Background: The hedgehog pathway plays a critical role in the development of prostate. However, the role of the hedgehog pathway in prostate cancer is not clear. Prostate cancer is the second most prevalent cause of cancer death in American men. Therefore, identification of novel therapeutic targets for prostate cancer has significant clinical implications.

The hedgehog pathway plays a critical role in the development of the foregut. However, the role of the hedgehog pathway in primary esophageal cancers is not well studied. Here, we report that elevated expression of hedgehog target genes occurs in 14 of 22 primary esophageal cancers. The hedgehog signaling activation is not associated with tumor subtypes, stages, or differentiation. While the sonic hedgehog (Shh) transcript is localized to the tumor tissue, expression of Gli1 and PTCH1 is observed both in the tumor and in the stroma. We discovered that 4 esophageal squamous cell carcinomas, which overexpress Shh, have genomic amplification of the Shh gene. Treatment of esophageal cancer cells with smoothened antagonist, KAAD‐cyclopamine, or the neutralizing antibodies of Shh reduces cell growth and induces apoptosis. Overexpression of Gli1 under the CMV promoter renders these cells resistant to the treatments. Thus, our results indicate that elevated expression of Shh and its target genes is quite common in esophageal cancers. Our data also indicate that downregulation of Gli1 expression may be an important mechanism by which KAAD‐cyclopamine inhibits growth and induces apoptosis in esophageal cancer cells (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html). © 2005 Wiley‐Liss, Inc.

The hedgehog (Hh) pathway plays a critical role during development of embryos and cancer. Although the molecular basis by which protein kinase A (PKA) regulates the stability of hedgehog downstream transcription factor cubitus interruptus, the Drosophila homologue of vertebrate Gli molecules, is well documented, the mechanism by which PKA inhibits the functions of Gli molecules in vertebrates remains elusive. Here, we report that activation of PKA retains Gli1 in the cytoplasm. Conversely, inhibition of PKA activity promotes nuclear accumulation of Gli1. Mutation analysis identifies Thr 374 as a major PKA site determining Gli1 protein localization. In the three-dimensional structure, Thr 374 resides adjacent to the basic residue cluster of the nuclear localization signal (NLS). Phosphorylation of this Thr residue is predicted to alter the local charge and consequently the NLS function. Indeed, mutation of this residue to Asp (Gli1/T374D) results in more cytoplasmic Gli1 whereas a mutation to Lys (Gli1/T374K) leads to more nuclear Gli1. Disruption of the NLS causes Gli1/T374K to be more cytoplasmic. We find that the change of Gli1 localization is correlated with the change of its transcriptional activity. These data provide evidence to support a model that PKA regulates Gli1 localization and its transcriptional activity, in part, through modulating the NLS function.

It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-B activation by lipopolysaccharide and tumor necrosis factor-␣ plays a pivotal role in the suppression of cyp3a4 through interactions of NF-B with the PXR⅐retinoid X receptor (RXR) complex. Inhibition of NF-B by NF-B-specific suppressor SRIB␣ reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-␣. Furthermore, we showed that NF-B p65 disrupted the association of the PXR⅐RXR␣ complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-B p65 directly interacted with the DNA-binding domain of RXR␣ and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR⅐RXR␣ complex. This mechanism of suppression by NF-B activation may be extended to other nuclear receptor-regulated systems where RXR␣ is a dimerization partner.Inflammatory responses and infections suppress the biotransformation of drugs and decrease the hepatointestinal capacity of drug clearance. This results in alterations of therapeutic indices and increases the toxicity of certain administered drugs. Inflammatory responses also play important roles in liver pathological conditions such as drug-induced hepatitis and cholestatic diseases (1, 2). The mechanisms of these clinically important effects have not been well understood.In human liver, the first pass of biotransformation is mainly carried out by cytochrome P-450 (CYP) 2 3A4, which is the predominant isoform of monooxygenases that are expressed in the adult hepatointestinal system. It is estimated that CYP3A4 is responsible for the metabolism of over 50% of drugs in use today, many of which are either metabolically activated and/or metabolically broken down (detoxified) through this enzyme. Therefore, transcriptional and post-transcriptional alterations of CYP3A4 activity have direct effects on the efficacy of drugs and detoxification of xenobiotics (reviewed in Refs. 3 and 4). Recent molecular and pharmacological studies have demonstrated that transcriptional activation of cyp3a4 is mediated by the nuclear receptor PXR (pregnane X receptor). The rodent PXR (5) and its human homolog hPXR (6), also known as steroid and xenobiotic receptor (7) or hPAR (8), were identified as xenobiotic receptors that can be activated by certain xenobiotics and endobiotics. PXR regulates the expression of cyp3a4 by associating with its obligate partner RXR, and the heterodimer binds to the nuclear receptor response elements found in the regulatory regions of these genes. Genes that are regulated by PXR include multiple drug-resistant genes such as MDR1 (9) and MRP2 (10) as wel...

