Studies of gene fusions in rhabdomyosarcoma

我们研究了在成肌细胞系统中 P3F 诱导的肿瘤发生过程中发生的分子变化，这可能最终使复发性肿瘤独立于融合蛋白。未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的比较揭示了原发肿瘤细胞和复发肿瘤细胞之间的许多差异。特别是，在缺乏 P3F 的情况下，原发性肿瘤细胞中 P3F 上调的大多数直接靶标在复发性肿瘤细胞中并未上调。 FGF8是复发性肿瘤中少数几个上调的P3F靶点之一，它编码成纤维细胞生长因子家族的成员，在涉及生长和肿瘤发生的多个信号通路的上游发挥作用。在表达 MYCN/P3F 的亲本和原发肿瘤细胞中，FGF8 受 P3F 上调，并且在复发肿瘤细胞中以不依赖 P3F 的方式以更高水平表达。我们研究了 FGF8 是否参与复发性肿瘤细胞的致癌表型。复发性肿瘤细胞中 FGF8 的 CRISPR-Cas9 敲除损害了体外转化活性和体内肿瘤发生，而亲代成肌细胞（用 MYCN 转导）中 FGF8 cDNA 的表达刺激了体外转化和体内肿瘤发生。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基。 FGF8蛋白存在于来自复发肿瘤细胞的条件培养基中，但不存在于亲本细胞中，并且来自复发肿瘤细胞的条件培养基能够转化亲本成肌细胞，而来自亲本细胞的培养基不具有这种作用。在条件培养基对亲本细胞转化的进一步测试中，亲本细胞中的FGF8过表达导致其条件培养基获得转化活性，而复发肿瘤细胞中的FGF8敲除或来自复发肿瘤细胞的培养基的FGF8免疫耗竭消除了该培养基的转化活性。为了研究 FGF8 在原发性肿瘤中的作用，我们检查了来自成肌细胞系统和人 FP RMS 系的原发性肿瘤细胞，它们均表现出 P3F 依赖性转化。当 P3F 被诱导时，原代肿瘤细胞表达 FGF8，并且大多数人 FP RMS 细胞系表达 FGF8。使用 CRISPR-Cas9 方法，FGF8 敲除会导致原发性肿瘤细胞和 FP RMS 系的增殖减少和转化丧失。我们还鉴定了 P3F 阳性 RH30 RMS 细胞系的一种变体，该细胞系自发地丧失了大部分 P3F 表达，导致细胞在培养物中生长但未转化。 FGF8表达构建体的转导导致体外转化活性恢复，而P3F表达没有任何变化。这些发现表明，FGF8 对于 P3F 的大部分致癌活性是必要且充分的，并强调了 FGF8 作为参与 P3F 依赖性致瘤性机制的下游 P3F 靶标的重要性。最后，这些综合结果表明，失调的 FGF8 表达可以维持不依赖于 P3F 的致瘤性，从而提供了不依赖于 P3F 的复发和对针对 P3F 的靶向治疗的耐药性的机制。

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.