Role of Ubc13 in Breast Cancer Metastasis

We recently found that Ubc13 controls breast cancer metastasis. However, the underlying molecular mechanisms remain elusive. In order to gain a better understanding of the signaling pathways controlled by Ubc13 in metastasis, we performed a gene array analysis on xenografts derived from orthotopically implanted MDA-MB-231 LM2 cells, which preferentially metastazize to lung in NOD/SCID mice. MDA-MB-231 LM2 cells were infected with lentivial shRNA constructs targeting either GFP (Control) or Ubc13 (ubc13). The resulting Control or Ubc13-silenced cells were implanted into fat pads of six weeks old female virgin NOD/SCID mice. After eight weeks, tumors were excised, followed by collagenase/hyaluronidase digestion to establish single cell suspension, from which epithelial cells were enriched by depleting mouse cells of hematopoietic, endothelial, and fibroblast origin (CD45, TER119, CD31 and BP-1). The enrihced epithelial cells (three Control and three Ubc13-KD cells) were lysed in TRIZOL to extract RNA, which were then used to run microarray.