Hiroaki Okuyama

Hypoxia-Inducible Factor-1-Dependent Repression of E-cadherin in von Hippel-Lindau Tumor Suppressor–Null Renal Cell Carcinoma Mediated by TCF3, ZFHX1A, and ZFHX1B

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.

Effects of Aging and Hypoxia-Inducible Factor-1 Activity on Angiogenic Cell Mobilization and Recovery of Perfusion After Limb Ischemia

Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1&agr;, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1&agr; gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.

Stromal Cell–Derived Factor-1α and CXCR4 Expression in Hemangioblastoma and Clear Cell-Renal Cell Carcinoma: von Hippel-Lindau Loss-of-Function Induces Expression of a Ligand and Its Receptor

The genetic hallmark of hemangioblastomas and clear cell-renal cell carcinomas (CC-RCCs) is loss-of-function of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL is required for oxygen-dependent degradation of hypoxia-inducible factor-1alpha (HIF-1alpha). In hemangioblastomas and CC-RCCs, HIF-1alpha is constitutively overexpressed leading to increased transcription of HIF-1-regulated genes, including vascular endothelial growth factor (VEGF). Because loss of VHL function is associated with increased expression of the chemokine receptor CXCR4 in CC-RCCs, we investigated the expression of HIF-1alpha, CXCR4, and its ligand stromal cell-derived factor-1alpha (SDF-1alpha) in hemangioblastomas and CC-RCCs. Immunohistochemistry revealed overexpression of both CXCR4 and SDF-1alpha within tumor cells and endothelial cells of hemangioblastomas and CC-RCCs. HIF-1alpha was detected in tumor cell nuclei of both hemangioblastomas and CC-RCCs. A specific ELISA showed that hemangioblastomas and CC-RCCs expressed SDF-1alpha protein at levels that were significantly higher than those found in normal tissue. Analysis of the VHL-null RCC line 786-0 revealed that SDF-1alpha mRNA levels were 100-fold higher than in a subclone transfected with the wild-type VHL gene. Expression of CXCR4 and SDF-1alpha mRNA was significantly decreased in HIF-1alpha-null compared with wild-type mouse embryo fibroblasts (MEFs). ELISA and Western blot studies for SDF-1alpha and CXCR4 protein expression confirmed the RNA findings in RCC lines and MEFs. These results suggest that loss-of-function of a single tumor suppressor gene can up-regulate the expression of both a ligand and its receptor, which may establish an autocrine signaling pathway with important roles in the pathogenesis of hemangioblastoma and CC-RCC.

Spontaneous Transformation of Cultured Mouse Bone Marrow–Derived Stromal Cells

Bone marrow-derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis.

Expression of vascular endothelial growth factor receptor 1 in bone marrow-derived mesenchymal cells is dependent on hypoxia-inducible factor 1.

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.

Cellular Hypoxia of Pancreatic β-Cells Due to High Levels of Oxygen Consumption for Insulin Secretion in Vitro

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.

Downregulation of c-MYC Protein Levels Contributes to Cancer Cell Survival under Dual Deficiency of Oxygen and Glucose

The c-MYC protein participates in energy-consuming processes such as proliferation and ribosome biosynthesis, and its expression is often dysregulated in human cancers. Cancer cells distant from blood vessels in solid tumors are in short supply of oxygen and nutrition yet can adapt to the microenvironment and survive under metabolic stress. The role and regulation of c-MYC protein in the tumor microenvironment of limited energy sources are poorly understood. Here, we show that c-MYC protein levels in cancer cells are strikingly reduced in the area distant from the blood vessels in vivo and also under oxygen- and glucose-deprived conditions in vitro. The rapid reduction of c-MYC protein levels requires low levels of both oxygen and glucose, and under these conditions, downregulation is mainly achieved by enhanced degradation. Suppression of c-MYC protein levels by small hairpin RNA decreases the necrotic cell death induced by oxygen and glucose deprivation. Thus, the environmental milieu regulates c-MYC protein levels, and downregulation of c-MYC might be a strategy for cancer cells to survive under conditions of limited energy sources.

