Western blot analysis, conclude that CUL3, an E3-ubiquitin ligase, is down regulated in lung cancer cell lines A549, H522, H2030, and H2009 compared to normal human bronchial epithelial cells. CUL3 is down regulation in lung cancer is supported by the Human Protein Atlas that shows, by immunohistochemistry, that human cancer tissue has weak expression of CUL3 compared to normal lung tissue.
We identified WAC, WW-domain containing adaptor with coiled-coil, in 3 forward genetic colorectal cancer screens. Knockdown of WAC by shRNA in HCT116 and human colonic epithelial cells leads to a decrease in colonies in soft agar. To knockout WAC, we designed WAC targeted TALENs and transfected them into HCT116 human colorectal cancer cells. The cells were single cell cloned and DNA sequenced. The sequencing resulted in cell strains with novel WAC truncations, deletions, and base pair substitutions. Western blot confirmed the genetic abnormalities altered WAC expression. These novel WAC mutants are being assayed by cell proliferation, apoptosis, and soft agar assays to determine if WAC mutations have cancer associated phenotypes.
Forward genetic screens and validation of the identified genes are important for understanding the genetics regulating cancer.