Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis that controls angiogenesis, erythropoiesis, and glycolysis via transcriptional activation of target genes under hypoxic conditions. O 2 -dependent binding of the von Hippel-Lindau (VHL) tumor suppressor protein targets the HIF-1␣ subunit for ubiquitination and proteasomal degradation. The activity of the HIF-1␣ transactivation domains is also O 2 regulated by a previously undefined mechanism. Here, we report the identification of factor inhibiting HIF-1 (FIH-1), a protein that binds to HIF-1␣ and inhibits its transactivation function. In addition, we demonstrate that FIH-1 binds to VHL and that VHL also functions as a transcriptional corepressor that inhibits HIF-1␣ transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH-1 provides a unifying mechanism for the modulation of HIF-1␣ protein stabilization and transcriptional activation in response to changes in cellular O 2 concentration.

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Pancreatic ductal adenocarcinoma is a devastating disease, characterized by a rapid progression and poor treatment response. Using gene expression profiling of pancreatic cancer tissues, we previously identified periostin as a potential diagnostic and therapeutic target. In this study, we report the overexpression of periostin in a larger set of pancreatic cancer tissues and show that although the periostin transcript is exclusively expressed in tumour cells, the protein product is only detected in the extracellular matrix adjacent to cancer cells. Using an enzyme-linked immunosorbent assay (ELISA) assay, we show significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. We demonstrate that periostin promotes the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhances the survival of tumour cells exposed to hypoxic conditions. At the molecular level, we provide evidence that the a 6 b 4 integrin complex acts as the cell receptor of periostin in pancreatic cancer cells and that interaction promotes phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) though activation of the PI3 kinase pathway, but not the RAS/MEK/ERK pathway. These findings suggest an important role of periostin in pancreatic cancer and provide a rationale to study periostin for diagnostic and therapeutic applications.

Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of oxygen homeostasis in metazoan species. HIF-1 mediates changes in gene transcription in response to changes in cellular oxygenation. The half-life of the HIF-1alpha subunit is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that OS-9, the protein product of a widely expressed gene, interacts with both HIF-1alpha and HIF-1alpha prolyl hydroxylases. OS-9 gain-of-function promotes HIF-1alpha hydroxylation, VHL binding, proteasomal degradation of HIF-1alpha, and inhibition of HIF-1-mediated transcription. OS-9 loss-of-function caused by RNA interference increases HIF-1alpha protein levels, HIF-1-mediated transcription, and VEGF mRNA expression under nonhypoxic conditions. These data indicate that OS-9 is an essential component of a multiprotein complex that regulates HIF-1alpha levels in an O2-dependent manner.

