gain a deep understanding of the role of MES and ADRN TFs in neuroblastoma and other cancers as potential indicators of disease prognosis, progression, and relapse. Methods: To that end, we first investigated the expression and mutational profile of MES and ADRN TFs in neuroblastoma. Moreover, we established their correlation with neuroblastoma risk groups and overall survival while establishing their extended networks with long non-coding RNAs (lncRNAs). Furthermore, we analysed the pan-cancer expression and mutational profile of these TFs and their correlation with patient survival and finally their network connectivity, using a panel of bioinformatic tools including GEPIA2, human pathology atlas, TIMER2, Omicsnet, and Cytoscape. Results: We show the association of multiple MES and ADRN TFs with neuroblastoma risk groups and overall survival and find significantly higher expression of various MES and ADRN TFs compared to normal tissues and their association with overall survival and disease-free survival in multiple cancers. Moreover, we report the strong correlation of the expression of these TFs with the infiltration of stromal and immune cells in the tumour microenvironment and with stemness and metastasis-related genes. Furthermore, we reveal extended pan-cancer networks comprising these TFs that influence the tumour microenvironment and metastasis and may be useful indicators of cancer prognosis and patient survival. Conclusion: Our meta-analysis shows the significance of MES and ADRN TFs as indicators of patient prognosis and the putative utility of these TFs as potential novel biomarkers.

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Stromal cells in the tumor microenvironment (TME) closely interact with tumor cells and affect tumor cell behavior in diverse manners. We herein investigated the mechanisms by which cancer-associated fibroblasts (CAFs) affect the functional polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) in vitro and in human cancer samples. The expression of CD68, CD14, CD163, CD200R, CD206, HLA-G, CD80, and CD86 was higher in CD14-positive cells co-cultured with the culture supernatants of CAFs established from OSCC specimens (CAF-educated cells) than in control cells. The gene expression level of ARG1, IL10, and TGFB1 was increased in CAF-educated cells. CAF-educated cells suppressed T cell proliferation more strongly than control cells, and the neutralization of TGF-β IL-10, or arginase I significantly restored T cell proliferation. We then investigated the relationship between the infiltration of CAFs and TAMs using tissue samples obtained from patients with OSCC. The infiltration of CAFs was associated with the numbers of CD68-positive and CD163-positive macrophages. It also correlated with lymphatic invasion, vascular invasion, lymph node involvement, and the TNM stage. The infiltration of CAFs was identified as an independent prognostic factor in OSCC. Our results indicate that CAFs play important roles in shaping the tumor immunosuppressive microenvironment in OSCC by inducing the protumoral phenotype of TAMs. Therapeutic strategies to reverse CAF-mediated immunosuppression need to be considered.
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Background Understanding the interactions between tumor and the host immune system is critical to finding prognostic biomarkers, reducing drug resistance, and developing new therapies. Novel computational methods are needed to estimate tumor-infiltrating immune cells and understand tumor–immune interactions in cancers. ResultsWe analyze tumor-infiltrating immune cells in over 10,000 RNA-seq samples across 23 cancer types from The Cancer Genome Atlas (TCGA). Our computationally inferred immune infiltrates associate much more strongly with patient clinical features, viral infection status, and cancer genetic alterations than other computational approaches. Analysis of cancer/testis antigen expression and CD8 T-cell abundance suggests that MAGEA3 is a potential immune target in melanoma, but not in non-small cell lung cancer, and implicates SPAG5 as an alternative cancer vaccine target in multiple cancers. We find that melanomas expressing high levels of CTLA4 separate into two distinct groups with respect to CD8 T-cell infiltration, which might influence clinical responses to anti-CTLA4 agents. We observe similar dichotomy of TIM3 expression with respect to CD8 T cells in kidney cancer and validate it experimentally. The abundance of immune infiltration, together with our downstream analyses and findings, are accessible through TIMER, a public resource at http://cistrome.org/TIMER. Conclusions We develop a computational approach to study tumor-infiltrating immune cells and their interactions with cancer cells. Our resource of immune-infiltrate levels, clinical associations, as well as predicted therapeutic markers may inform effective cancer vaccine and checkpoint blockade therapies.
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In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and sub-type specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here we show by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a programme of motor neuronal differentiation characterised by expression of Islet1 and of the cholinergic transmitter phenotype, in line with our previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations.
