
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

69721
10.1038/s41598-024-69721-9
Article
On discovery of novel hub genes for ER+ and TN breast cancer types through RNA seq data analyses and classification models
Saddiqa Alishbah 1
Zakir Mahrukh 1
Sheikh Mawara 2
Muneer Zahid 1
Hassan Arsalan 1
Ali Iqra 1
Haq Ihtisham Ul 3
Khan Azmat Ali 4
Malik Abdul amoinuddin@ksu.edu.sa

4
Siddiqi Abdul Rauf araufsiddiqi@comsats.edu.pk

1
1 https://ror.org/00nqqvk19 grid.418920.6 0000 0004 0607 0704 Computational Biology and Bioinformatics Group (CBBG), Department of Biosciences, COMSATS University Islamabad, Park Road Islamabad, Islamabad, Pakistan
2 https://ror.org/04eq9g543 grid.419165.e 0000 0001 0775 7565 Pakistan Agriculture Research Council, Islamabad, Pakistan
3 https://ror.org/02dyjk442 grid.6979.1 0000 0001 2335 3149 Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, M. Strzody 9, 44-100 Gliwice, Poland
4 https://ror.org/02f81g417 grid.56302.32 0000 0004 1773 5396 Department of Pharmaceutics, College of Pharmacy, King Saud University, 1145111451 Riyadh, Saudi Arabia
6 9 2024
6 9 2024
2024
14 208406 3 2024
7 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Breast cancer (BC) is a malignant neoplasm which is classified into various types defined by underlying molecular factors such as estrogen receptor positive (ER+), progesterone receptor positive (PR+), human epidermal growth factor positive (HER2+) and triple negative (TNBC). Early detection of ER+ and TNBC is crucial in the choice of diagnosis and appropriate treatment strategy. Here we report the key genes associated to ER+ and TNBC using RNA-Seq analysis and machine learning models. Three ER+ and TNBC RNA seq datasets comprising 164 patients in-toto were selected for standard NGS hierarchical data processing and data analyses protocols. Enrichment pathway analysis and network analysis was done and finally top hub genes were identified. To come with a reliable classifier which could distinguish the distinct transcriptome patterns associated to ER+ and TNBC, ML models were built employing Naïve Bayes, SVM and kNN. 1730 common DEG’s exhibiting significant logFC values with 0.05 p-value threshold were identified. A list of top ten hub genes were screened on the basis of maximal clique centrality (MCC) which included CDC20, CDK1, BUB1, AURKA, CDCA8, RRM2, TTK, CENPF, CEP55 and NDC80.These genes were found to be involved in crucial cell cycle pathways. k-Nearest Neighbor (kNN) model was observed to be best classifier with accuracy 84%, specificity 66% and sensitivity 95% to differentiate between ER+ and TNBC RNA-Seq transcriptomes. Our screened list of 10 hub genes can thus help unearth novel molecular signatures implicated in ER+ and TNBC onset, prognosis and design of novel protocols for breast cancer diagnostics and therapeutics.

Keywords

Breast cancer
TN
ER+ 
Machine learning
Differentially expressed genes
Hub genes
Subject terms

Cancer
Computational biology and bioinformatics
Oncology
issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Breast cancer (BC) is a multifactorial heterogenous disease which is characterized by uncontrolled cell proliferation1,2. BC is the most prevalent cancer type which primarily affects women contributing a huge health burden on public and individual spendings. Breast cancer accounts for nearly 38.9% of all human cancer types. A GLOBOCAN survey for 2022 found that 11.6% of new cases of BC occurred in females, and the death rate was almost 6.9%3. The prevalence of breast cancer in Asia is about 40%4, and Pakistan reported about 1.38 million cases of breast cancer in 20155. Multiple genetical, hormonal and environmental factors are involved in causing breast cancer. Breast cancer affects mostly the females, originating from germ line mutations. Key genes found to be involved in breast cancer include BRCA1/BRCA26, TP537, PTEN8, STK119, CDH110,11.

Complex and diverse BC subtypes make it difficult to study the underlying pathways and risk factors responsible for the onset of the disease. This necessitates a comprehensive understanding of the various pathways responsible for onset and proliferation of the disease; it also implicates that the genes involved in these pathways could be used for prevention, early detection, and personalized treatment approaches.

Aberrations in the expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human epidermal growth factor receptor 2 (HER2) have been often associated to three distinct subtypes of breast cancer which are observed both clinically and in molecular expression of hormonal imbalance. ER+ breast cancer is of considerable significance for several impacting factors associated to its diagnosis, prognosis, and treatment. ER+ breast cancer has been found to typically respond well to endocrine therapy in about 70% of cases12.

On the other hand triple negative breast cancer (TNBC) is another type of BC whose molecular characteristics vary from the aforementioned BC types, exhibiting no significant variation in behavior of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER-2)13,14. Approximately, 15–20% of all BCs diagnosed lies in the category of TNBC tumors15. Compared with other types of tumors, TNBC tumors have an aggressive appearance, a poor prognosis, and high recurrence rates16–23. Therefore, accurate identification of differentially expressed genes (DEG) networks is needed for comprehensive understanding and distinct characterization of various breast cancer types.

This study is aimed at identifying the potential hub genes that contribute to both ER+ and TNBC development and progression. In order to determine the molecular basis of biological differences, integrated bioinformatics analyses were performed including classification of the BC types based on machine learning models. DEG analyses were performed to delineate the transcriptomic profiles associated uniquely to ER+ and TNBC types on the basis of LogFC and P values; in the end hub gene were identified for both the types of BC which might serve as biomarkers for the disease. Our findings will contribute to better understanding of distinct phenotypes associated to ER+ and TNBC oncogenesis, and the development of novel diagnostic and therapeutic alternatives against the disease.

Materials and methods

RNA Seq datasets of ER+ and TNBC patients were retrieved from ArrayExpress. The datasets were quality checked, aligned; the duplicate reads were removed, and differentially expressed genes were identified on GALAXY suit24. DAVID and Cytoscape were employed to analyze pathways and networks associated with the disease and to determine which genes are involved in the pathogenesis of breast cancer25,26. Machine learning classifiers including Support Vector Machine, Naïve Bayes and k-Nearest Neighbor were employed for generating a classification model to distinguish both the BC subtypes.

Dataset description

ER+ and TNBC RNA Seq datasets were obtained from ArrayExpress27 repository which is a curated database for high-throughput sequenced data. The datasets used in the study are E-GEOD-58135, E-MTAB-4993 and E-GEOD-45419 and the description of dataset is provided in (Table 1). ArrayExpress is linked to European Nucleotide Archive (ENA), a nucleotide database that provides nucleotide sequencing data, sequence assembly information, and functional annotations. The datasets were uploaded on Galaxy server (https://usegalaxy.eu/) for processing via ENA28. An overview of the layout of various processes employed in the study is presented in (Fig. 1).Table 1 Datasets description.

