Serum hepatocyte growth factor and interleukin-6 are effective prognostic markers for non-small cell lung cancer.

AIM: We surveyed prognostic biomarkers for resectable non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: We obtained preoperative serum from 109 patients, and measured the levels of hepatocyte growth factor (HGF), interleukin-6 (IL-6), and nicotinamide N-methytransferase (NNMT) in the sera. RESULTS: The median HGF and IL-6 contents were 860 pg/ml and 2.7 pg/ml, respectively. Analysis of survival curves indicated that an HGF or IL-6 level higher than the median was associated with poor overall survival (HGF, p=0.019; IL-6, p=0.002). In addition, we analyzed stage III lung cancer alone. Higher HGF and IL-6 levels were associated with poor overall survival (HGF, p=0.016; IL-6, p=0.013). Disease-free survival was not statistically significantly affected by these cytokine contents. The tumor status (pT factor) and nodal status (pN factor) were not associated with the survival of stage III patients. CONCLUSION: The levels of HGF and IL-6 in serum could be useful prognostic indicators of the survival of patients with stage III NSCLC undergoing surgery and chemotherapy.

Serum levels of nicotinamide N-methyltransferase in patients with lung cancer.

PURPOSE: The aim of this study was to determine if nicotinamide N-methyltransferase (NNMT) is useful as a biomarker of lung cancer (stages I-III). METHODS: We established an ELISA system for NNMT. We determined the levels of NNMT and carcinoembryonic antigen (CEA) in sera of 113 non-small cell lung cancer (NSCLC) patients undergoing surgery and sera of 50 non-neoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) and of 24 healthy donors. RESULTS: The serum levels of NNMT were significantly higher in lung cancer patients than in COPD patients and healthy donors. The relationship between the specificity and sensitivity of NNMT and CEA measurements for the detection of lung cancer was analyzed by means of receiver-operating characteristic curves. The corresponding areas under the curves were 0.703 for NNMT and 0.621 for CEA, indicating slightly better sensitivity of NNMT. With 90% specificity, the sensitivities of NNMT and CEA as lung cancer markers were 25 and 24%, respectively. There was no significant correlation between NNMT and CEA. Therefore, the sensitivity of NSCLC detection at 90% specificity increased from 25 to 32% when NNMT was used in combination with CEA. CONCLUSION: The NNMT serum level may have significance in the early detection of NSCLC patients.

Stat3 up-regulates expression of nicotinamide N-methyltransferase in human cancer cells.

PURPOSE: To discover new molecular targets for cancer therapy and diagnosis, we surveyed signal transducers and activators of transcription 3 (Stat3)-regulated genes, because constitutive activation of Stat3 is associated with a wide variety of human malignancies. METHODS: We investigated the Stat3-regulated genes in 293 cells with cDNA microarray analysis and found that Nicotinamide N-methyltransferase (NNMT) was induced on stimulation of the cells with leukemia inhibitory factor. We examined the expression of NNMT in several types of cancer cells by real-time quantitative RT-PCR. To examine the role of Stat3, Hep-G2 hepatocellular carcinoma cells were transfected with NNMT promoter-luciferase reporter construct together with conditionally active Stat3 (Stat3ER) or dominant-negative Stat3 expression vector and NNMT promoter activity was determined. The expression of NNMT and activated Stat3 in 88 colon cancer tissues and 17 normal colon tissues was examined with immunohistochemical analysis. RESULTS: In Hep-G2 cells and SW480 colon cancer cells, NNMT expression increased on stimulation of the cells with interleukin 6. NNMT promoter activity in Hep-G2 cells was dependent on the activation of Stat3. MDA-MB-468 breast cancer cells and HT29 colon cancer cells expressed constitutively a high level of NNMT. Treatment of these cells with Stat3 siRNA or curcumin, which inhibited Stat3 phosphorylation, resulted in reduction of the NNMT level. We found a correlation between the expression of NNMT and activated Stat3 (P<0.001) in the colon cancer tissues. CONCLUSION: NNMT is a novel Stat3-regulated gene. Its expression is enhanced with the activation of Stat3 in colon cancer tissues. NNMT may be a potential candidate for a tumor marker of various kinds of cancers.

Generation of inhibitory DNA aptamers against human hepatocyte growth factor.

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.

The human hepatocyte growth factor (HGF) gene is transcriptionally activated by leukemia inhibitory factor through the Stat binding element.

We found that human melanoma SEKI and neuroepithelioma NAGAI cells, which are known to secrete high concentrations of leukemia inhibitory factor (LIF), also secrete high levels of hepatocyte growth factor (HGF). We therefore examined the role of LIF in HGF expression and examined the human HGF promoter. The expression of both LIF and HGF mRNA is very low in HEK293 cells. Treatment of these cells with LIF stimulated the expression of HGF mRNA. The cis-acting regulatory element of the HGF promoter in SEKI and 293 cells was analysed by means of a transient expression assay. By deletion analysis, we showed that the region comprising the -181 to -73 bp was required for full activity of the HGF promoter in SEKI cells and for LIF responsiveness of 293 cells. This region contains putative consensus sequences for the Stat and NF-IL6 (C/EBP beta) transcription factors. The activity of the HGF promoter was abolished by mutation of the Stat site at -99/-91, while the activity only slightly decreased on mutation of the NF-IL6 site. Treatment with anti-LIF antibodies or interruption of Stat3 signaling by dominant-negative Stat3 also reduced the HGF promoter activity. Stat3 activation was constitutive in SEKI cells and induced on treatment of 293 cells with LIF. These results suggest that cytokines, growth factors and oncogenes (v-Src, etc.) that activate Stat3 are important regulators of HGF expression.