Effects of chronic stress on the behavior of MMTV-PyMT mice. A. Example of Open Field Test, the central area of the open field is designated within the red rectangle, while the outer area is the peripheral zone. B. The trajectory of mice in the open field. C. Example of Tail Suspension Test. CS: chronic stress. NC: negative control.

Sequences of the siRNA oligos targeting Cebpb

DEGs in HoxB8 neutrophils with or without knockout of C/EBPβ. A. Differentially expressed genes (DEGs) in zymosan-induced HoxB8 neutrophils between wild type (WT) and knockout (KO) of Cebpb. DEGs with p < 0.005, fold change >2 were highlighted in red. B. Expression level of Ccl3 and Ccl4 in WT and Cebpb-KO HoxB8 neutrophils. C. Gene set enrichment analysis (GSEA) analysis of cell chemotaxis in WT and Cebpb-KO HoxB8 neutrophils. D. Bubble plot displaying the top 10 activated and top 10 suppressed pathways in GSEA-GO analysis of DEGs between WT and Cebpb-KO HoxB8 neutrophils.

Conditional knockout of Ccl3/Ccl4 in neutrophils using an optimized CRISPR/Cas9 approach. A. Schematic representation of the main functional elements in the CRISPR plasmid designed to specially target Ccl3 and Ccl4 in neutrophils. pSpCas9(BB)-2A-Puro (PX459) was used as the vector backbone. B. Sequences of Guide RNAs (gRNAs) designed to target the mouse Ccl3 and Ccl4 genes. C. Western blot analysis of CCL3 and CCL4 in primary neutrophils from the lungs of breast tumor-burden mice in both NC and CS groups with or without knockout of Ccl3/Ccl4.

Neutrophils in CS mice promoted breast cancer cell metastasis in the lung. A. Flow cytometry analysis of neutrophils isolated from the lungs of MMTV-PyMT mice. B. Transwell assays of 4T1 breast cancer cells by co-culturing with primary lung neutrophils from NC or CS mice in the bottom chamber. CS: chronic stress. NC: negative control. Data are presented as the mean ± SEM (n = 3, T-test).

Effects of chronic stress on lung metastasis in MMTV-PyMT mice. A. Image of the whole lung in each mouse in the NC or CS group. B. Hematoxylin and Eosin (HE) staining of the paraffin-embedded lung slide from each mouse in the group of NC or CS. CS: chronic stress. NC: negative control.

Sequence of the primers for promoter region of Ccl3 and Ccl4

scRNA-seq analysis of MoMacDc cell subpopulations in the lung of NC and CS MMTV-PyMT mice. A. UMAP plot showing different population of MoMacDc cells from the single-cell RNA sequencing analysis of the lung. B. Comparison of the fraction of each MoMacDc cell subpopulation between the CS and NC groups. (Fisher exact test, ***p < 0.001). C. Heatmap showing the expression of marker genes in each subpopulation of MoMacDc cells. D. Flow cytometry analysis of Macrophages (stained with F4/80) in the of lung of mice with or without receiving chronic stress stimulation. E. Quantitative analysis of Macrophages in D (T test). F. Volcano plot displaying differentially expressed genes (DEGs) in dendritic cells between the CS and NC groups. Those MHC Class II Protein Complex-related genes were highlighted in red. G. Bubble plot displaying the top 10 significantly downregulated pathways (NES <0) identified in the GSEA analysis of dendritic cells (CS vs. NC). H. GSEA analysis showing enrichment of the MHC Class II Protein Complex Binding Pathway in dendritic cells from CS mice, compared to NC. CS: chronic stress. NC: negative control.

Effects of chronic stress on body weight in MMTV-PyMT mice. Body weight in MMTV-PyMT mice from week 5 to week 14 with or without receiving chronic stress stimulation. CS: chronic stress. NC: negative control. Data are presented as the mean ± SEM.

Kaplan Meier Analysis showing a poorer survival correlated with the higher expression of CCR1 in breast cancer patients. A. Correlation analysis between the expression levels of CCR1 and recurrence-free survival (RFS) in total 4,929 breast cancer patients. B-C. Correlation analysis between the expression levels of CCR1 and RFS in 2,638 lymph node negative (B) and 1,656 lymph node positive (C) breast cancer patients.

