Notch functions as an oncogene or tumor inhibitor in various cancers, and decreases in Notch2 expressionare associated with increasing grade of human breast cancer. We constitutively activated Notch signaling with intracellular domain (ICD) expression in the human adenocarcinoma line MDA-MB-231. Notch2 signaling increased apoptosis, whereas Notch4ICD (int3) significantly increased cell proliferation and growth. Cells with activated Notch2 or Notch4 were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth was significantly altered depending on the receptor activated. Notch2ICD potently suppressed tumor take and growth, leading to a 60% decrease in tumors and significantly smaller, necrotic tumors. Despite this, Notch2ICD tumors were highly vascularized, although the vessels were smaller and comprised a more immature network compared with Notch4ICD tumors. Notch4ICD tumors were highly aggressive and well vascularized, indicating a role for Notch4 signaling in the promotion of the malignant phenotype in addition to its transforming ability. Although both NotchICD groups expressed angiogenic factors, Notch4ICD had selective vascular endothelial growth factor-D in both tumor and host stroma, suggesting a differential regulation of cytokines that may impact vascular recruitment and autocrine tumor signaling. Our results demonstrate that Notch2 signaling is a potent inhibitory signal in human breast cancer xenografts. (Am J

Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGFdependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when coexpressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels.

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.

BACKGROUND Estrogen receptor (ER) remains one of the most important biomarkers for breast cancer subtyping and prognosis, and comparative genome hybridization has greatly contributed to the understanding of global genetic imbalance. The authors used single-nucleotide polymorphism (SNP) arrays to compare overall copy number aberrations (CNAs) as well as loss of heterozygosity (LOH) of the entire human genome in ER-positive and ER-negative breast carcinomas. METHODS DNA was extracted from frozen tumor sections of 21 breast carcinoma specimens and analyzed with a proprietary 50K XbaI SNP array. Copy number and LOH probability values were derived for each sample. Data were analyzed using bioinformatics and computational software, and permutation tests were used to estimate the significance of these values. RESULTS There was a global increase in CNAs and LOH in ER-negative relative to ER-positive cancers. Gain of the long arm of chromosome 1 (1q) and 8q were the most obvious changes common in both subtypes: An increase in the chromosome 1 short arm (1p)/1q ratio was observed in ER-negative samples, and an increased 16p/16q ratio was observed in ER-positive samples. Significant CNAs (adjusted P<.05) in ER-negative relative to ER-positive tumors included 5q deletion, loss of 15q, and gain of 2p and 21q. Copy-neutral LOH (cnLOH) common to both ER-positive and ER-negative samples included 9p21, the p16 tumor suppressor locus, and 4q13, the RCHY1 (ring finger and CHY zinc finger domain-containing 1) oncogene locus. Of particular interest was an enrichment of 17q LOH among the ER-negative tumors, potentially suggesting breast cancer 1 gene (BRCA1) mutations. CONCLUSIONS SNP array detected both genetic imbalances and cnLOH and was capable of discriminating ER-negative breast cancer from ER-positive breast cancer.