Regulation of differentiation and invasion in RMS by ASAP1

与Paul Randazzo博士合作，我们发现敲除ArfGAP ASAP1及其同系物ASAP2和ASAP3可以阻断曲美替尼诱导融合阴性横纹肌肉瘤(FN-RMS)细胞的分化。我们假设这是由于它作为GTP酶激活蛋白(GAP)对小的GTP酶Arf1和Arf5起作用。为了验证这一假设，我们推翻了Arf1和Arf5。作为一种GAP，ASAP1与活性Arf结合，催化GTP水解为GDP，终止Arf信号转导。因此，如果GAP活性对于ASAP1介导的分化调控是必不可少的，那么敲除Arf1或Arf5将具有与敲除ASAP1相反的效果。然而，我们发现Arf1和Arf6的敲除对分化的阻断程度与ASAP1相似。因此，我们推测ASAP1也可能是Arf的效应因子。为了验证这一假设，我们将用不能结合Arf的间隙死亡的ASAP1和ASAP1突变体挽救ASAP1击倒，用不能结合ASAP1的Arf突变体挽救Arf击倒，并确定这些挽救对FN-RMS细胞分化的影响。我们还发现，ASAP1、ASAP2和ASAP3基因敲除通过抑制生肌转录因子Mygenin或MEF2C的表达来阻断分化。然而，ASAP1是一种膜相关蛋白，其调控转录因子表达的机制尚不清楚。我们推测，ASAP家族可能通过运输膜锚定蛋白来调节转录。ASAP1与整合素和受体酪氨酸激酶的运输有关。我们将通过对FN-RMS细胞在增殖和分化条件下的亚细胞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靶标的亚细胞定位。我们还将在存在和不存在ASAP1基因敲除的情况下，通过活细胞成像追踪囊泡的运输。对ASAP介导的转录调控的另一种假说是，ASAP对肌动蛋白细胞骨架的调节改变了MAP激酶信号。为了验证这一假设，我们将用在Src或非肌肉肌球蛋白IIA结合区突变的ASAP1击倒ASAP1，并检测其对分化、MAPK信号转导和肌源性转录因子表达的影响。最近，我们通过基因工程使RMS细胞在分化过程中表达荧光蛋白。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 Tanner博士合作)，以评估分化和侵袭/转移之间的联系。此外，在与Roberto Weigert博士合作的工作中，我们发现RMS细胞系异种移植细胞在将细胞原位注射到舌头而不是后肢时，会自发转移到局部区域的淋巴和肺部。我们计划使用这个模型来研究MAPK通路和ASAP1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.