Iroquois Homeobox 1 Acts as a True Tumor Suppressor in Multiple Organs by Regulating Cell Cycle Progression

Abstract

Iroquois homeobox 1 (IRX1) belongs to the Iroquois homeobox family known to play an important role during embryonic development. Interestingly, however, recent studies have suggested that IRX1 also acts as a tumor suppressor. Here, we use homozygous knockout mutants of zebrafish to demonstrate that the IRX1 gene is a true tumor suppressor gene and mechanism of the tumor suppression is mediated by repressing cell cycle progression. In this study, we found that knockout of zebrafish Irx1 gene induced hyperplasia and tumorigenesis in the multiple organs where the gene was expressed. On the other hands, overexpression of the IRX1 gene in human tumor cell lines showed delayed cell proliferation of the tumor cells. These results suggest that the IRX1 gene is truly involved in tumor suppression. In an attempt to identify the genes regulated by the transcription factor IRX1, we performed microarray assay using the cRNA obtained from the knockout mutants. Our result indicated that the highest fold change of the differential genes fell into the gene category of cell cycle regulation, suggesting that the significant canonical pathway of IRX1 in antitumorigenesis is done by regulating cell cycle. Experiment with cell cycle blockers treated to IRX1 overexpressing tumor cells showed that the IRX1 overexpression actually delayed the cell cycle. Furthermore, Western blot analysis with cyclin antibodies showed that IRX1 overexpression induced decrease of cyclin production in the cancer cells. In conclusion, our in vivo and in vitro studies revealed that IRX1 gene functionally acts as a true tumor suppressor, inhibiting tumor cell growth by regulating cell cycle.

Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

“…Homeobox domains are characteristic protein folds that bind DNA and regulate the expression of target genes during development and differentiation. Jung et al analyzed the function of IRX1 in the development of zebrafish and they demonstrated that IRX1 acts a tumor suppressor in multiple organs by regulating the cell cycle progression [20]. Here, we revealed by GSEA that IRX1 expression is negatively correlated with E2F-and Myc-targets in several lung cancer data sets (Table 1).…”

“…The promoter region of IRX1 is located in a CpG island ( Figure 1b) and hypermethylation of IRX1 has been found in gastric cancer [15], head and neck squamous carcinoma [16], and other cancer entities [17][18][19]. Functional data indicate that IRX1 acts as an epigenetically regulated tumor suppressor in different types of cancer [15,20].…”

Genetic Engineering of Zebrafish in Cancer Research

“…Despite a recent implementation, the CRISPR/Cas9 system has been already applied to the zebrafish cancer research field [82][83][84][85][86][87][88][89][90] (Table 4). Iroquois homeobox 1 (IRX1) is an homeobox-containing transcription factor playing a role in embryonic development and suspected to act as a tumor suppressor gene in head and neck squamous cell carcinoma, gastric cancers and glioma [106][107][108][109].…”

“…In zebrafish, Irx1 is encoded by two ohnologues irx1a and irx1b. The group of Seung Woo Park (Yonsei University College of Medicine, Seoul, Korea) used the TALEN technology to inactivate irx1a, while irx1b mutants were generated using the CRISPR/Cas9 system [82]. Homozygous mutants for both irx1a and irx1b (irx1a -/-; irx1b -/-) are malformed and die within 6 months of age.…”

“…In sum, programmable site-specific endonucleases have been successfully used in zebrafish (i) to generate models harboring mutations in tumor suppressor genes (e.g., nf1, rb1; [76,78]); (ii) to engineer novel genetic alterations in tumor suppressor genes (e.g., tp53 del/del ; [81]); (iii) to demonstrate the tumor suppressor function of novel candidate genes (e.g., irx1, spred1, arid1a [82,83,87]); (iv) to characterize the role of genes involved in tumor development (e.g., twist3; [90]); (v) as well as to investigate the cooperation between different mutations in the tumorigenesis onset (e.g., tp53 and nf1 with atrx or suz12 [85,86]). The use of programmable site-specific endonucleases can be applied to the engineering of zebrafish mutant lines [76,77], but also to the rapid generation of F0 mosaic somatic mutants [78,79,83,84,90].…”