Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis. ### Competing Interest Statement The authors have declared no competing interest. * LD : Light and Dark CRD : Circadian Rhythm Disruption LILRB4 : leukocyte immunoglobulin-like receptor 4 TNBC : Triple-negative Breast Cancer ER : Estrogen receptor PR : Progesterone receptor HER2 : Human Epidermal Growth Factor Receptor 2 SCN : Suprachiasmatic nucleus CLOCK : Circadian locomotor output cycles kaput BMAL-1 : Brain and muscle Arnt-like protein-1 PAS : Per-Arnt-Sim domain PER : Period CRY : Cryptochrome TME : Tumor microenvironment GEMM : Genetically engineered mouse model FVB : Friend LeukemiaVirus B TEB : Terminal end bud H&E : Hematoxylin and Eosin AGR : Albumin-to-globulin ratio scRNA-seq : Single-cell RNA sequence CD : Cluster of differentiation KLF2 : Krüppel-like factors2 SLC2A1 : Solute Carrier Family 2 Member 1 TRPS1 : Transcriptional Repressor GATA Binding 1 TOP2A : Topoisomerase II Alpha FTH1 : Ferritin heavy chain 1 NES : Nestin CLEC11A : C-Type Lectin Domain Containing 11A S100A4 : S100 Calcium Binding Protein A4 RPL35A : Ribosomal Protein L35a FXYD3 : FXYD Domain Containing Ion Transport Regulator 3 COL1A2 : Collagen Type I Alpha 2 Chain EPCAM : Epithelial cellular adhesion molecule CDK : Cyclin-dependent kinase M1 : Type1 macrophage M2 : Type2 macrophage CAFs : Cancer-associated fibroblasts L-R : Ligand-receptor FGF : Fibroblast growth factor PDGF : Platelet-derived growth factor TNF : Tumor necrosis factor NOTCH : Neurogenic locus notch homolog protein TGFβ : transforming growth factor-beta CoAp : Coenzyme A MxIF : Multiplex immunofluorescence Treg : Regulatory T-cell α-SMA : Alpha-smooth muscle actin FN1 : fibronectin 1 FACS : Fluorescence-activated cell sorting FoxP3 : forkhead box P3 CSC : Cancer stem cell MDSCs : Myeloid-derived suppressor cells TAMs : Tumor-associated macrophages IFN : Interferon IL : Interleukin G-CSF : Granulocyte colony-stimulating factor CCL2/12 : Chemokine (C-C motif) ligand 2/12 CXCL5 : C-X-C motif chemokine 5 PCR : Polymerase chain reaction TCGA : The Cancer Genome Atlas DGE : Differential gene expression APOE : apolipoprotein E SHP-2 : SH2 domain-containing protein tyrosine phosphatase-2 uPAR : Urokinase-like plasminogen activator ARG1 : Arginase 1 AML : Acute Myeloid Leukemia mIHC : multiplex immunofluorescence histochemistry ARNT2 : Aryl hydrocarbon receptor nuclear translocator 2 NR1D1 : Nuclear Receptor Subfamily 1 Group D Member 1 RORB & C : RAR Related Orphan Receptor B and C ITIMs : Immunoreceptor tyrosine-based inhibitory motifs ITAMs : Immunoreceptor tyrosine-based activating motifs APCs : Antigen-presenting cells JAK/STAT : Janus kinase/signal transducers and activators of transcription