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The human epidermal growth factor receptor (EGFR) promoter is activated by both wild-type and tumorderived mutant p53. In this communication, we demonstrate that EGFR promoter sequence requirements for transactivation by wild-type and mutant p53 are different. Transient-expression assays with EGFR promoter deletions identified a wild-type human p53 response element, 5-AGCTAGACGTCCGGGCAGCCCCCGGCG -3, from positions ؊265 to ؊239. Electrophoretic mobility shift analysis and DNase I footprinting assays indicated that wild-type p53 binds sequence specifically to the response element. Using circularly permuted DNA fragments containing the p53-binding site, we show that wild-type p53 binding induces DNA bending at this site. We further show that the EGFR promoter is also activated by tumor-derived p53 mutants p53-143A, p53-175H, p53-248W, p53-273H, and p53-281G. However, the transactivation by mutant p53 does not require the wild-type p53-binding site. The minimal EGFR promoter from positions ؊104 to ؊20 which does not contain the wild-type p53-binding site is transactivated by the p53 mutants but not by the wild-type protein, showing a difference in the mechanism of transactivation by wild-type and mutant p53. Transactivation of the EGFR promoter by p53 may represent a novel mechanism of cell growth regulation.

Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not Wild type (WT) mice, with N-nitrosodiethylamine (DEN), resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of pro-survival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance towards senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media (CM) from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated association of Factor XII (FXII) mRNA with polysomes resulting in increased translation. siRNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis. Conclusion We uncover novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence and activation of coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.

Lysophosphatidic acid (LPA) is a ligand of multiple G protein–coupled receptors. The LPA1–3receptors are members of the endothelial cell differentiation gene (Edg) family. LPA4/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA5/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa4in mice. Although LPA4-deficient mice displayed no apparent abnormalities, LPA4-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA4, LPA4deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA4converted LPA4-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA4strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA1in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA4attenuated LPA1-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA4is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53 ؊/؊ mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53؊/؊ ). ras/p53 ؊/؊ mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53 ؉/؉ and ras/p53 ؉/؊ mice were of mammary origin, ras/p53 ؊/؊ mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53 ؊/؊ mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53 ؉/؉ mice. Interestingly, the increased growth rates of ras/p53 ؊/؊ tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.