Spatial and Single-Cell Analyses Reveal the Pro-Invasiveness Role of NUAK1 in Breast Cancer through EMT Regulation

Spatial and Single-Cell Analyses Reveal the Pro-Invasiveness Role of NUAK1 in Breast Cancer through EMT Regulation | 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 Spatial and Single-Cell Analyses Reveal the Pro-Invasiveness Role of NUAK1 in Breast Cancer through EMT Regulation Jiani Wang, Jiumei Yang, Cuicui Li, Dongbo Qiu, Baoyu Zhang, Peng Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5300363/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 8 You are reading this latest preprint version Abstract Objective Breast carcinoma is a leading malignancy in women, and the role of Novel (nua) kinase family 1 (NUAK1) in its progression is not well-defined. This study aimed to investigate the biological significance of NUAK1 in breast cancer and its potential as a prognostic marker. Methods We assessed the expression levels of NUAK1 in breast cancer tissues and cell lines using RT-qPCR and cultured cell assays. Statistical analysis was conducted to correlate NUAK1 expression levels with clinicopathological features. Survival analysis was performed to determine the prognostic value of NUAK1 in breast cancer. Additionally, Transwell invasion assays and the evaluation of EMT (epithelial-mesenchymal transition)-related proteins were conducted to ascertain the impact of NUAK1 on cellular invasion and EMT. Furthermore, spatial transcriptomic analysis utilizing the CROST dataset and single-cell RNA sequencing data were employed to dissect the expression patterns of NUAK1 and its association with the TME Results NUAK1 was found to be upregulated in breast cancer tissues and cell lines compared to non-cancerous controls. High expression of NUAK1 was significantly associated with poorer patient survival and was an independent prognostic factor. Transwell assays demonstrated that NUAK1 overexpression significantly enhanced cellular invasion. Overexpression of NUAK1 also induced EMT, as evidenced by decreased expression of epithelial markers and increased expression of the mesenchymal marker Vimentin. Single-cell analysis across various datasets highlighted NUAK1's expression in endothelial cells and its correlation with the TNM stage. Spatial transcriptomic analysis revealed that NUAK1 expression, particularly in B-Nai cells, was associated with a distinct immune cell landscape and communication patterns within the TME, influencing TGFβ and WNT signaling pathways. Conclusions Our findings indicate that NUAK1 is upregulated in breast cancer and serves as an independent prognostic marker. NUAK1 promotes breast cancer cell invasion through the induction of EMT and is implicated in the modulation of the TME. The single-cell analysis and spatial transcriptomic data provide novel insights into the cellular and molecular mechanisms underlying NUAK1's role in breast carcinogenesis, suggesting its potential as a therapeutic target. NUAK1 breast cancer prognosis epithelial-mesenchymal transition invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Breast carcinoma is one of the most common malignant tumors in women and is also the leading cause of death related to malignancy in women 1 , 2 . Due to the improvement of diagnosis and treatment, the mortality rate of breast cancer showed a continuous downward trend 3 . However, not all patients can benefit from it. The main reason is that breast carcinoma is a highly heterogeneous malignancy in clinical and biological 4 . At present, according to the state of ER, PR, HER2 and Ki-67, breast carcinoma is mainly divided into 4 different subtypes. Different subtypes have different clinical treatment protocols 5 . Although researchers have been committed to the progress of treatment and the discovery of new drugs, the treatment effect of metastatic breast cancer patients is still stubborn and almost no significant improvement. In fact, distant metastasis accounts for most of the breast cancer related mortality 6 . To enhance the clinical management of breast cancer, it is very important to identify new biomarkers which can facilitate risk stratification, and prognostic assessment. Therefore, there is an urgent need to develop novel biomarkers to predict the clinical outcomes and guide clinical practice in breast cancer. Novel (nua) kinase family 1 (NUAK1), also known as the fifth member of adenosine monophosphate-activated protein kinase (AMPK)-related kinase family (ARK5), can mediate cell survival and differentiation, and plays multiple roles in regulating cellular adhesion, metabolism and response 7 . Clinically, nuak1 can promote tumor development, and tumor patients with high expression of nuak1 have lower survival rate and poor prognosis. Studies have shown that nuak1 is an "energy monitoring molecule" in tumor cells, which can monitor the energy balance in cells 8 . During tumor nutritional starvation, nuak1 relies on caspase 8 to inhibit Akt and promote tumor growth 8 – 10 . Nuak1 can inhibit tumor cell apoptosis caused by factors such as glucose starvation and cytokine TNF-α (tumor necrosis factor-α) 9 , also NUAK1 can promote tumor cell survival through TGF – β receptor to tolerate nutritional starvation 11 ; nuak1 activation can also make liver cancer cells tolerate glucose starvation through G1/S cell cycle arrest 12 ; nuak1 can also downregulate apoptosis factor (FAS) ,inhibit the activities of Fas receptor and caspase-6 10 . In addition, NUAK1 is closely related to tumor invasion and metastasis. It is reported that Epithelial to mesenchymal transition (EMT) plays an important role in tumor drug resistance 13 , 14 . Drug resistant tumor cells has high malignant, migration and invasion behavior. NUAK1 induces EMT to make hepatocarcinoma cell drug resistance 15 . Likewise, NUAK1 is also suggested involving in cell migration and invasion via EMT in gastric carcinoma 10 . Forthoremore, NUAK1 is closely associated with migration and metastatic potential in various other carcinoma, including pancreatic carcinoma (12) 12 , lung carcinoma 13 and ovarian carcinoma 14 , which suggested NUAK1 as a potential therapeutic target in many malignant carcinomas. In the present study, we evaluate the expression of NUAK1 in breast carcinoma illuminate significance in tumor prognosis. Also, through cellular experiment, we explore that NUAK1 regulates EMT process and affects cell migration ability. Materials and methods Patients and tissue specimen s This study was approved by the Institutional Research Ethics Committee of Sun Yatsen University, and written informed consent was obtained prior from each patient. A total of 160 patients with breast carcinoma who were histopathologically diagnosed and underwent curative surgery from Dec 2000 to Feb 2014 in the Third Affiliated Hospital of Sun Yatsen University were enrolled in this study. Clinicopathological classification and tumor-node- metastasis (TNM) staging system were defined on the basis of the eighth edition American Joint Committee on Cancer (AJCC). There were 16 (10.0%), 95 (59.4%), 49 (30.6%) patients belonging to stage I, II and III respectively. Detailed clinicopathological information of these patients was shown in Table 1 . The overall survival rate (OS) was calculated from the date of first operation to the date of patient death and considered as censored for the patients who were still alive at last follow-up. The followup time ranged from 2 to162 months (median followup time 127 months). Of these 160 patients with breast carcinoma, 26 paired adjacent noncancerous tissues (at least 2cm distant from the edge of the tumor) were conserved immediately in surgery. Cell lines and cell culture Human breast cancer cell lines MDA-MB-231, SK-BR-3, MDA-MB-435, MCF-7 and T47D were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS, Hyclone Laboratories, Logan, UT) and 100 units/ml penicillin and streptomycin at 37℃ in humidified 5% CO 2 . All cell lines were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China. Human mammary epithelial cell (HMEC) was obtained from primary cultured fresh breast epithelial tissues. The procedure was described in our previously published articles(16) 16 .HMEC was cultured in Keratinocyte-SFM medium (Invitrogen, Grand Island, NY) supplemented with bovine pituitary extract at 37°C in 5% CO 2 . Quantitative real-time polymerase chain reaction (qRT-PCR) analysis Total RNA extracted from breast cancer and adjacent noncancerous tissues using Trizol reagent (Invitrogen) was reversed transcription into complementary DNA (cDNA) by the Super Script H III First-Strand Synthesis System (Invitrogen). NUAK1 mRNA expression was detected by qPCR using iQTM SYBR® Green Supermix with the CFX384 Real-Time System (both Bio-Rad Laboratories, Inc). The expression level of NUAK1 mRNA was normalized to the endogenous expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. The primer sequences were as follows: NUAK1 sense 5’-GCTCGAGGACTCATACGGTG-3’, antisense 5’-GAGGGCATCACAGTCACACA-3’, ‘GAPDH sense 5’-TGTTGCC ATCAATGACCCCTT-3’, antisense 5’-CTCCACGACGTACTCAGCG-3’. Western blotting assay Western blotting assay Cells were extracted using RIPA buffer, and protein concentration was detected by BCA protein assay kit (Bicinchoninic Acid Kit, Sigma-Aldrich, USA). Equal amount of protein samples were loaded into 10.0% resolving SDS-PAGE gel electrophoresis, and then transferred onto polyvinylidene difluoride membrane. After blocking the non-specific binding with 5% non-fat milk in TBS-T solution, the membranes were probed with a primary Polyclonal antibody to NUAK1 (Affinity Biosciences LTD. DF10340), followed by incubation with secondary antibodies (dilution 1:1000; Cell signaling, Billerica, MA). The protein signals were visualized with ECL solution (Beyotime Institute of Biotechnology). GAPDH (dilution 1:1000; Cell signaling, Billerica, MA) was used as loading control. Immunohistochemistry (IHC) and Score Evaluation Tissue sections (4-mm thick) were deparaffinized with xylenes and rehydrated in a series of graded alcohol before blocked with 0.3% hydrogen peroxide at room temperature for 15 min. The slides were boiled in ethylenediamine tetraacetic acid buffer (EDTA, pH = 8.0) in a microwave to retrieve antigens for 30 min. The slides were then incubated with the rabbit polyclonal anti-NUAK1 antibody (ab718714, 1:50, Abacam, Cambridge, UK) in a moist chamber at 4°C overnight. Next day, the tissue sections were treated with horseradish peroxidase (DAKO ChemMate™ EnVision™ Detection Kit, Copenhagen, Danmark) for 30 min at 37° C and subsequently stained with the 3,3′-diaminobenzidine (DAB) for visualization. Finally, nuclear counterstain was conducted with freshly diluted Mayer’s hematoxylin. Normal rabbit IgG was used instead of the primary antibody as negative control. Three independent pathologists blinded to the patients’ clinicopathological data conducted the score assessment for NUAK1 expression. Scoring criteria was calculated as follows: staining score = intensity score + percentage score. The intensity was graded as: 0, negative; 1, weak; 2, moderate and 3, strong. The proportion of NUAK 1 (+) cells was graded as:0, 0–5%; 1, 6–25%; 2,26–50%; 3,51–75%; and 4, 76–100%. The optimal cutoff value was determined: a final score ranging from 0 to 4 was defined as NUAK1 low expression and ranging from 5 to 12 was defined as NUAK1 high expression. Plasmid Construction and Retroviral Infection Plncx2 plasmid vector was used to generate plncx2-NUAK1. The plasmid construction was verified by DNA sequencing. Production of retrovirus was performed according to the instructions, SKBR3 cells and MCF-7 cells were subjected with infection of retrovirus expressing NUAK1 or vector. For establishing stable cell lines with overexpression of NUAK1, 1×10 5 cells were seeded into 6 cm tissue culture plates with complete growth medium supplemented with G418 at 48 h post-transfection and selected for 14 days. Transwell invasion assays Invasion of cells was evaluated in Transwell cell culture chambers with filters (8-µm pore size, Costar,Cambridge, MA). The membrane was pre-coated with Matrigel (Falcon BD). MCF-7 and SKBR3 cells were seeded to the upper chamber, which was placed into the lower chamber containing 600 µL culture medium supplemented with 20% FBS. After 24 h incubation at 37°C, the cells on the upper chambers were removed with a cotton swab. The cells on the lower surface were fixed with methanol for 10 min, then stained with 0.2% crystal violet solution for 1 h and imaged under an inverted microscope. Five random microscopic fields (×100) per well were counted and the mean was determined. All assays were performed three times. