Abstract
Our previous study found that Notch3 knockout mice exhibit defects in mammary gland development. To elucidate the underlying mechanism, tissue samples were subjected to RNA-seq, GO, and KEGG enrichment analyses and qRT-PCR validation. Of enriched pathways, chemokine signaling pathway and cytokine–cytokine receptor interaction were noticed in both Notch3wt/wt/Notch3wt/– and Notch3wt/wt/Notch3–/– mice, in which the expression of chemokine ligand 2 (CCL2) was sharply reduced in Notch3wt/– and Notch3–/– mammary gland tissues. The Mouse ENCODE transcriptome data reveal that the mammary gland fat pad exhibits a high CCL2, CCR2, and CCR4 expression, indicating that these molecules play important roles during mammary gland development. Specifically, defective mammary glands in Notch3 knockout mice could be partially rescued by CCL2 overexpression lentivirus through intraductal injection. An in vitro study showed that CCL2 overexpression promoted the proliferation, migration, and cancerous acinar formation of 4T1 cells, which could rescue the defective migration of 4T1 cells caused by Notch3 knockdown. We also found that Notch3 transcriptionally regulated the expression of CCL2 in a classical pattern. Our findings illustrated that Notch3-regulating CCL2/CCR4 axis should be an important signaling pathway for mammary gland development and should be a candidate target for breast cancer therapy.
Highlights
Compared with other major organs, the mammary gland is a unique glandular organ because it reaches full development only after birth
We report that Notch family receptor member 3 (Notch3) knockdown can inhibit the proliferation and the migration of 4T1 murine mammary carcinoma cells via the C-C motif chemokine ligand 2 (CCL2)/C-C chemokine receptor type 4 (CCR4) axis
Like normal developing mammary glands, the rudimentary ductal had formed in Notch3wt/wt mice, and the ducts and the branches were elongated toward the lymph node, and the terminal end buds (TEBs) were numerous at 3 weeks of age
Summary
Compared with other major organs, the mammary gland is a unique glandular organ because it reaches full development only after birth. Its development can be divided into three main stages throughout the lifetime, namely, embryonic, pubertal, and adult (Macias and Hinck, 2012). During these different stages, the epithelial cells of the mammary gland will undergo many rounds of Abbreviations: Notch, Notch family receptor member 3; CCL2, C-C motif chemokine ligand 2; CCR2, C-C chemokine receptor type 2; CCR4, C-C chemokine receptor type 4; DEGs, differentially expressed genes; KEGG analysis, Kyoto Encyclopedia of Genes and Genomes analysis; GO, gene ontology. Many dysregulated pathways and malignant biological processes observed in breast cancer progression mimic those observed during normal mammary gland development and tissue remodeling. Based on the model system of mouse mammary gland, many researchers constantly explore developmental mechanisms and some basic questions in cancer biology by knocking in/down certain gene(s), which have been extrapolated to humans (Lanigan et al, 2007)
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