No	Datasets	ArrayExpress accession no	ENA accession no	ER+ samples	TN samples	
1	Breast cancer RNA seq	E-GEOD-58135	ENA-SRP042620	45	42	
2	Whole transcriptome profiling of 63 breast cancer tumors	E-MTAB-4993	ENA-ERP016798	51	12	
3	An integrated model of the transcriptome landscape of HER-2 positive breast cancer	E-GEOD-45419	ENA-SRP019936	8	8	

Figure 1 Data processing and analysis workflow.

Data pre-processing

Data preprocessing was done by using FASTQC and FASTQ Groomer on the samples. HISAT2 was used for dataset alignment because of its high efficiency. “MarkDuplicates” was used to compare sequences and reads in the SAM file by measuring 5` positions of the reads or sequences or paired reads. Afterwards, “RmDup” was used to remove the duplicate reads. An RNA-expression analysis of datasets was conducted by “featureCounts”, which counts both DNA and RNA expression.

Identification of differential expressed genes

A quality-controlled normalized data set was used for supervised analysis comparing gene expression levels between ER+ and TNBC samples using DESeq2. Genes with p value < 0.05 and fold change < −1 and > 1 were statistically considered significant.

Machine learning

Expression file data was used to build mathematical models employing supervised machine learning classifiers. Three different supervised ML classifiers including SVM29, Naïve bayes30 and kNN30 were used to predict the accuracy, sensitivity and specificity of the model.

Functional enrichment analysis

Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) was used for the functional annotation of GO terms and the analysis of KEGG pathway enrichment. DAVID is a widely used resource for evaluating the functional significance of quantitative gene expression profiles25. The analysis of molecular or biological function GO terms and enrichment of pathways analysis was performed for candidate DEGs with a p-value cutoff of < 0.05 were considered significant. An online tool called REVIGO (Available online: http://revigo.irb.hr/) was utilized to summarize and visualize long lists of GO terms31. The GO terms were clustered and represented in a scatter plot using a semantic similarity measure.

Network analysis

To evaluate the interactive relationships among DEGs, STRING (Available online: https://string-db.org/) was utilized to construct a network of PPI (protein-protein interactions)32,33. The cutoff standard was set to a confident interaction score of > 0.4 to eliminate PPI interactions that are inconsistent. Thus, a PPI network with a strong degree of confidence was obtained. The STRING tool results were then combined with Cytoscape software34 to visualize PPI interactions of statistically significant DEGs35. Cytohubba was used to constructs a sub-network of hub genes based on maximal clique centrality (MCC) algorithm in such a way that molecular species are represented as nodes and their intermolecular interactions are known as links or edges between those nodes36. Thickness of lines between nodes and edges represents the affinity of interaction. Thicker the line stronger will be the interaction and vice versa.

Expression of hub genes

Using cancer data analysis portal (UALCAN, (https://ualcan.path.uab.edu/analysis.html) a web-based tool for analyzing hub gene expression, and clinical data from The Cancer Genome Atlas (TCGA), a box and whisker plot was generated showing gene expression levels in different cancers and their subtypes at various levels of sub-stages37. CDK1,CDC20,CDCA8,RRM2,NDC80,CEP55,CENPF,BUB1,TTK and AURKA were significantly overexpressed in breast cancer tissues based on menopause status than in normal tissues38.

Ethics approval and consent to participate

We further confirm that any aspect of the work covered in this manuscript has not involved human patients and thus requires no ethical approval of any relevant body.

Results

Result of differential expression

The raw read data was aligned against Hg38Chr using HISAT2; the duplicates were identified and removed using MarkDuplicates and RmDup, respectively. R package DESeq2 was employed to figure out the differentially expressed genes from feature count files of SAM format. DESeq2 generated histogram, MA and PC plot for each dataset shown in (Figs. 2–4). The common DEG’s among three RNA Seq datasets were obtained by Venny tool (https://bioinfogp.cnb.csic.es/tools/venny/)39, 1730 overlapping genes were identified among three datasets as shown in (Fig. 5).Figure 2 The PC plot (A), Dispersion estimates (B), histogram (C) and MA plot (D) were created by DESeq2 tool of E-GEOD-45419 dataset. (A) PC plot shows two phenotypes: ER+ and TN. They are grouped on the basis of expression. (B) Dispersion estimates quantify the level of variability in gene expression across samples. Blue dots represent low dispersion estimates of genes and it shows the gene expression is relatively stable while the black dots represent high dispersion estimates. The red line shows the mean or median dispersion estimates. The blue dot close to red line indicated stable expression and the black dots close to red line suggests that the expression values are more variable. (C) Histogram shows the DEG’s grouped into bins or the frequency of genes. (D) MA plot the differences between measurements based on ER+ and TN by transforming the data by using log ratio and mean average. The red color shows the dispersion of differentially expressed genes while grey color shows no variation.

Figure 3 The PC plot (A) Dispersion estimates (B) histogram (C) and MA plot (D) were created by DESeq2 tool of E-MTAB-4993 dataset. (A) PC plot shows two phenotypes: ER+ and TN. They are grouped on the basis of expression. (B) Dispersion estimates quantify the level of variability in gene expression across samples. Blue dots represent low dispersion estimates of genes and it shows the gene expression is relatively stable while the black dots represent high dispersion estimates. The red line shows the mean or median dispersion estimates. The blue dot close to red line indicated stable expression and the black dots close to red line suggests that the expression values are more variable.(C) Histogram shows the DEG’s grouped into bins or the frequency of genes. (D)MA plot the differences between measurements based on ER+ and TN by transforming the data by using log ratio and mean average. The red color shows the dispersion of differentially expressed genes while grey color shows no variation.

Figure 4 The PC plot (A) Dispersion estimates (B) histogram (C) and MA plot (D) were created by DESeq2 tool of E-MTAB-58135 dataset. (A) PC plot shows two phenotypes: ER+ and TN. They are grouped on the basis of expression. (B) Dispersion estimates quantify the level of variability in gene expression across samples. Blue dots represent low dispersion estimates of genes and it shows the gene expression is relatively stable while the black dots represent high dispersion estimates. The red line shows the mean or median dispersion estimates. The blue dot close to red line indicated stable expression and the black dots close to red line suggests that the expression values are more variable. (C) Histogram shows the DEG’s grouped into bins or the frequency of genes. (D) MA plot the differences between measurements based on ER+ and TN by transforming the data by using log ratio and mean average. The red color shows the dispersion of differentially expressed genes while grey color shows no variation.

Figure 5 The Venn diagram shows that 1730 common DEG’s were found in the datasets.

