A High-Fat Diet Promotes Mammary Gland Myofibroblast Differentiation through MicroRNA 140 Downregulation

Abstract

Human breast adipose tissue is a heterogeneous cell population consisting of mature white adipocytes, multipotent mesenchymal stem cells, committed progenitor cells, fibroblasts, endothelial cells, and immune cells. Dependent on external stimulation, adipose-derived stem cells differentiate along diverse lineages into adipocytes, chondrocytes, osteoblasts, fibroblasts, and myofibroblasts. It is currently not fully understood how a high-fat diet reprograms adipose-derived stem cells into myofibroblasts. In our study, we used mouse models of a regular diet and of high-fat-diet-induced obesity to investigate the role of dietary fat on myofibroblast differentiation in the mammary stromal microenvironment. We found that a high-fat diet promotes myofibroblast differentiation by decreasing microRNA 140 (miR-140) expression in mammary adipose tissue through a novel negative-feedback loop. Increased transforming growth factor ␤1 (TGF-␤1) in mammary adipose tissue in obese mice activates SMAD3 signaling, causing phospho-SMAD3 to bind to the miR-140 locus and inhibit miR-140 transcription. This prevents miR-140 from targeting SMAD3 for degradation, resulting in amplified TGF-␤1/SMAD3 signaling and miR-140 downregulation-dependent myofibroblast differentiation. Using tissue and coculture models, we found that myofibroblasts and the fibrotic microenvironment created by myofibroblasts impact the stemness and proliferation of normal ductal epithelial cells and early-stage breast cancer invasion and stemness.KEYWORDS Obesity, breast cancer, fibrosis, miRNA, myofibroblasts T he heterogeneous cell population found within the mammary stromal vascular fraction (SVF) is essential for extracellular matrix (ECM) remodeling and disease development. Despite the importance of the mammary adipose tissue, surprisingly little is known about normal tissue homeostasis. The SVF consists of multipotent mesenchymal stem cells, committed progenitor cells, fibroblasts, endothelial cells, and immune cells. Under the influence of a variety of hormones, growth factors, and external factors, mesenchymal stem cells (MSCs) in the stromal vascular fraction are able to differentiate into a multitude of cell types, including adipocytes, chondrocytes, osteoblasts, fibroblasts, and myofibroblasts. For example, to commit MSCs into the adipogenic lineage in vitro, the master regulators peroxisome proliferator-activated receptor gamma (PPAR␥) and C/EBP␣ are induced using a combination of dexamethasone, indomethacin, insulin, and methylisobutylxanthine, whereas in osteoblast differentiation, RUNX2 is activated using a mixture of ascorbate, bone morphogenetic protein, dexamethasone, and 1,25-dihydroxy vitamin D 3 (1). While these cocktails can be used to induce differentiation, the complex signaling pathways regulating adipose MSCs are not fully elucidated.Fibroblasts are critical for the production and maintenance of the extracellular matrix through secretion of ECM proteins, including fibronectin and collagen as well as

Effect of high-fat diet feeding and associated transcriptome changes in the peak lactation mammary gland in C57BL/6 dams

“…Lactation imposes a significant energy demand by the mammary gland. Additionally, HFD feeding has been reported to cause significant effects on mammary gland adipose tissue (39) and myofibroblast differentiation (64). At the computational level, three genes (DDR1, Atp2b2, and Csf1) with a reported role in mammary gland ductal development were confirmed to exhibit significant differential isoform expression between HFD and LFD dams.…”

“…Additionally, breastmilk composition changes due to nutritional modification have been proven to have long-term effects on offspring, further highlighting the importance of breastmilk in early life (21). Some studies have shown transcriptomic changes in milk or mammary gland in animal models fed an HFD (20,64). A recent study found that mice fed an HFD before and during lactation have significantly altered microRNA expression lev-els as well as whole transcriptome changes in milk (14).…”

Impact of miR‐140 Deficiency on Non‐Alcoholic Fatty Liver Disease

“…To further examine extracellular matrix deposition, we performed IHC staining for fibronectin, a primary component of the basement membrane. We recently identified fibronectin as a direct target of miR‐140, and found that chow‐fed miR‐140 knockout mice had upregulated expression of fibronectin compared to WT . As demonstrated in Figure A, there was a small, insignificant increase in central vein fibronectin expression in WTHFD mice compared to WTLFD.…”

“…Through utilization of a diet‐induced obesity mouse model, we found that obesity downregulates miR‐140 expression in the stromal mammary gland through TGFβ induced SMAD3 signaling. Moreover, we demonstrated that loss of miR‐140 results in a feedback loop that drives mammary myofibroblast differentiation, promoting the development of a fibrotic mammary microenvironment . As we demonstrated that high‐fat diet resulted in miR‐140 downregulation, we wanted to determine whether miR‐140 loss is sufficient to serve as one of the necessary “hits” in the development of NAFLD.…”

MicroRNAs as regulators of mitochondrial dysfunction and obesity

“…During apoptotic stimuli, miR-140 is upregulated, contributing to the suppression of Mfn1 expression during apoptosis (63). Interestingly, miR-484 and miR-140 have been reported to be reduced in obesity (81,116).…”