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics (Version 22.0; IBM Corp., New York, USA) and GraphPad Prism 8.0 (San Diego, CA, USA). Two tailed Student’s t-test were used to compare the difference of NUAK1 mRNA levels between breast cancer and paired adjacent noncancerous tissues. The χ2 test or the Fisher’s exact test was employed to evaluate the association between NUAK1and other clinicopathological characteristics of patients with breast cancer. The overall survival curves were plotted by Kaplan-Meier method and compared by log-rank test. Relative risks (RRs) associated with NUAK1 and other clinicopathological features were evaluated by univariate and multivariate Cox proportional hazards regression analyses. Variations for the trans-well assay were assessed by the two-tailed Student’s t test. A p value less than 0.05 based on two-tailed tests was considered as statistically significant. Results The expression of NUAK1 is elevated in breast cancer tissues and cell lines We first investigated the expression of NUAK1in 26 paired breast cancer and adjacent non‑cancerous tissues by RT-qPCR assay. The results revealed that the mRNA level of NUAK1 was up-regulated in breast cancer tissues, compared with adjacent non‑cancerous tissues (Figure 1A). To further detect the endogenic NUAK1 expression in breast cancer cell lines, we cultured primary cultured human mammary epithelia cell (HEMC) and breast cancer cell lines (MDA-MB-231, SKBR3, MDA-MB-435, MCF-7, and T47D). The expression of NUAK1 was higher in the breast cancer cells compared with normal breast epithelia cell (Figure 1B). Figure 1 Expression of NUAK1 is elevated in breast cancer tissues. (A) RT-qPCR was performed to determine the expression of NUAK1 in the breast cancer and adjacent non‑cancerous tissues. (B) Western blotting analysis of NUAK1 protein expression in breast cancer cells and primary cultured human mammary epithelia cell (HEMC), (C) Immunochemistry analyses of NUAK1 expression in breast cancer tissue samples. Representative images of Negative staining, (D) weakly positive staining (+), (E) positive staining (++), and (F) strongly positive staining (+++) of NUAK1. The magnification was 400×. NUAK1 overexpression is associated with patient survival To test the expression of NUAK1 protein in breast cancer, we performed immunohistochemistry (IHC) staining of 160 breast cancer tissue samples. NUAK1 immunostaining was in cytoplasmic and nucleus. Different intensities of staining are shown in Figure 1C, D, E, F 87 of 160 (54.4%) paraffin-embedded breast cancer tissues showed high expression of NUAK1. There was no statistical difference in mean age (p = 0.291), T classification (p = 0.155), differentiation (p = 0.263), expression of ER (p = 0.703), expression of PR (p = 0.196) and expression of Her-2 (p = 0.221) between NUAK1 high and low expression groups. While NUAK1 expression was significantly associated with clinical stage (p =0.004), and N classification (p < 0.001) (Table 1). Patients with high NUAK1 expression had a shorter OS (p = 0.021) than low NUAK1 expression (Figure 2A). The univariate analysis model revealed that Clinical stage (p = 0.039), ER expression (p = 0.008), PR expression (p = 0.011), and NUAK1 expression (p =0.024) showed prognostic implication for the predication of breast cancer patients. In the multivariate Cox regression model, ER expression (p = 0.004), and NUAK1 expression (p=0.014) were independent prognostic factors for OS (Table 2). We also analyzed the prognostic value of NUAK1 in selective patient subgroups stratified by Clinical stage, T classification, N classification and Grade, respectively. The impact on the OS associated with the expression of NUAK1 showed no statistical significance in early‑stage tumors (Figure 2B; p=0.051) or late‑stage tumors (Figure 2C; p=0.735). The impact on the OS continued to be no statistical significance in T1‑2 subgroups (Figure 2D; p=0.060), and T3 subgroup (Figure 2E; p=0.125). For patients in N1-3 subgroups, the expression of NUAK1 was strongly asso­ciated with OS duration (Figure 2G; p=0.022), but not for patients in N0 subgroups (Figure 2F; p=0.271). When it was evaluated according to Grade, the impact on the OS associated with the expression of NUAK1 showed no statistical significance in Grade1 tumors (Figure 2H; p=0.899), but obviously significance in Grade2-3 tumors (Figure 2I; p=0.007). Figure 2 Kaplan-Meier survival curves with log-rank test of the 160 patients with breast cancer. (A) OS rates for cases with high NUAK1 expression vs. low NUAK1 expression in all patients, (B) OS rates for early clinical stage cases (stage I/Ⅱ) with high NUAK1 expression vs. those with low NUAK1 expression levels, (C) OS rates for late‑stage cases (stage Ⅲ) with high NUAK1 expression vs. those with low NUAK1 expression levels, (D) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with T1‑2‑grade breast tumors, (E) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with T3‑grade breast tumors. (F) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1expression level in patients without lymphatic metastasis (N0), (G) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with lymphatic metastasis (N1‑3), (H) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with Grade1 breast tumors, (I) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with Grade2-3 breast tumors. NUAK1 Might Affect the Invasion Ability of Breast Cancer Cells To further investigate whether NUAK1 could affect the proliferation, invasion, migration and colony formation ability of breast cancer cells, we established stable SKBR3 and MCF-7 cell line that expressed NUAK1 (SKBR3-NUAK1 or MCF-7- NUAK1) or empty vector (SKBR3- vector or MCF-7- vector). As shown in Figure 3A, the expression level of NUAK1 was significantly increased in SKBR3-NUAK1 or MCF-7- NUAK1cells compared with empty vector control cells. Transwell invasion assays were performed to determine the effect of NUAK1 on cellular invasion. The results showed that the overexpression of NUAK1 in SKBR3 and MCF-7 cells caused significant promotion of cellular invasion, as indicated by increase number of transmembrane cells. (Figure 3B, C). This finding suggested that NUAK1 promoted breast cancer cell invasion. Figure 3 NUAK1 promotes invasion ability in breast cancer cells. A. Western blotting analysis of NUAK1 protein expression in NUAK1-overexpressed MCF-7 cell and SKBR3 cell or empty vector cell lines. B Transwell invasion assay was performed between control and NUAK1 overexpression cells (MCF-7 and SKBR3), C. Quantification results for cell invasion. Overexpression of NUAK1 Induces the Expression of EMT-Related Proteins To test whether the promotion of invasion ability by NUAK1 overexpression is related to the epithelial-mesenchymal transition (EMT), we performed western blotting of SKBR3-NUAK1, MCF-7-NUAK1, SKBR3-vector and MCF-7-vector for EMT markers, including E-cadherin, β-catenin and Vimentin. The result showed that overexpression of NUAK1 significantly reduced expression of epithelial markers, such as E-cadherin and β-catenin, and triggered the expression of Vimentin, a mesenchymal marker (Figure 4). These data indicated that overexpression of NUAK1 induced EMT in breast cancer cells Figure 4 Overexpression of NUAK1induced EMT in breast cancer cells. Western blotting analysis of EMT markers E-cadherin, β-catenin and Vimentin in SKBR3-NUAK1, MCF-7-NUAK1, SKBR3-vector and MCF-7-vector. GAPDH was used as a loading control. Overexpression of NUAK1 and Tumor Microenvironment The observed induction of EMT by NUAK1 overexpression in breast cancer cells suggests a potential reprogramming of the tumor microenvironment (TME), which is known to play a critical role in cancer progression. The TME is a complex and dynamic entity, comprising not only cancer cells but also a variety of immune cells, fibroblasts, and extracellular matrix components. Among these components, the immune cell landscape has emerged as a significant factor influencing tumor growth, invasion, and metastasis. To gain a deeper understanding of how NUAK1 overexpression influences the TME, we utilized CIBERSORT. This computational approach estimates the relative abundance of various immune cell subsets within the tumor tissue by analyzing gene expression profiles. Utilizing data from TCGA BRCA cohort, we investigated the association between NUAK1 expression levels and immune cell infiltration. Figure 5A offers a comparative insight into the distribution of 22 specific immune cell types across groups categorized by low and high NUAK1 expression. The histogram in Figure 5B delineates the distribution of immune cell content. Our analysis revealed that the high NUAK1 expression group demonstrated significantly diminished levels of memory B cells, CD8+ T cells, activated CD4+ memory T cells, follicular helper T cells, regulatory T cells (Tregs), activated NK cells, and activated dendritic cells when juxtaposed with the low expression group. In stark contrast, there was a notable elevation in the levels of naive B cells, resting CD4+ memory T cells, M2 macrophages, resting dendritic cells, resting mast cells, and neutrophils within the high expression group. Subsequently, we assessed the correlation between NUAK1 expression and immune cell composition. This analysis highlighted significant positive correlations with resting CD4+ memory T cells, resting mast cells, M2 macrophages, naïve B cells, resting dendritic cells, and neutrophils. Conversely, negative correlations were observed with follicular helper T cells, Tregs, CD8+ T cells, activated NK cells, memory B cells, and activated CD4+ memory T cells, as depicted in Figure 5C. Moreover, NUAK1 expression correlated positively with the ESTIMATE score, encapsulating the overall TME, and the stromal score, which isolates the stromal aspect of the TME (Figure 5D). Collectively, these correlations imply that elevated NUAK1 expression is linked to a TME that may foster a more aggressive tumor phenotype, potentially through its influence on the recruitment and activation of specific immune cells. Figure 5 Immune infiltration analysis. (A) Immune cell content stacking rlot between high NUAK1 expression and low NUAK1 expression. (B) Immune cell content histogram comparing high and low NUAK1 expression groups. (C) Correlation between NUAK1 expression level and the relative abundances of 22 immune cells. (D) Correlation of NUAK1 expression with stromal, ESTIMATE, and immune scores. Functional Enrichment Analysis of NUAK1 To further delineate the biological functions of NUAK1 and its role in tumorigenesis, 50 potential binding proteins of NUAK1 were identified from the STRING database (Figure 6A). These proteins are likely involved in a variety of biological processes that could impact the tumor microenvironment. Subsequently, we conducted GO enrichment analysis (Figure 6B-D) and KEGG pathway enrichment analysis (Figure 6E) on these 50 target proteins. The GO enrichment analysis revealed key biological processes, notably encompassing cell-cell signaling mediated by Wnt, regulation of the Wnt signaling pathway, and the proteasome-mediated ubiquitin-dependent protein catabolic process. These processes include the intrinsic apoptotic signaling pathway, particularly those regulated by P53 class mediators and SCF-dependent proteasomal degradation. The KEGG pathway enrichment analysis highlighted pathways associated with the Hippo signaling pathway, ubiquitin-mediated proteolysis, Wnt signaling pathway, proteoglycans in cancer, cellular senescence, focal adhesion, Hedgehog signaling pathway, and regulation of the actin cytoskeleton (Figure 6E). To explore the potential diseases associated with these proteins, we searched the DisGeNET database for related disease information on these genes. The results indicated significant associations between these genes and various types of cancer, including breast cancer (Figure 6F). Figure 6 Enrichment analysis of NUAK1. (A) A network of NUAK1 and its 50 potential co-interaction proteins. (B-D) GO enrichment analysis of the NUAK1 co-interaction proteins reveals enriched biological functions (BP), cellular components (CC), and molecular functions (MF). (E) KEGG pathway enrichment analysis. (F) Enrichment analysis in DisGeNET. Single-cell analysis of NUAK1 Based on the TISCH2 17, 18 , we conducted an investigation into the correlation between NUAK1 and TME across 10 different single-cell RNA datasets, as shown in Figure 7A. It is noteworthy that in five of the breast cancer single-cell datasets, NUAK1 demonstrated high expression levels primarily in endothelial cells. In order to further explore this relationship, we selected the GSE114727 dataset for a more detailed analysis, employing the umap visualization technique to depict various cell types including immune and stromal cells, as well as specific cell types such as CD8 T cells, dendritic cells (DCs), endothelial cells, fibroblasts, mast cells, and NK cells, as depicted in Figures 7B and 7C. In the GSE114727 cohort, NUAK1 was predominantly expressed in endothelial