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

Abstract 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, and dendritic cell 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. Footnotes We performed a new set of experiments on the role of CRD on tumor microenvironment. We analyzed a panel of immune cells via flow cytometry and further confirmed the data using immunostaining. List of abbreviations - 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 - CoA - 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