KEGG pathways analysis of those highly expressed genes in the Neu_CP (A) or Neu_CSP (B) subtypes of neutrophils from the lung of MMTV-PyMT mice. Pathways of interest were indicated with red ellipses.

Effects of chronic stress on immune cells in the peripheral blood of MMTV-PyMT mice. A-B. Flow cytometry analysis of total granulocytes, total lymphocytes, total T cells, CD8+ T cells and CD4+ T cells (A), neutrophils (B) in the peripheral blood of MMTV-PyMT tumor mice with or without receiving chronic stress stimulation. C. The proportions of total granulocytes and total lymphocytes among all white cells in blood. CS: chronic stress. NC: negative control. Data are presented as the mean ± SEM (n = 5).

Analysis of endothelial permeability in the lung of NC and CS MMTV-PyMT mice. A. Bubble plot illustrating the expression of 3 genes related to Tight Junction (Cldn, Ocln, and Tjp1) in lung endothelial cells from NC and CS mice. B. Heatmap showing the GSEA enrichment scores of Tight Junction, Gap Junction, and Adherens Junction pathways in lung endothelial cells from NC and CS mice. C. Boxplot showing the ModuleScore of the endothelial barrier in lung endothelial cells from NC and CS mice.

Mental stress is widely recognized as a significant risk factor for breast cancer, exerting detrimental effects on both progression and prognosis. Herein, we investigated the role of stress in regulating breast cancer metastasis. In genetically engineered and transplantation breast cancer mouse models, chronic stress stimulation increased tumor growth and lung metastasis. Single-cell RNA sequencing analysis of the premetastatic lung microenvironment revealed induction of a previously unrecognized subtype of cancer stress-primed (CSP) neutrophils, characterized by the overexpression of Ccl3, Ccl4, Cxcl2, Il1r2, and Cebpb. Pseudotime trajectory analysis demonstrated that chronic stress caused a shift of neutrophils from the cancer-primed neutrophil subtype to the CSP subtype in the lung. Activation of the glucocorticoid receptor NR3C1 by the stress hormone corticosterone induced expression of Cebpb in neutrophils, which then promoted transcription of Ccl3 and Ccl4. The differentiation of neutrophils into the CSP subtype promoted lung metastasis of CCR1+ breast cancer cells via CCL3/CCL4-mediated recruitment. Targeting this axis using an anti-Ly6G antibody to deplete neutrophils, a CRISPR/Cas9-mediated approach to conditionally knock out Ccl3/Ccl4 in neutrophils, and BX471 treatment to inhibit CCR1 in cancer cells all significantly reduced breast cancer lung metastasis. Together, this study not only demonstrates a stress-neutrophil-cancer axis that promotes lung metastasis in breast cancer but also provides potential strategies for reducing lung metastasis by targeting CSP neutrophils or CCR1+ breast cancer cells.Significance: Stress induces a neutrophil subtype in the lungs that secretes CCL3 and CCL4 to stimulate metastasis of breast cancer cells by activating CCR1, offering potential strategies for preventing or treating metastasis.

Heatmap displaying the top 5 active transcription factors in each subtype of neutrophils (analyzed using SCENIC). C/EBPβ showed the strongest activity in the Neu_CSP (Neu2) subtype of neutrophils.

Representative hematoxylin and eosin (H&E) staining of paraffin-embedded lung tissue sections from individual mice in the NC or CS group treated with either IgG or anti-Ly6G antibody.