cells, with expression also observed in fibroblasts and myofibroblasts, as shown in Figures 7D and 7E. We then proceeded to analyze the expression of NUAK1 in relation to the TNM stage, with the results presented in Figure 7F-H. NUAK1 exhibited differential expression across various stages of stromal cells, demonstrating higher expression in stages I and III, and comparatively lower levels in stage II. This pattern of expression was consistent across endothelial cells, fibroblasts, monocytes/macrophages, and myofibroblasts, indicating a potential correlation between NUAK1 expression and the progression of the TNM stage. Subsequent Gene Set Enrichment Analysis (GSEA) identified NUAK1 as being highly expressed in regions associated with the TGF-BETA signaling, apoptosis, and P53 pathway models, as seen in Figures 7I, 7J, and 7M. Additionally, there was a general increase in the epithelial-mesenchymal transition and WNT/β-catenin signaling pathways, as demonstrated in Figures 7K and 7L, respectively. Figure 7 Single-cell analysis of NUAK1 in TME. (A) Heatmap displaying relative expression of NUAK1 in endothelial cells across 10 single-cell BRCA datasets. (B) The scRNA-seq data from the GSE114727 dataset. (C) The immune cells and stromal cells within the BRCA dataset (GSE114727) were subjected to single-cell analysis using TISCH2. Umap (D) and violin plot (E) to explore the expression profiles of NUAK1 in GSE114727. (F) Violin plot of NUAK1 expression in immune and stromal cells across TNM stages in the GSE114727 BRCA single-cell Dataset. (G) UMAP plot illustrating cellular distribution across TNM stages in the GSE114727 BRCA single-cell Dataset. (H) Violin plot of NUAK1 expression across TNM stages in 11 cell types from BRCA GSE114727 dataset. (I-M) Gene Set Enrichment Analysis (GSEA) of TGF-BETA signaling, apoptosis, epithelial-mesenchymal transition, WNT beta catenin signaling, and the P53 pathway in BRCA. Spatial Transcriptomic Analysis of NUAK1 Expression Employing the CROST dataset 19, 20 for our spatial transcriptomic analysis, we commenced with an examination that leveraged the dataset's integrated quality control measures, which included assessments of count, gene, mitochondrial, and ribosomal statistics (Figure 8 A-D). This preliminary filtration of data led to the spatial clustering analysis (Figure 8E and F), distinguishing twelve unique cellular clusters within the breast cancer tissue microenvironment. The cell type deconvolution (Figure 8G and H) within this context exposed B-Nai cells as a predominant cell type, alluding to their substantial influence. A striking observation was the heightened expression levels of the NUAK1 gene within B-Nai cells, with expression also noted in mast cells, CD8+ T-Nai, and macrophages (Figure 8I), which may be pivotal for breast cancer development. Spatial correlation analysis (Figure 8J) illustrated B-Nai cells in negative correlation with several cell types, suggesting intricate intercellular interactions. The spatial colocalization analysis (Figure 8M) further delineated a network of communications, with B-Nai cells being central to this dialogue, particularly with T-Nai, T-FH, NK, and pDC cells. Cluster-cluster communication analysis (Figure 8K, L) and cell-cell communication analysis (Figure 8N, O) provided insights into the interactions among different cellular populations, with B-Nai cells and mast cells, macrophages, and B-Lym cells identified as key communicators within the tumor microenvironment. Analysis of the TGFβ signaling pathway through a heatmap (Figure 8P) and spatial images (Figure 8Q) showed that CD8+ T-Nai cells were profoundly involved in all signaling roles. B-Nai cells were highlighted as significant receivers and influencers, while mast cells were identified as key influencers with a critical regulatory function within the TGFβ signaling network. Macrophages were distinguished by their active participation as receivers, potentially acting as nexus points for immune cell communication. Heatmap analysis (Figure 8R) and spatial images (Figure 8S) of the WNT signaling pathway revealed distinct patterns of involvement among immune cell types. B-Nai cells were particularly highlighted in the roles of receivers, mediators, and influencers, suggesting a significant role in the reception, mediation, and modulation of WNT signaling, potentially impacting adaptive immune responses. In contrast, mast cells exhibited a profound level of participation across all assessed signaling roles, implying a multifaceted role in immune cell communication and the initiation, transmission, and regulation of WNT signaling events. Figure 8 NUAK1 expression of spatially transcriptomic-defined clusters in BRCA. (A) Count statistics. (B) Gene statistics. (C) Mitochondrion statistics. (D) Ribosome statistics. (E, F) Spatial clustering analysis. (G, H) Cell type deconvolution. (I) The spatial location and gene changes of NUAK1. (J) Spatial correlation analysis. (K) Cluster-cluster communication analysis in number of interactions (K) and strength of interactions (L). (M) Spatial colocalization analysis. Cell–cell communication analysis in number of interactions (N) and strength of interactions (O). (P) Heatmap of signaling roles (senders, receivers, mediators and influencers) for TGFb signaling pathway. (Q) Spatial images of intercellular communication networks for TGFb signaling pathway. (R) Heatmap of signaling roles (senders, receivers, mediators and influencers) for WNT signaling pathway. (S) Spatial images of intercellular communication networks for WNT signaling pathway. Discussion According to the data in 2020, the number and mortality of breast carcinoma among women in the world ranked first 2 . It is the most commonly diagnosed malignant tumor with more than 2,100,000 new cases worldwide and more than 626,000 deaths 21 and also the principal cause of cancer-related death among women over the world 3 , 22 . Looking back at the history of breast cancer treatment, it is easy to see that the enlargement of surgical scope cannot improve the survival rate. In recent years, the improvement of survival rate of breast cancer is mainly due to early detection, early diagnosis and continuous improvement of postoperative comprehensive adjuvant therapy. Nevertheless, there are still some patients who cannot avoid tumor recurrence and metastasis. Basically, breast cancer is a heterogeneous malignancy with different histological types, diverse molecular profiles and varied clinical responses to therapy. It is divided into 5 molecular subtypes based on the genome and transcriptome sequences, including Luminal A phenotype, Luminal B phenotype, HER-2 enriched phenotype, Basal-like phenotype and Claudin-low phenotype 23 . It is difficult to accurately predict prognosis in breast cancer due to its phenotypic and molecular diversity. Therefore, it is imperative to explore new risk genes and therapeutic targets to obtain new drugs and treatment strategies. In 2003, Suzuki et al. discovered NUAK1 as a member of AMPK (adenosine activated protein kinase / Amp activated protein kinase) family 8 . The protein has a molecular weight of 74 KD and contains a highly conserved active T ring 8 , 24 . It is known that activation of NUAK1 kinase activity requires threonine 211 phosphorylation by LKB1 or serine 600 phosphorylation by Akt kinase. Like most AMPK regulated kinases, the activity of NUAK1 is increased by 10 to 20 times by LKB1 phosphorylation through threonine on its T ring 24 . The NUAK1 signaling pathway has been successively proved to play an important physiological and pathological role in vivo, such as promoting cell growth, inhibiting apoptosis, regulating angiogenesis, promoting cell proliferation and participating in glucose metabolism, and is related to the initiation of malignant tumors 25 . Suzuki confirmed that NUAK1 is the main factor affecting the survival and migration of Akt dependent tumor cells through in vitro research 9 . It was found that NUAK1 could induce the survival of tumor cells through Akt protein dependent nutritional starvation, and enhance the invasion ability of tumor cells through the Akt signaling pathway mediated by IGF-1 (insulin-like growth factor-1) 26 . NUAK1 acts on the basement membrane type 1-matrix metalloproteinases (MT1 MMPs) to inhibit apoptosis and stimulate the occurrence of invasive behavior 27 . Studies have found that NUAK1 promotes the invasion and metastasis of pancreatic carcinoma and colon carcinoma by regulating the secretion of MMP-2 and MMP-9 28 .Roh SA also reported that NUAK1 is strongly correlated with MMP-2, MMP-9 and S100A4 29 . Therefore, Akt / NUAK1 signaling pathway may be a new pathway closely related to tumor formation to induce cell survival. Some studies reported that up-regulation of NUAK1 was closely related to advanced clinical stage, enhanced distant metastasis and short survival period in colorectal cancer, nasopharyngeal carcinoma 30 , gastric cancer and ovarian cancer 28 , 31 , 32 . In current study, we assessed the expression of NUAK1 in breast carcinoma and its prognostic significance. Firstly, we compare the expression level of NUAK1 in breast carcinoma cell lines and tissue samples compared with normal human breast epithelial cells, and adjacent noncancerous tissues, respectively. The results revealed that NUAK1 expression was obviously up-regulated in tumor samples compared with the adjacent non-cancerous tissue samples, suggesting that NUAK1 is an important biomarker for the oncogenesis of breast carcinoma (Fig. 1 A). The endogenic NUAK1 expression in breast cancer cells were also higher compared with human breast epithelia cell (Fig. 1 B). Furthermore, we analyzed the relationship of NUAK1 expression with clinicopathologic features in 160 patients with breast carcinoma. As showed in Table 1 , overexpression of NUAK1 had obvious correlation with advanced TNM staging and lymph node metastasis. Simultaneously, no significant correlation was found between NUAK1 and age, T classification, histological grading, ER expression, PR expression or HER2 expression. Correspondently, NUAK1 was proved to be closely correlated with the tumor invasion and lymph node metastasis in head and neck cancer(29) 33 . Therefore, we reasonably speculate that NUAK1 works as a critical factor promoting tumor invasion and distant metastasis in breast carcinoma, and also may serve as therapeutic target. However, further investigation is needed to verify our speculation. Additionally, it has been shown by Cox-regression analyses those patients with excessive NUAK1 expression had a significant worse overall survival rate, and that the status of NUAK1 was independent prognostic index influencing overall survival (Table 2 ). Subgroup study stratified by T classification, N classification Clinical stage and Grade suggested that the up-regulation of NUAK1 was closely associated with OS in patient with invasive clinicopathological characteristics, including lymph node metastasis and poor differentiation (Fig. 2 G, I). These findings suggested the possibility of up-regulation of NUAK1 to become a new biomarker for unfavorable prognosis in breast cancer patients. Through CIBERSORT immune infiltration analysis, we observed a negative correlation between high expression of NUAK1 and cells with anti-tumor activity, such as CD8 + T cells and activated NK cells 34 – 37 . Conversely, there was a positive correlation with cells that promote tumor progression, including Tregs and M2 macrophages 38 – 41 . Notably, the latter are known to sustain a Th2 response, which has been shown to be conducive to cancer growth 41 . These findings suggest that NUAK1 may play a key role in the regulation of the breast cancer microenvironment, facilitating tumor invasion and metastasis. At the single-cell level, our analysis revealed that NUAK1 is predominantly expressed in endothelial cells, with lower levels of expression also observed in fibroblasts and myofibroblasts. Furthermore, GSEA indicated higher expression of NUAK1 in regions associated with TGFβ and WNT signaling pathways, which are known to play crucial roles in EMT and tumor progression 42 – 45 . Spatial transcriptomics analysis provided additional insights into the role of NUAK1 within the TME. Our findings confirmed the elevated expression of NUAK1 specifically within the B cell naive subgroup. This observation is in concordance with the CIBERSORT immune infiltration analysis from our results section, which indicated a higher proportion of naive B cells in samples with high NUAK1 expression. Moreover, the spatial analysis unveiled intricate intercellular communication networks, highlighting interactions between B cell naive and cells involved in TGFβ and WNT signaling pathways. The WNT and TGFβ signaling pathways are key regulators in the EMT process, promoting tumor invasion and metastasis by affecting