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, 21 (10), 1003-1014

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

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

In Hye Jung et al. Neoplasia.

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.

Figures

Figure 1
Figure 1
Generation of Irx1 knock-out zebrafish. Targeted gene mutation was performed by TALEN and CRISPR/Cas9 knockout methods for Irx1a (A) and for Irx1b gene (B), respectively. Frameshift mutation was caused by the knockout strategies. Insertion or deletion of the nucleotides was indicated by red letterings. (C) Three-day-old embryos of the zebrafish mutants. While either Irx1a or Irx1b homozygote mutants did not show abnormal morphology during embryonic development, some siblings (homozygote for both Irx1a and Irx1b) from the breeding of Irx1a−/−/1b+/− and Irx1a+/−/1b−/− showed severe morphologic abnormality. This suggests that majority of homozygote mutants of Irx1a and Irx1b are developmentally defective. A few of Irx1a−/−/1b−/− zebrafish, however, were found to survive up to 3 months of age but were not fertile and died within 6 months of age.
Figure 2
Figure 2
Expression of zebrafish Irx1 gene. (A) Whole mount ISH expression in embryogenic zebrafish. Irx1 gene expression is found highly in brain area at early developmental stage (see 48 hpf, hours postfertilizaton) and later distributed to internal organs (5 dpf, days postfertilization). (B-D) Images of ISH in 3-month-old adult tissue sections. (B) Expression in intestine and kidney. Both Irx1a and Irx1b genes are expressed at the crypt base of intestines and renal tubular cells. (C) ISH observation of Irx1 gene expression in reproductive organs, typically in primordial germ cells and oocytes (insets indicate 40× low-power images). (D) Expression in the liver and bile ducts. While none of the hepatocytes express Irx1 gene, meticulous examination revealed obvious expression in the biliary tree including extrahepatic (arrows) and intrahepatic bile ducts (arrowheads).
Figure 3
Figure 3
Abnormal phenotypes developed in multiple organs caused by Irx1 knockout in zebrafish (long-term observation up to 18 months). (A, B, and E) Control. (C and D, F-L) Irx1a knockout (Irx1b data not shown due to similar images). Hyperplasia or tumor developed in the multiple organs where certain degree of Irx1 expression was noted on the ISH experiment. Note the testicular (black arrowheads), renal (red arrowheads), and ovarian tumors (arrow). (L) Cystic tumor of ovary (Inset indicates IHC image of CK19).
Figure 4
Figure 4
Bile duct phenotypes and tumor invasions found in the Irx1a knockout (Irx1b data not shown due to similar images). (A and B) A large liver mass (boundary by red arrowheads) showing positivity for PCNA. The tumors often invaded the liver (C and K), intestine (E), and pancreas (D, G, and L). The bile duct tumor was often multifocal (H and I) (black arrowheads). (M) Tumor cells revealed robust expression of PCNA suggesting enhanced proliferation. (N) IHC for pancytokeratin in tumor cells. (O and P) ISH images for Irx1 mRNA showing positive expression of both Irx1a and Irx1b genes in the tumor cells. As the bile duct cells in the knockout lines also produce Irx1 mRNAs that harbor mutations, the positivity on ISH is supportive finding that the tumors are originated from bile duct cells. P, pancreas; L, liver.
Figure 5
Figure 5
cRNA microarray analysis. (A and B) Differential genes were explored by microarray (Agilent Gene Chip) analysis using the visceral organs of 3-month-old zebrafish from AB, Irx1a−/−, and Irx1a−/−/b−/−. The microarray revealed 687 upregulated 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. The upper colored bar indicates the gene expression, and the metric used was Euclidean distance, with average linkage for distance between clusters. (C) Real-time RT-PCR recapitulated the microarray results.
Figure 6
Figure 6
Transduction of human IRX1 gene into human cholangiocarcinoma cell lines. (A) Western blot experiment showed Irx1 expression in four different cholangiocarcinoma cell lines. Among these, HuCCT1 (high Irx1 expression cell line) and SNU1196 (low Irx1 expression cell line) were selected for overexpression study. (B) Lentiviral transduction of IRX1-GFP fusion gene (right). Note the nuclear localization of IRX1-GFP in Irx1 transduced cells whereas GFP-alone transduced cell showed diffused expression (left). (C) Western blot experiment confirmed the transduced protein, tGFP. Fused IRX1-tGFP protein bands are seen with larger size. (D) Cell proliferation assay. The results showed that IRX1 overexpression resulted in decreased cellular proliferation in both HuCCT1 and SNU1196 cell lines. (E) Flow cytometry analysis for cell death induced by H2O2 treatment showing increased susceptibility to H2O2 in the Irx1-expressing cells. (F) Flow cytometry for cell cycle change. IRX1-expressing cells showed decreased mitotic fraction measured by phosphohistone H3 (pHH3).
Figure 7
Figure 7
Cell cycle synchronization study. (A and B) Cell synchronization was obtained by treatment with hydroxyuria and nocodazole, respectively, for G1/S and G2/M phase arrest. Arrest was then released, detecting the cell cycle progression. Dotted red lines indicate 2N and 4N cells. The result showed that mitotic exit is markedly delayed by IRX1 overexpression, while S phase progression is slightly delayed. Numbers in upper and lower lines indicate G1/S and G2/M fractions, respectively. (C) The mitosis marked by pHH3. G2/M phase-blocked cells were released and stained for pHH3 at indicated time points. Yellow to white cells (red arrowheads) represent pHH3- and GFP-positive cells. Numbers of percentage indicate fractions of the pHH3-positive cells. At 3 and 6 hours after release, higher cell fractions are still positive for pHH3 in IRX1-expressing cells. (D) Western blot analysis against cyclins. The result showed that levels of various cyclin expression are decreased by IRX1 expression.
Supplementary Fig. 1
Supplementary Fig. 1
cRNA microarray analysis. High-resolution image of Figure 5A.

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