Characterization of tenascin-W, an emerging player in the metastatic bone marrow niche

Tumors are heterogeneous organ-like tissues including not only tumor cells themselves but also auxiliary cells such as, endothelial cells, fibroblasts, inflammatory cells, and bone marrow derived stem or stromal cells (BMSCs), which collectively create the surrounding microenvironment also referred to as stromal compartment. By now the active role of the tumor-stroma in driving the dissemination phase and the following engraftment of tumor cells in secondary organs is widely accepted. Indeed, the perpetual activation of stromal cells is extended beyond the local primary tumors and they can take part in preparing a permissive environment at distant anatomic sites by providing oxygen and nutrients essential for tumor growth and invasion. Tenascin-W (TNW) is a matricellular protein with a dynamically changing pattern of expression during development and disease. Its pronounced presence in developing bones implies a function in osteogenesis. In adults, tenascin-W is mostly restricted to stem cell niches, and is also expressed in the microenvironment of solid cancers. These distinct expression patterns imply a complex regulation of tenascin-W gene expression at the transcriptional level. Here we analyzed tenascin-W expression in a xenograft model of breast cancer metastasis to the bone. Quantitative mRNA analysis revealed an upregulation of tenascin-W in mouse osteoblast populations sorted from bones harboring human breast cancer metastases. Long bone sections containing metastases exhibit expression of mouse tenascin-W protein proving that tenascin-W is supplied by the metastatic niche and not by the tumor cells. Transwell and co-culture studies show that bone marrow stem cells (BMSCs) express tenascin-W protein after exposure to factors secreted by MDA-MB231-1833 breast cancer cells. These findings prompted us to investigate the cis and trans-acting elements that drive tenascin-W . 5’RAC analysis of mRNA from human breast cancer, glioblastoma, and bone tissue showed a single tenascin-W transcript with a transcription start site (TSS) at a non-coding first exon upstream of exon2, which contains the translation start codon (ATG). The promoter region between -957bp and -79bp influences transcription and the minimal promoter sequence is contained within 79bp from the TSS. Computational analysis shows the presence of Smad4 nuclear transcription factor binding site at -61bp from the TSS in proximity of a TATA box sequence. Site-directed mutagenesis of the Smad4-binding site strongly impaired the SEAP reporter gene expression driven by the basal promoter. Furthermore, we found three evolutionary conserved regions in the first intron harboring glucocorticoid response elements (GRE), which negatively affect 10 I. Summary transcription initiation from the basal promoter (-79bp). Therefore, we assessed whether TGF1 and glucocorticoids (GCs) act on tenascin-W gene expression in the tumor context. We identified TGF1 as an important factor inducing human tenascin-W gene transcription in BMSCs through activation of ALK5. Preincubation of BMSCs with the ALK5 inhibitor, SB431542, abolished tenascin-W induction by TGF1. Moreover, GCs impaired tenascin-W mRNA expression in BMSCs. Finally, recombinant tenascin-W protein stimulated MDA-MB231-1833 cell proliferation and migration in vitro assays. Our experiments suggest that tenascin-W acts as a niche component for breast cancer metastasis to the bone by supporting cell migration and cell proliferation of the breast cancer cells. The analysis of the tumor bed contribution to cancer progression is a new frontier to unravel. It will lead to novel approaches to interfere with mechanism implicated in drug resistance, tumor relapse and metastatic spread.