intercellular junctions and altering cell phenotype 46 – 48 . The communication between B cell naive and these signaling pathways implies that NUAK1 may exert its signaling functions not in isolation but through a complex interplay with other cellular components of the TME. Therefore, we propose that NUAK1 may serve as a critical regulatory factor that promotes the progression of breast cancer by influencing the behavior of naive B cells and other immune cells. Specifically, the high expression of NUAK1 in naive B cells may impact the EMT process by secreting specific cytokines or through direct cell-to-cell contact, potentially providing a favorable microenvironment for the invasion and metastasis of breast cancer cells. EMT paticipates in various biological and pathological processes, it has closely relationship with anti-apoptosis and chemotherapeutic resistance 49 . It is the process by which epithelial cells with compact cell-cell adhesion convert to mesenchymal cells (13) 13 . In this process, the gene expression undergoes multiple changes: Epithelial markers (tight junction protein 1, E-cadherin and occludin) were down-regulated and mesenchymal markers (fibronectin, N-cadherin and vimentin ) were up-regulated 50 . Research on ovarian cancer reported that NUAK1 was up-regulated in cancer cells and was verified to be closely related with EMT 51 . Moreover, NUAK1 was revealed to regulate EMT in multiple tumors 14 , 15 , 52 , 53 . In the NUAK1 knockdowngastric cells, the expression of E-cadherin was increased, together with down-regulation of Vimentin. Furthermore, NUAK1 was positively correlated with Vimentin in gastric cancer, but negatively associated with E-cadherin 32 . In our study, upregulation of NUAK1 expression increase cell invasion ability. At the same time, EMT related β-catenin and E-cadherin expression was decreased, and the expression of mesenchymal marker vimentin was increased. From the above results, it makes sense that the functional role of NUAK1 on breast cancer invasion and distant metastasis may be interpteted to its induction in EMT. This study revealed that the expression of NUAK1 is elevated in breast carcinoma tissues, it is also a predictor of prognosis. Moreover, we found NUAK1 promoted breast cancer cell invasion, which is related to its function of induction to EMT in breast cancer cells, thus indicating that NUAK1 may have potential as a valuable prognostic index and novel therapeutic target for patients with breast carcinoma. Conclusion In summary, our research uncovers the multifaceted roles of NUAK1 in breast cancer, including the promotion of cellular invasion, regulation of EMT, and potential effects within the tumor microenvironment. High expression of NUAK1 correlates with poor prognosis and may serve as a valuable prognostic indicator and therapeutic target. Future studies are needed to further explore the molecular mechanisms of NUAK1 in the development of breast cancer, as well as its precise role in the WNT, TGFβ, and EMT signaling pathways, to provide new strategies for the treatment of breast cancer. Declarations CONFLICT OF INTEREST STATEMENT The authors have no conflict of interest. Patient consent for publication Not applicable. Competing Interests The authors have declared that no competing interest exists. Funding This study was supported by grants from the Natural Science Foundation of Guangdong Province, China (2021A1515012493). Guangzhou Science and Technology Plan Project: 2023A03J0195. Author Contribution We declare that all the listed authors have participated actively in the study and all meet the requirements of the authorship. Drs. Peng Zhang and Baoyu Zhang designed the study and wrote the protocol, Drs. Jiani Wang, Jiumei Yang and Cuicui Li acquired the manuscript, analyzed the data, and wrote the first draft of the manuscript and mainly revised the manuscript. Dongbo Qiu analyzed the data. All authors approved the final version of the manuscript. Acknowledgements None declared. DATA AVAILABILITY STATEMENT The data generated in the present study may be requested from the corresponding author. 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Supplementary Files table1.docx table2.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 05 May, 2026 Reviews received at journal 14 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 08 Mar, 2025 Reviewers invited by journal 22 Jan, 2025 Editor assigned by journal 21 Nov, 2024 Submission checks completed at journal 26 Oct, 2024 First submitted to journal 20 Oct, 2024 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. 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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-5300363","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370695541,"identity":"efd1e373-506e-4094-8400-8d43b08d2d3c","order_by":0,"name":"Jiani Wang","email":"","orcid":"","institution":"The Third Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Jiani","middleName":"","lastName":"Wang","suffix":""},{"id":370695542,"identity":"70c17d3b-8191-4f3f-88b5-ffba9ed35e36","order_by":1,"name":"Jiumei Yang","email":"","orcid":"","institution":"Guangdong Second Provincial General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiumei","middleName":"","lastName":"Yang","suffix":""},{"id":370695543,"identity":"82ae60fe-c9bd-4851-8e62-52f226be3b25","order_by":2,"name":"Cuicui Li","email":"","orcid":"","institution":"the Fifth Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cuicui","middleName":"","lastName":"Li","suffix":""},{"id":370695544,"identity":"e7f1fa73-9d66-41d4-a5f1-43761705bf1a","order_by":3,"name":"Dongbo Qiu","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Dongbo","middleName":"","lastName":"Qiu","suffix":""},{"id":370695545,"identity":"65788a0c-3325-447f-b799-cc8a4dd6b1ce","order_by":4,"name":"Baoyu Zhang","email":"","orcid":"","institution":"The Third Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Baoyu","middleName":"","lastName":"Zhang","suffix":""},{"id":370695546,"identity":"b36b1995-0de7-4db5-94b7-6d7913961cbe","order_by":5,"name":"Peng Zhang","email":"data:image/png;base64,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","orcid":"","institution":"The Third Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-10-21 01:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5300363/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5300363/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69084266,"identity":"0c531485-05eb-4acb-98ac-e175c99268cf","added_by":"auto","created_at":"2024-11-15 12:26:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":286503,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of NUAK1 is elevated in breast cancer tissues. (A) RT-qPCR was performed to determine the expression of NUAK1 in the breast cancer and adjacent non‑cancerous tissues. (B) Western blotting analysis of NUAK1 protein expression in breast cancer cells and primary cultured human mammary epithelia cell (HEMC), (C) Immunochemistry analyses of NUAK1expression in breast cancer tissue samples. Representative images of Negative staining, (D) weakly positive staining (+), (E) positive staining (++), and (F) strongly positive staining (+++) of NUAK1. The magnification was 400×.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/2df1750dbe30feb0f1275a35.jpeg"},{"id":69083168,"identity":"c3811f6e-dbac-4711-8c06-68ac796e7347","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57211,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier survival curves with log-rank test of the 160 patients with breast cancer. (A) OS rates for cases with high NUAK1 expression vs. low NUAK1expression in all patients, (B) OS rates for early clinical stage cases (stage I/Ⅱ) with high NUAK1 expression vs. those with low NUAK1 expression levels, (C) OS rates for late‑stage cases (stage Ⅲ) with high NUAK1 expression vs. those with low NUAK1 expression levels, (D) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with T1‑2‑grade breast tumors, (E) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with T3‑grade breast tumors. (F) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1expression level in patients without lymphatic metastasis (N0), (G) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with lymphatic metastasis (N1‑3), (H) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with Grade1 breast tumors, (I) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with Grade2-3 breast tumors.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/a86cc9e9bd7b11fbb4ed0c6f.png"},{"id":69083172,"identity":"1e8ee973-6e7b-41cd-bced-88667c48aa22","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":400874,"visible":true,"origin":"","legend":"\u003cp\u003eNUAK1 promotes invasion ability in breast cancer cells. A. Western blotting analysis of NUAK1 protein expression in NUAK1-overexpressed MCF-7 cell and SKBR3 cell or empty vector cell lines. B Transwell invasion assay was performed between control and NUAK1 overexpression cells (MCF-7 and SKBR3), C. Quantification results for cell invasion.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/3c261c6bdbe19338127639d1.png"},{"id":69083173,"identity":"0ab87322-1501-4304-bc13-5a826dc566fb","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25739,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of NUAK1induced EMT in breast cancer cells. Western blotting analysis of EMT markers E-cadherin, β-catenin and Vimentin in SKBR3-NUAK1, MCF-7-NUAK1, SKBR3-vector and MCF-7-vector. GAPDH was used as a loading control.\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/e43085b84d5932ab0c89c8f1.png"},{"id":69083174,"identity":"0634e35d-a395-4465-b9be-bf3773aaba37","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2698027,"visible":true,"origin":"","legend":"\u003cp\u003eImmune infiltration analysis. (A) Immune cell content stacking rlot between high NUAK1 expression and low NUAK1 expression. (B) Immune cell content histogram comparing high and low NUAK1 expression groups. (C) Correlation between NUAK1 expression level and the relative abundances of 22 immune cells. (D) Correlation of NUAK1 expression with stromal, ESTIMATE, and immune scores.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/006f490ba6ef6ef3da7dd81e.png"},{"id":69083175,"identity":"da51816d-71c0-442a-9cda-fca99a775b7f","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5190115,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment analysis of NUAK1. (A) A network of NUAK1 and its 50 potential co-interaction proteins. (B-D) GO enrichment analysis of the NUAK1 co-interaction proteins reveals enriched biological functions (BP), cellular components (CC), and molecular functions (MF). (E) KEGG pathway enrichment analysis. (F) Enrichment analysis in DisGeNET.\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/f28050a59bd5066d41d90aac.png"},{"id":69084267,"identity":"9bb14758-9df9-4fa2-94e6-83e69c7af218","added_by":"auto","created_at":"2024-11-15 12:26:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10719697,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-cell analysis of NUAK1 in TME. (A) Heatmap displaying relative expression of NUAK1 in endothelial cells across 10 single-cell BRCA datasets. (B) The scRNA-seq data from the GSE114727 dataset. (C) The immune cells and stromal cells within the BRCA dataset (GSE114727) were subjected to single-cell analysis using TISCH2. Umap (D) and violin plot (E) to explore the expression profiles of NUAK1 in GSE114727. (F) Violin plot of NUAK1 expression in immune and stromal cells across TNM stages in the GSE114727 BRCA single-cell Dataset. (G) UMAP plot illustrating cellular distribution across TNM stages in the GSE114727 BRCA single-cell Dataset. (H) Violin plot of NUAK1 expression across TNM stages in 11 cell types from BRCA GSE114727 dataset. (I-M) Gene Set Enrichment Analysis (GSEA) of TGF-BETA signaling, apoptosis, epithelial-mesenchymal transition, WNT beta catenin signaling, and the P53 pathwayin BRCA.\u003c/p\u003e","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/c4795cb3254738ebb26a3621.png"},{"id":69083176,"identity":"b5bfe371-d8ba-46f2-bc70-362570666d18","added_by":"auto","created_at":"2024-11-15 12:18:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":36217183,"visible":true,"origin":"","legend":"\u003cp\u003eNUAK1 expression of spatially transcriptomic-defined clusters in BRCA. (A) Count statistics. (B) Gene statistics. (C) Mitochondrion statistics. (D) Ribosome statistics. (E, F) Spatial clustering analysis. (G, H) Cell type deconvolution. (I) The spatial location and gene changes of NUAK1. (J) Spatial correlation analysis. (K) Cluster-cluster communication analysis in number of interactions (K) and strength of interactions (L). (M) Spatial colocalization analysis. Cell–cell communication analysis in number of interactions (N) and strength of interactions (O). (P) Heatmap of signaling roles (senders, receivers, mediators and influencers) for TGFb signaling pathway. (Q) Spatial images of intercellular communication networks for TGFb signaling pathway. (R) Heatmap of signaling roles (senders, receivers, mediators and influencers) for WNT signaling pathway. (S) Spatial images of intercellular communication networks for WNT signaling pathway.