Inhibitor of DNA binding-1, a master regulator of vasculogenic mimicry by cancer cells.

Cancer cells within solid tumours routinely access the blood supply by promoting endothelium-dependent angiogenesis, but tumour vasculature can also be formed by the cancer cells themselves via vasculogenic mimicry (VM). With increasing evidence that VM correlates with cancer progression, understanding the regulatory factors that underpin VM could provide new therapeutic strategies to improve patient outcomes. Herein we investigate changes to the gene expression profile of breast cancer cells during the early stages of VM formation. Results identify the transcriptional regulator Inhibitor of DNA binding 1 (ID1) to be elevated ~10-fold within the first two hours of VM formation by MDA-MB-231-LM2 cells. A role for ID1 in promoting VM was supported by ID1-targeting siRNA in breast and pancreatic cancer cells. Similarly, administration of the pan-ID inhibitor AGX-51, interrupted VM formation by MDA-MB-231-LM2 and BxPC-3 cells. Inhibition of ID1 also reduced the production of pro-angiogenic and pro-cancerous proteins, e.g. DKK-1, VEGF, CD31, MMP9, PDGF-AA, angiogenin, and IL-8. Regulatory control of these genes by ID1 was further implicated through identification of the requisite DNA binding motifs upstream of the gene start site. Finally, in silico analysis of barcoded VM-competent MDA-MB-231 cells injected into the mammary fat pad of mice enabled gene expression profiling of cancer cells in the primary tumour and metastatic sites. Elevated expression of ID1 was observed in the cancer cells that has metastasised to the lung and the liver and was associated with an enrichment of pro-angiogenic genes. Taken together, this study further implicates ID1 as an important master regulator of cancer progression via its role in the formation of VM structures, protein production and metastasis. Overall design: This study aims to identify genes of early VM regulatory events. To address this, the human TNBC cell line MDA-MB-231-LM2 was cultured in either two-dimensions (2D, tissue culture flasks) or in 3D as VM-structures in Matrigel and harvested at early time points of VM formation (i.e. two and six hours) followed by bulk RNA sequencing and comparative gene expression analysis.