Regulation of differentiation and invasion in RMS by ASAP1

在与Paul兰达佐博士的合作中，我们发现ArfGAP ASAP 1及其同源物ASAP 2和ASAP 3的敲低阻断了曲美替尼诱导的融合阴性横纹肌肉瘤（FN-RMS）细胞的分化。我们假设这是由于其作为GTP酶激活蛋白（GAP）对小GTP酶Arf 1和Arf 5的功能。为了验证这个假设，我们敲除了Arf 1和Arf 5。作为GAP，ASAP 1与活性Arf结合，催化GTP水解为GDP，并终止Arf信号传导。因此，如果GAP活性对于ASAP 1介导的分化调节是必需的，那么Arf 1或Arf 5的敲低将具有与ASAP 1敲低相反的效果。然而，我们发现Arf 1和Arf 6的敲低阻断分化的程度与ASAP 1相似。因此，我们推测ASAP 1也可能是Arf的效应子。为了检验这一假设，我们将用GAP死亡的ASAP 1和不能结合Arf的ASAP 1突变体来拯救ASAP 1敲低，并用不能结合ASAP 1的Arf突变体来拯救Arf敲低，并确定这些拯救对FN-RMS细胞分化的影响。我们还发现ASAP 1、ASAP 2和ASAP 3敲低通过抑制生肌转录因子myogenin或MEF 2C的表达来阻断分化。然而，ASAP 1，一种膜相关蛋白，调节转录因子表达的机制尚不清楚。我们推测ASAP家族可能通过膜锚定蛋白的运输来调节转录。ASAP 1与整合素和受体酪氨酸激酶的运输有关。我们将通过在增殖和分化条件下对FN-RMS细胞进行亚细胞分级分离来验证这一假设，并通过免疫印迹法确定ASAP及其整合素和RTK靶点的亚细胞定位。我们还将在存在和不存在ASAP 1敲低的情况下通过活细胞成像来跟踪囊泡运输。ASAP介导的转录调节的另一种假设是ASAP对肌动球蛋白细胞骨架的调节改变了MAP激酶信号传导。为了验证这一假设，我们将拯救ASAP 1敲低与ASAP 1突变的Src或非肌肉肌球蛋白IIA结合域，并检查分化，MAPK信号传导，和生肌转录因子表达的影响。最近，我们已经设计了RMS细胞在分化时表达荧光蛋白。我们计划在斑马鱼模型中使用这些细胞（与LCDS斑马鱼核心设施和Kandice坦纳博士合作）来评估分化和侵袭/转移之间的联系。此外，在与Roberto Weigert博士合作完成的工作中，我们已经证明，当细胞原位注射到舌头而不是后肢时，RMS细胞系异种移植物自发转移到局部淋巴结和肺部。我们计划使用这个模型来研究MAPK通路和ASAP 1在RMS转移中的作用。

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.