\u003c/p\u003e","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/dfdf3cee519d4fd5a3ff454a.png"},{"id":69084822,"identity":"9ae197f8-b129-4f8d-92f0-0863f1e308f4","added_by":"auto","created_at":"2024-11-15 12:34:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9783243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/4d7e59c8-0916-44e2-bd9e-5b7c7a2c30e9.pdf"},{"id":69083171,"identity":"2dec8703-bb60-42b8-b69e-1ce42bb4978a","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18807,"visible":true,"origin":"","legend":"","description":"","filename":"table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/a864bbadd69b9e9ab7463012.docx"},{"id":69083170,"identity":"087eac57-73e3-43ce-8735-a0d667e9a741","added_by":"auto","created_at":"2024-11-15 12:18:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18418,"visible":true,"origin":"","legend":"","description":"","filename":"table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5300363/v1/470988e91535d4b5ffd74c84.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spatial and Single-Cell Analyses Reveal the Pro-Invasiveness Role of NUAK1 in Breast Cancer through EMT Regulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast carcinoma is one of the most common malignant tumors in women and is also the leading cause of death related to malignancy in women\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Due to the improvement of diagnosis and treatment, the mortality rate of breast cancer showed a continuous downward trend\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, not all patients can benefit from it. The main reason is that breast carcinoma is a highly heterogeneous malignancy in clinical and biological\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. At present, according to the state of ER, PR, HER2 and Ki-67, breast carcinoma is mainly divided into 4 different subtypes. Different subtypes have different clinical treatment protocols\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although researchers have been committed to the progress of treatment and the discovery of new drugs, the treatment effect of metastatic breast cancer patients is still stubborn and almost no significant improvement. In fact, distant metastasis accounts for most of the breast cancer related mortality\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To enhance the clinical management of breast cancer, it is very important to identify new biomarkers which can facilitate risk stratification, and prognostic assessment. Therefore, there is an urgent need to develop novel biomarkers to predict the clinical outcomes and guide clinical practice in breast cancer.\u003c/p\u003e \u003cp\u003eNovel (nua) kinase family 1 (NUAK1), also known as the fifth member of adenosine monophosphate-activated protein kinase (AMPK)-related kinase family (ARK5), can mediate cell survival and differentiation, and plays multiple roles in regulating cellular adhesion, metabolism and response\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Clinically, nuak1 can promote tumor development, and tumor patients with high expression of nuak1 have lower survival rate and poor prognosis. Studies have shown that nuak1 is an \"energy monitoring molecule\" in tumor cells, which can monitor the energy balance in cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. During tumor nutritional starvation, nuak1 relies on caspase 8 to inhibit Akt and promote tumor growth\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Nuak1 can inhibit tumor cell apoptosis caused by factors such as glucose starvation and cytokine TNF-α (tumor necrosis factor-α) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, also NUAK1 can promote tumor cell survival through TGF \u0026ndash; β receptor to tolerate nutritional starvation \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e; nuak1 activation can also make liver cancer cells tolerate glucose starvation through G1/S cell cycle arrest\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e; nuak1 can also downregulate apoptosis factor (FAS) ,inhibit the activities of Fas receptor and caspase-6\u003csup\u003e10\u003c/sup\u003e. In addition, NUAK1 is closely related to tumor invasion and metastasis.\u003c/p\u003e \u003cp\u003eIt is reported that Epithelial to mesenchymal transition (EMT) plays an important role in tumor drug resistance\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Drug resistant tumor cells has high malignant, migration and invasion behavior. NUAK1 induces EMT to make hepatocarcinoma cell drug resistance\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Likewise, NUAK1 is also suggested involving in cell migration and invasion via EMT in gastric carcinoma\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Forthoremore, NUAK1 is closely associated with migration and metastatic potential in various other carcinoma, including pancreatic carcinoma (12)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, lung carcinoma\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and ovarian carcinoma\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, which suggested NUAK1 as a potential therapeutic target in many malignant carcinomas.\u003c/p\u003e \u003cp\u003eIn the present study, we evaluate the expression of NUAK1 in breast carcinoma illuminate significance in tumor prognosis. Also, through cellular experiment, we explore that NUAK1 regulates EMT process and affects cell migration ability.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003ePatients and tissue specimen\u003c/b\u003e \u003cem\u003es\u003c/em\u003e \u003c/p\u003e \u003cp\u003e This study was approved by the Institutional Research Ethics Committee of Sun Yatsen University, and written informed consent was obtained prior from each patient. A total of 160 patients with breast carcinoma who were histopathologically diagnosed and underwent curative surgery from Dec 2000 to Feb 2014 in the Third Affiliated Hospital of Sun Yatsen University were enrolled in this study.\u003c/p\u003e \u003cp\u003e Clinicopathological classification and tumor-node- metastasis (TNM) staging system were defined on the basis of the eighth edition American Joint Committee on Cancer (AJCC). There were 16 (10.0%), 95 (59.4%), 49 (30.6%) patients belonging to stage I, II and III respectively. Detailed clinicopathological information of these patients was shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The overall survival rate (OS) was calculated from the date of first operation to the date of patient death and considered as censored for the patients who were still alive at last follow-up. The followup time ranged from 2 to162 months (median followup time 127 months).\u003c/p\u003e \u003cp\u003eOf these 160 patients with breast carcinoma, 26 paired adjacent noncancerous tissues (at least 2cm distant from the edge of the tumor) were conserved immediately in surgery.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eHuman breast cancer cell lines MDA-MB-231, SK-BR-3, MDA-MB-435, MCF-7 and T47D were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS, Hyclone Laboratories, Logan, UT) and 100 units/ml penicillin and streptomycin at 37℃ in humidified 5% CO\u003csub\u003e2\u003c/sub\u003e. All cell lines were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China.\u003c/p\u003e \u003cp\u003eHuman mammary epithelial cell (HMEC) was obtained from primary cultured fresh breast epithelial tissues. The procedure was described in our previously published articles(16)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.HMEC was cultured in Keratinocyte-SFM medium (Invitrogen, Grand Island, NY) supplemented with bovine pituitary extract at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative real-time polymerase chain reaction (qRT-PCR) analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA extracted from breast cancer and adjacent noncancerous tissues using Trizol reagent (Invitrogen) was reversed transcription into complementary DNA (cDNA) by the Super Script H III First-Strand Synthesis System (Invitrogen). NUAK1 mRNA expression was detected by qPCR using iQTM SYBR\u0026reg; Green Supermix with the CFX384 Real-Time System (both Bio-Rad Laboratories, Inc). The expression level of NUAK1 mRNA was normalized to the endogenous expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. The primer sequences were as follows: NUAK1 sense 5\u0026rsquo;-GCTCGAGGACTCATACGGTG-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003eantisense 5\u0026rsquo;-GAGGGCATCACAGTCACACA-3\u0026rsquo;, \u0026lsquo;GAPDH sense 5\u0026rsquo;-TGTTGCC ATCAATGACCCCTT-3\u0026rsquo;, antisense 5\u0026rsquo;-CTCCACGACGTACTCAGCG-3\u0026rsquo;.\u003c/p\u003e\n\u003ch3\u003eWestern blotting assay\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting assay\u003c/div\u003e \u003cp\u003eCells were extracted using RIPA buffer, and protein concentration was detected by BCA protein assay kit (Bicinchoninic Acid Kit, Sigma-Aldrich, USA). Equal amount of protein samples were loaded into 10.0% resolving SDS-PAGE gel electrophoresis, and then transferred onto polyvinylidene difluoride membrane. After blocking the non-specific binding with 5% non-fat milk in TBS-T solution, the membranes were probed with a primary Polyclonal antibody to NUAK1 (Affinity Biosciences LTD. DF10340), followed by incubation with secondary antibodies (dilution 1:1000; Cell signaling, Billerica, MA). The protein signals were visualized with ECL solution (Beyotime Institute of Biotechnology). GAPDH (dilution 1:1000; Cell signaling, Billerica, MA) was used as loading control.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC) and Score Evaluation\u003c/h3\u003e\n\u003cp\u003eTissue sections (4-mm thick) were deparaffinized with xylenes and rehydrated in a series of graded alcohol before blocked with 0.3% hydrogen peroxide at room temperature for 15 min. The slides were boiled in ethylenediamine tetraacetic acid buffer (EDTA, pH\u0026thinsp;=\u0026thinsp;8.0) in a microwave to retrieve antigens for 30 min. The slides were then incubated with the rabbit polyclonal anti-NUAK1 antibody (ab718714, 1:50, Abacam, Cambridge, UK) in a moist chamber at 4\u0026deg;C overnight. Next day, the tissue sections were treated with horseradish peroxidase (DAKO ChemMate\u0026trade; EnVision\u0026trade; Detection Kit, Copenhagen, Danmark) for 30 min at 37\u0026deg; C and subsequently stained with the 3,3\u0026prime;-diaminobenzidine (DAB) for visualization. Finally, nuclear counterstain was conducted with freshly diluted Mayer\u0026rsquo;s hematoxylin. Normal rabbit IgG was used instead of the primary antibody as negative control.\u003c/p\u003e \u003cp\u003eThree independent pathologists blinded to the patients\u0026rsquo; clinicopathological data conducted the score assessment for NUAK1 expression. Scoring criteria was calculated as follows: staining score\u0026thinsp;=\u0026thinsp;intensity score\u0026thinsp;+\u0026thinsp;percentage score. The intensity was graded as: 0, negative; 1, weak; 2, moderate and 3, strong. The proportion of NUAK 1 (+) cells was graded as:0, 0\u0026ndash;5%; 1, 6\u0026ndash;25%; 2,26\u0026ndash;50%; 3,51\u0026ndash;75%; and 4, 76\u0026ndash;100%. The optimal cutoff value was determined: a final score ranging from 0 to 4 was defined as NUAK1 low expression and ranging from 5 to 12 was defined as NUAK1 high expression.\u003c/p\u003e\n\u003ch3\u003ePlasmid Construction and Retroviral Infection\u003c/h3\u003e\n\u003cp\u003ePlncx2 plasmid vector was used to generate plncx2-NUAK1. The plasmid construction was verified by DNA sequencing.\u003c/p\u003e \u003cp\u003eProduction of retrovirus was performed according to the instructions, SKBR3 cells and MCF-7 cells were subjected with infection of retrovirus expressing NUAK1 or vector. For establishing stable cell lines with overexpression of NUAK1, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded into 6 cm tissue culture plates with complete growth medium supplemented with G418 at 48 h post-transfection and selected for 14 days.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTranswell invasion assays\u003c/h2\u003e \u003cp\u003eInvasion of cells was evaluated in Transwell cell culture chambers with filters (8-\u0026micro;m pore size, Costar,Cambridge, MA). The membrane was pre-coated with Matrigel (Falcon BD).