KIF5B-RET基因重排发生在约1%的肺腺癌中。最近,抑制RET磷酸化的靶向药物已经在一些临床研究中进行了评估;然而,对这种基因融合在肺癌中的作用知之甚少。采用免疫组化法检测FOXA2蛋白在肺腺癌患者肿瘤组织中的表达。KIF5B-RET融合细胞以粘性形式增殖,并紧密地生长在可变大小的集落中。RET及其下游信号分子的表达,包括p-BRAF,p-ERK,和p-AKT,增加。在KIF5B-RET融合细胞中,胞内p-ERK的表达在胞浆中高于胞核中。两个转录因子,STAT5A和FOXA2在mRNA水平表现出显著不同的表达,最终被选中。p-STAT5A在细胞核和细胞质中高表达,而FOXA2蛋白的表达较低;然而,它在细胞核中比在细胞质中高得多。与FOXA2在RET重排野生型NSCLC中的表达(45.0%)相比,在大多数RET重排NSCLC中观察到高表达(3+)(94.4%)。同时,在2D细胞培养中,KIF5B-RET融合细胞从第7天开始延迟增加,仅在第9天增加了一倍。然而,注射KIF5B-RET融合细胞的小鼠肿瘤从第26天开始迅速增加。在细胞周期分析中,G0/G1期KIF5B-RET融合细胞在第4天增加(50.3±2.6%),而空细胞(39.3±5.2%;P=0.096)。细胞周期蛋白D1和E2表达减少,而CDK2表达略有增加。与空细胞相比,pRb和p21表达减少,TGF-β1mRNA高表达,蛋白质主要积累在细胞核中。TwistmRNA和蛋白表达增加,而SnailmRNA和蛋白表达降低。特别是,在用FOXA2siRNA处理的KIF5B-RET融合细胞中,TGF-β1mRNA表达明显降低,Twist1和SnailmRNA表达升高。我们的数据表明,KIF5B-RET融合细胞中的细胞增殖和侵袭性受STAT5A和FOXA2通过连续激活多个RET下游信号级联的上调调节,包括ERK和AKT信号通路。我们发现TGF-β1mRNA,在KIF5B-RET融合细胞中观察到显著的增量,在转录水平上由FOXA2调节。
KIF5B-RET gene rearrangement occurs in ~1% of lung adenocarcinomas. Recently, targeted agents that inhibit RET phosphorylation have been evaluated in several clinical studies; however, little is known about the role of this gene fusion in driving lung cancer. Immunohistochemistry was used to evaluate the expression of the FOXA2 protein in tumor tissues of patients with lung adenocarcinoma. KIF5B-RET fusion cells proliferated in a cohesive form and grew tightly packed with variable-sized colonies. The expression of RET and its downstream signaling molecules, including p-BRAF, p-ERK, and p-AKT, increased. In KIF5B-RET fusion cells, the intracellular expression of p-ERK was higher in the cytoplasm than in the nucleus. Two transcription factors,
STAT5A and FOXA2, exhibiting significantly different expressions at the mRNA level, were finally selected. p-
STAT5A was highly expressed in the nucleus and cytoplasm, whereas the expression of the FOXA2 protein was lower; however, it was much higher in the nucleus than in the cytoplasm. Compared with the expression of FOXA2 in the RET rearrangement-wild NSCLC (45.0%), high expression (3+) were observed in most RET rearrangement NSCLCs (94.4%). Meanwhile, KIF5B-RET fusion cells began to increase belatedly from day 7 and only doubled on day 9 in 2D cell culture. However, tumors in mice injected with KIF5B-RET fusion cells began to rapidly increase from day 26. In cell cycle analyses, the KIF5B-RET fusion cells in G0/G1 were increased on day 4 (50.3 ± 2.6%) compared with the empty cells (39.3 ± 5.2%; P = 0.096). Cyclin D1 and E2 expressions were reduced, whereas CDK2 expression slightly increased. pRb and p21 expression was diminished compared with the empty cells, TGF-β1 mRNA was highly expressed, and the proteins were accumulated mostly in the nucleus. Twist mRNA and protein expression was increased, whereas Snail mRNA and protein expression was decreased. Particularly, in KIF5B-RET fusion cells treated with FOXA2 siRNA, the expression of TGF-β 1 mRNA was remarkably reduced but Twist1 and Snail mRNA were increased. Our data suggest that cell proliferation and invasiveness in KIF5B-RET fusion cells are regulated by the upregulation of
STAT5A and FOXA2 through the continuous activation of multiple RET downstream signal cascades, including the ERK and AKT signaling pathways. We found that TGF-β1 mRNA, where significant increments were observed in KIF5B-RET fusion cells, is regulated at the transcriptional level by FOXA2.