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Objective: The overexpression of transcription factor Sine oculis homeobox 1 (SIX1) is discovered in various malignant tumors and has been known to be closely associated with tumorigenesis, progression and prognosis. This study aims to determine the role of SIX1 in endometrial cancer (EC). Methods: In this study, we analyzed the SIX1 expression profile and the correlation with the corresponding clinical characteristics of EC samples from the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases. Wilcoxon signed-rank test was applied to analyze the difference between tumor group and control group. The potential biological processes or signaling pathways related to SIX1 activity in EC was also assessed. Results: The results showed that SIX1 was overexpressed in EC tissues compared to normal tissues (P=2.029e-15, P=6.25e-6). The SIX1 level was correlated with tumor grade (P=2.91e-4), peritoneal cytology (P=0.005), and the subsequent tumor surgery (P=1.169e-4). SIX1 expression was negatively associated with overall survival rate (P=4.241e-4, P=0.000241) and served as an independent factor that affected EC overall survival rate (P=0.005063), similar to other factors such as age, Figo stage, and tumor (T) stage. SIX1 participates in cancer pathogenesis through gene regulation that involves PI3K/AKT/MTOR signaling, mitotic spindle, G2M checkpoint, E2F targets, NOTCH signaling, glycolysis, cholesterol homeostasis, DNA repair and early estrogen response. Conclusions: Our data demonstrate that SIX1 is overexpressed in EC and associated with adverse clinicopathological outcomes, which can function as an independent factor for EC prognosis.
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The long non-coding RNA (lncRNA) LIFR-AS1 has been shown to be involved in the development of several human cancers. This study was designed to determine the expression profile and role of lncRNA-LIFR-AS1 in human thyroid cancer. The results showed significant (p < 0.05) upregulation of LncRNA-LIFR-AS1 in thyroid cancer tissues and cells. However, silencing of LncRNA-LIFR-AS1 inhibited the viability and proliferation of human thyroid cancer cells inducing G2/M cell cycle arrest. The G2/M phase cells increased from 8.56% in negative control (NC) to around 35.03% in si-LIFR-AS1. This was also found to be concomitant with the downregulation of cyclin B1 and CDK1 expressions. The thyroid cancer cells exhibited remarkably lower invasion and migration under transcriptional knockdown of lncRNA-LIFR-AS1 which was also associated with downregulation of MMP-2 and MMP-9 expression. Importantly, transcriptional silencing of lncRNA-LIFR-AS1 inhibited thyroid cancer tumorigenesis, in vivo. Collectively, the results suggest the tumor-promoting role of lncRNA-LIFR-AS1 in thyroid cancer and highlight its potential as therapeutic target.
Article
Objective: Long non-coding RNA DBH-AS1 (DBH-AS1) has emerged as a novel regulator in cancer initiation and progression of several tumors. However, the expression of DBH-AS1 in osteosarcoma and its effect on the tumorigenesis of osteosarcoma are unclear. The purpose of this study was to determine the role of DBH-AS1 in osteosarcoma progression. Patients and methods: The expression level of DBH-AS1 in 119 pairs of osteosarcoma tissues and five cell lines was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The association of DBH-AS1 expression with clinicopathological factors and prognosis was also analyzed. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8), EdU and cell colony formation assays and apoptosis in MG63 and U2OS cells was examined by flow cytometry. Following that, transwell invasion and wound-healing assays were used to explore cell migration and invasion, respectively. The expression of the PI3K/Akt pathway-related proteins was examined by Western blot analysis. Results: We observed that DBH-AS1 was distinctly overexpressed in osteosarcoma tissue and cells, and associated with lymph node status and metastasis status. Osteosarcoma patients with a higher DBH-AS1 expression showed significantly poorer overall survival than those with lower DBH-AS1 expression. Multivariate analysis demonstrated that high DBH-AS1 expression was an independent poor prognostic factor for osteosarcoma patients. Functional assays revealed that knockdown of DBH-AS1 inhibited cell proliferation, migration and invasion, while promoted apoptosis in osteosarcoma. Moreover, suppression of DBH-AS1 could inhibit the activation of the PI3K/Akt pathway, which was demonstrated by examining the expression levels of p-PI3K and p-Akt. Conclusions: Our data first reported that DBH-AS1 may act as an oncogenic lncRNA by modulating the PI3K/Akt pathway in osteosarcoma, which may serve as a candidate prognostic biomarker and target for new therapies in osteosarcoma.