\u003c/p\u003e \u003cp\u003eMCF-7 and SKBR3 cells were seeded to the upper chamber, which was placed into the lower chamber containing 600 \u0026micro;L culture medium supplemented with 20% FBS. After 24 h incubation at 37\u0026deg;C, the cells on the upper chambers were removed with a cotton swab. The cells on the lower surface were fixed with methanol for 10 min, then stained with 0.2% crystal violet solution for 1 h and imaged under an inverted microscope. Five random microscopic fields (\u0026times;100) per well were counted and the mean was determined. All assays were performed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using IBM SPSS Statistics (Version 22.0; IBM Corp., New York, USA) and GraphPad Prism 8.0 (San Diego, CA, USA). Two tailed Student\u0026rsquo;s t-test were used to compare the difference of NUAK1 mRNA levels between breast cancer and paired adjacent noncancerous tissues. The χ2 test or the Fisher\u0026rsquo;s exact test was employed to evaluate the association between NUAK1and other clinicopathological characteristics of patients with breast cancer. The overall survival curves were plotted by Kaplan-Meier method and compared by log-rank test. Relative risks (RRs) associated with NUAK1 and other clinicopathological features were evaluated by univariate and multivariate Cox proportional hazards regression analyses. Variations for the trans-well assay were assessed by the two-tailed Student\u0026rsquo;s t test. A \u003cem\u003ep\u003c/em\u003e value less than 0.05 based on two-tailed tests was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe expression of NUAK1 is elevated in breast cancer tissues and cell lines\u003c/p\u003e\n\u003cp\u003eWe first investigated the expression of NUAK1in 26 paired breast cancer and adjacent non‑cancerous tissues by RT-qPCR assay. The results revealed that the mRNA level of NUAK1 was up-regulated in breast cancer tissues, compared with adjacent non‑cancerous tissues (Figure 1A). To further detect the endogenic NUAK1 expression in breast cancer cell lines, we cultured primary cultured human mammary epithelia cell (HEMC) and breast cancer cell lines (MDA-MB-231, SKBR3, MDA-MB-435, MCF-7, and T47D). The expression of NUAK1 was higher in the breast cancer cells compared with normal breast epithelia cell (Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 1 Expression of NUAK1 is elevated in breast cancer tissues. (A) RT-qPCR was performed to determine the expression of NUAK1 in the breast cancer and\u0026nbsp;adjacent non‑cancerous tissues. (B)\u0026nbsp;Western blotting analysis of NUAK1 protein expression in breast cancer cells and primary cultured human mammary epithelia cell (HEMC), (C)\u0026nbsp;Immunochemistry analyses of NUAK1 expression in breast cancer tissue samples. Representative images of Negative staining, (D) weakly positive staining (+), (E) positive staining (++), and (F) strongly positive staining (+++) of NUAK1. The magnification was 400\u0026times;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNUAK1 overexpression is associated with patient survival\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the expression of NUAK1 protein in breast cancer, we performed immunohistochemistry (IHC) staining of 160 breast cancer tissue samples. NUAK1 immunostaining was in cytoplasmic and nucleus. Different intensities of staining are shown in Figure 1C, D, E, F 87 of 160 (54.4%) paraffin-embedded breast cancer tissues showed high expression of NUAK1. There was no statistical difference in mean age (p = 0.291), T classification (p = 0.155), differentiation (p = 0.263), expression of ER (p = 0.703), expression of PR (p = 0.196) and expression of Her-2 (p = 0.221) between NUAK1 high and low expression groups. While NUAK1 expression was significantly associated with clinical stage (p =0.004), and N classification (p \u0026lt; 0.001) (Table 1). Patients with high NUAK1 expression had a shorter OS (p = 0.021) than low NUAK1 expression (Figure\u0026nbsp;2A). The univariate analysis model revealed that Clinical stage (p = 0.039), ER expression (p = 0.008), PR expression (p = 0.011), and NUAK1 expression (p =0.024) showed prognostic implication for the predication of breast cancer patients. In the multivariate Cox regression model, ER expression (p = 0.004), and NUAK1 expression (p=0.014) were independent prognostic factors for OS (Table\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003eWe also analyzed the prognostic value of NUAK1 in selective patient subgroups stratified by Clinical stage, T classification, N classification and Grade, respectively. The impact on the OS associated with the expression of NUAK1 showed no statistical significance in early‑stage tumors (Figure 2B; p=0.051) or late‑stage tumors (Figure 2C; p=0.735). The impact on the OS continued to be no statistical significance in T1‑2 subgroups (Figure 2D; p=0.060), and T3 subgroup (Figure 2E; p=0.125). For patients in N1-3 subgroups, the expression of NUAK1 was strongly asso\u0026shy;ciated with OS duration (Figure 2G; p=0.022), but not for patients in N0 subgroups (Figure 2F; p=0.271). When it was evaluated according to Grade, the impact on the OS associated with the expression of NUAK1 showed no statistical significance in Grade1 tumors (Figure 2H; p=0.899), but obviously significance in Grade2-3 tumors (Figure 2I; p=0.007).\u003c/p\u003e\n\u003cp\u003eFigure 2 Kaplan-Meier survival curves with log-rank test of the 160 patients with breast cancer. (A) OS rates for cases with high NUAK1 expression vs. low NUAK1 expression in all patients, (B) OS rates for early clinical stage cases (stage I/Ⅱ) with high NUAK1 expression vs. those with low NUAK1 expression levels, (C) OS rates for late‑stage cases (stage Ⅲ) with high NUAK1 expression vs. those with low NUAK1 expression levels, (D) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with T1‑2‑grade breast tumors, (E) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with T3‑grade breast tumors. (F) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1expression level in patients without lymphatic metastasis (N0), (G) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with lymphatic metastasis (N1‑3), (H) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with Grade1 breast tumors, (I) OS rates for cases with high NUAK1 expression vs. cases with low NUAK1 expression levels in patients with Grade2-3 breast tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNUAK1 Might Affect the Invasion Ability of Breast Cancer Cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate whether NUAK1 could affect the proliferation, invasion, migration and colony formation ability of breast cancer cells, we established stable SKBR3 and MCF-7 cell line that expressed NUAK1 (SKBR3-NUAK1 or MCF-7- NUAK1) or empty vector (SKBR3- vector or MCF-7- vector). As shown in Figure 3A, the expression level of NUAK1 was significantly increased in SKBR3-NUAK1 or MCF-7- NUAK1cells compared with empty vector control cells.\u003c/p\u003e\n\u003cp\u003eTranswell invasion assays were performed to determine the effect of NUAK1 on cellular invasion. The results showed that the overexpression of NUAK1 in SKBR3 and MCF-7 cells caused significant promotion of cellular invasion, as indicated by increase number of transmembrane cells. (Figure 3B, C). This finding suggested that NUAK1 promoted breast cancer cell invasion.\u003c/p\u003e\n\u003cp\u003eFigure 3 NUAK1 promotes invasion ability in breast cancer cells. A. Western blotting analysis of NUAK1 protein expression in NUAK1-overexpressed MCF-7 cell and SKBR3 cell or empty vector cell lines. B Transwell invasion assay was performed between control and NUAK1 overexpression cells (MCF-7 and SKBR3), C. Quantification results for cell invasion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOverexpression of NUAK1 Induces the Expression of EMT-Related Proteins\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether the promotion of invasion ability by NUAK1 overexpression is related to the epithelial-mesenchymal transition (EMT), we performed western blotting of SKBR3-NUAK1, MCF-7-NUAK1, SKBR3-vector and MCF-7-vector for EMT markers, including E-cadherin, \u0026beta;-catenin and Vimentin. The result showed that overexpression of NUAK1 significantly reduced expression of epithelial markers, such as E-cadherin and \u0026beta;-catenin, and triggered the expression of Vimentin, a mesenchymal marker (Figure 4). These data indicated that overexpression of NUAK1 induced EMT in breast cancer cells\u003c/p\u003e\n\u003cp\u003eFigure 4\u0026nbsp;Overexpression of NUAK1induced EMT in breast cancer cells.\u0026nbsp;Western blotting analysis\u0026nbsp;of EMT markers E-cadherin,\u0026nbsp;\u0026beta;-catenin and Vimentin in\u0026nbsp;SKBR3-NUAK1, MCF-7-NUAK1, SKBR3-vector and MCF-7-vector.\u0026nbsp;GAPDH was used as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOverexpression of NUAK1 and Tumor Microenvironment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe observed induction of EMT by NUAK1 overexpression in breast cancer cells suggests a potential reprogramming of the tumor microenvironment (TME), which is known to play a critical role in cancer progression. The TME is a complex and dynamic entity, comprising not only cancer cells but also a variety of immune cells, fibroblasts, and extracellular matrix components. Among these components, the immune cell landscape has emerged as a significant factor influencing tumor growth, invasion, and metastasis.\u003c/p\u003e\n\u003cp\u003eTo gain a deeper understanding of how NUAK1 overexpression influences the TME, we utilized CIBERSORT. This computational approach estimates the relative abundance of various immune cell subsets within the tumor tissue by analyzing gene expression profiles. Utilizing data from TCGA BRCA cohort, we investigated the association between NUAK1 expression levels and immune cell infiltration. Figure 5A offers a comparative insight into the distribution of 22 specific immune cell types across groups categorized by low and high NUAK1 expression. The histogram in Figure 5B delineates the distribution of immune cell content.\u003c/p\u003e\n\u003cp\u003eOur analysis revealed that the high NUAK1 expression group demonstrated significantly diminished levels of memory B cells, CD8+ T cells, activated CD4+ memory T cells, follicular helper T cells, regulatory T cells (Tregs), activated NK cells, and activated dendritic cells when juxtaposed with the low expression group. In stark contrast, there was a notable elevation in the levels of naive B cells, resting CD4+ memory T cells, M2 macrophages, resting dendritic cells, resting mast cells, and neutrophils within the high expression group.\u003c/p\u003e\n\u003cp\u003eSubsequently, we assessed the correlation between NUAK1 expression and immune cell composition. This analysis highlighted significant positive correlations with resting CD4+ memory T cells, resting mast cells, M2 macrophages, na\u0026iuml;ve B cells, resting dendritic cells, and neutrophils. Conversely, negative correlations were observed with follicular helper T cells, Tregs, CD8+ T cells, activated NK cells, memory B cells, and activated CD4+ memory T cells, as depicted in Figure 5C.\u003c/p\u003e\n\u003cp\u003eMoreover, NUAK1 expression correlated positively with the ESTIMATE score, encapsulating the overall TME, and the stromal score, which isolates the stromal aspect of the TME (Figure 5D). Collectively, these correlations imply that elevated NUAK1 expression is linked to a TME that may foster a more aggressive tumor phenotype, potentially through its influence on the recruitment and activation of specific immune cells.\u003c/p\u003e\n\u003cp\u003eFigure 5 Immune infiltration analysis. (A) Immune cell content stacking rlot between high NUAK1 expression and low NUAK1 expression. (B) Immune cell content histogram comparing high and low NUAK1 expression groups. (C) Correlation between NUAK1 expression level and the relative abundances of 22 immune cells. (D) Correlation of NUAK1 expression with stromal, ESTIMATE, and immune scores.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunctional Enrichment Analysis of NUAK1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further delineate the biological functions of NUAK1 and its role in tumorigenesis, 50 potential binding proteins of NUAK1 were identified from the STRING database (Figure 6A). These proteins are likely involved in a variety of biological processes that could impact the tumor microenvironment.\u003c/p\u003e\n\u003cp\u003eSubsequently, we conducted GO enrichment analysis (Figure 6B-D) and KEGG pathway enrichment analysis (Figure 6E) on these 50 target proteins. The GO enrichment analysis revealed key biological processes, notably encompassing cell-cell signaling mediated by Wnt, regulation of the Wnt signaling pathway, and the proteasome-mediated ubiquitin-dependent protein catabolic process. These processes include the intrinsic apoptotic signaling pathway, particularly those regulated by P53 class mediators and SCF-dependent proteasomal degradation.