Studies of gene fusions in rhabdomyosarcoma

我们研究了在P3F诱导成肌细胞系统肿瘤形成过程中发生的分子变化，最终可能使复发的肿瘤不依赖于融合蛋白。比较未经处理的亲代成肌细胞与复发肿瘤细胞和表达MYCN/P3F的原发肿瘤细胞的表达谱发现，原发肿瘤细胞和复发肿瘤细胞之间存在许多差异。特别是，在没有P3F的情况下，原发肿瘤细胞中P3F上调的大多数直接靶点在复发肿瘤细胞中没有上调。FGF8是在部分复发肿瘤中上调的为数不多的P3F靶点之一，它编码成纤维细胞生长因子家族的一个成员，该家族的功能位于几个参与生长和肿瘤发生的信号通路的上游。FGF8在表达MYCN/P3F的亲代和原代肿瘤细胞中被P3F上调，在复发的肿瘤细胞中以P3F不依赖的方式高水平表达。我们研究了FGF8是否与复发肿瘤细胞的致癌表型有关。CRISPR-Cas9基因敲除复发性肿瘤细胞中的FGF8基因可抑制体外转化活性和体内成瘤作用，而在亲代成肌细胞(转导MYCN)中表达FGF8基因则可促进体外转化和体内成瘤。为了确定FGF8是否以自分泌方式发挥作用，我们研究了这些细胞系的条件培养液。FGF8蛋白存在于复发性肿瘤细胞的条件培养液中，但不存在于亲代细胞中，复发性肿瘤细胞的条件培养液能够转化亲代成肌细胞，而亲代细胞的条件培养液则不具有这种作用。在亲本细胞条件培养液的进一步转化试验中，FGF8在亲本细胞中的过表达导致其条件培养液的转化活性增强，而在复发肿瘤细胞中FGF8基因敲除或FGF8免疫耗竭使该条件培养液的转化活性丧失。为了研究FGF8在原发肿瘤中的作用，我们研究了来自成肌细胞系统和人FP RMS系的原代肿瘤细胞，这两种细胞都表现出P3F依赖的转化。原代肿瘤细胞在P3F诱导时表达FGF8，大多数人FP RMS细胞系表达FGF8。使用CRISPR-Cas9方法，FGF8基因敲除导致原代肿瘤细胞和FP RMS系的增殖减少和转化丧失。我们还发现了P3F阳性的RH30 RMS细胞系的一个变种，它自发地失去了大部分P3F的表达，导致细胞在培养中生长但不转化。转导FGF8表达载体可在不改变P3F表达的情况下恢复体外转化活性。这些发现表明，FGF8对P3F的大部分致癌活性是必要和充分的，并突显了FGF8作为下游P3F靶点的重要性，参与了P3F依赖的致瘤机制。最后，这些联合结果表明，解除调控的FGF8表达可以维持P3F非依赖性致瘤性，从而为P3F非依赖性复发和针对P3F的靶向治疗耐药提供了一种机制。

We investigated the molecular changes occurring during P3F-induced tumorigenesis in the myoblast system that may ultimately allow a recurrent tumor to be independent of the fusion protein. Comparison of expression profiles of untreated parental myoblasts with recurrent tumor cells and MYCN/P3F-expressing primary tumor cells revealed numerous differences between primary and recurrent tumor cells. In particular, most direct targets up-regulated by P3F in primary tumor cells were not up-regulated in recurrent tumor cells in the absence of P3F. One of the few P3F targets up-regulated in a subset of recurrent tumors is FGF8, which encodes a member of the fibroblast growth factor family that functions upstream of several signaling pathways involved in growth and oncogenesis. FGF8 is up-regulated by P3F in MYCN/P3F-expressing parental and primary tumor cells, and is expressed at higher levels in recurrent tumor cells in a P3F-independent manner. We investigated whether FGF8 is involved in the oncogenic phenotype of recurrent tumor cells. CRISPR-Cas9 knockout of FGF8 in recurrent tumor cells impaired in vitro transforming activity and in vivo tumorigenesis whereas expression of an FGF8 cDNA in parental myoblasts (transduced with MYCN) stimulated in vitro transformation and vivo tumorigenesis. To determine if FGF8 is acting in an autocrine fashion, we studied conditioned medium from these cell lines. FGF8 protein is present in conditioned medium from recurrent tumor cells but not parental cells, and conditioned medium from recurrent tumor cells is able to transform parental myoblasts whereas medium from parental cells does not have this effect. In further tests of conditioned medium on transformation of parental cells, FGF8 overexpression in parental cells results in gain of transforming activity by its conditioned medium whereas FGF8 knockout in recurrent tumor cells or FGF8 immunodepletion of medium from recurrent tumor cells abrogates transforming activity of this medium. To study the role of FGF8 in primary tumors, we examined primary tumor cells from the myoblast system and human FP RMS lines, which both show P3F-dependent transformation. The primary tumor cells express FGF8 when P3F is induced and most human FP RMS cell lines express FGF8. Using a CRISPR-Cas9 approach, FGF8 knockout results in decreased proliferation and loss of transformation in primary tumor cells and FP RMS lines. We also identified a variant of the P3F-positive RH30 RMS cell line which spontaneously lost most P3F expression, resulting in cells which grow in culture but are not transformed. Transduction of a FGF8 expression construct resulted in recovery of in vitro transforming activity without any change in P3F expression. These findings show that FGF8 is necessary and sufficient for much of the oncogenic activity of P3F, and highlight the importance of FGF8 as a downstream P3F target involved in the mechanism of P3F-dependent tumorigenicity. Finally, these combined results indicate that deregulated FGF8 expression can sustain P3F-independent tumorigenicity, thus providing a mechanism for P3F-independent recurrence and resistance to targeted therapy directed against P3F.