We obtained evidence suggesting that Trp53 directly influences the steady state level of the Notch4 intracellular domain (NICD4) through the action of MDM2. Using cancer cell culture models we explored the relationship between Trp53, MDM2 and Notch and document an antagonistic relationship between Notch and Trp53, both at a molecular as well as a functional level, consistent with previous reports. Our data support a direct physical interaction between Notch and Trp53 and suggest a model wherein Trp53 inhibits Notch signaling through a post-translational mechanism involving the formation of a Notch-MDM2 that leads to Notch ubiquitination and its degradation. NICD also can upregulate MDM2 mRNA levels leading to the degradation of Trp53. Our study raises the possibility that Trp53 plays a pivotal role in the suppression of Notch-associated tumorigenesis since 30% (4/14) of the mammary tumors in NICD4 transgenic mice as well as 14% (1/7) NICD1 mammary tumors have Trp53 mutations whereas, WAP-INT6, and WAP-CRIPTO mammary tumors have no Trp53 mutations. Currently a manuscript is being prepared describing this work. Rspo2 was identified as a novel common integration site for the mouse mammary tumor virus. Here we show that Rspo2 potentiates Wnt/β-catenin signaling in mouse mammary epithelial cells. Stable expression of Rspo2 in mammary epithelial lines altered their morphology and slowed their rate of proliferation on plastic in contrast to Wnt1, which enhanced cell growth. Co-expression of both factors resulted in an intermediate phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three- dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/β-catenin pathway, or short hairpin RNA targeting β-catenin expression demonstrated that the invasive activity was not mediated by β-catenin. Individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Our results indicate that Rspo2 and Wnt1 individually have contrasting effects on mammary epithelial cell growth. While they act synergistically in the β-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. Currently a manuscript is being prepared describing this work.
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