Abstract 4480: Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration

The Iroquois homeobox 1 (IRX1) belongs to Iroquois homeobox family and plays an important role during embryonic development. Recent studies have suggested that IRX1 acts as a tumor suppressor in gastric cancer and head and neck carcinoma. The zebrafish allows easy genetic modification at a lower cost, thus being a valuable model for cancer biology. For functional annotation we established homozygous knockout mutants of Irx1a and Irx1b and followed to evaluate long-term phenotypes. Homozygous knockout zebrafish for either Irx1a or Irx1b are vital and survived long enough to see long-term phenotypes. However, majority of the homozygotes for both Irx1a /b were severely malformed, could not survive longer than 6 month, and were not fertile. Surprisingly, hyperplasia and tumorigenesis developed in homozygous Irx1a mutants in multiple organs, including bile duct, intestine, kidney, ovary, and testis where irx1 genes are expressed in adults when evaluated by in situ hybridization. Bile duct hyperplasia or tumor was the most common phenotype, occurring in half and two thirds at 6 and 12 months, respectively. Irx1b mutants also developed similar tumorigenesis but showed less prevalent phenotypes than Irx1a mutants, suggesting functional preservation and redundancy. Analysis of differential genes (Wild type vs. Irx1a/b knockout) by cRNA microarray (Agilent Zebrafish v3 GE 4X44 Microarrays) revealed 687 up- and 963 downregulated genes by Irx1a/b knockout. Significant canonical pathways were cyclins and cell cycle regulation, mitotic roles of polo-like kinase, FXR/RXR activation, estrogen-mediated S-phase entry, and cell cycle control of chromosomal replication, suggesting aberrant cell cycle regulation as an important mechanism of tumorigenesis. Real-time RT PCR validated the differential expression of cell cycle-related genes, especially genes involved in G2/M phase progression. Differential genes included CCND1, ARAF, CDKN2C, and FGFR1 among the upregulated genes, and ANAPC7, CCNA2, CCNB1, 2, HAUS1, 3, 6, CDC7, CDC20, CDCA8, FBXO5, 32, 43 and PLK3 among the downregulated genes. Overexpression by transfection decreased cell proliferation in cholangiocarcinoma cells (SNU-1196 and HuCCT1) without altering sensitivity to H2O2-induced apoptosis on annexin V flow cytometry. Western blot revealed decreased expression of CCND1, CCNE, CCNA1, CCNB1 by IRX1 expression. Flow cytometry using propidium iodide and Alexa647-anti-pHH3 showed decreased mitotic fraction by IRX1 expression. On cell cycle synchronization and release using hydroxyuria and nocodazole showed slight delay in G1-S progression and marked delay in G2-M progression and entry into G1. In conclusion, this study identified Irx1 gene as an important tumor-suppressor gene at various organs, and knockout or Irx1 leads to tumorigenesis by altering cell cycle regulation, especially at G2/M phase. Citation Format: Inhye Jung, Dawoon E. Jung, DoHee Kim, Dong Hee Kim, Yong Yoon Chung, Seung Woo Park. Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4480.