Regulation of differentiation and invasion in RMS by ASAP1

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

In collaboration with Dr. Paul Randazzo, we discovered that knockdown of the ArfGAP ASAP1 and its homologues ASAP2 and ASAP3 block trametinib-induced differentiation of fusion negative rhabdomyosarcoma (FN-RMS) cells. We hypothesized that this was due to its function as a GTPase-Activating Protein (GAP) toward the small GTPases Arf1 and Arf5. To test this hypothesis, we knocked down Arf1 and Arf5. As a GAP, ASAP1 binds to active Arf, catalyzes the hydrolysis of GTP to GDP, and terminates Arf signaling. Therefore, if GAP activity is essential for ASAP1-meditated regulation of differentiation, knockdown of Arf1 or Arf5 would have the opposite effect of knockdown of ASAP1. However, we discovered that knockdown of Arf1 and Arf6 block differentiation to a similar degree as ASAP1. Therefore, we hypothesize that ASAP1 may also be an effector of Arf. To test this hypothesis, we will rescue ASAP1 knockdown with GAP-dead ASAP1 and ASAP1 mutants that are not able to bind Arf, and rescue Arf knockdown with Arf mutants that are not able to bind ASAP1 and determine the effect of these rescues on differentiation in FN-RMS cells. We also discovered that ASAP1, ASAP2 and ASAP3 knockdowns block differentiation by suppressing expression of the myogenic transcription factors myogenin or MEF2C. However, the mechanism by which ASAP1, a membrane-associated protein, is regulating transcription factor expression is unknown. We hypothesize that the ASAP family may regulate transcription through trafficking of membrane-anchored proteins. ASAP1 has been associated with trafficking of both integrins and receptor tyrosine kinases. We will test this hypothesis by subcellular fractionation of FN-RMS cells in proliferative and differentiation conditions and determine the subcellular localization of ASAP and its integrin and RTK targets by immunoblot. We will also track vesicle trafficking by live cell imaging in the presence and absence of ASAP1 knockdown. An alternative hypothesis for ASAP-mediated regulation of transcription is that ASAP's regulation of the actomyosin cytoskeleton alters MAP kinase signaling. To test this hypothesis, we will rescue ASAP1 knockdown with ASAP1 mutated at the Src or non-muscle myosin IIA binding domains and examine the effect on differentiation, MAPK signaling, and myogenic transcription factor expression. Recently, we have engineered RMS cells to express fluorescent protein when undergoing differentiation. We plan to use these cells in zebrafish models (in collaboration with the LCDS zebrafish core facility and Dr. Kandice Tanner) to evaluate the link between differentiation and invasion/metastasis. In addition, in work done in collaboration with Dr. Roberto Weigert, we have shown that RMS cell line xenografts spontaneously metastasize to locoregional lymph nodes and the lungs when the cells are injected orthotopically into the tongue as opposed to the hindlimb. We plan to use this model to study the role of the MAPK pathway and ASAP1 in RMS metastasis.