Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis. It represents an attractive therapeutic target in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis. In HIF-1alpha knockdown DLD-1 colon cancer cells (DLD-1(HIF-kd)), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1alpha (DLD-1(HIF-wt)). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1(HIF-kd) but not DLD-1(HIF-wt) cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-kappaB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1(HIF-kd) but not DLD-1(HIF-wt) xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1alpha may be most effective when IL-8 is simultaneously targeted.

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1␣ was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between ؊418 and ؊223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between ؊300 and ؊251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at ؊271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1␣ but instead requires the activation of PI3K/Rho/ROCK and c-Myc.

Current classifications of human gastroenteropancreatic neuroendocrine tumors (NETs) are inconsistent and based upon histopathologic but not molecular features. We sought to compare a molecular classification with the World Health Organization (WHO) histologic classification, identify genes that may be important for tumor progression, and determine whether gastrointestinal NETs (GI-NETs) differ in their molecular profile from pancreatic NETs (PNETs). DNA microarray analysis was performed to identify differentially expressed genes in PNETs and GI-NETs. Confirmation of expression levels was obtained by quantitative real-time PCR. Immunoblotting and mutational analysis were performed for selected genes. Hierarchical clustering of 19 PNETs revealed a 'benign' and 'malignant' cluster that corresponded well with the WHO categories of welldifferentiated endocrine tumor (WDET) and well-differentiated endocrine carcinoma (WDEC) respectively. FEV, adenylate cyclase 2 (ADCY2), nuclear receptor subfamily 4, group A, member 2 (NR4A2), and growth arrest and DNA-damage-inducible, beta (GADD45b) were the most highly up-regulated genes in the malignant group of PNETs. Platelet-derived growth factor receptor (PDGFR) was expressed in both WDETs and WDECs, and phosphorylation of PDGFR-b was observed in 83% of all PNETs. Malignant ileal GI-NETs exhibited a distinctive gene expression profile, and extracellular matrix protein 1 (ECM), vesicular monoamine member 1 (VMAT1), galectin 4 (LGALS4), and RET Proto-oncogene (RET) were highly up-regulated genes. Gene expression profiles reflect the current WHO classification and can distinguish benign from malignant PNETs and also PNETs from GI-NETs. This suggests that molecular profiling may enhance tumor classification schemes. Potential gene targets have also been identified, and PDGFR and RET are candidates that may represent novel therapeutic targets.

The addition of sorafenib to gemcitabine did not demonstrate improved efficacy in advanced BTC patients. Biomarker subgroup analysis suggested that some patients might benefit from combined treatment.