Classification outcomes

Classification models were built to differentiate BC samples based on DEG’s identified byDESeq2 tool. The classification algorithms including SVM, Naïve Bayes and kNN were employed on the training dataset of 134 samples and test dataset comprising of 32 samples. The train and test datasets were used as input data for the classifier. The accuracy level rose up to 84% in the validation stage of the kNN algorithm whereas the accuracy achieved in SVM was the lowest as 71% while the accuracy of Naïve Bayes was observed to be 81%. All the samples were successfully classified by the models, the results are shown in (Fig. 6) and (Table 2) respectively.Figure 6 Results of SVM (A), Naïve Bayes (B) and kNN (C) respectively.

Table 2 Classifier’s results.

Classifier	Sensitivity	Specificity	Accuracy	Accuracy Percentage (%)	
SVM	0.666	0.85	0.71875	72	
kNN	0.95	0.6666	0.84375	84	
Naive byes	0.667	0.9	0.8125	81	

Pathway analysis

Gene enrichment analysis and KEGG pathways of 1730 common DEG’s were identified by using DAVID tool. The biological processes (BP), molecular functions (MF) and cell components (CC) were obtained which are shown in (Tables 3–5) respectively. Genes were involved in different biological pathways including mammary gland alveolus development (GO:0060749), response to drug (GO:0042493), natural killer cell mediated cytotoxicity (GO:0042267), regulation of insulin secretion (GO:0050796), peripheral nervous system development (GO:0007422), cAMP-mediated signaling (GO:0019933), and regulation of cell growth (GO:0001558), as detailed in (Table 3). The GO molecular function analysis revealed the involvement of DEGs in phosphatidylinositol phospholipase C activity (GO:0004435), mRNA 5’ UTR binding (GO:0048027), and calcium ion binding (GO:0005509), Table 4. In addition, CC group genes were mainly enriched in the extracellular space (GO:0005615), basolateral plasma membrane (GO:0016323), and extracellular region (GO:0005576), (Table 5). Furthermore, we classified DEGs associated with different biological pathways according to the KEGG reference database using the DAVID method (P < 0.05; FDR < 0.05). The KEGG pathway analysis showed the association of DEG’s in cell cycle, Insulin secretion, pathways in cancer and prostate cancer. The results are exhibited in (Table 6). REVIGO was used to visualize gene ontology in form of scatter plot. The scatter plot depicts semantic similarity between GO terms on x-axis, whereas the y-axis indicates p-value or significance, the plot is shown in (Fig. 7). The x-axis shows that terms that are functionally closely related. A lower p-value indicates a greater significance for terms positioned higher on the y-axis. GO hierarchies can be represented by different colors in the scatter plot.Table 3 Biological processes in which genes are involved.

Category	Term	Description	Count	P-value	
BP	GO:0060749	Mammary gland alveolus development	6	5.90E-04	
BP	GO:0042493	Response to drug	27	7.21E-04	
BP	GO:0042267	Natural killer cell mediated cytotoxicity	6	7.88E-04	
BP	GO:0050796	Regulation of insulin secretion	10	0.002232	
BP	GO:0007422	Peripheral nervous system development	6	0.003154	
BP	GO:0007626	Locomotory behavior	11	0.003233	
BP	GO:0008284	Positive regulation of cell proliferation	34	0.003703	
BP	GO:0019933	cAMP-mediated signaling	7	0.005342	
BP	GO:0001558	Regulation of cell growth	10	0.007388	

Table 4 Molecular Functions in which genes are involved.

Category	Term	Description	Count	P-value	
MF	GO:0004435	Phosphatidylinositol phospholipase C activity	6	0.005084	
MF	GO:0046703	Natural killer cell lectin-like receptor binding	4	0.005309	
MF	GO:0048027	mRNA 5'-UTR binding	4	0.005309	
MF	GO:0001158	Enhancer sequence-specific DNA binding	5	0.009123	
MF	GO:0005509	Calcium ion binding	45	0.009493	
MF	GO:0015254	Glycerol channel activity	4	0.012641	
MF	GO:0043565	Sequence-specific DNA binding	34	0.013999	
MF	GO:0008236	Serine-type peptidase activity	8	0.017159	
MF	GO:0008134	Transcription factor binding	21	0.019617	

Table 5 Cellular Components in which genes are involved.

Category	Term	Description	Count	P-value	
CC	GO:0005615	Extracellular space	96	8.27E-07	
CC	GO:0016323	Basolateral plasma membrane	19	7.34E-04	
CC	GO:0005576	Extracellular region	91	0.005143	
CC	GO:0005581	Collagen trimer	11	0.005922	
CC	GO:0005578	Proteinaceous extracellular matrix	21	0.011515	
CC	GO:0019897	Extrinsic component of plasma membrane	5	0.020575	

Table 6 KEGG Pathways of DEGs.

Category	Term	Description	Count	P-value	
KEGG pathway	hsa04110	Cell cycle	18	8.72E-06	
KEGG pathway	hsa04911	Insulin secretion	13	1.40E-04	
KEGG pathway	hsa05200	Pathways in cancer	32	2.37E-04	
KEGG pathway	hsa04925	Aldosterone synthesis and secretion	12	3.76E-04	
KEGG pathway	hsa04024	cAMP signaling pathway	20	3.81E-04	
KEGG pathway	hsa05214	Glioma	10	0.001105	
KEGG pathway	hsa04114	Oocyte meiosis	13	0.001653	
KEGG pathway	hsa04916	Melanogenesis	12	0.002226	
KEGG pathway	hsa04915	Estrogen signaling pathway	11	0.006429	
KEGG pathway	hsa04923	Regulation of lipolysis in adipocytes	8	0.006937	
KEGG pathway	hsa05215	Prostate cancer	10	0.008809	
KEGG pathway	hsa04020	Calcium signaling pathway	15	0.013324	
KEGG pathway	hsa04115	p53 signaling pathway	8	0.017886	

Figure 7 The Scatterplot represents the cluster representatives (i.e. terms remaining after the redundancy reduction) in a two-dimensional space derived by applying multidimensional scaling to a matrix of the GO term semantic similarities.

Network analysis

In network analysis, the gene interacting network was constructed by STRING and was visualized by Cystoscape. Network was constructed for 1730 differentially expressed genes which consisted of 1505 nodes and 9714 edges, (Fig. 8); the interaction between the two nodes determined the co-relation. In Cytohubba, the MCC algorithm measures the centrality of nodes by analyzing their involvement in large cliques. A network structure and connectivity can be determined by identifying hub genes. The top 10 Hub genes identified were: CDC20, CDK1, BUB1, AURKA, CDCA8, RRM2, TTK, CENPF, CEP55 and NDC80, the network involving the aforementioned genes is shown in(Fig. 9).Figure 8 Network of differentially expressed genes by String. The thick lines indicate significant association, functional similarity or co-regulation between the genes while thin lines represent low level interactions. Genes associated with thin lines still exhibit level of association but the significance is relatively low.