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Diffuse large B‐cell lymphoma (DLBC) is a subtype of lymphoma with the worst prognosis. Existing treatment methods are not effective enough due to its high occurrence of metastasis. Therefore, identification of effective therapeutic targets is becoming increasingly important. In this research, lncRNA Dopamine β hydroxylase antisense RNA 1 (DBH‐AS1) was found to be upregulated in DLBC tissues and cells. Knockdown of DBH‐AS1 suppressed the proliferation, migration and invasion of cancer cells. Afterwards, RBP BUD13 homolog (BUD13) was found to be upregulated in cancer tissues and cells while binding to DBH‐AS1. Fibronectin 1 (FN1) was the downstream mRNA of BUD13. FN1 was upregulated in DLBC and was positively correlated with DBH‐AS1. Further rescue assays proved that DBH‐AS1 mediated FN1 expression by recruiting BUD13. In the meantime, BUD13 stabilized FN1 mRNA to promote FN1 expression. In this way DBH‐AS1/BUD13/FN1 axis was confirmed. A set of rescue assays proved that DBH‐AS1 regulated DLBC progression via BUD13 and FN1. The function and mechanism of DBH‐AS1 was investigated for the first time in DLBC. DBH‐AS1 might become a therapeutic target in lymphoma treatment in the future. This article is protected by copyright. All rights reserved.
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Background Existing meta-analysis have shown that the miR-200 family can be taken as a prognostic biomarker for many tumors. However, great heterogeneity was shown in predicting overall survival (OS) and progression-free survival (PFS). Emerging studies indicate that the expression levels of members of the miR-200 family are tissue-specific among various tumor tissues, which may be the main reason of the heterogeneity in predicting survival prognosis of tumor patients with the miR-200 family as biomarkers. By further analysis of heterogeneity of the miR-200 family as a biomarker for predicting survival prognosis of patients with different tumors, we expected to provide an accurate basis for the clinical application of the miR-200 family to predict the prognosis of patients with different tumors. Methods Eligible published studies were identified by searching the databases of PubMed, Embase and Web of Science. The clinical data of patients in the studies were pooled, and pooled hazard ratios (HR) with 95% confidence intervals (95% CI) were used to calculate the strength of this association. The expressions of miRNAs were extracted from The Cancer Genome Atlas (TCGA). We presented the expressions of each member in miR-200 family in 15 types of cancer by boxplot, and analyzed the correlation among the members of miR-200 family by Spearman method. Different subgroup analyses were then performed based on the correlation among the members of miR-200 family, and the publication bias was assessed using the funnel plot of the Egger bias indicator test. Results Of 36 articles, including 15 tumor types and 4644 patients were included to perform meta-analysis. It was found that miR-200 family members can be used as independent protective factors in patients with various tumors but the miR-200 family has a higher heterogeneity in predicting prognosis: OS (HR = 0.82, 95% CI: 0.66–1.03, I² = 85%, P < 0.01) and PFS (HR = 0.81, 95% CI: 0.57–1.16, I² = 97%, P < 0.01). The data from TCGA database were used to analyze the expression levels of the miR-200 family and the results showed that the expression of miR-429 in different cancers is very different, and there are significant differences in expression levels compared with other miR-200 family members; the expression levels of miR-200a and miR-200b in various tumor tissues were similar to each other, respectively; miR-200c and miR-141 showed similar expression levels in each of most types of cancer tissues except ovarian cancer (OC). The expression levels of members of the miR-200 family in breast cancer (BRCA), cervical cancer (CESC), colon cancer (COAD), esophageal cancer (ESCA), head and neck cancer (HNSC), lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are relatively stable, but great variations can be found in the expression levels of miR-200 family members in ovarian cancer (OC), liver cancer (LIHC), renal clear cell carcinoma (KIRC) and renal papillary cell carcinoma (KIRP). Cluster analysis of expression of target genes of miR-200 family in different cancers yielded similar results to the expression level of the miR-200 family. Subgroup analysis of OC, LIHC, GC and LUAD based on expression levels and clustering results reduced or even eliminated the heterogeneity of miR-200 family members in predicting patient outcomes. Conclusions Our results convincingly demonstrated that the miR-200 family could serve as a prognostic biomarker for cancers mentioned above and has potential value in clinical practice. MiR-200 family as prognostic biomarkers needs to be performed according to different tumor tissues and correlation between members in miR-200 family.
