Hiroshi Kijima

MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis

The molecular mechanism of tissue-specific metastasis in tumors endogenously expressing members of the vascular endothelial growth factor (VEGF) family is not yet clear. Here we demonstrate that MMP9 is specifically induced in premetastatic lung endothelial cells and macrophages by distant primary tumors via VEGFR-1/Flt-1 tyrosine kinase (TK) and that it significantly promotes lung metastasis. In a genetic approach using mice, suppression of MMP9 induction by deletion of either VEGFR-1TK or MMP9 markedly reduced lung metastasis. Furthermore, the MMP9 levels in endothelial cells of normal lung lobes from patients carrying distant tumors were significantly elevated as compared with those from patients without tumors. Thus, a block of MMP9 induction via VEGFR-1 inhibition could be useful for the prevention of tumor metastasis in lung.

Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer.

Vascular endothelial growth factor (VEGF) is a well known factor that induces angiogenesis. Four isoforms, i.e. VEGF206, 189, 165, and 121, have been identified. We examined the isoform patterns of VEGF mRNA using reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. All the colon cancers examined expressed VEGF121. The isoform patterns were classified into three groups: type 1, VEGF121; type 2, VEGF121 + VEGF165; type 3, VEGF121 + VEGF165 + VEGF189. Three of the 61 colon cancers examined showed type 1 expression, 26 showed type 2 expression and 32 showed the type 3 pattern. The patients with liver metastases showed the type 3 isoform expression pattern at a significantly higher incidence (12 of 16, 75%) than those without liver metastasis (20 of 45, 44%) (P=0.036). The type 3 isoform pattern was significantly associated with M1 stage (P=0.019). The patients with colon cancer and the type 3 isoform pattern showed significantly poor prognosis (P < 0.01, Cox-Mantel). The colon cancers with the type 3 pattern showed a significantly higher involvement of veins (P=0.006). These observations suggest that the aberrant type 3 expression pattern of VEGF189 mRNA isoforms is correlated with liver metastasis, M stage, and poor prognosis in colon cancer.

Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma.

Vascular endothelial growth factor (VEGF) has five isoforms (VEGF206, 189, 165, 145 and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the growth of RCC.

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization.

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both co-culture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.

Cell-retained Isoforms of Vascular Endothelial Growth Factor (VEGF) are Correlated with Poor Prognosis in Osteosarcoma

Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121 + VEGF165, and 16 (53%) for VEGF121 + VEGF165 + VEGF189. Patients with osteosarcomas with VEGF165 (n = 24) had significantly poorer prognosis in comparison with those without VEGF165 (P = 0.022, Wilcoxons test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P < 0.001, Mann-Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm.

Neuropilin 1 and neuropilin 2 co-expression is significantly correlated with increased vascularity and poor prognosis in nonsmall cell lung carcinoma.

Cell‐retained isoforms of vascular endothelial growth factor A (VEGF‐A) have been reported to play an essential role in tumor progression through stromal neovascularization in malignant solid tumors. While more than 95% of nonsmall cell lung carcinoma (NSCLC) expresses cell‐retained VEGF‐A isoform, the clinicopathologic implications of neuropilin (NRP), considered the specific receptor for limited types of VEGF‐A isoform, are not well understood.

Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer

SummaryTwo subtypes of thrombospondin (TSP-1 and TSP-2) have inhibitory roles in angiogenesis in vitro, although the biological significance of these TSP isoforms has not been determined in vivo. We examined TSP-1 and TSP-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. Thirty-eight of these 61 colon cancers were positive for TSP-2 expression and showed hepatic metastasis at a significantly lower incidence than those without TSP-2 expression (P = 0.02). TSP-2 expression was significantly associated with M0 stage in these colon cancers (P = 0.03), whereas TSP-1 expression showed no apparent correlation with these factors. The colon cancer patients with TSP-2 expression showed a significantly low frequency of liver metastasis correlated with the cell-associated isoform of vascular endothelial growth factor (VEGF-189) (P = 0.0006). Vascularity was estimated by CD34 staining, and TSP-2(–)/VEGF-189(+) colon cancers showed significantly increased vessel counts and density in the stroma (P < 0.0001). TSP-2(–)/VEGF-189(+) colon cancer patients also showed significantly poorer prognosis compared with those with TSP-2(+) / VEGF-189(–) (P = 0.0014). These results suggest that colon cancer metastasis is critically determined by angiogenesis resulting from the balance between the angioinhibitory factor TSP-2 and angiogenic factor VEGF-189.

Methylation of the KEAP1 gene promoter region in human colorectal cancer

BackgroundThe Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.MethodsWe used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1.ResultsDNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs.ConclusionHypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

Clinical implications of interleukin (IL)-10 induced by non-small-cell lung cancer

BACKGROUND The type 2 cytokine interleukin (IL)-10 has been reported to inhibit the antitumour activity of the regional immunity against various neoplasms. Certain lung cancers produce IL-10, but the clinical significance of IL-10 expression is not well understood. PATIENTS AND METHODS We examined IL-10 and IL-10 receptor (IL-10R) mRNA expression in 82 non-small-cell lung cancers (NSCLC) by reverse transcription-polymerase chain reaction (RT-PCR) assay. Immunohistochemistry (IHC) and enzyme immunoassay (EIA) were applied to evaluate the cellular localisation and the serum levels of IL-10. RESULTS RT-PCR assay revealed IL-10 mRNA expression in 68 (83%) of 82 NSCLC surgical specimens (40 of 50 adenocarcinomas, 22 of 26 squamous cell carcinomas, 5 of 5 large-cell carcinomas, 1 of 1 adenosquamous-cell carcinoma). RT-PCR assay also revealed IL-10R mRNA expression in 79 cases of NSCLC (96.1%). IL-10 expression was confirmed within tumour cells by IHC. EIA showed no significant serum IL-10 elevation in the 12 NSCLC positive for IL-10 mRNA expression (0-2.99 pg/ml). The NSCLC patients with IL-10 production showed significantly poorer prognosis than those without IL-10 production (P < 0.05, Kaplan Meier, log-rank test). CONCLUSIONS These results suggested that the cytoplasmic IL-10 correlated to clinical prognosis, and that IL-10 expression is a prognostic factor for NSCLC.

Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression

DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic‐helix‐loop‐helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up‐regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up‐regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia‐inducible factor 1α (HIF‐1α) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co‐immunoprecipitated with HIF‐1α but not with ARNT1. The binding of HIF‐1α to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over‐expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.