\u003c/p\u003e\n\u003cp\u003eThe KEGG pathway enrichment analysis highlighted pathways associated with the Hippo signaling pathway, ubiquitin-mediated proteolysis, Wnt signaling pathway, proteoglycans in cancer, cellular senescence, focal adhesion, Hedgehog signaling pathway, and regulation of the actin cytoskeleton (Figure 6E). To explore the potential diseases associated with these proteins, we searched the DisGeNET database for related disease information on these genes. The results indicated significant associations between these genes and various types of cancer, including breast cancer (Figure 6F).\u003c/p\u003e\n\u003cp\u003eFigure 6 Enrichment analysis of NUAK1. (A) A network of NUAK1 and its 50 potential co-interaction proteins. (B-D) GO enrichment analysis of the NUAK1 co-interaction proteins reveals enriched biological functions (BP), cellular components (CC), and molecular functions (MF). (E) KEGG pathway enrichment analysis. (F) Enrichment analysis in DisGeNET.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSingle-cell analysis of NUAK1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the TISCH2\u003csup\u003e17, 18\u003c/sup\u003e, we conducted an investigation into the correlation between NUAK1 and TME across 10 different single-cell RNA datasets, as shown in Figure 7A. It is noteworthy that in five of the breast cancer single-cell datasets, NUAK1 demonstrated high expression levels primarily in endothelial cells. In order to further explore this relationship, we selected the GSE114727 dataset for a more detailed analysis, employing the umap visualization technique to depict various cell types including immune and stromal cells, as well as specific cell types such as CD8 T cells, dendritic cells (DCs), endothelial cells, fibroblasts, mast cells, and NK cells, as depicted in Figures 7B and 7C. In the GSE114727 cohort, NUAK1 was predominantly expressed in endothelial cells, with expression also observed in fibroblasts and myofibroblasts, as shown in Figures 7D and 7E. We then proceeded to analyze the expression of NUAK1 in relation to the TNM stage, with the results presented in Figure 7F-H. NUAK1 exhibited differential expression across various stages of stromal cells, demonstrating higher expression in stages I and III, and comparatively lower levels in stage II. This pattern of expression was consistent across endothelial cells, fibroblasts, monocytes/macrophages, and myofibroblasts, indicating a potential correlation between NUAK1 expression and the progression of the TNM stage. Subsequent Gene Set Enrichment Analysis (GSEA) identified NUAK1 as being highly expressed in regions associated with the TGF-BETA signaling, apoptosis, and P53 pathway models, as seen in Figures 7I, 7J, and 7M. Additionally, there was a general increase in the epithelial-mesenchymal transition and WNT/\u0026beta;-catenin signaling pathways, as demonstrated in Figures 7K and 7L, respectively.\u003c/p\u003e\n\u003cp\u003eFigure 7 Single-cell analysis of NUAK1 in TME. (A) Heatmap displaying relative expression of NUAK1 in endothelial cells across 10 single-cell BRCA datasets. (B) The scRNA-seq data from the GSE114727 dataset. (C) The immune cells and stromal cells within the BRCA dataset (GSE114727) were subjected to single-cell analysis using TISCH2. Umap (D) and violin plot (E) to explore the expression profiles of NUAK1 in GSE114727. (F) Violin plot of NUAK1 expression in immune and stromal cells across TNM stages in the GSE114727 BRCA single-cell Dataset. (G) UMAP plot illustrating cellular distribution across TNM stages in the GSE114727 BRCA single-cell Dataset. (H) Violin plot of NUAK1 expression across TNM stages in 11 cell types from BRCA GSE114727 dataset. (I-M) Gene Set Enrichment Analysis (GSEA) of TGF-BETA signaling, apoptosis, epithelial-mesenchymal transition, WNT beta catenin signaling, and the P53 pathway in BRCA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatial Transcriptomic Analysis of NUAK1 Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmploying the CROST dataset\u003csup\u003e19, 20\u003c/sup\u003e for our spatial transcriptomic analysis, we commenced with an examination that leveraged the dataset\u0026apos;s integrated quality control measures, which included assessments of count, gene, mitochondrial, and ribosomal statistics (Figure 8 A-D). This preliminary filtration of data led to the spatial clustering analysis (Figure 8E and F), distinguishing twelve unique cellular clusters within the breast cancer tissue microenvironment. The cell type deconvolution (Figure 8G and H) within this context exposed B-Nai cells as a predominant cell type, alluding to their substantial influence. A striking observation was the heightened expression levels of the NUAK1 gene within B-Nai cells, with expression also noted in mast cells, CD8+ T-Nai, and macrophages (Figure 8I), which may be pivotal for breast cancer development.\u003c/p\u003e\n\u003cp\u003eSpatial correlation analysis (Figure 8J) illustrated B-Nai cells in negative correlation with several cell types, suggesting intricate intercellular interactions. The spatial colocalization analysis (Figure 8M) further delineated a network of communications, with B-Nai cells being central to this dialogue, particularly with T-Nai, T-FH, NK, and pDC cells.\u003c/p\u003e\n\u003cp\u003eCluster-cluster communication analysis (Figure 8K, L) and cell-cell communication analysis (Figure 8N, O) provided insights into the interactions among different cellular populations, with B-Nai cells and mast cells, macrophages, and B-Lym cells identified as key communicators within the tumor microenvironment.\u003c/p\u003e\n\u003cp\u003eAnalysis of the TGF\u0026beta; signaling pathway through a heatmap (Figure 8P) and spatial images (Figure 8Q) showed that CD8+ T-Nai cells were profoundly involved in all signaling roles. B-Nai cells were highlighted as significant receivers and influencers, while mast cells were identified as key influencers with a critical regulatory function within the TGF\u0026beta; signaling network. Macrophages were distinguished by their active participation as receivers, potentially acting as nexus points for immune cell communication. Heatmap analysis (Figure 8R) and spatial images (Figure 8S) of the WNT signaling pathway revealed distinct patterns of involvement among immune cell types. B-Nai cells were particularly highlighted in the roles of receivers, mediators, and influencers, suggesting a significant role in the reception, mediation, and modulation of WNT signaling, potentially impacting adaptive immune responses. In contrast, mast cells exhibited a profound level of participation across all assessed signaling roles, implying a multifaceted role in immune cell communication and the initiation, transmission, and regulation of WNT signaling events.\u003c/p\u003e\n\u003cp\u003eFigure 8 NUAK1 expression of spatially transcriptomic-defined clusters in BRCA. (A) Count statistics. (B) Gene statistics. (C) Mitochondrion statistics. (D) Ribosome statistics. (E, F) Spatial clustering analysis. (G, H) Cell type deconvolution. (I) The spatial location and gene changes of NUAK1. (J) Spatial correlation analysis. (K) Cluster-cluster communication analysis in number of interactions (K) and strength of interactions (L). (M) Spatial colocalization analysis. Cell\u0026ndash;cell communication analysis in number of interactions (N) and strength of interactions (O). (P) Heatmap of signaling roles (senders, receivers, mediators and influencers) for TGFb signaling pathway. (Q) Spatial images of intercellular communication networks for TGFb signaling pathway. (R) Heatmap of signaling roles (senders, receivers, mediators and influencers) for WNT signaling pathway. (S) Spatial images of intercellular communication networks for WNT signaling pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccording to the data in 2020, the number and mortality of breast carcinoma among women in the world ranked first\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is the most commonly diagnosed malignant tumor with more than 2,100,000 new cases worldwide and more than 626,000 deaths\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and also the principal cause of cancer-related death among women over the world \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Looking back at the history of breast cancer treatment, it is easy to see that the enlargement of surgical scope cannot improve the survival rate. In recent years, the improvement of survival rate of breast cancer is mainly due to early detection, early diagnosis and continuous improvement of postoperative comprehensive adjuvant therapy. Nevertheless, there are still some patients who cannot avoid tumor recurrence and metastasis. Basically, breast cancer is a heterogeneous malignancy with different histological types, diverse molecular profiles and varied clinical responses to therapy. It is divided into 5 molecular subtypes based on the genome and transcriptome sequences, including Luminal A phenotype, Luminal B phenotype, HER-2 enriched phenotype, Basal-like phenotype and Claudin-low phenotype \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. It is difficult to accurately predict prognosis in breast cancer due to its phenotypic and molecular diversity. Therefore, it is imperative to explore new risk genes and therapeutic targets to obtain new drugs and treatment strategies.\u003c/p\u003e \u003cp\u003eIn 2003, Suzuki et al. discovered NUAK1 as a member of AMPK (adenosine activated protein kinase / Amp activated protein kinase) family\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The protein has a molecular weight of 74 KD and contains a highly conserved active T ring\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. It is known that activation of NUAK1 kinase activity requires threonine 211 phosphorylation by LKB1 or serine 600 phosphorylation by Akt kinase. Like most AMPK regulated kinases, the activity of NUAK1 is increased by 10 to 20 times by LKB1 phosphorylation through threonine on its T ring\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The NUAK1 signaling pathway has been successively proved to play an important physiological and pathological role in vivo, such as promoting cell growth, inhibiting apoptosis, regulating angiogenesis, promoting cell proliferation and participating in glucose metabolism, and is related to the initiation of malignant tumors\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Suzuki confirmed that NUAK1 is the main factor affecting the survival and migration of Akt dependent tumor cells through in vitro research\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It was found that NUAK1 could induce the survival of tumor cells through Akt protein dependent nutritional starvation, and enhance the invasion ability of tumor cells through the Akt signaling pathway mediated by IGF-1 (insulin-like growth factor-1) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. NUAK1 acts on the basement membrane type 1-matrix metalloproteinases (MT1 MMPs) to inhibit apoptosis and stimulate the occurrence of invasive behavior\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Studies have found that NUAK1 promotes the invasion and metastasis of pancreatic carcinoma and colon carcinoma by regulating the secretion of MMP-2 and MMP-9\u003csup\u003e28\u003c/sup\u003e.Roh SA also reported that NUAK1 is strongly correlated with MMP-2, MMP-9 and S100A4\u003csup\u003e29\u003c/sup\u003e. Therefore, Akt / NUAK1 signaling pathway may be a new pathway closely related to tumor formation to induce cell survival.\u003c/p\u003e \u003cp\u003eSome studies reported that up-regulation of NUAK1 was closely related to advanced clinical stage, enhanced distant metastasis and short survival period in colorectal cancer, nasopharyngeal carcinoma\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, gastric cancer and ovarian cancer\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In current study, we assessed the expression of NUAK1 in breast carcinoma and its prognostic significance. Firstly, we compare the expression level of NUAK1 in breast carcinoma cell lines and tissue samples compared with normal human breast epithelial cells, and adjacent noncancerous tissues, respectively. The results revealed that NUAK1 expression was obviously up-regulated in tumor samples compared with the adjacent non-cancerous tissue samples, suggesting that NUAK1 is an important biomarker for the oncogenesis of breast carcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The endogenic NUAK1 expression in breast cancer cells were also higher compared with human breast epithelia cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFurthermore, we analyzed the relationship of NUAK1 expression with clinicopathologic features in 160 patients with breast carcinoma. As showed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, overexpression of NUAK1 had obvious correlation with advanced TNM staging and lymph node metastasis. Simultaneously, no significant correlation was found between NUAK1 and age, T classification, histological grading, ER expression, PR expression or HER2 expression. Correspondently, NUAK1 was proved to be closely correlated with the tumor invasion and lymph node metastasis in head and neck cancer(29)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Therefore, we reasonably speculate that NUAK1 works as a critical factor promoting tumor invasion and distant metastasis in breast carcinoma, and also may serve as therapeutic target. However, further investigation is needed to verify our speculation.