Article
Background: The expression of β-catenin and paired-like homeobox 2B (PHOX2B) expression were assessed in Neuroblastoma (NB) patients as a diagnostic, prognostic and/or predictive markers. Methods: Bone marrow (BM) samples of 52 NB patients were assessed for the expression of β-catenin by immunohistochemistry (IHC), and PHOX2B by real time PCR (RT-PCR), compared to 12 healthy normal controls (NC). The data were correlated to the clinic-pathological features of the patients, response to treatment and disease relapse. Results: β-catenin was expressed in 40 (76.92%) patients (P < .001). While PHOX2B was expressed in 32/52 (61.5%) patients, with a fold change of 0.29 (0.01-40.0, P = .096). β-catenin expression associated significantly with advanced tumor stage, high risk, positive results by MIBG and bone scan (P = .002, P < .001, P = .006, P = .013; respectively). Also it associated significantly with synaptophysin expression in the BM biopsy (P < .001), with a significant concordance (K = 0.519, P < .001). The expression of β-catenin associated significantly with PHOX2B gene expression [28/32 (87.5%), P = .04], and its fold change (P = .027), with a significant measure of agreement (K = 0.297, P = .022). The fold change of PHOX2B gene expression associated significantly with the high risk of the patients (P = .04). Poor response to treatment associated significantly with the expression of neuron specific enolase (NSE), β-catenin and PHOX2B in NB patients (P = .021, P = .019 and P = .040; respectively). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of synaptophysin for the diagnosis of BM metastasis in NB patients were (69%, 65.2%, 71.4%, 62.5%; respectively, P = .024). While with β-catenin (93.1%, 43.5%, 67.5%, 83.3%; respectively, P = .003), and PHOX2B expression (65.5%, 34.5%, 59.4%, 50%; respectively, P = .574). Conclusion: β-Catenin could be used as a sensitive and reliable marker for detection of BM metastasis and also a good predictor for resistance to treatment in NB patients. While, PHOX2B gene expression in BM aspirate could be a marker for high risk patients and poor response to treatment.
Article
Activity of the human long interspersed nuclear elements‐1 (LINE‐1) retrotransposon occurs mainly in early embryonic development and during hippocampal neurogenesis. SOX‐11, a transcription factor relevant to neuronal development, has unknown functions in the control of LINE‐1 retrotransposon activity during neuronal differentiation. To study the dependence of LINE‐1 activity on SOX‐11 during neuronal differentiation, we induced differentiation of human SH‐SY5Y neuroblastoma cells and adult adipose mesenchymal stem cells (hASCs) to a neuronal fate and found increased LINE‐1 activity. We also show that SOX‐11 protein binding to the LINE‐1 promoter is higher in differentiating neuroblastoma cells, while knock‐down of SOX‐11 inhibits the induction of LINE‐1 transcription in differentiating conditions. These results suggest that activation of LINE‐1 retrotransposition during neuronal differentiation is mediated by SOX‐11. This article is protected by copyright. All rights reserved.
Article
Background: It has been reported that dysregulated lncRNAs are associated with the pathogenesis of human tumors including hepatocellular carcinoma (HCC). LncRNA DBH-AS1 was reported to be an oncogene in HCC. However, the molecular mechanisms of DBH-AS1 in HCC progression are largely undefined. Methods: The expressions of DBH-AS1 and miR-138 in HCC tissues and cells were examined by qRT-PCR. The effects of DBH-AS1 and miR-138 on cell viability, colony formation, apoptosis, and FAK/Src/ERK signaling pathway were determined by CCK-8, colony formation, flow cytometry, and western blot analyses, respectively. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to explore the interaction between miR-138 and DBH-AS1. Tumor xenograft assay was performed to confirm the function and mechanism of DBH-AS1 in the progression of HCC in vivo. Results: DBH-AS1 expression was upregulated and miR-138 expression was downregulated in HCC tissues and cells. DBH-AS1 silencing and miR-138 overexpression reduced cell viability, inhibited colony formation, and induced apoptosis. Moreover, DBH-AS1 acted as a molecular sponge of miR-138 to downregulate miR-138 expression. Also, DBH-AS1 overexpression attenuated miR-138-mediated anti-proliferation and pro-apoptosis effects. Additionally, miR-138 overexpression gave rise to a blockage on FAK/Src/ERK pathway, while this effect was undermined by increased DBH-AS1. Furthermore, DBH-AS1 promoted tumor growth and induced the activation of FAK/Src/ERK pathway by targeting miR-138 in vivo. Conclusion: DBH-AS1 facilitated the development of HCC via miR-138/FAK/Src/ERK pathway, establishing the molecular basis of DBH-AS1 in clinical application for HCC.
