Ewing sarcoma (EWS) is a rare and highly aggressive cancer arising in both bone and soft tissue that mainly affects children, adolescents, and young adults. EWS has a 70-80% survival rate for patients with localized disease, which drastically reduces to ~30% upon disease progression to a metastatic state. EWS is characterized by the fusion of a FET gene family and an ETS transcription factor—the most common being EWS-FLI1 (~85% of all EWS). Utilizing genetic, epigenetic, and bioinformatics tools, a novel and highly specific EWS-FLI1 regulated gene, LOXHD1, with a potential oncogenic role was previously discovered by my lab group. Since solid tumors, like EWS, have been known to develop a hypoxic microenvironment—which quite often leads the cancer cells to metastasize and migrate to other parts of the human body—I hypothesized that preventing hypoxic microenvironment by inhibiting LOXHD1 can reduce the metastatic potential of EWS. The CRISPR dCas9-KRAB system was first used to repress the transcriptional activity ofLOXHD1 in two EWS cell lines, SK-N-MC and RD-ES. Upon hypoxia treatment, these knockdown cells underwent RNA-seq and gene set enrichment analysis (GSEA), which showed a very weak hypoxic stress response and a strong negative enrichment for the Hallmark Hypoxia signature. Western blot analysis further showed a decrease in HIF1α (a hallmark of cellular hypoxic-stress response) protein expression. Furthermore, immunofluorescent staining of F-actin in the knockdown cells showed highly disorganized and chaotic actin filaments, and cell invasion and migration assays demonstrated a ~2-fold reduction in invasion potential—thus, highlighting the role of LOXHD1 as a regulator of cytoskeletal assembly and metastasis. Overall, these in vitro studies showed that LOXHD1 plays a significant role in Ewing sarcoma metastasis in hypoxic conditions.
