Nadja Redd; Gloria Slattum; Jody Rosenblatt; Franco Jin, University of Utah
Epithelia encase most of our internal organs and, indeed, the whole body. To serve as an effective barrier against pathogens, epithelia must regulate cell numbers even as cells continuously divide, grow, and die. Cell extrusion happens when a cell fated to die signals neighboring cells to squeeze it out of the epithelium using an actomyosin ring. The extruded cell may undergo apoptosis, a highly regulated process of programmed cell death, or be a live cell that later dies through anoikis, or cell death through loss of contact with the basement membrane and survival signals. Typically, cells extrude apically, away from the basal membrane, and are removed from the lumen of an organ or gland. However, some cells are able to extrude basally and survive post-extrusion, a strategy that an oncogene-bearing tumor cell might use to achieve metastasis. KRAS is one such oncogene that is often mutated in human cancer. The wildtype gene governs multiple pro-growth and survival pathways in the cell. A mutation in one amino acid, called KRasV12, converts it to an oncogene and allows the protein to be constitutively active, inappropriately enhancing its function. This often leads to runaway cell proliferation and tumorigenesis. In prior tissue culture studies we observed that KRasV12 cells are significantly more likely to extrude basally than apically, and could possibly migrate and invade. We therefore hypothesized that KRasV12-driven basal extrusion leads to metastasis, a novel idea that has never been demonstrated. We sought to test our hypothesis in a zebrafish model of epithelial cancer. We injected one-cell embryos with EGFP-labeled KRasV12 or CAAX control, sorted for embryos positive for transgenes at 24 hpf, and fixed the embryos at 48 hpf. We performed immunostaining for EGFP, E-cadherin (periderm marker) or p63 (basal cell marker), and the nucleus (DAPI). We used the expression of caspase-3, a protein activated during apoptosis, and nuclear morphology to determine cell death. Using E-cadherin or p63 expression to mark the borders of the developing epidermis, we quantified live or dying cells either on the epidermal surface or internalized into the fish body. Indeed we find that embryos with the KRasV12 oncogene have a significantly higher percentage of live internalized cells compared to control, suggesting that these may later give rise to metastases. We are currently performing live imaging studies to ascertain if KRasV12 cells spread via basal extrusion and make it to the bloodstream.