Article
Neuroblastoma (NB) is the most common extracranial solid tumor in children with contrasting outcomes. Precise risk assessment contributes to prognosis prediction, which is critical for treatment strategy decisions. In this study, we developed a 3-protein predictor model, including the neural stem cell marker Msi1, neural differentiation marker ID1, and proliferation marker proliferating cell nuclear antigen (PCNA), to improve clinical risk assessment of patients with NB. Kaplan-Meier analysis in the microarray data (GSE16476) revealed that low expression of ID1 and high expression of Msi1 and PCNA were associated with poor prognosis in NB patients. Combined application of these 3 markers to constitute a signature further stratified NB patients into different risk subgroups can help obtain more accurate prediction performance. Survival prognostic power of age and Msi1_ID1_PCNA signature by receiver operating characteristics analysis showed that this signature predicted more effectively and sensitively compared with classic risk stratification system, compensating for the deficiency of the prediction function of the age. Furthermore, we validated the expressions of these 3 proteins in neuroblastic tumor spectrum tissues by immunohistochemistry revealed that Msi1 and PCNA exhibited increased expression in NB compared with intermedial ganglioneuroblastoma and benign ganglioneuroma, whereas ID1 levels were reduced in NB. In conclusion, we established a robust risk assessment predictor model based on simple immunohistochemistry for therapeutic decisions of NB patients. Copyright
Article
Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.
Article
p>We present NDEx 2.0, the latest release of the Network Data Exchange (NDEx) online data commons (www.ndexbio.org) and the ways in which it can be used to (i) improve the quality and abundance of biological networks relevant to the cancer research community; (ii) provide a medium for collaboration involving networks; and (iii) facilitate the review and dissemination of networks. We describe innovations addressing the challenges of an online data commons: scalability, data integration, data standardization, control of content and format by authors, and decentralized mechanisms for review. The practical use of NDEx is presented in the context of a novel strategy to foster network-oriented communities of interest in cancer research by adapting methods from academic publishing and social media. Cancer Res; 77(21); e58–61. ©2017 AACR .</p
Article
Background: Neuroblastomas (NBs) are the most common solid cancer of childhood and infancy; however, in poorly differentiated forms, they present diagnostic challenges. GATA3 has been implicated functionally in NB differentiation, and limited data support its use as an immunohistochemical biomarker for NBs in resection specimens. Methods: GATA3 was tested retrospectively in 30 consecutive archival NB samples, including archival cytopathology needle cores and cell blocks (n = 6), scant surgical biopsy specimens and 2-mm NB tissue cores (n = 16), and air-dried touch imprints (n = 8) to evaluate the utility of this marker. Immunostaining was performed per the institutional standard, Clinical Laboratory Improvement Amendments-compliant automated staining protocol. GATA3 nuclear staining was scored qualitatively for its intensity and proportion of positivity. Results: All 30 NB specimens showed diffuse nuclear positivity with GATA3. Each sample revealed either strong (n = 26) or moderate nuclear staining (n = 4) in more than 75% of NB cells, regardless of the presence or lack of stromata or necrosis or the degree of differentiation. Conclusions: GATA3 is a reliable diagnostic marker for NBs not only in scant/limited surgical specimens but also in cytologic samples, including air-dried touch imprints, which have previously been undescribed for this marker. Cancer (Cancer Cytopathol) 2017. © 2017 American Cancer Society.
Article
Background: Smad4 and GATA3 proteins are known prognostic markers in various cancers. Smad4 is a mediator linked to both tumour suppression and progression. GATA3 is a regulator of development and morphogenesis of the mammary gland. We assessed and compared the predictive performance of Smad4 and GATA3 for clinical outcomes in patients with breast cancer. Methods: The combined expression pattern based on Smad4+/- and GATA3+/- was evaluated by immunostaining using breast cancer tissue microarray, and the relationships between protein expression and clinicopathological variables were analysed. Results: Smad4 expression was only associated with an ill-defined tumour border, whereas GATA3 was associated with several good prognostic factors. On analysis of combined markers, there was a significant difference in the expression of fascin (an important factor for cancer invasiveness) between the Smad4+/GATA3- and Smad4-/GATA3+ groups. Smad4+/GATA3- was correlated with worse clinicopathological parameters, relapse-free survival (RFS), and overall survival (OS), compared to Smad4-/GATA3+. Conclusion: Combined markers of Smad4/GATA3 showed a superior performance compared to single markers for predicting RFS and OS in patients with breast cancer.