Figure 9 Top 10 hub genes are identified based on MCC algorithm.

Hub genes expression analysis

Transcriptional and translational expression levels of all hub genes were significantly higher (P = 0.05) in cancerous tissues compared with normal tissues. Furthermore, based on patient menopause status, hub gene expression levels were significantly higher in breast cancer samples than in normal samples in patients at different cancer stages as shown by box and whisker plots at (Fig. 10).Figure 10 Box and whisker plot exhibiting expression profiles of ten hub-genes at various menopausal stages shows statistically significant differences among premenopausal, perimenopausal and postmenopausal patients compared to normal controls based on data from The cancer genome atlas (TCGA) database.

Discussion

In this study three RNA-Seq datasets comprising of ER+ and TNBC samples were studied, having been-processed, aligned, screened and filtered for duplicates, and finally processed for calculation of expression counts; thus 1730 overlapping DEG’s were identified which served as the training and test dataset for classification models to identify transcriptomic patterns which may help differentiate between ER+ and TNBC. The DEG’s of ER+ and TN samples were filtered on the basis of logFC and p-values. Pathway and network analysis of the selected DEG’s was performed at DAVID25 and Cytoscape26. Classification models were built based on three different algorithms to successfully differentiate between ER+ and TNBC types. The accuracy, sensitivity and specificity of the classifiers were estimated. Highest accuracy was exhibited by kNN classifier that is 84% as compared to other two classifiers SVM and Naïve Bayes whose accuracy was 72 and 81% respectively. Thus, kNN was found to be a best classifier between ER+ and TNBC types.

The DEG’s were identified across three RNA-Seq datasets, and three classification models, Support Vector Machine (SVM), Naïve Bayes, and k-Nearest Neighbors (kNN), were built to distinguish between ER+ and TNBC samples which is clinically extremely important for diagnosis and the choice of therapeutic alternatives. Often a miss diagnosis of TNBC i.e. false negative TNBC cases, which are mistakenly diagnosed as ER+ , lead to a lot of clinical complications and vice versa We therefore improvised ml classifiers training upon aforementioned DEG data to come up with a protocol which could help improving the current methodology for BC. To evaluate the effectiveness of each model in correctly discriminating between ER+ and TNBC cases and minimizing false positives, performance metrics such as accuracy, sensitivity and specificity were used. This comprehensive analysis not only elucidates the key molecular signatures which could serve to discriminate ER+ from TNBC but also underscores the utility of machine learning methodologies in enhancing the accuracy of BC diagnosis.

Results of GO analyses including CC, MF, BP showed that these overlapping DEG’s were primarily enriched in extracellular space and are associated with cell cycle, positive regulation of cell proliferation, cAMP-mediated signaling, transcription factor binding, sequence-specific DNA binding, calcium ion binding. In addition, the KEGG pathway enrichment analysis indicated that these overlapping DEGs were significantly enriched in pathways in cancer, cAMP signaling pathway, cell cycle, oocyte meiosis, estrogen signaling pathway, p53 signaling pathway and calcium signaling pathway. These enriched gene function and KEGG pathways provide insights regarding the molecular mechanism of ER+ and TNBC progression. Our analyses led to the inference that CDC20, CDK1, BUB1, AURKA, CDCA8, RRM2, TTK, CENPF, CEP55, and NDC80 serve as hub genes in the progression of ER+ and TN and also a predictor for the worst survival rates of BC patients. As illustrated in TCGA analysis, the breast cancer samples in multiple clinicopathological subgroups, the ten hub genes were consistently overexpressed (p0.05) in patients.

Previous studies have revealed that CDK1, BUB1, AURKA, CDCA8, RRM2, TTK, CENPF, CEP55 and NDC80 are implicated in cell cycle and associated with tumorigenesis. The CDK1, also known as CDC2, is involved in the precise division of cells40. In the TNBC clinical subtype of breast cancer, inhibiting CDK1 expression can suppress tumor cell growth and induce apoptosis41. In addition, BUB1 is one of the key mitotic checkpoint genes whose expression level is closely correlated with the proliferation of carcinoma cells42–44. RRM2, a breast cancer hub gene has been found to be closely associated with tumor growth, invasion, angiogenesis, tumor metastasis, as well as the prognosis of patients with breast cancer45,46. Furthermore, protein kinase TTK is capable of phosphorylating both serine and threonine simultaneously. The TTK plays a crucial role in cell division and is highly expressed in a wide variety of malignant tumors47.

Approximately 73% of patients with breast cancer overexpress Aurora kinase A (AURKA), a kinase essential to cell division and particularly the process of chromosome segregation during mitosis48,49. AURKA plays an important role in spindle assembly, centrosome maturation, and chromosome alignment49. Breast cancer development is negatively affected by the overexpression of AURKA. Similarly, CDCA8, also known as cell division cycle associated 8, is a part of the chromosomal passenger complex. It plays a crucial role in mitosis by regulating chromosome alignment and segregation at the centromeres50. Centromere protein F (CENPF) has previously been reported to be a marker of cell proliferation in several human malignancies, including breast cancer51,52. The centrosome protein 55 (CEP55) is an important microtubule-binding protein that is located in the centrosome of interphase cells and in the midbody of metaphase cells. It has been observed that CEP55 is overexpressed in several cancer types, such as colon, lung, and breast cancer53. It has been shown that NDC80, CDK1, and CCNB1 play key roles in breast cancer pathophysiology, such as regulating the growth and invasion of the cancer54. In accordance with our research, these hub genes might serve as potential biomarkers for the early-stage diagnosis and prognosis of ER+ and TNBC breast cancer. Thus, aberrations in their expression level (logFC) can be associated to the onset of breast cancer. As a consequence of this inference, we also pursued to develop ML models which could successfully distinguish the RNA Seq profile of an ER+ or TNBC affected individual from the normal healthy individuals, as our datasets include the patients both in the early-stage metastasis stage of the BC. Seven of the ten hub genes identified in the study, CDK1, CDC20, CEP55, CENPF, BUB1, TTK and AURKA have been associated with ER+ immune signature in various studies but they have not been research for their association in TNBC as of now. This study comes up with another three hub genes CDCA80, RRM2 and NDC80 which may help to potentially re-refine the unique immune signature for ER+ and TNBC. Putatively the ten hub genes identified here may also help revise the immune signatures for TNBC and also to distinguish it from rest of BC types.

Of course, these genes have been reported earlier on the basis of various gene association studies to be immune signatures of TNBC. But this study is first of its kind which clearly illustrates that association on the basis of experimental evidence as exhibited by transcriptomic datasets. The analysis also hypothesizes that the key features in variation in expression of these hub genes may also be associated to the BC.