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We present a computational study of tissue transcriptomic data of 14 cancer types to address: what may drive cancer cell division? Our analyses point to that persistent disruption of the intracellular pH by Fenton reactions may be at the root of cancer development. Specifically, we have statistically demonstrated that Fenton reactions take place in cancer cytosol and mitochondria across all the 14 cancer types, based on cancer tissue gene-expression data integrated via the Michaelis–Menten equation. In addition, we have shown that (i) Fenton reactions in cytosol of the disease cells will continuously increase their pH, to which the cells respond by generating net protons to keep the pH stable through a combination of synthesizing glycolytic ATPs and consuming them by nucleotide syntheses, which may drive cell division to rid of the continuously synthesized nucleotides; and (ii) Fenton reactions in mitochondria give rise to novel ways for ATP synthesis with electrons ultimately coming from H2O2, largely originated from immune cells. A model is developed to link these to cancer development, where some mutations may be selected to facilitate cell division at rates dictated by Fenton reactions.

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.

Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. At present, the molecular mechanism of Hh signaling-mediated carcinogenesis is not completely understood. Using a mouse model (K14cre/ R26SmoM2) of SMO-mediated basal cell carcinoma development, we identified TGF␤2 as a major Hh-regulated gene. TGF␤2 expression was high in the keratinocytes, with activated TGF␤ signaling (indicated by elevated expression of phosphorylated SMAD2/3) detected in both tumor and stroma. The significance of TGF␤ signaling for SMO function was demonstrated in two assays. Down-regulation of TGF␤2 expression prevented Hh signaling-dependent osteoblast differentiation and motor neuron differentiation. Furthermore, inhibition of TGF␤ signaling by TGF␤ receptor I inhibitor SD208 significantly reduced tumor area in K14cre/R26SmoM2 mice. Tumor shrinkage in mice was associated with an increased number of lymphocytes, suggesting an immune suppression role of TGF␤ signaling. The relevance of our results to human cancer is reflected by the fact that human basal cell carcinomas, which almost always harbor activated Hh signaling, have activated TGF␤ signaling, as indicated by high levels of phosphorylated SMAD2 and SMAD3 in tumor and stroma. Together, our data indicate that TGF␤ signaling is critical for Hh signaling-mediated carcinogenesis.The Hedgehog pathway plays an important role in cell differentiation, tissue polarity, cell proliferation, and carcinogenesis (1-4). The seven-transmembrane domain-containing protein Smoothened (SMO) 4 serves as the key player for signal transduction of this pathway, whose function is inhibited by another transmembrane protein, Patched (PTC), in the absence of Hh ligands. Binding of Hh to its receptor PTC releases this inhibition, allowing SMO to signal downstream, leading to formation of active forms of Gli transcription factors. As transcription factors, Gli molecules can regulate target gene expression by direct association with a specific consensus sequence located at the promoter region of the target genes (5). In addition to the canonical pathways (ligand overexpression, altered expression of Hh signaling molecules, or gene mutations), recent studies indicate that Hh signaling can also be activated by other signaling pathways, such as K-Ras. Both canonical and non-canonical Hh signaling activation are found in many types of human cancer, including brain tumors, gastrointestinal, prostate, lung, and breast cancers (6 -8).Mounting evidence indicates that Hh signaling activation occurs frequently in a number of human cancers (9), but the underlying molecular basis remains largely elusive. To understand the molecular basis by which Hh signaling regulates carcinogenesis, we analyzed gene expression of a mouse model of basal cell carcinoma in which an activated form of SMO (SmoM2) replaces the wild type SMO allele and is expressed under the control of the keratin 14 promoter. Our results indicated that ...