Involvement of Mutation-based Inhibition of β-Catenin Phosphorylation at Ser33 in the Malignant Progression of Lung (Pre)neoplastic Lesions Induced by N-nitrosobis(2-hydroxypropyl)amine in Male Fischer 344 Rats

In this study we investigated the Ser33 phosphorylation status of β-catenin protein in relation to genomic mutations in lung (pre)neoplastic lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Fischer 344 rats. Six-week-old animals received 2000 ppm of BHP in the drinking water for 8 weeks and were sacrificed 12 weeks thereafter. Histopathologically, 69 of 75 rats demonstrated multiple lung (pre)neoplastic lesions, classified into 27 slight and 33 advanced hyperplasias (preneoplasms) and 61 neoplasms, including adenomas, adenocarcinomas, and adenosquamous carcinomas. Nucleotide mutation analysis of the β-catenin gene detected a total of 33 mutations in 12 assessed lung (pre)neoplastic lesions. The mutations tended to accumulate in positions near the phosphorylation region of the gene, between codons 33 and 45. Immunohistochemical analysis showed β-catenin protein expression to be increased and its localization changed from the cell membrane to the cytoplasm and finally the nuclei with advancing malignancy of the lung lesions. In contrast, the expression of phosphorlyated β-catenin protein at Ser33 was weakened in lung (pre)neoplastic lesions compared to normal lung tissues. These results suggest that BHP-induced mutation of the β-catenin gene results in amino acid conversions in its product protein, which in turn lead to inhibition of phosphorylation of the protein and escape from protein degradation. These phenomena might contribute to the malignant progression of the lung (pre)neoplastic lesions, which start from the relatively early stage in lung carcinogenesis.