RNA seq analyses of three datasets comprising of 134 samples, also illustrates that these genes may serve as biomarkers or immune signatures distinctly for ER+ and TNBC types. Therefore, we not only report the transcriptomic attributes associated to TNBC etiology but also a set of genes which are also associated to the other uncontrolled BC type such as ER+ . Our models along with identified hub genes provide for key features exclusively associated with both of the BC types.

Globally, breast cancer is one of the most prevalent cancers affecting women. In advanced stages of breast cancer, the disease can spread to the entire body through blood vessels and lymphatics, resulting in death directly caused by the disease. In spite of the promising results of advanced therapies for controlling breast cancer prior to metastasis, the treatment of advanced stage breast cancer remains a challenge. The therapies for preventing breast cancer recurrence and metastasis are also scarce. Hence, finding biomarkers which could help improving the diagnosis strategies, monitoring the metastasis of breast cancer, and understanding its peculiar mechanisms is of utmost importance.

Conclusion

The current study, involving three extensive datasets containing 134 ER+ and TNBC transcriptomes, led to the identification of 1730 differentially expressed genes uniquely associated to ER+ and TNBC individuals. The hub genes can serve as biomarkers for the diagnosis and/or prognosis of ER+ and TNBC patients. Pathway enrichment analysis and network analysis revealed the key signaling pathways implicated by these genes. Classification models based on SVM, Naïve Bayes and kNN were built on datasets. These models were ranked on the basis of accuracy, specificity and sensitivity. kNN was ranked as best classifier with sensitivity of 95%, accuracy of 84%, and specificity of 66%,. We successfully demonstrated that transcriptome analysis integrated with ML classifiers can be used to improve diagnosis of ER+ and TNBC patients.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Alishbah Saddiqa and Mahrukh Zakir. The formal analysis was done by Mawara Sheikh and Arsalan Hassan. Iqra Ali, Zahid Muneer and Ihtisham Ul Haq assisted in writeup manuscript submission and formal analysis. Azmat Ali Khan and Abdul Malik assisted in writing—review & editing whereas Abdul Rauf Siddiqi has done conceptualization and supervision of the study. All authors read and approved the final manuscript. We, the undersigned authors of the manuscript titled “On Discovery of Novel Hub Genes for ER+ and TN Breast Cancer Types through RNA Seq Data Analyses and Classification Models”, hereby confirm that we have all read and approved the final version of the manuscript for publication in Cancer Causes and Control Journal.

Funding

This work was funded by the Researchers Supporting Project Number (RSP2024R376) at King Saud University, Riyadh 11451, Saudia Arabia. We would like to appreciate King Saud University, Saudi Arabia, for funding this work through the research.