Following the publication of the above article, the authors drew to the Editor's attention that they has misclassified some of their original data, and this led to the erroneous compilation of the cell invasion and scratch wound assay data shown in Fig. 5A and B respectively on p. 604. Moreover, the authors realized that the same GAPDH control western blotting data had inadvertently been included in Fig. 4A and G on p. 603, where these data were correctly shown only for Fig. 4G. However, the authors had retained their original data for these figures, and the revised versions of Figs. 4 and 5, now showing the correct data for the GAPDH bands in Fig. 4A and the correct data for Fig. 5A and B, are shown on the next two pages. Note that the errors made in terms of the assembly of the data in these figures did not affect the overall conclusions reported in the paper. The authors are grateful to the Editor of Oncology Reports for granting them this opportunity to publish a Corrigendum, and apologize to both the Editor and the readership for any inconvenience caused. [Oncology Reports 38: 598-606, 2017; DOI: 10.3892/or.2017.5667].

Mental stress has been shown to negatively impact the development and progression of human cancer, including breast cancer. However, its effects on the tumor microenvironment (TME) remain unclear. In this study, we applied single-cell sequencing analysis to tumor tissues from MMTV-PyMT transgenic mice with mammary gland tumors with or without exposure to mental stress. In association with a significant promotion of the cell cycle and tumor growth induced by mental stress, we observed the dedifferentiation of luminal subtype of tumor cells into a subgroup of cancer stem cell-like basal cells, as well as enhanced cell proliferation in epithelial tumor cells, endothelial cells, and fibroblasts. In addition, stress stimulation led to an increase in tumor-associated neutrophils (TANs) and tumor-infiltrating dendritic cells (TIDCs), while suppressing immune cells such as cytotoxic T lymphocytes (CTLs), naïve T cells, B cells, and NK cells within the TME. We also observed a shift in macrophages from the M1 to the M2 phenotype. Furthermore, pathway enrichment analysis of differentially expressed genes, gene signature U score analysis, and immunofluorescence staining of the tumor tissue sections were conducted for further validation. The current study not only systematically elucidates the impact of mental stress on mammary gland tumors and the TME in vivo, but also provides insights into the mechanism underlying mental stress-induced tumor growth and progression in breast cancer.

In oral squamous cell carcinoma (OSCC), a highly aggressive and frequently lethal malignancy, the role and action mechanism of the microtubule regulatory protein CDK5RAP2 have not been fully understood. Here, we show that CDK5RAP2 is highly expressed in OSCC and its expression correlates with clinical stage and lymph node metastasis of the disease. The expression of CDK5RAP2 is regulated by the Wnt signaling pathway. Depletion of CDK5RAP2 inhibits the tumorigenesis and migration of OSCC cells and alters the OSCC cancer stem (-like) cell (CSC) signature. Notably, suppression of CDK5RAP2 expression disrupts spindle orientation during mitosis. Collectively, these results identify CDK5RAP2 as a potential CSC marker and reveal a mechanism that controls the CSC population in OSCC.

Breast cancer is the most common cancer in women around the world. Emerging evidence has indicated the important roles that non-coding RNAs play in regulating tumor development and progression in breast cancer. Herein, we found a dual function of long non-coding RNA (LncRNA) CCAT2 in the luminal subtype of breast cancer, depending on its subcellular distribution. CCAT2 showed an overall downregulation in the tumor tissues from luminal breast cancer patients. Transient overexpression of CCAT2 in the luminal subtype of breast cancer cell MCF-7 or T47D significantly suppressed cell proliferation in vitro and inhibited tumor growth in vivo. Gene expression analysis of cancer stem cell markers including OCT4, NANOG, h-TERT, SOX2 and KLF4; flow cytometry analysis of breast cancer stem cell population, and mammosphere formation assay demonstrated inhibition of cancer cell stemness with transient transfection of CCAT2 in which exogenous CCAT2 mainly distributed in the cytoplasm and regulated miR-221-p27 signaling via RNA sequence interaction. However, overexpression of CCAT2 in MCF-7 cells through pMX retroviral nuclear expression vector accumulated CCAT2 in the nucleus, leading to upregulation of OCT4-PG1, a pseudogene of stem gene OCT4, thereby promoting the cancer cell stemness. In conclusion, the current study, for the first time, revealed a dual function of lncRNA CCAT2 as a tumor suppressor or oncogene depending upon its subcellular distribution. It also demonstrated the regulatory mechanism of cytoplasmic CCAT2 in suppressing tumorigenesis in the luminal subtype of breast cancer.