FOXA2 and STAT5A regulate oncogenic activity of KIF5B-RET fusion

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.