Genomic analyses identify marker molecules and processes in metastatic breast cancer tissues

Breast cancer metastasis is the major reason for deaths from breast cancer. Identication of breast cancer metastasis is of great importance for the management and prediction of cancer progression. However, the key genes and signaling pathways remain unclear in metastatic breast cancers. Our objective is to nd the key molecules and signaling pathways by analyzing the RNA-sequence data. The GSE189411 was constructed by the Illumina NovaSeq 6000 (Mus musculus). The KEGG and GO analyses showed the cytokine−cytokine receptor interaction and human papillomavirus infection are the two major processes during the liver metastasis of breast cancer cells. Moreover, we discovered ten key relevant molecules including ITGB2, FCGR3A, CD86, CD80, FOXP3, SYK, CCR5, VCAM1, RAC2, ICAM1. Our study may provide novel insights for the early diagnosis of breast cancer metastasis.


Introduction
Breast cancer is the most malignancy and common death reason in women 1 . Due to the early detection and systemic treatments, the mortality from breast cancer in the US has declined 2 . However, breast cancer is still the most common reason for death in developing countries such as Africa and Asia 3 . Early breast cancer without metastases is a curable disease 4 . Primary surgery is not the optimal choice for all patients with breast cancer 5 . Though targeted therapies have increased the survival rate, the tumor relapses are caused by the drug resistance mechanisms 6 .
Breast cancer metastasis is characterized by local invasion and transferring cancer cells to other organs 7 .
Evidence shows that metastasis can originate from genetic and epigenetic changes 8 . The genetic alterations occur in the DNA sites, but the epigenetic alterations are associated with DNA methylation and histone acetylation 9 .
In this study, we analyzed the metastatic tissues from breast cancer mouse models by using the RNA-seq data. We found a variety of DEGs and signi cant biological processes. We also performed the gene enrichment and constructed the protein-protein interaction (PPI) network and biological processes map to gure out the relationships among the DEGs. The DEGs and functional processes will help the early diagnosis of breast cancer metastasis.

Data resources
Gene dataset GSE189411 was downloaded from the GEO database. The data was produced by the Illumina NovaSeq 6000 (Mus musculus) (Guangxi medical university, Shuangyong Road, Nanning, China). The analyzed dataset includes 3 groups of controls and 3 groups of metastatic liver tissues.

Data acquisition and processing
The data were organized and analyzed by the R package as previously described [10][11][12][13][14] . We used a classical t-test to identify DEGs with P< 0.05 and fold change ≥1.5 as being statistically signi cant.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) KEGG and GO analyses were conducted by the R package (ClusterPro ler) and Reactome. P<0.05 was considered statistically signi cant.

Protein-protein interaction (PPI) networks
The Molecular Complex Detection (MCODE) of Cytoscope software (US) was used to construct the PPI networks. The signi cant modules were produced from constructed PPI networks and String networks.

Results
Identi cation of DEGs in liver tissue after seeding the breast cancer cells To identify the impacts of cancer cells' metastasis in the liver, we analyzed the RNA-seq data from the liver tissues with the transplantation of cancer cells in the axilla. A total of 925 genes were identi ed with the threshold of P < 0.05. The top up-and-down-regulated genes were shown by the heatmap and volcano plot ( Figure 1). The top ten DEGs were selected in Table 1.

PPI network analysis
To determine the relationships of the DEGs, we constructed the PPI network by using the 811 nodes and 2055 edges with Cytoscope software (combine score > 0.4). Table 2 showed the top ten interactive genes with the highest degree scores. The top two modules were indicated in Figure 3. We further analyzed the PPI and DEG network by Reactome map (Figure 4) and identi ed the top ten functional processes including "Chemokine receptors bind chemokines", "WNT ligand biogenesis and tra cking", "Interleukin-10 signaling", "YAP1-and WWTR1 (TAZ)-stimulated gene expression", "Rhesus blood group biosynthesis", "Common Pathway of Fibrin Clot Formation", "RND1 GTPase cycle", "Neutrophil degranulation", "Nef mediated downregulation of MHC class I complex cell surface expression", and "BH3-only proteins associate with and inactivate anti-apoptotic BCL-2 members" (Supplemental Table S1).

Discussion
The knowledge of the molecular mechanisms can improve the clinical treatment of breast cancers. Recent studies showed primary breast tumors that initiated metastases can be distinguished by their gene-expression pro les from those that remained localized 15 . Therefore, our study is to nd out the potential marker genes in the metastasis tissues to improve the diagnosis of breast cancer in the early stage.
We gured out the "cytokine−cytokine receptor interaction" and "human papillomavirus infection" are the two major processes during the liver metastasis of breast cancer cells. Marcela Esquivel-Velázquez et al found a number of in ammatory cytokines such as IL6, IL17, and TNF are important for breast cancer initiation, promotion, angiogenesis, and metastasis 16  We also identi ed ten signi cant relevant molecules to the metastasis of breast cancer. ITGB2 is a prognostic marker gene for patients with breast cancer 18,19 . Patrick G Gavin et al found that the nucleotide polymorphisms in FCGR3A are strongly associated with breast cancer 20  unfolded protein response, immune response, and aging [23][24][25][26][27][28][29][30][31][32][33][34] . Interestingly, FOXP3 was found to be controlled by the circadian clock in the T cell, which may further affect the microenvironment in cancers 35 . SYK was found to possess the promotor and repressor activities of breast cancers. David J Lamb et al found the SYK inhibitor BI1002494 showed no increased proliferation of breast cancer cells 36 .
Xuanmao Jiao et al found CCR5 is considered as a new therapeutic target for metastatic breast cancer 37 . G protein-coupled receptor (GPCR) related signaling pathways involve different physiological and pathophysiological processes including metabolism, immune, and cancers [38][39][40][41][42][43][44][45][46][47][48][49] . Strikingly, CCR5 is a critical GPCR protein, which regulates several immune cells such as T-lymphocytes, macrophages, and dendritic cells 50 . Xiaoqin Huang et al found patients with breast cancer and diabetes can promote cancer adhesion to vascular endothelium via ICAM1 and VCAM1 51 . Yi Zhang et al reported that RAC2 is the negative coe cient that was correlated with a better prognosis 52 .
In summary, our study identi ed the potential gene markers for the metastasis of breast cancer. The cytokine−cytokine receptor interaction and human papillomavirus infection are the mainly affected processes during the metastasis of breast cancer. This study may provide knowledge in the early diagnosis of metastatic breast cancer.

Funding
This work was not supported by any funding.

Declarations of interest
There is no con ict of interest to declare. Tables 1-2   Tables 1-2