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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005148-30
Application #
7965001
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
30
Fiscal Year
2009
Total Cost
$1,095,394
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Callahan, Robert; Mudunur, Uma; Bargo, Sharon et al. (2012) Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors. Oncotarget 3:1320-34
Klauzinska, Malgorzata; Baljinnyam, Bolormaa; Raafat, Ahmed et al. (2012) Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells. J Cell Physiol 227:1960-71
Baljinnyam, Bolormaa; Klauzinska, Malgorzata; Saffo, Saad et al. (2012) Recombinant R-spondin2 and Wnt3a up- and down-regulate novel target genes in C57MG mouse mammary epithelial cells. PLoS One 7:e29455
Raafat, Ahmed; Strizzi, Luigi; Lashin, Karim et al. (2012) Effects of age and parity on mammary gland lesions and progenitor cells in the FVB/N-RC mice. PLoS One 7:e43624
Sun, Youping; Klauzinska, Malgorzata; Lake, Robert J et al. (2011) Trp53 regulates Notch 4 signaling through Mdm2. J Cell Sci 124:1067-76
Chiluiza, David; Bargo, Sharon; Callahan, Robert et al. (2011) Expression of truncated eukaryotic initiation factor 3e (eIF3e) resulting from integration of mouse mammary tumor virus (MMTV) causes a shift from cap-dependent to cap-independent translation. J Biol Chem 286:31288-96
Raafat, Ahmed; Goldhar, Anita S; Klauzinska, Malgorzata et al. (2011) Expression of Notch receptors, ligands, and target genes during development of the mouse mammary gland. J Cell Physiol 226:1940-52
Bargo, Sharon; Raafat, Ahmed; McCurdy, David et al. (2010) Transforming acidic coiled-coil protein-3 (Tacc3) acts as a negative regulator of Notch signaling through binding to CDC10/Ankyrin repeats. Biochem Biophys Res Commun 400:606-12
Raafat, A; Lawson, S; Bargo, S et al. (2009) Rbpj conditional knockout reveals distinct functions of Notch4/Int3 in mammary gland development and tumorigenesis. Oncogene 28:219-30
Li, Sheng-Jian; Yen, Ten-Yang; Endo, Yoshimi et al. (2009) Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function. Cell Signal 21:916-25

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