Abstract 1066: Wee1 deficiency in the mammary gland results in genomic instability and tumorigenesis by affecting cell cycle coordination and mitotic spindle morphogenesis

Abstract

Abstract Previous studies have shown that tyrosine kinase Wee1 controls the timing of entry into mitosis both in fission and budding yeast cells by working as a cell-size checkpoint protein, whereas in Drosophila embryogenesis it regulates mitotic entry during the first divisions that occur in a common cytoplasm called a syncytium. However, the role of Wee1 in somatic cells in mammals has not yet been investigated. The objective of our study was to unravel the effect of Wee1 deletion specifically in the mammary gland by using a Cre-loxP system to disrupt the kinase activity of Wee1 by deleting exons 9 and 10. Since Wee1 is implicated in controlling mitotic entry, first we examined the cell cycle progression. Our data revealed that Wee1 deficient mammary glands displayed higher levels of BrdU-stained cells compared to wild type in the absence of apoptosis and this could account for the enhanced ductal branching observed. When mammary sections were stained for both phosphorylated histone H3 (phospho-H3), and Aurora B, higher number of cells from Wee1 mutant mammary glands showed the characteristic large punctate foci at the periphery of the nucleus, implying that these cells are cycling through mitosis. Interestingly, majority of these phospho-H3 positive cells displayed BrdU incorporation within the same nucleus. In contrast no cells were costained for BrdU and phospho-p27, a marker of late G1. This observation indicates that Wee1 mutated cells prematurely enter mitosis prior to the completion of S-phase. The miscoordination of cell cycle in the mutated mammary gland triggers the DNA damage response (DDR) pathway since we noticed increased levels of DNA damage foci formation relative to wild-type tissues as revealed by indirect immunofluorescence with antibodies against mouse 53BP1 and phospho-H2AX. Using time lapse microscopy to follow cell behavior, we found that 30% of Wee1 mutant MEF cells in mitosis arrested at metaphase and suffered mitotic abortion. We further showed that Wee1 mutated cells exhibited chromosome condensation and segregation problems, mitotic spindle defects and centrosome inactivation. These phenotypes could contribute to genomic instability by increasing the number of aneuploid/polyploid cells. Indeed, Wee1 haploinsufficincy in the mammary gland is sufficient to induce tumorigenesis with a median time to tumor latency of about 20 months. In accordance with the role of Wee1 in completing mitosis chromosome spreads of primary tumor cells showed that majority of the cells exhibited variation in ploidy levels with near tetraploid to hypertetraploid cells. In conclusion, these data suggest that Wee1 coordinates distinct cell-division events in somatic cells to allow correct segregation of genetic information into daughter cells and maintenance of genomic integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1066.