\u003c/p\u003e \u003cp\u003eAdditionally, it has been shown by Cox-regression analyses those patients with excessive NUAK1 expression had a significant worse overall survival rate, and that the status of NUAK1 was independent prognostic index influencing overall survival (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Subgroup study stratified by T classification, N classification Clinical stage and Grade suggested that the up-regulation of NUAK1 was closely associated with OS in patient with invasive clinicopathological characteristics, including lymph node metastasis and poor differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, I). These findings suggested the possibility of up-regulation of NUAK1 to become a new biomarker for unfavorable prognosis in breast cancer patients.\u003c/p\u003e \u003cp\u003eThrough CIBERSORT immune infiltration analysis, we observed a negative correlation between high expression of NUAK1 and cells with anti-tumor activity, such as CD8\u0026thinsp;+\u0026thinsp;T cells and activated NK cells\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Conversely, there was a positive correlation with cells that promote tumor progression, including Tregs and M2 macrophages\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Notably, the latter are known to sustain a Th2 response, which has been shown to be conducive to cancer growth\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings suggest that NUAK1 may play a key role in the regulation of the breast cancer microenvironment, facilitating tumor invasion and metastasis. At the single-cell level, our analysis revealed that NUAK1 is predominantly expressed in endothelial cells, with lower levels of expression also observed in fibroblasts and myofibroblasts. Furthermore, GSEA indicated higher expression of NUAK1 in regions associated with TGFβ and WNT signaling pathways, which are known to play crucial roles in EMT and tumor progression\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Spatial transcriptomics analysis provided additional insights into the role of NUAK1 within the TME. Our findings confirmed the elevated expression of NUAK1 specifically within the B cell naive subgroup. This observation is in concordance with the CIBERSORT immune infiltration analysis from our \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section, which indicated a higher proportion of naive B cells in samples with high NUAK1 expression. Moreover, the spatial analysis unveiled intricate intercellular communication networks, highlighting interactions between B cell naive and cells involved in TGFβ and WNT signaling pathways. The WNT and TGFβ signaling pathways are key regulators in the EMT process, promoting tumor invasion and metastasis by affecting intercellular junctions and altering cell phenotype\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The communication between B cell naive and these signaling pathways implies that NUAK1 may exert its signaling functions not in isolation but through a complex interplay with other cellular components of the TME. Therefore, we propose that NUAK1 may serve as a critical regulatory factor that promotes the progression of breast cancer by influencing the behavior of naive B cells and other immune cells. Specifically, the high expression of NUAK1 in naive B cells may impact the EMT process by secreting specific cytokines or through direct cell-to-cell contact, potentially providing a favorable microenvironment for the invasion and metastasis of breast cancer cells.\u003c/p\u003e \u003cp\u003eEMT paticipates in various biological and pathological processes, it has closely relationship with anti-apoptosis and chemotherapeutic resistance\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. It is the process by which epithelial cells with compact cell-cell adhesion convert to mesenchymal cells (13)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this process, the gene expression undergoes multiple changes: Epithelial markers (tight junction protein 1, E-cadherin and occludin) were down-regulated and mesenchymal markers (fibronectin, N-cadherin and vimentin ) were up-regulated\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Research on ovarian cancer reported that NUAK1 was up-regulated in cancer cells and was verified to be closely related with EMT\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Moreover, NUAK1 was revealed to regulate EMT in multiple tumors\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In the NUAK1 knockdowngastric cells, the expression of E-cadherin was increased, together with down-regulation of Vimentin. Furthermore, NUAK1 was positively correlated with Vimentin in gastric cancer, but negatively associated with E-cadherin\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In our study, upregulation of NUAK1 expression increase cell invasion ability. At the same time, EMT related β-catenin and E-cadherin expression was decreased, and the expression of mesenchymal marker vimentin was increased. From the above results, it makes sense that the functional role of NUAK1 on breast cancer invasion and distant metastasis may be interpteted to its induction in EMT. This study revealed that the expression of NUAK1 is elevated in breast carcinoma tissues, it is also a predictor of prognosis. Moreover, we found NUAK1 promoted breast cancer cell invasion, which is related to its function of induction to EMT in breast cancer cells, thus indicating that NUAK1 may have potential as a valuable prognostic index and novel therapeutic target for patients with breast carcinoma.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our research uncovers the multifaceted roles of NUAK1 in breast cancer, including the promotion of cellular invasion, regulation of EMT, and potential effects within the tumor microenvironment. High expression of NUAK1 correlates with poor prognosis and may serve as a valuable prognostic indicator and therapeutic target. Future studies are needed to further explore the molecular mechanisms of NUAK1 in the development of breast cancer, as well as its precise role in the WNT, TGFβ, and EMT signaling pathways, to provide new strategies for the treatment of breast cancer.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCONFLICT OF INTEREST STATEMENT\u003c/h2\u003e \u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e \u003ch2\u003ePatient consent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from the Natural Science Foundation of Guangdong Province, China (2021A1515012493). Guangzhou Science and Technology Plan Project: 2023A03J0195.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWe declare that all the listed authors have participated actively in the study and all meet the requirements of the authorship. Drs. Peng Zhang and Baoyu Zhang designed the study and wrote the protocol, Drs. Jiani Wang, Jiumei Yang and Cuicui Li acquired the manuscript, analyzed the data, and wrote the first draft of the manuscript and mainly revised the manuscript. Dongbo Qiu analyzed the data. All authors approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNone declared.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY STATEMENT\u003c/h2\u003e \u003cp\u003eThe data generated in the present study may be requested from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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EMBO Mol Med. 2019;11(11):e10515. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.15252/emmm.201910515\u003c/span\u003e\u003cspan address=\"10.15252/emmm.201910515\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Lai Q, He C, Fang Y, Yan Q, Zhang Y, Wang X, Gu C, Wang Y, Ye L, et al. RUNX1 promotes tumour metastasis by activating the Wnt/β-catenin signalling pathway and EMT in colorectal cancer. J Exp Clin Cancer Res. 2019;38(1):334. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13046-019-1330-9\u003c/span\u003e\u003cspan address=\"10.1186/s13046-019-1330-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Y, Geng J, Wang X, Meng Q, Xu S, Lang Y, Zhou Y, Qi L, Wang Z, Wei Z, et al. 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Oncol Lett. 2015;9(6):2675\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NUAK1, breast cancer, prognosis, epithelial-mesenchymal transition, invasion","lastPublishedDoi":"10.21203/rs.3.rs-5300363/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5300363/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eBreast carcinoma is a leading malignancy in women, and the role of Novel (nua) kinase family 1 (NUAK1) in its progression is not well-defined. This study aimed to investigate the biological significance of NUAK1 in breast cancer and its potential as a prognostic marker.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe assessed the expression levels of NUAK1 in breast cancer tissues and cell lines using RT-qPCR and cultured cell assays. Statistical analysis was conducted to correlate NUAK1 expression levels with clinicopathological features. Survival analysis was performed to determine the prognostic value of NUAK1 in breast cancer. Additionally, Transwell invasion assays and the evaluation of EMT (epithelial-mesenchymal transition)-related proteins were conducted to ascertain the impact of NUAK1 on cellular invasion and EMT. Furthermore, spatial transcriptomic analysis utilizing the CROST dataset and single-cell RNA sequencing data were employed to dissect the expression patterns of NUAK1 and its association with the TME\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNUAK1 was found to be upregulated in breast cancer tissues and cell lines compared to non-cancerous controls. High expression of NUAK1 was significantly associated with poorer patient survival and was an independent prognostic factor. Transwell assays demonstrated that NUAK1 overexpression significantly enhanced cellular invasion. Overexpression of NUAK1 also induced EMT, as evidenced by decreased expression of epithelial markers and increased expression of the mesenchymal marker Vimentin. Single-cell analysis across various datasets highlighted NUAK1's expression in endothelial cells and its correlation with the TNM stage. Spatial transcriptomic analysis revealed that NUAK1 expression, particularly in B-Nai cells, was associated with a distinct immune cell landscape and communication patterns within the TME, influencing TGFβ and WNT signaling pathways.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings indicate that NUAK1 is upregulated in breast cancer and serves as an independent prognostic marker. NUAK1 promotes breast cancer cell invasion through the induction of EMT and is implicated in the modulation of the TME. The single-cell analysis and spatial transcriptomic data provide novel insights into the cellular and molecular mechanisms underlying NUAK1's role in breast carcinogenesis, suggesting its potential as a therapeutic target.\u003c/p\u003e","manuscriptTitle":"Spatial and Single-Cell Analyses Reveal the Pro-Invasiveness Role of NUAK1 in Breast Cancer through EMT Regulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 12:17:57","doi":"10.21203/rs.3.rs-5300363/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T01:35:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T21:40:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194228858267098232821266408186820971038","date":"2025-09-02T17:51:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267814425272372837412060725635526420505","date":"2025-03-08T13:53:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-22T08:08:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-21T14:13:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-26T13:36:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2024-10-21T01:34:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14b46969-cb42-4b13-9107-f0b8e97bc9f0","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T01:40:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-15 12:17:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5300363","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5300363","identity":"rs-5300363","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}