Abstract
BackgroundBone is one of the most frequent metastatic sites of advanced breast cancer. Current therapeutic agents aim to inhibit osteoclast-mediated bone resorption but only have palliative effects. During normal bone remodeling, the balance between bone resorption and osteoblast-mediated bone formation is essential for bone homeostasis. One major function of osteoblast during bone formation is to secrete type I procollagen, which will then be processed before being crosslinked and deposited into the bone matrix.MethodsSmall RNA sequencing and quantitative real-time PCR were used to detect miRNA levels in patient blood samples and in the cell lysates as well as extracellular vesicles of parental and bone-tropic MDA-MB-231 breast cancer cells. The effects of cancer cell-derived extracellular vesicles isolated by ultracentrifugation and carrying varying levels of miR-218 were examined in osteoblasts by quantitative real-time PCR, Western blot analysis, and P1NP bone formation marker analysis. Cancer cells overexpressing miR-218 were examined by transcriptome profiling through RNA sequencing to identify intrinsic genes and pathways influenced by miR-218.ResultsWe show that circulating miR-218 is associated with breast cancer bone metastasis. Cancer-secreted miR-218 directly downregulates type I collagen in osteoblasts, whereas intracellular miR-218 in breast cancer cells regulates the expression of inhibin β subunits. Increased cancer secretion of inhibin βA results in elevated Timp3 expression in osteoblasts and the subsequent repression of procollagen processing during osteoblast differentiation.ConclusionsHere we identify a twofold function of cancer-derived miR-218, whose levels in the blood are associated with breast cancer metastasis to the bone, in the regulation of type I collagen deposition by osteoblasts. The adaptation of the bone niche mediated by miR-218 might further tilt the balance towards osteolysis, thereby facilitating other mechanisms to promote bone metastasis.
Highlights
Bone is one of the most frequent metastatic sites of advanced breast cancer
Results miR-218 is associated with breast cancer bone metastasis To search for miRNAs that might be functionally relevant to breast cancer bone metastasis, we obtained sera from 47 stage IV breast cancer patients with (n = 33) or without (n = 14) bone metastases and performed small RNA sequencing
To identify miRNAs characteristically secreted by bone-metastasizing breast cancer cells, we profiled the miRNAs in the Extracellular vesicle (EV) secreted by the metastatic breast cancer cell line MDAMB-231 (MDA-231) as well as its bone-seeking variant designated as MDA-231-bone [29]. hsa-miR-218-5p was significantly higher in the sera from patients with bone metastases compared to those without (Additional file 3: Table S3), and was > 5-fold higher in the EVs from MDA-231-bone cells compared to those from parental MDA-231 (Additional file 4: Table S4)
Summary
Bone is one of the most frequent metastatic sites of advanced breast cancer. Current therapeutic agents aim to inhibit osteoclast-mediated bone resorption but only have palliative effects. One major function of osteoblast during bone formation is to secrete type I procollagen, which will be processed before being crosslinked and deposited into the bone matrix. The bone tropism of breast cancer cells is in part mediated by chemokines and their receptors, exemplified by C-X-C chemokine receptor type 4 (CXCR4) expressed. Our lab has previously shown that breast cancer-secreted EVs target endothelial cells and fibroblasts to induce vascular permeability and metabolic reprogramming, respectively [7,8,9]. Stromal cells in the metastatic niche can secrete EVs to regulate cancer cells [10]. Bone marrow mesenchymal stem cells secrete miR-23b-containing exosomes, which induce breast cancer dormancy by targeting a cell cycle regulator myristoylated alanine rich protein kinase C substrate (MARCKS) [11]. A recent study found that exosomes exhibited organotropism, which was regulated by the integrin profile of these exosomes [12]
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