No abstract

The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1a expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1a expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1a expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer. ' 2006 Wiley-Liss, Inc.Key words: hypoxia; tumor-stromal interaction; pancreatic cancer; hepatocyte growth factor; c-Met Most aggressive tumors have acquired the ability to develop their own blood vessels as they grow. However, the tumor cells grow faster than the endothelial cells, so the structure and function of the tumor vasculature is highly disorganized in comparison to normal tissues. 1,2 In this microenvironment, within tumor tissues, cancer cells are exposed to hypoxia. 3 Recent studies have revealed that hypoxia-inducible factor-1 (HIF-1) plays a key role in tumor progression under hypoxia. 4,5 HIF-1 is a heterodimeric basic helix-loop-helix PER/ARNT/SIM (HLH-PAS) transcription factor that consists of HIF-1a and a constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) known as HIF-1b. Under normoxic condition, HIF-1a is bound to the tumor suppressor Von Hippel-Lindau (VHL) protein. This protein complex causes HIF-1a to be targeted by proteasomes, thus leading to rapid protein degradation. Conversely, under hypoxic conditions, HIF-1a is stabilized and dimerized with HIF-1b, translocates to the nucleus and transactivates various gene expressions. HIF-1 is reported to regulate various gene expressions by binding to the hypoxia-responsive element on the target genes. More than 40 genes that are important for glucose transpo...

Hepatocyte growth facor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. Our previous study demonstrated that tumor-stromal interaction under hypoxia augments the aggressive invasive features of pancreatic cancer line PK8 through activated HGF/c-Met signaling. The present study investigated whether or not hypoxia increases HGFA expression in PK8 cells and promotes the processing of HGF, and leads to c-Met activation. Moreover, HGFA promoter assays were performed to define whether hypoxia inducible factor-1 alpha (HIF-1a) directly activates the HGFA promoter in a hypoxia-dependent fashion. As a result, hypoxia induced the HGFA mRNA and protein expression in PK8 and the elevation under hypoxia was inhibited by the transfection of HIF-1a siRNA, thus indicating HIF-1a-dependent induction of HGFA. The transfection of siRNA against HGFA to PK8 cells suppressed the conversion to the active HGF, which is secreted from fibroblast MRC5. H epatocyte growth factor (HGF) is a multifunctional cytokine produced from stromal cells, and it functions as a mitogen, motogen, and morphogen, and in angiogenesis.(1-3)The various effects of HGF are mediated through binding to the specific receptor, c-Met receptor tyrosine kinase, which is expressed on the cell surfaces of epithelial origin. HGF is mainly produced in cells of mesenchymal origin and secreted as a single-chain precursor form (pro-HGF). The proteolytic conversion of pro-HGF to the two-chain heterodimeric active form (mature HGF) is essential in its biological activity.(4) The most potent enzyme involved in the activation of pro-HGF is HGF activator (HGFA), a serine protease discovered in serum that is related to coagulation factor XIIa.(5) The liver is the main source of HGFA, and produces HGFA as an inactive form (pro-HGFA), which is converted to the active form by thrombin. (6) The extrahepatic expression of HGFA has also been reported, and recent studies have demonstrated HGFA to be involved in the activation of pro-HGF in several tumors. (7)(8)(9)(10) Hypoxia is a common feature of various solid tumors due to inadequacies in their vasculature.(11) The hypoxic environment, in which hypoxia-inducible factor-1 (HIF-1) plays a key role, might be associated with both malignant progression and a poor response to various treatments. (12,13) HIF-1 is a heterodimeric basic helix-loop-helix PER/ARNT/SIM (HLH-PAS) transcription factor that consists of HIF-1α and a constitutively expressed aryl hydrocarbon receptor nuclear translocator known as HIF-1β. Under normoxic conditions, HIF-1α is bound to the tumor suppressor Von Hippel-Lindau protein. This protein complex causes HIF-1α to be targeted by proteasomes, thus leading to rapid protein degradation. In contrast, under hypoxic condition, HIF-1α is stabilized and dimerized with HIF-1β, translocates to the nucleus, and transactivates expressions of various genes. (14,15) The hypoxia-responsive element (HRE) consists of a pair of consecutive transcription factor binding sites, ...

These data suggest that the HGF/c-Met signaling via HIF-1alpha ?may therefore negatively affect the prognosis in patients with pancreatic cancer, and targeting tumor stroma under hypoxia might thus be potentially useful as a novel therapy for this cancer.