The Calcineurin-NFAT-Angiopoietin 2 signaling axis in lung endothelium is critical for the establishment of lung metastases

The pre-metastatic niche is a pre-determined site of metastases, awaiting the influx of tumor cells. Here we demonstrate that the calcineurin-NFAT pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. We previously showed that DSCR-1 functions in a negative feedback loop to attenuate calcineurin signaling. Upregulation of the calcineurin pathway via loss of Dscr-1 leads to a significant increase in lung metastasis due to the increased expression of a newly identified NFAT target, Angiopoietin (Ang)-2. An increase in VEGF levels specifically in the lung versus other organ microenvironments triggers a threshold of calcineurin-NFAT signaling that transactivates Ang2 in lung endothelium. Further, we demonstrate that overexpression of DSCR-1 or the Ang-2 receptor, soluble Tie2, prevents activation of the lung endothelium inhibiting lung metastases in our mouse models. Our studies provide insights into mechanisms underlying angiogenesis in the pre-metastatic niche and offers novel targets for lung metastases. Whole lung total RNA were harvested from wildtype or Dscr-1 null mutation mice (C57BL6/J background), and subjected to Affymetrix Mouse 430 2.0 array in duplicate conditions. Alternatively, primary cultured human lung endothelial cells (LONZA) were transfected with either siRNA, control or DSCR-1, or adenovirus expressing DSCR-1 or mock control. Adenovirally transfected lung endothelial cells were serum-starved (EBM-2 (LONZA) plus 0.5% FBS) and treated with 50ng/ml VEGF for indicated time points. After the treatments, total RNA was harvested and subjected to Affymetrix Human U133 Plus 2.0 array.