Data availability

Datasets are available on ArrayExpress database with following Accession numbers. Accession No: E-GEOD-58135, E-MTAB-4993 and E-GEOD-45419.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Downie FP Mar Fan HG Houédé-Tchen N Yi Q Tannock IF Cognitive function, fatigue, and menopausal symptoms in breast cancer patients receiving adjuvant chemotherapy: Evaluation with patient interview after formal assessment Psycho Oncol. J. Psychol. Soc. Behav. Dimens. Cancer 2006 15 921 930
Downie, F. P., Mar Fan, H. G., Houédé-Tchen, N., Yi, Q. & Tannock, I. F. Cognitive function, fatigue, and menopausal symptoms in breast cancer patients receiving adjuvant chemotherapy: Evaluation with patient interview after formal assessment. Psycho Oncol. J. Psychol. Soc. Behav. Dimens. Cancer 15, 921–930 (2006).
2. Fan HGM Houédé-Tchen N Yi Q-L Chemerynsky I Downie FP Sabate K Tannock IF Fatigue, menopausal symptoms, and cognitive function in women after adjuvant chemotherapy for breast cancer: 1-and 2-year follow-up of a prospective controlled study J. Clin. Oncol. 2005 23 31 8025 8032 10.1200/JCO.2005.01.6550 16258100
Fan, H. G. M. et al. Fatigue, menopausal symptoms, and cognitive function in women after adjuvant chemotherapy for breast cancer: 1-and 2-year follow-up of a prospective controlled study. J. Clin. Oncol. 23(31), 8025–8032 (2005).16258100 10.1200/JCO.2005.01.6550
3. Bray F Laversanne M Sung H Ferlay J Siegel RL Soerjomataram I Jemal A Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J. Clin. 2024 74 3 229 263 10.3322/caac.21834 38572751
Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 74(3), 229–263 (2024).38572751 10.3322/caac.21834
4. Youn HJ Han W A review of the epidemiology of breast cancer in Asia: Focus on risk factors Asian Pac. J. Cancer Prev. APJCP 2020 21 4 867 10.31557/APJCP.2020.21.4.867 32334446
Youn, H. J. & Han, W. A review of the epidemiology of breast cancer in Asia: Focus on risk factors. Asian Pac. J. Cancer Prev. APJCP 21(4), 867 (2020).32334446 10.31557/APJCP.2020.21.4.867
5. Menhas R Shumaila U Breast cancer among Pakistani women Iran. J. Pub. Health 2015 44 4 586 26056679
Menhas, R. & Shumaila, U. Breast cancer among Pakistani women. Iran. J. Pub. Health 44(4), 586 (2015).26056679
6. Rosen EM Fan S Pestell RG Goldberg ID BRCA1 gene in breast cancer J. Cell. Physiol. 2003 196 1 19 41 10.1002/jcp.10257 12767038
Rosen, E. M., Fan, S., Pestell, R. G. & Goldberg, I. D. BRCA1 gene in breast cancer. J. Cell. Physiol. 196(1), 19–41 (2003).12767038 10.1002/jcp.10257
7. Olivier M Hollstein M Hainaut P TP53 mutations in human cancers: Origins, consequences, and clinical use Cold Spring Harb. Perspect. Biol. 2010 2 1 a001008 10.1101/cshperspect.a001008 20182602
Olivier, M., Hollstein, M. & Hainaut, P. TP53 mutations in human cancers: Origins, consequences, and clinical use. Cold Spring Harb. Perspect. Biol. 2(1), a001008 (2010).20182602 10.1101/cshperspect.a001008
8. Maehama T Dixon JE The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3, 4, 5-trisphosphate J. Biol. Chem. 1998 273 22 13375 13378 10.1074/jbc.273.22.13375 9593664
Maehama, T. & Dixon, J. E. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3, 4, 5-trisphosphate. J. Biol. Chem. 273(22), 13375–13378 (1998).9593664 10.1074/jbc.273.22.13375
9. Gill R Yang S Meerzaman D Mechanic L Bowman E Jeon H Roy Chowdhuri S Shakoori A Dracheva T Hong K Frequent homozygous deletion of the LKB1/STK11 gene in non-small cell lung cancer Oncogene 2011 30 35 3784 3791 10.1038/onc.2011.98 21532627
Gill, R. et al. Frequent homozygous deletion of the LKB1/STK11 gene in non-small cell lung cancer. Oncogene 30(35), 3784–3791 (2011).21532627 10.1038/onc.2011.98
10. Campeau PM Foulkes WD Tischkowitz MD Hereditary breast cancer: new genetic developments, new therapeutic avenues Hum. Genet. 2008 124 31 42 10.1007/s00439-008-0529-1 18575892
Campeau, P. M., Foulkes, W. D. & Tischkowitz, M. D. Hereditary breast cancer: new genetic developments, new therapeutic avenues. Hum. Genet. 124, 31–42 (2008).18575892 10.1007/s00439-008-0529-1
11. Pharoah PD, Guilford P, Caldas C, Consortium IGCL Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families Gastroenterology 2001 121 6 1348 1353 10.1053/gast.2001.29611 11729114
Pharoah PD, Guilford P, Caldas C, Consortium IGCL. Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology 121(6), 1348–1353 (2001).11729114 10.1053/gast.2001.29611
12. Kurosky SK Mitra D Zanotti G Kaye JA Treatment patterns and outcomes of patients with metastatic ER+/HER-2− breast cancer: A multicountry retrospective medical record review Clin. Breast Cancer 2018 18 4 e529 e538 10.1016/j.clbc.2017.10.008 29199086
Kurosky, S. K., Mitra, D., Zanotti, G. & Kaye, J. A. Treatment patterns and outcomes of patients with metastatic ER+/HER-2− breast cancer: A multicountry retrospective medical record review. Clin. Breast Cancer 18(4), e529–e538 (2018).29199086 10.1016/j.clbc.2017.10.008
13. Gonçalves H Jr Guerra MR Duarte Cintra JR Fayer VA Brum IV Bustamante Teixeira MT Survival study of triple-negative and non-triple-negative breast cancer in a Brazilian cohort Clin. Med. Insights Oncol. 2018 12 1179554918790563 10.1177/1179554918790563 30083066
Gonçalves, H. Jr. et al. Survival study of triple-negative and non-triple-negative breast cancer in a Brazilian cohort. Clin. Med. Insights Oncol. 12, 1179554918790563 (2018).30083066 10.1177/1179554918790563
14. Wu J Hicks C Breast cancer type classification using machine learning J.Personal. Med. 2021 11 2 61 10.3390/jpm11020061
Wu, J. & Hicks, C. Breast cancer type classification using machine learning. J.Personal. Med. 11(2), 61 (2021).10.3390/jpm11020061
15. Perou C Molecular stratification of triple-negative breast cancers Oncologist 2010 15 39 48 10.1634/theoncologist.2010-S5-39 21138954
Perou, C. Molecular stratification of triple-negative breast cancers. Oncologist 15, 39–48 (2010).21138954 10.1634/theoncologist.2010-S5-39
16. Joyce D Murphy D Lowery A Curran C Barry K Malone C McLaughlin R Kerin M Prospective comparison of outcome after treatment for triple-negative and non-triple-negative breast cancer Surgeon 2017 15 5 272 277 10.1016/j.surge.2016.10.001 28277293
Joyce, D. et al. Prospective comparison of outcome after treatment for triple-negative and non-triple-negative breast cancer. Surgeon 15(5), 272–277 (2017).28277293 10.1016/j.surge.2016.10.001
17. Li X Yang J Peng L Sahin AA Huo L Ward KC O’Regan R Torres MA Meisel JL Triple-negative breast cancer has worse overall survival and cause-specific survival than non-triple-negative breast cancer Breast Cancer Res. Treat. 2017 161 279 287 10.1007/s10549-016-4059-6 27888421
Li, X. et al. Triple-negative breast cancer has worse overall survival and cause-specific survival than non-triple-negative breast cancer. Breast Cancer Res. Treat. 161, 279–287 (2017).27888421 10.1007/s10549-016-4059-6
