Studies of gene fusions in rhabdomyosarcoma

横纹肌肉瘤基因融合的研究

• 批准号: 10486830
• 负责人: Frederic Barr
• 金额: $ 70.45万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486830
• 关键词: Animals; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Cell Culture System; Cell Culture Techniques; Cell Line; Cell fusion; Cells; Childhood; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complementary DNA; Conditioned Culture Media; Development; Doxycycline; Event; Excision; FGF8 gene; FOXO1A gene; Family; Fibroblast Growth Factor; Fusion Oncogene Proteins; Gene Fusion; Genes; Genetic Transcription; Goals; Growth; Human; Impairment; In Vitro; Knock-out; Laboratory mice; MYCN gene; Maintenance; Malignant Neoplasms; Modeling; Molecular; Myoblasts; Neoplasm Metastasis; Oncogenes; Oncogenic; PAX3 gene; Pathogenicity; Pathway interactions; Patients; Phenotype; Point Mutation; Population; Primary Neoplasm; Proteins; Recovery; Recurrence; Recurrent tumor; Resistance; Rhabdomyosarcoma; Role; Signal Pathway; System; Testing; Tumorigenicity; Variant; autocrine; cDNA Expression; experimental study; genetic analysis; genome-wide; in vivo; member; neoplastic cell; novel; overexpression; resistance mechanism; soft tissue; targeted treatment; therapeutic target; transcription factor; translational approach; tumor; tumor progression; tumorigenesis; tumorigenic

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.

我们研究了成肌细胞系统中P3 F诱导的肿瘤发生过程中发生的分子变化，这些变化最终可能使复发性肿瘤独立于融合蛋白。比较未处理的亲本成肌细胞与复发肿瘤细胞和表达MYCN/P3 F的原发性肿瘤细胞的表达谱，揭示了原发性和复发性肿瘤细胞之间的许多差异。特别是，在原发性肿瘤细胞中由P3 F上调的大多数直接靶点在不存在P3 F的复发性肿瘤细胞中不上调。在复发性肿瘤的子集中上调的少数P3 F靶点之一是FGF 8，其编码成纤维细胞生长因子家族的成员，其在参与生长和肿瘤发生的几种信号传导途径的上游起作用。FGF 8在表达MYCN/P3 F的亲本和原发肿瘤细胞中被P3 F上调，并且以P3 F非依赖性方式在复发肿瘤细胞中以更高水平表达。我们研究了FGF 8是否参与复发性肿瘤细胞的致癌表型。复发性肿瘤细胞中FGF 8的CRISPR-Cas9敲除损害了体外转化活性和体内肿瘤发生，而亲本成肌细胞（用MYCN转导）中FGF 8 cDNA的表达刺激了体外转化和体内肿瘤发生。为了确定FGF 8是否以自分泌方式起作用，我们研究了来自这些细胞系的条件培养基。FGF 8蛋白存在于复发肿瘤细胞的条件培养基中，但不存在于亲代细胞中，并且复发肿瘤细胞的条件培养基能够转化亲代成肌细胞，而亲代细胞的培养基则不具有这种作用。在条件培养基对转化亲本细胞的进一步测试中，亲本细胞中的FGF 8过表达导致其条件培养基获得转化活性，而复发性肿瘤细胞中的FGF 8敲除或复发性肿瘤细胞中培养基的FGF 8免疫耗竭消除了该培养基的转化活性。为了研究FGF 8在原发性肿瘤中的作用，我们检测了来自成肌细胞系统和人FP RMS系的原发性肿瘤细胞，这两种细胞都显示出P3 F依赖性转化。当P3 F被诱导时，原代肿瘤细胞表达FGF 8，并且大多数人FP RMS细胞系表达FGF 8。使用CRISPR-Cas9方法，FGF 8敲除导致原代肿瘤细胞和FP RMS系中增殖减少和转化丧失。我们还鉴定了P3 F阳性RH 30 RMS细胞系的一种变体，其自发地丧失了大部分P3 F表达，导致细胞在培养物中生长但不转化。FGF 8表达构建体的转导导致体外转化活性的恢复，而P3 F表达没有任何变化。这些发现表明FGF 8对于P3 F的大部分致癌活性是必需的和足够的，并且突出了FGF 8作为参与P3 F依赖性致瘤性机制的下游P3 F靶的重要性。最后，这些综合结果表明，FGF 8表达失调可以维持P3 F非依赖性致瘤性，从而提供了P3 F非依赖性复发和对针对P3 F的靶向治疗的抗性的机制。