Notch receptors and their ligands play critical roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal controlling mechanism is mediated by physical interactions between Mdm2 and Notch4 and suggest the existence of a trimeric complex between Trp53, Notch and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4 induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 plays a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland. Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, 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. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into nave mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, 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/beta-catenin pathway, or short hairpin RNA targeting beta-catenin expression demonstrated that the invasive activity was not mediated by beta-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the beta-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. Integration of mouse mammary tumor virus (MMTV) at the common integration siteInt6 occurs in the gene encoding eIF3e, the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation, but by an unknown mechanism. Since eIF3e makes up at least part of the binding site for eIF4G, we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G, signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP, c-Myc, CYR61, and Pim-1, which are translated in a cap-independent manner, were shifted to heavier polysomes while mRNAs encoding GAPDH, actin, L32, and L34, which are translated in a cap-dependent manner, were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between upregulation of cap-independent translation and MMTVinduced tumorigenesis contrasts with the well established model for malignant transformation involving upregulation of highly cap-dependent translation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005148-32
Application #
8348867
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
32
Fiscal Year
2011
Total Cost
$1,014,966
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
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
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
Watanabe, Kazuhide; Nagaoka, Tadahiro; Lee, Joseph M et al. (2009) Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1. J Cell Biol 187:343-53

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