Congenital aortic valve stenosis (AVS), coarctation of the aorta (COA) and hypoplastic left heart syndrome (HLHS) are congenital cardiovascular malformations that all involve the left ventricular outflow tract (LVOT). They are presumably caused by a similar developmental mechanism involving the developing endothelium. The exact etiology for most LVOT malformations is unknown, but a strong genetic component has been established. We demonstrate here that mutations in the gene NOTCH1, coding for a receptor in a developmentally important signaling pathway, are found across the spectrum of LVOT defects. We identify two specific mutations that reduce ligand (JAGGED1) induced NOTCH1 signaling. One of these mutations perturbs the S1 cleavage of the receptor in the Golgi. These findings suggest that the levels of NOTCH1 signaling are tightly regulated during cardiovascular development, and that relatively minor alterations may promote LVOT defects. These results also establish for the first time that AVS, COA and HLHS can share a common pathogenetic mechanism at the molecular level, explaining observations of these defects co-occurring within families.

Mdm2 is an essential regulator of the p53 tumor suppressor. Mdm2 is modified at transcriptional, post-transcriptional, and post-translational levels to control p53 activity in normal versus stressed cells. Importantly, errors in these regulatory mechanisms can result in aberrant Mdm2 expression and failure to initiate programmed cell death in response to DNA damage. Such errors can have severe consequences as evidenced by tumor phenotypes resulting from amplification at the Mdm2 locus and changes in post-transcriptional and post-translational regulation of Mdm2. Although Mdm2 mediated inhibition of p53 is well characterized, Mdm2 interacts with many additional proteins and also targets many of these for proteosomal degradation. Mdm2 also has E3-ligase independent functions and p53-independent functions that have important implications for genome stability and cancer.

Zinc deficiency in humans is associated with an increased risk of upper aerodigestive tract (UADT) cancer. In rodents, zinc deficiency predisposes to carcinogenesis by causing proliferation and alterations in gene expression. We examined whether in zinc-deficient rodents, targeted disruption of the cyclooxygenase (COX)-2 pathway by the COX-2 selective inhibitor celecoxib or by genetic deletion prevent UADT carcinogenesis. Tongue cancer prevention studies were conducted in zinc-deficient rats previously exposed to a tongue carcinogen by celecoxib treatment with or without zinc replenishment, or by zinc replenishment alone. The ability of genetic COX-2 deletion to protect against chemically-induced forestomach tumorigenesis was examined in mice on zinc-deficient versus zinc-sufficient diet. The expression of 3 predictive biomarkers COX-2, nuclear factor (NF)-j B p65 and leukotriene A 4 hydrolase (LTA 4 H) was examined by immunohistochemistry. In zinc-deficient rats, celecoxib without zinc replenishment reduced lingual tumor multiplicity but not progression to malignancy. Celecoxib with zinc replenishment or zinc replenishment alone significantly lowered lingual squamous cell carcinoma incidence, as well as tumor multiplicity. Celecoxib alone reduced overexpression of the 3 biomarkers in tumors slightly, compared with intervention with zinc replenishment. Instead of being protected, zinc-deficient COX-2 null mice developed significantly greater tumor multiplicity and forestomach carcinoma incidence than wild-type controls. Additionally, zinc-deficient COX-22/2 forestomachs displayed strong LTA 4 H immunostaining, indicating activation of an alternative pathway under zinc deficiency when the COX-2 pathway is blocked. Thus, targeting only the COX-2 pathway in zinc-deficient animals did not prevent UADT carcinogenesis. Our data suggest zinc supplementation should be more thoroughly explored in human prevention clinical trials for UADT cancer. ' 2007 Wiley-Liss, Inc.Key words: zinc deficiency; upper aerodigestive tract cancer; chemoprevention; COX-2; COX-2 null mice Upper aerodigestive tract (UADT) cancer, including esophageal and oral cancer, is an important cause of morbidity and mortality worldwide. 1 With a 5-year survival of 10%, esophageal cancers are deadly. The prognosis of oral cancer, the major site being the tongue, is equally dismal, with an increasing incidence worldwide, particularly in young adults. 2 Patients with oral cancer have a high mortality rate, because of field cancerization effects that result in second primary tumors, particularly in the esophagus. 3,4 Thus, new chemopreventive and therapeutic approaches are much needed to prevent and treat these deadly cancers.While chronic alcohol consumption and tobacco use are the major risk factors for UADT cancer, epidemiologic and clinical studies have implicated dietary zinc deficiency (ZD) in the etiology of esophageal squamous cell carcinoma (SCC) and head and neck SCC. [5][6][7][8][9] In 2005, Abnet et al. 10 provided the strongest evidence of an a...

Summary Somites are embryonic precursors of the axial skeleton and skeletal muscles, and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated both at the transcriptional and post-transcriptional levels for proper clock function. Here we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3′UTR, and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that miRNAs function in the post-transcriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of miRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight post-transcriptional regulation.

Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1G661S and NOTCH1A683T) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1A683T exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1G661S exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.

Many human tumors express high levels of the p53 inhibitor Mdm2, resulting from amplification of the Mdm2 locus or aberrant post-translational regulation of the Mdm2 protein. While the importance of Mdm2 in regulating p53 is clear, Mdm2 also has p53-independent roles. For example, overexpression of Mdm2 results in genomic instability in a p53-independent manner. In addition, Mdm2 has many additional binding partners; some such as the tumor suppressor p73 have also been implicated in genomic instability. In this study, cells and tumors with Mdm2 overexpression and p73 loss exhibit increased genomic instability as compared to either alteration alone and cooperate in development of B-cell lymphomagenesis. Cytogenetic analyses of mouse embryonic fibroblasts and pre-malignant B-cells demonstrate that loss of p73 exacerbates the chromosome breaks and fusions observed in Mdm2Tg cells. B-cell lymphomas from Mdm2Tg;p73+/− mice retain the remaining p73 allele, and exhibit elevated levels of the anti-apoptotic protein Bcl2 and thus dampen apoptosis. In summary, Mdm2 overexpression and p73 loss cooperate in genomic instability and tumor development, indicating that the oncogenic functions of Mdm2 are a combined effect of inhibiting p53 and p73 functions. Given that p73 is lost or silenced in human B-cell lymphomas [1–4], the Mdm2Tg;p73+/− mouse serves as a model for human disease and may provide additional insight into the pathways that contribute to B-cell lymphomagenesis.

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