HYPUSINATION IN INTESTINAL EPITHELIAL CELLS PREVENTS COLON CARCINOGENESIS ASSOCIATED WITH APC DELETION

Introduction: The enzyme deoxyhypusine synthase (DHPS) conjugates the N-terminal moiety of the polyamine spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form a unique amino acid, hypusine. This hypusination leads to EIF5A activation and facilitates translation of specific mRNAs. We recently reported that mice with deletion of Dhps in intestinal epithelial cells exhibit spontaneous colitis and are prone to colon tumorigenesis when treated with the carcinogen azoxymethane. Here, we investigated the role of hypusination in a model of sporadic colorectal cancer (CRC). Methods: C57BL/6 mice with tamoxifen (TAM)-inducible disruption of Apc were used by crossing the intestinal epithelial cell-specific driver CDX2P-CreER^T2 to Apc^fl/fl mice. These animals were crossed to C57BL/6 Dhps^fl/fl mice to generate inducible epithelial disruption of both Apc and Dhps (Apc^fl/fl;Dhps^fl/fl). Male mice were injected with 4.5 mg/kg TAM or not, and clinical parameters monitored. After 35 days, mice were sacrificed. Tumors in the proximal and distal colon were counted and measured. Colons were Swiss-rolled for histology. Genotyping and gene expression were determined by PCR and RT-qPCR, respectively. Results: Deletion of Apc was only detected in tumor tissues of Apc^fl/fl and Apc^fl/fl;Dhps^fl/fl mice treated with TAM, while the Dhps mutant allele was found in the non-tumor and tumor tissues of Apc^fl/fl;Dhps^fl/fl mice, but not Apc^fl/fl mice. We found that 0/28 and 4/26 Apc^fl/fl and Apc^fl/fl;Dhps^fl/fl mice died, respectively, during the 35 days after TAM (P=0.0329; Mantel-Cox test). TAM-treated Apc^fl/fl;Dhps^fl/fl mice lost more weight than animals with Apc deletion only (P<0.0001). At sacrifice, 28.6% of Apc^fl/fl mice did not develop tumors after TAM, whereas all Apc^fl/fl;Dhps^fl/fl mice developed tumors (P<0.01). These animals had more tumors (P<0.001) and more total tumor burden (P<0.01) than TAM-treated Apc^fl/fl mice. Tumor number and burden were significantly increased in Apc^fl/fl;Dhps^fl/fl mice in both the proximal and distal colon. There were more mice with histologic adenomas (P<0.001) and with low-grade or high-grade dysplasia (P=0.0195) in the Apc^fl/fl;Dhps^fl/fl + TAM group versus the Apc^fl/fl + TAM group. Expression of the β-catenin-target genes Myc, Axin2, and Mmp7 was increased at the same level in the tumors from TAM-treated Apc^fl/fl;Dhps^fl/fl and Apc^fl/fl mice. Conclusions: Our data indicate that tumorigenesis associated with Apc deletion is worsened by the ablation of the Dhps gene in intestinal epithelial cells. However, when tumors are formed, the oncogenic signaling is not dependent on DHPS activity. Therefore, epithelial hypusination is essential to dampen the initiation of adenoma formation and strategies to enhance hypusination, such as by spermidine supplementation, have potential for chemoprevention of CRC.