Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

Hepatitis B viral transactivator HBx alleviates p53-mediated repression of alpha-fetoprotein gene expression.

Chronic infection with hepatitis B virus (HBV) is associated with development of hepatocellular carcinoma (HCC). The exact mechanism by which chronic infection with HBV contributes to onset of HCC is unknown. However, previous studies have implicated the HBV transactivator protein, HBx, in progression of HCC through its ability to bind the human tumor suppressor protein, p53. In this study, we have examined the ability of HBx to modify p53 regulation of the HCC tumor marker gene, alpha-fetoprotein (AFP). By utilizing in vitro chromatin assembly of DNA templates prior to transcription analysis, we have demonstrated that HBx functionally disrupts p53-mediated repression of AFP transcription through protein-protein interaction. HBx modification of p53 gene regulation is both tissue-specific and dependent upon the p53 binding element. Our data suggest that the mechanism by which HBx alleviates p53 repression of AFP transcription is through an association with DNA-bound p53, resulting in a loss of p53 interaction with liver-specific transcriptional co-repressors.