Li Fangda, Meyer April N, Peiris Malalage N, Nelson Katelyn N, Donoghue Daniel J
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0367, USA.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0367, USA; UCSD Moores Cancer Center and Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0367, USA.
Transl Oncol. 2020 Dec;13(12):100853. doi: 10.1016/j.tranon.2020.100853. Epub 2020 Aug 24.
Chromosomal translocations such as t(10;12)(q26,q12) are associated with intrahepatic cholangiocarcinoma, a universally fatal category of liver cancer. This translocation creates the oncogenic fusion protein of Fibroblast Growth Factor Receptor 2 joined to Periphilin 1. The aims of this study were to identify significant features required for biological activation, analyze the activation of downstream signaling pathways, and examine the efficacy of the TKIs BGJ398 and TAS-120, and of the MEK inhibitor Trametinib.
These studies examined FGFR2-PPHLN1 proteins containing a kinase-dead, kinase-activated, or WT kinase domain in comparison with analogous FGFR2 proteins. Biological activity was assayed using soft agar colony formation in epithelial RIE-1 cells and focus assays in NIH3T3 cells. The MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways were examined for activation. Membrane association was analyzed by indirect immunofluorescence comparing proteins altered by deletion of the signal peptide, or by addition of a myristylation signal.
Biological activity of FGFR2-PPHLN1 required an active FGFR2-derived tyrosine kinase domain, and a dimerization domain contributed by PPHLN1. Strong activation of canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT signaling pathways was observed. The efficacy of the tyrosine kinase inhibitors BGJ398 and TAS-120 was examined individually and combinatorially with the MEK inhibitor Trametinib; heterogeneous responses were observed in a mutation-specific manner. A requirement for membrane localization of the fusion protein was also demonstrated.
Our study collectively demonstrates the potent transforming potential of FGFR2-PPHLN1 in driving cellular proliferation. We discuss the importance of sequencing-based, mutation-specific personalized therapeutics in treating FGFR2 fusion-positive intrahepatic cholangiocarcinoma.
染色体易位，如t(10;12)(q26,q12)，与肝内胆管癌相关，肝内胆管癌是一种普遍致命的肝癌类型。这种易位产生了与外周蛋白1连接的成纤维细胞生长因子受体2的致癌融合蛋白。本研究的目的是确定生物激活所需的显著特征，分析下游信号通路的激活情况，并研究酪氨酸激酶抑制剂BGJ398和TAS-120以及MEK抑制剂曲美替尼的疗效。
这些研究检测了含有激酶失活、激酶激活或野生型激酶结构域的FGFR2-PPHLN1蛋白，并与类似的FGFR2蛋白进行比较。使用上皮RIE-1细胞的软琼脂集落形成试验和NIH3T3细胞的焦点试验检测生物活性。检测MAPK/ERK、JAK/STAT3和PI3K/AKT信号通路的激活情况。通过间接免疫荧光分析膜结合情况，比较通过缺失信号肽或添加肉豆蔻酰化信号而改变的蛋白质。
FGFR2-PPHLN1的生物活性需要一个由FGFR2衍生的活性酪氨酸激酶结构域，以及一个由PPHLN1贡献的二聚化结构域。观察到经典的MAPK/ERK、JAK/STAT3和PI3K/AKT信号通路有强烈激活。分别检测了酪氨酸激酶抑制剂BGJ398和TAS-120以及与MEK抑制剂曲美替尼联合使用时的疗效；观察到以突变特异性方式出现的异质性反应。还证明了融合蛋白膜定位的必要性。
我们的研究共同证明了FGFR2-PPHLN1在驱动细胞增殖方面具有强大的转化潜力。我们讨论了基于测序的、突变特异性个性化疗法在治疗FGFR2融合阳性肝内胆管癌中的重要性。