Purpose: Although chemotherapy with gemcitabine is a common mode of treatment of pancreatic cancer, 75% of patients do not benefit from this therapy. It is likely that the sensitivity of cancer cells to gemcitabine is determined by a number of different factors. Experimental Design: To identify genes that might contribute to resistance to gemcitabine, 15 pancreatic cancer cell lines were subjected to gemcitabine treatment. Simultaneously, gene expression profiling using a cDNA microarray to identify genes responsible for gemcitabine sensitivity was performed. Results: The pancreatic cancer cell lines could be classified into three groups: a gemcitabine ''sensitive,'' an ''intermediate sensitive,'' and a ''resistant'' group. Microarray analysis identified 71 genes that show differential expression between gemcitabine-sensitive and -resistant cell lines including 27 genes relatively overexpressed in sensitive cell lines whereas 44 genes are relatively overexpressed in resistant cell lines. Among these genes, 7 genes are potentially involved in the phosphatidylinositol 3-kinase/Akt pathway. In addition to this major signaling pathway, Bcl2/adenovirus E1B 19 kDa protein interacting protein (BNIP3), a Bcl-2 family proapoptotic protein, was identified as being expressed at lower levels in drug-resistant pancreatic cancer cell lines. In an analysis of 21 pancreatic cancer tissue specimens, more than 90% showed down-regulated expression of BNIP3.When expression of BNIP3 was suppressed using small interfering RNA, gemcitabine-induced cytotoxicity in vitro was much reduced. Conclusions: These results suggest that BNIP3 and the phosphatidylinositol 3-kinase/Akt pathway may play an important role in the poor response to gemcitabine treatment in pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease that is characterized by a particularly marked resistance to chemotherapy. We previously showed an association between decreased expression of BNIP3 and chemoresistance in PDAC cell lines. To further explore the molecular basis of chemoresistance in PDAC, we analyzed microarray data obtained from normal pancreas and PDAC tumor samples to identify genes exhibiting a negative correlation with the expression profile of BNIP3. This analysis identified several S100 family proteins, of which two, S100A2 and S100A4, showed in vitro the ability to repress exogenous BNIP3 promoter activity. We subsequently showed that RNA interference-mediated S100A4 knockdown resulted in an elevated expression of BNIP3 in PDAC cell lines that possess an unmethylated BNIP3 promoter, suggesting that, in addition to hypermethylation, S100A4 overexpression may represent an alternative mechanism for inhibiting BNIP3 function in PDAC. S100A4 knockdown also resulted in an increased sensitivity of PDAC cell lines to gemcitabine treatment, which was coupled with an increase in apoptosis and cell cycle arrest. To investigate the underlying mechanisms mediating these effects, we studied the effect of silencing the expression of S100A4 on the induction of apoptosis, cell cycle arrest, and the activation of apoptotic mediators. Knockdown of S100A4 clearly induced apoptosis with increased fragmentation of DNA and phosphatidyl serine externalization; activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase; and release of cytochrome c into the cytosol. These findings provide evidence that supports a novel role for S100A4 as a prosurvival factor in pancreatic cancer. [Cancer Res 2007;67(14):6786-95]

The mechanism of localisation of metallothionein-I (MT-I) mRNA was studied in transfected cells by in situ hybridisation and cell fractionation. Hepatoma cells were transfected with the 5'-untranslated region and coding region of the beta-globin gene alone or linked to either the beta-globin 3'-untranslated region (3'-UTR) or the MT-I 3'-UTR. The wild-type beta-globin mRNA and the beta-globin mRNA lacking its native 3'-UTR were present in free and cytoskeletal-bound polysomes to a similar extent and showed no localisation. Chimaeric globin-metallothionein transcripts were significantly enriched in cytoskeletal-bound polysomes and were localised in the perinuclear cytoplasm. Chimaeric globin-metallothionein and wild-type globin transcripts were of similar stability. Chinese Hamster Ovary cells were transfected with constructs in which the MT-I 5'-untranslated region and coding sequences were linked to either the endogenous 3'-UTR or the glutathione peroxidase 3'-UTR. Wild-type MT-I transcripts were localised in the perinuclear cytoplasm but the chimaeric MT-I-glutathione peroxidase transcripts showed no distinct localisation. The results indicate that the 3'-UTR of MT-I mRNA contains a localisation signal which promotes both the association of the mRNA with the cytoskeleton and its perinuclear localisation.

Pyruvate dehydrogenase kinase (PDK) is a pivotal enzyme in cellular energy metabolism that has previously been implicated in cancer through both RNAi based studies and clinical correlations with poor prognosis in several cancer types.Here, we report the discovery of a novel and selective ATP competitive pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, VER-246608 increased pyruvate dehydrogenase complex (PDC) activity, oxygen consumption and attenuated glycolytic activity. However, these effects were only observed under D-glucose-depleted conditions and required almost complete ablation of PDC E1α subunit phosphorylation. VER-246608 was weakly anti-proliferative to cancer cells in standard culture media; however, depletion of either serum or combined D-glucose/L-glutamine resulted in enhanced cellular potency. Furthermore, this condition-selective cytostatic effect correlated with reduced intracellular pyruvate levels and an attenuated compensatory response involving deamination of L-alanine. In addition, VER-246608 was found to potentiate the activity of doxorubicin. In contrast, the lipoamide site inhibitor, Nov3r, demonstrated sub-maximal inhibition of PDK activity and no evidence of cellular activity.These studies suggest that PDK inhibition may be effective under the nutrient-depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy.