Lee Sora, Qiao Jingbo, Paul Pritha, O'Connor Kathleen L, Evers Mark B, Chung Dai H
Departments of Pediatric Surgery, Vanderbilt University Medical Center1, Nashville, TN, USA.
Oncotarget. 2012 Dec;3(12):1576-87. doi: 10.18632/oncotarget.732.
Neuroblastomas express increased levels of gastrin-releasing peptide receptor (GRP-R). However, the exact molecular mechanisms involved in GRP-R-mediated cell signaling in neuroblastoma growth and metastasis are unknown. Here, we report that focal adhesion kinase (FAK), as a critical downstream target of GRP-R, is an important regulator of neuroblastoma tumorigenicity. We found that FAK expression correlates with GRP-R expression in human neuroblastoma sections and cell lines. GRP-R overexpression in SK-N-SH cells increased FAK, integrin α3 and β1 expressions and cell migration. These cells demonstrated flatter cell morphology with broad lamellae, in which intense FAK expression was localized to the leading edges of lamellipodia. Interestingly, FAK activation was, in part, dependent on integrin α3 and β1 expression. Conversely, GRP-R silencing decreased FAK as well as Mycn levels in BE(2)-C cells, which displayed a denser cellular morphology. Importantly, rescue experiments in GRP-R silenced BE(2)-C cells showed FAK overexpression significantly enhanced cell viability and soft agar colony formation; similarly, FAK overexpression in SK-N-SH cells also resulted in increased cell growth. These effects were reversed in FAK silenced BE(2)-C cells in vitro as well as in vivo. Moreover, we evaluated the effect of FAK inhibition in vivo. FAK inhibitor (Y15) suppressed GRP-induced neuroblastoma growth and metastasis. Our results indicate that FAK is a critical downstream regulator of GRP-R, which mediates tumorigenesis and metastasis in neuroblastoma.
神经母细胞瘤中胃泌素释放肽受体（GRP-R）水平升高。然而，GRP-R介导的细胞信号传导在神经母细胞瘤生长和转移中的确切分子机制尚不清楚。在此，我们报告粘着斑激酶（FAK）作为GRP-R的关键下游靶点，是神经母细胞瘤致瘤性的重要调节因子。我们发现，在人神经母细胞瘤切片和细胞系中，FAK表达与GRP-R表达相关。SK-N-SH细胞中GRP-R的过表达增加了FAK、整合素α3和β1的表达以及细胞迁移。这些细胞表现出更扁平的细胞形态，具有宽阔的片状伪足，其中强烈的FAK表达定位于片状伪足的前缘。有趣的是，FAK的激活部分依赖于整合素α3和β1的表达。相反，GRP-R沉默降低了BE(2)-C细胞中FAK以及Mycn的水平，这些细胞呈现出更致密的细胞形态。重要的是，在GRP-R沉默的BE(2)-C细胞中进行的拯救实验表明，FAK过表达显著增强了细胞活力和软琼脂集落形成；同样，SK-N-SH细胞中FAK过表达也导致细胞生长增加。在体外和体内，FAK沉默的BE(2)-C细胞中这些效应均被逆转。此外，我们评估了FAK抑制在体内的作用。FAK抑制剂（Y15）抑制了GRP诱导的神经母细胞瘤生长和转移。我们的结果表明，FAK是GRP-R的关键下游调节因子，其介导神经母细胞瘤的肿瘤发生和转移。