18. Pan X-B Qu S Jiang Y-M Zhu X-D Triple negative breast cancer versus non-triple negative breast cancer treated with breast conservation surgery followed by radiotherapy: A systematic review and meta-analysis Breast Care 2015 10 6 413 416 10.1159/000441436 26989362
Pan, X.-B., Qu, S., Jiang, Y.-M. & Zhu, X.-D. Triple negative breast cancer versus non-triple negative breast cancer treated with breast conservation surgery followed by radiotherapy: A systematic review and meta-analysis. Breast Care 10(6), 413–416 (2015).26989362 10.1159/000441436
19. Ye J Xia X Dong W Hao H Meng L Yang Y Wang R Lyu Y Liu Y Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel–cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines Int. J. Nanomed. 2016 11 4125 4140 10.2147/IJN.S113638
Ye, J. et al. Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel–cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines. Int. J. Nanomed. 11, 4125–4140 (2016).10.2147/IJN.S113638
20. Qiu J Xue X Hu C Xu H Kou D Li R Li M Comparison of clinicopathological features and prognosis in triple-negative and non-triple negative breast cancer J. Cancer 2016 7 2 167 10.7150/jca.10944 26819640
Qiu, J. et al. Comparison of clinicopathological features and prognosis in triple-negative and non-triple negative breast cancer. J. Cancer 7(2), 167 (2016).26819640 10.7150/jca.10944
21. Podo F Santoro F Di Leo G Manoukian S De Giacomi C Corcione S Cortesi L Carbonaro LA Trimboli RM Cilotti A Triple-negative versus non–triple-negative breast cancers in high-risk women: Phenotype features and survival from the HIBCRIT-1 MRI-including screening study Clin. Cancer Res. 2016 22 4 895 904 10.1158/1078-0432.CCR-15-0459 26503945
Podo, F. et al. Triple-negative versus non–triple-negative breast cancers in high-risk women: Phenotype features and survival from the HIBCRIT-1 MRI-including screening study. Clin. Cancer Res. 22(4), 895–904 (2016).26503945 10.1158/1078-0432.CCR-15-0459
22. Nabi M Ahangar A Wahid M Kuchay S Clinicopathological comparison of triple negative breast cancers with non-triple negative breast cancers in a hospital in North India Niger. J. Clin. Pract. 2015 18 3 381 386 10.4103/1119-3077.153248 25772923
Nabi, M., Ahangar, A., Wahid, M. & Kuchay, S. Clinicopathological comparison of triple negative breast cancers with non-triple negative breast cancers in a hospital in North India. Niger. J. Clin. Pract. 18(3), 381–386 (2015).25772923 10.4103/1119-3077.153248
23. Koshy N Quispe D Shi R Mansour R Burton GV Cisplatin–gemcitabine therapy in metastatic breast cancer: Improved outcome in triple negative breast cancer patients compared to non-triple negative patients Breast 2010 19 3 246 248 10.1016/j.breast.2010.02.003 20227277
Koshy, N., Quispe, D., Shi, R., Mansour, R. & Burton, G. V. Cisplatin–gemcitabine therapy in metastatic breast cancer: Improved outcome in triple negative breast cancer patients compared to non-triple negative patients. Breast 19(3), 246–248 (2010).20227277 10.1016/j.breast.2010.02.003
24. Lei X Lin H Wang J Ou Z Ruan Y Sadagopan A Chen W Xie S Chen B Li Q Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression Nat. Commun. 2022 13 1 3882 10.1038/s41467-022-31417-x 35794100
Lei, X. et al. Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression. Nat. Commun. 13(1), 3882 (2022).35794100 10.1038/s41467-022-31417-x
25. Huang DW Sherman BT Lempicki RA Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat. Protoc. 2009 4 1 44 57 10.1038/nprot.2008.211 19131956
Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4(1), 44–57 (2009).19131956 10.1038/nprot.2008.211
26. Kohl, M., Wiese, S. & Warscheid, B.Cytoscape: software for visualization and analysis of biological networks. Data Min. Proteom. Stand. Appl. 696, 291–303 (2011).
27. Sarkans U Füllgrabe A Ali A Athar A Behrangi E Diaz N Fexova S George N Iqbal H Kurri S From arrayexpress to biostudies Nucleic Acids Res. 2021 49 D1 D1502 D1506 10.1093/nar/gkaa1062 33211879
Sarkans, U. et al. From arrayexpress to biostudies. Nucleic Acids Res. 49(D1), D1502–D1506 (2021).33211879 10.1093/nar/gkaa1062
28. Leinonen R Akhtar R Birney E Bower L Cerdeno-Tárraga A Cheng Y Cleland I Faruque N Goodgame N Gibson R The European nucleotide archive Nucleic Acids Res. 2010 39 D28 D31 10.1093/nar/gkq967 20972220
Leinonen, R. et al. The European nucleotide archive. Nucleic Acids Res. 39, D28–D31 (2010).20972220 10.1093/nar/gkq967
29. Zararsız G Goksuluk D Korkmaz S Eldem V Zararsiz GE Duru IP Ozturk A A comprehensive simulation study on classification of RNA-Seq data PloS ONE 2017 12 8 e0182507 10.1371/journal.pone.0182507 28832679
Zararsız, G. et al. A comprehensive simulation study on classification of RNA-Seq data. PloS ONE 12(8), e0182507 (2017).28832679 10.1371/journal.pone.0182507
30. Jabeen, A., Ahmad, N. & Raza, K. Machine learning-based state-of-the-art methods for the classification of rna-seq data. Class. BioApps Automat. Decis. Mak. 26, 133–172 (2018).
31. Supek F Bošnjak M Škunca N Šmuc T REVIGO summarizes and visualizes long lists of gene ontology terms PloS ONE 2011 6 7 e21800 10.1371/journal.pone.0021800 21789182
Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PloS ONE 6(7), e21800 (2011).21789182 10.1371/journal.pone.0021800
32. Szklarczyk D Morris J Cook H Kuhn M Wyder S Simonovic M Santos A Doncheva NT Roth A Bork P Jensen LJ von Mering C The STRING database in 2017: Quality-365 controlled protein-protein association networks made broadly accessible Nucleic Acids Res. 2017 45 D36 D368 10.1093/nar/gkw937
Szklarczyk, D. et al. The STRING database in 2017: Quality-365 controlled protein-protein association networks made broadly accessible. Nucleic Acids Res. 45, D36–D368 (2017).10.1093/nar/gkw937
33. Udhaya Kumar S Thirumal Kumar D Bithia R Sankar S Magesh R Sidenna M George Priya Doss C Zayed H Analysis of differentially expressed genes and molecular pathways in familial hypercholesterolemia involved in atherosclerosis: A systematic and bioinformatics approach Front. Genet. 2020 11 734 10.3389/fgene.2020.00734 32760426
Udhaya Kumar, S. et al. Analysis of differentially expressed genes and molecular pathways in familial hypercholesterolemia involved in atherosclerosis: A systematic and bioinformatics approach. Front. Genet. 11, 734 (2020).32760426 10.3389/fgene.2020.00734
34. Saito R Smoot ME Ono K Ruscheinski J Wang P-L Lotia S Pico AR Bader GD Ideker T A travel guide to cytoscape plugins Nat. Methods 2012 9 11 1069 1076 10.1038/nmeth.2212 23132118
Saito, R. et al. A travel guide to cytoscape plugins. Nat. Methods 9(11), 1069–1076 (2012).23132118 10.1038/nmeth.2212
35. Shannon P Markiel A Ozier O Baliga NS Wang JT Ramage D Amin N Schwikowski B Ideker T Cytoscape: A software environment for integrated models of biomolecular interaction networks Genome Res. 2003 13 11 2498 2504 10.1101/gr.1239303 14597658
Shannon, P. et al. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 13(11), 2498–2504 (2003).14597658 10.1101/gr.1239303