Regulation of cultured rat hepatocyte proliferation by stellate cells

BACKGROUND/AIMSnThis study using primary culture models was aimed to reveal the stellate cell-derived factors that regulate hepatocyte proliferation.nnnMETHODSnRat hepatocytes and stellate cells were cultured in serum-free Williams-E medium. We prepared hepatocyte mono-culture and two different co-cultures of hepatocytes and stellate cells; (1) co-culture on the same surface (Co-mix.) and (2) co-culture without contact between hepatocytes and stellate cells using a culture insert (Co-sep.). The change in the number and the DNA synthesis of hepatocytes was evaluated.nnnRESULTSnThe number of hepatocytes decreased to 76% of the original number after 48 h of starting mono-culture, while it remained at 106% in mixed co-culture (Co-mix.) and increased to 135% in separated co-culture (Co-sep.). The hepatocyte DNA synthesis was enhanced by carbenoxolone in Co-mix. and reduced by NK1 in each co-culture. PD153035 had no effect. Heparitinase-I (20 mU/ml) and sodium chrolate (25 mM) reduced the hepatocyte DNA synthesis in Co-sep. to 71.8 and 61.6%, respectively. Activation of mitogen-activated protein kinase was induced in hepatocytes stimulated by conditioned mediums.nnnCONCLUSIONSnHepatocyte proliferation was stimulated in the presence of stellate cells through hepatocyte growth factor, extracellular heparan sulfate (HS), and HS proteoglycan, and might be negatively regulated by gap junction-dependent mechanism.

Carbenoxolone inhibits DNA synthesis and collagen gene expression in rat hepatic stellate cells in culture

BACKGROUND/AIMSnThis study using primary-cultured rat hepatic stellate cells (HSCs) was aimed to reveal the effect of carbenoxolone and the other gap-junction blockers on the proliferation and activation of HSCs.nnnMETHODSnHSC morphology was microscopically evaluated. DNA synthesis was determined by [3H]thymidine incorporation. Expression of HSC activation markers and cell cycle-related proteins was evaluated by Western blot. Collagen alpha1(I) mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction.nnnRESULTSnCarbenoxolone triggered the morphological change of activated HSCs without inducing apoptosis. Culture-induced DNA synthesis was suppressed to 22.6 and 8.51%, respectively, by 40 and 80 microM carbenoxolone. The other gap-junction blockers failed to affect the morphology and the DNA synthesis of activated HSCs. Carbenoxolone decreased the expression of cyclins D1/2 and cyclin-dependent kinases 4/6. Platelet-derived growth factor (PDGF)-BB-elicited DNA synthesis was reduced to 45.6 and 3.27%, respectively, by 40 and 80 microM carbenoxolone. Phosphorylation of c-Raf, MEK and mitogen-activated protein kinase, but not PDGF receptor beta, under PDGF-BB stimulation was attenuated by carbenoxolone. Collagen alpha1(I) mRNA expression was significantly reduced. In addition, carbenoxolone suppressed the activation process of quiescent HSCs.nnnCONCLUSIONSnCarbenoxolone reduced the DNA synthesis and the expression of collagen alpha1(I) mRNA in activated HSCs independently of its pharmacological action as gap-junction blocker.

Regulation of 18F-FDG Accumulation in Colorectal Cancer Cells with Mutated KRAS

KRAS gene mutations occur in approximately 40% of colorectal cancers (CRCs) and are associated with resistance to anti–epidermal growth factor receptor antibody therapy. We previously demonstrated that 18F-FDG accumulation in PET was significantly higher in CRCs with mutated KRAS than in those with wild-type KRAS in a clinical setting. Here, we investigated the mechanisms by which mutated KRAS increased 18F-FDG accumulation. Methods: Using paired isogenic human CRC cell lines that differ only in the mutational status of the KRAS gene, we measured 18F-FDG accumulation in these cells in vitro and in vivo. We also investigated the roles of proteins that have a function in 18F-FDG accumulation. Finally, we examined the relationship among mutated KRAS, hypoxia-inducible factor 1α (HIF-1α), and maximum standardized uptake value with 51 clinical CRC samples. Results: In the in vitro experiments, 18F-FDG accumulation was significantly higher in KRAS-mutant cells than in wild-type controls under normoxic conditions. The expression levels of glucose transporter 1 (GLUT1) and hexokinase type 2 (HK2) were higher in KRAS-mutant cells, and 18F-FDG accumulation was decreased by knockdown of GLUT1. Hypoxic induction of HIF-1α was higher in KRAS-mutant cells than in wild-type controls; in turn, elevated HIF-1α resulted in higher GLUT1 expression and 18F-FDG accumulation. In addition, HIF-1α knockdown decreased 18F-FDG accumulation under hypoxic conditions only in the KRAS-mutant cells. Small-animal PET scans showed in vivo 18F-FDG accumulation to be significantly higher in xenografts with mutated KRAS than in those with wild-type KRAS. The immunohistochemistry of these xenograft tumors showed that staining of GLUT1 was consistent with that of HIF-1α and pimonidazole. In a retrospective analysis of clinical samples, KRAS mutation exhibited a significantly positive correlation with expressions of GLUT1 and HIF-1α and with maximum standardized uptake value. Conclusion: Mutated KRAS caused higher 18F-FDG accumulation possibly by upregulation of GLUT1; moreover, HIF-1α additively increased 18F-FDG accumulation in hypoxic lesions. 18F-FDG PET might be useful for predicting the KRAS status noninvasively.