36. Chin CH Chen SH Wu HH Ho CW Ko MT Lin CY cytoHubba: Identifying hub objects and sub-networks from complex interactome BMC Syst. Biol. 2014 8 4 1 7 24393148
Chin, C. H. et al. cytoHubba: Identifying hub objects and sub-networks from complex interactome. BMC Syst. Biol. 8(4), 1–7 (2014).24393148
37. Chandrashekar DS Bashel B Balasubramanya SAH Creighton CJ Ponce-Rodriguez I Chakravarthi BV Varambally S UALCAN: A portal for facilitating tumor subgroup gene expression and survival analyses Neoplasia 2017 19 8 649 658 10.1016/j.neo.2017.05.002 28732212
Chandrashekar, D. S. et al. UALCAN: A portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia 19(8), 649–658 (2017).28732212 10.1016/j.neo.2017.05.002
38. Liu Z Zhou J Wang Z Zhou Z Analysis of SEC24D gene in breast cancer based on UALCAN database Open Life Sci. 2019 14 1 707 711 10.1515/biol-2019-0080 33817210
Liu, Z., Zhou, J., Wang, Z. & Zhou, Z. Analysis of SEC24D gene in breast cancer based on UALCAN database. Open Life Sci. 14(1), 707–711 (2019).33817210 10.1515/biol-2019-0080
39. Oliveros JC: VENNY. An interactive tool for comparing lists with Venn Diagrams. http://bioinfogp.cnb.csic.es/tools/venny/index.html (2007).
40. Kang J Sergio CM Sutherland RL Musgrove EA Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells BMC Cancer 2014 14 1 1 13 10.1186/1471-2407-14-32 24383403
Kang, J., Sergio, C. M., Sutherland, R. L. & Musgrove, E. A. Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells. BMC Cancer 14(1), 1–13 (2014).24383403 10.1186/1471-2407-14-32
41. Liu Y Zhu Y-H Mao C-Q Dou S Shen S Tan Z-B Wang J Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles J. Controll. Release 2014 192 114 121 10.1016/j.jconrel.2014.07.001
Liu, Y. et al. Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles. J. Controll. Release 192, 114–121 (2014).10.1016/j.jconrel.2014.07.001
42. Yuan B Xu Y Woo J-H Wang Y Bae YK Yoon D-S Wersto RP Tully E Wilsbach K Gabrielson E Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability Clin. Cancer Res. 2006 12 2 405 410 10.1158/1078-0432.CCR-05-0903 16428479
Yuan, B. et al. Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability. Clin. Cancer Res. 12(2), 405–410 (2006).16428479 10.1158/1078-0432.CCR-05-0903
43. Shigeishi H Yoneda S Taki M Nobumori T Ohta K Higashikawa K Yasui W Kamata N Correlation of human Bub1 expression with tumor-proliferating activity in salivary gland tumors Oncol. Rep. 2006 15 4 933 938 16525682
Shigeishi, H. et al. Correlation of human Bub1 expression with tumor-proliferating activity in salivary gland tumors. Oncol. Rep. 15(4), 933–938 (2006).16525682
44. Ouyang B Knauf JA Ain K Nacev B Fagin JA Mechanisms of aneuploidy in thyroid cancer cell lines and tissues: Evidence for mitotic checkpoint dysfunction without mutations in BUB1 and BUBR1 Clin. Endocrinol. 2002 56 3 341 350 10.1046/j.1365-2265.2002.01475.x
Ouyang, B., Knauf, J. A., Ain, K., Nacev, B. & Fagin, J. A. Mechanisms of aneuploidy in thyroid cancer cell lines and tissues: Evidence for mitotic checkpoint dysfunction without mutations in BUB1 and BUBR1. Clin. Endocrinol. 56(3), 341–350 (2002).10.1046/j.1365-2265.2002.01475.x
45. Bell R Barraclough R Vasieva O Gene expression meta-analysis of potential metastatic breast cancer markers Curr. Mol. Med. 2017 17 3 200 210 10.2174/1566524017666170807144946 28782484
Bell, R., Barraclough, R. & Vasieva, O. Gene expression meta-analysis of potential metastatic breast cancer markers. Curr. Mol. Med. 17(3), 200–210 (2017).28782484 10.2174/1566524017666170807144946
46. Chen WX Yang LG Xu LY Cheng L Qian Q Sun L Zhu YI Bioinformatics analysis revealing prognostic significance of RRM2 gene in breast cancer Biosci. Rep. 2019 39 BSR20182062 10.1042/BSR20182062 30898978
Chen, W. X. et al. Bioinformatics analysis revealing prognostic significance of RRM2 gene in breast cancer. Biosci. Rep. 39, BSR20182062 (2019).30898978 10.1042/BSR20182062
47. Yao W Jiang M Zhang M Zhang H Liang X TTK: A promising target in malignant tumors J. Cell. Signal. 2021 2 3 212 220
Yao, W., Jiang, M., Zhang, M., Zhang, H. & Liang, X. TTK: A promising target in malignant tumors. J. Cell. Signal. 2(3), 212–220 (2021).
48. Tang A Gao K Chu L Zhang R Yang J Zheng J Aurora kinases: Novel therapy targets in cancers Oncotarget 2017 8 14 23937 10.18632/oncotarget.14893 28147341
Tang, A. et al. Aurora kinases: Novel therapy targets in cancers. Oncotarget 8(14), 23937 (2017).28147341 10.18632/oncotarget.14893
49. Wang F Zhang H Wang H Qiu T He B Yang Q Combination of AURKA inhibitor and HSP90 inhibitor to treat breast cancer with AURKA overexpression and TP53 mutations Med. Oncol. 2022 39 12 180 10.1007/s12032-022-01777-x 36071247
Wang, F. et al. Combination of AURKA inhibitor and HSP90 inhibitor to treat breast cancer with AURKA overexpression and TP53 mutations. Med. Oncol. 39(12), 180 (2022).36071247 10.1007/s12032-022-01777-x
50. Bu Y Shi L Yu D Liang Z Li W CDCA8 is a key mediator of estrogen-stimulated cell proliferation in breast cancer cells Gene 2019 703 1 6 10.1016/j.gene.2019.04.006 30953709
Bu, Y., Shi, L., Yu, D., Liang, Z. & Li, W. CDCA8 is a key mediator of estrogen-stimulated cell proliferation in breast cancer cells. Gene 703, 1–6 (2019).30953709 10.1016/j.gene.2019.04.006
51. Sun J Huang J Lan J Zhou K Gao Y Song Z Deng Y Liu L Dong Y Liu X Overexpression of CENPF correlates with poor prognosis and tumor bone metastasis in breast cancer Cancer Cell Int. 2019 19 1 1 11 10.1186/s12935-019-0986-8 30622437
Sun, J. et al. Overexpression of CENPF correlates with poor prognosis and tumor bone metastasis in breast cancer. Cancer Cell Int. 19(1), 1–11 (2019).30622437 10.1186/s12935-019-0986-8
52. Landberg G Erlanson M Roos G Tan EM Casiano CA Nuclear autoantigen p330d/CENP-F: A marker for cell proliferation in human malignancies Cytom. J. Int. Soc. Anal. Cytol. 1996 25 1 90 98
Landberg, G., Erlanson, M., Roos, G., Tan, E. M. & Casiano, C. A. Nuclear autoantigen p330d/CENP-F: A marker for cell proliferation in human malignancies. Cytom. J. Int. Soc. Anal. Cytol. 25(1), 90–98 (1996).
53. Yin Y Cai J Meng F Sui C Jiang Y MiR-144 suppresses proliferation, invasion, and migration of breast cancer cells through inhibiting CEP55 Cancer Biol. Ther. 2018 19 4 306 315 10.1080/15384047.2017.1416934 29561704
Yin, Y., Cai, J., Meng, F., Sui, C. & Jiang, Y. MiR-144 suppresses proliferation, invasion, and migration of breast cancer cells through inhibiting CEP55. Cancer Biol. Ther. 19(4), 306–315 (2018).29561704 10.1080/15384047.2017.1416934
54. Wei R Wang Z Zhang Y Wang B Shen N Li X Shang L Shang Y Yan W Bioinformatic analysis revealing mitotic spindle assembly regulated NDC80 and MAD2L1 as prognostic biomarkers in non-small cell lung cancer development BMC Med. Genom. 2020 13 1 14 10.1186/s12920-020-00762-5
Wei, R. et al. Bioinformatic analysis revealing mitotic spindle assembly regulated NDC80 and MAD2L1 as prognostic biomarkers in non-small cell lung cancer development. BMC Med. Genom. 13, 1–14 (2020).10.1186/s12920-020-00762-5
