The role of HGF/MET and FGF/FGFR in fibroblast-derived growth stimulation and lapatinib-resistance of esophageal squamous cell carcinoma.

BACKGROUND: Although advanced esophageal squamous-cell carcinoma (ESCC) is treated using a multidisciplinary approach, outcomes remain unsatisfactory. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of malignancies. We explored the effect of supernatant from esophageal fibroblasts on the cell growth and chemo-resistance of ESCC cell lines. METHODS: We used 22 ESCC cell lines, isolated primary human esophageal fibroblasts and immortalized fibroblasts. We first examined cell proliferation induced by fibroblast supernatant. The effect of supernatant was evaluated to determine whether paracrine signaling induced by fibroblasts can influence the proliferation of cancer cells. Next, we examined the effects of adding growth factors HGF, FGF1, FGF7, and FGF10, to the culture medium of cancer cells. These growth factors are assumed to be present in the culture supernatants of fibroblasts and may exert a paracrine effect on the proliferation of cancer cells. We also examined the intrinsic role of HGF/MET and FGFs/FGFR in ESCC proliferation. In addition, we examined the inhibitory effect of lapatinib on ESCC cell lines and studied whether the fibroblast supernatants affect the inhibitory effect of lapatinib on ESCC cell proliferation. Finally, we tested whether the FGFR inhibitor PD-173074 could eliminate the rescue effect against lapatinib that was induced by fibroblast supernatants. RESULTS: The addition of fibroblast supernatant induces cell proliferation in the majority of cell lines tested. The results of experiments to evaluate the effects of adding growth factors and kinase inhibitors suggests that the stimulating effect of fibroblasts was attributable in part to HGF/MET or FGF/FGFR. The results also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines. Though lapanitib at 1 µM inhibits cell proliferation by more than 50% in the majority of the ESCC cell lines, fibroblast supernatant can rescue the growth inhibition of ESCC cells. However, the rescue effect is abrogated by co-treatment with FGFR inhibitor. CONCLUSION: These results demonstrate that cell growth of ESCC depends on diverse receptor tyrosine kinase signaling, in both cell-autonomous and cell-non-autonomous manners. The combined inhibition of these signals may hold promise for the treatment of ESCC.

Lung cancer with loss of BRG1/BRM, shows epithelial mesenchymal transition phenotype and distinct histologic and genetic features.

BRG1 and BRM, two core catalytic subunits in SWI/SNF chromatin remodeling complexes, have been suggested as tumor suppressors, yet their roles in carcinogenesis are unclear. Here, we present evidence that loss of BRG1 and BRM is involved in the progression of lung adenocarcinomas. Analysis of 15 lung cancer cell lines indicated that BRG1 mutations correlated with loss of BRG1 expression and that loss of BRG1 and BRM expression was frequent in E-cadherin-low and vimentin-high cell lines. Immunohistochemical analysis of 93 primary lung adenocarcinomas showed loss of BRG1 and BRM in 11 (12%) and 16 (17%) cases, respectively. Loss of expression of BRG1 and BRM was frequent in solid predominant adenocarcinomas and tumors with low thyroid transcription factor-1 (TTF-1, master regulator of lung) and low cytokeratin7 and E-cadherin (two markers for bronchial epithelial differentiation). Loss of BRG1 was correlated with the absence of lepidic growth patterns and was mutually exclusive of epidermal growth factor receptor (EGFR) mutations. In contrast, loss of BRM was found concomitant with lepidic growth patterns and EGFR mutations. Finally, we analyzed the publicly available dataset of 442 cases and found that loss of BRG1 and BRM was frequent in E-cadherin-low, TTF-1-low, and vimentin-high cases and correlated with poor prognosis. We conclude that loss of either or both BRG1 and BRM is involved in the progression of lung adenocarcinoma into solid predominant tumors with features of epithelial mesenchymal transition and loss of the bronchial epithelial phenotype. BRG1 loss was specifically involved in the progression of EGFR wild-type, but not EGFR-mutant tumors.

Bifunctional triterpene/sesquarterpene cyclase: tetraprenyl-ß-curcumene cyclase is also squalene cyclase in Bacillus megaterium.

This study demonstrates that a tetraprenyl-ß-curcumene cyclase, which was originally identified as a sesquarterpene cyclase that converts a head-to-tail type of monocycle to a pentacycle, also cyclizes a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-hydroxypolypoda-13,17,21-triene. The 8α-hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium. It was also demonstrated that cyclizations of both tetraprenyl-ß-curcumene and squalene occurred with a purified B. megaterium TC homologue in the same reaction mixture. These results suggest that the tetraprenyl-ß-curcumene cyclase is bifunctional, cyclizing both tetraprenyl-ß-curcumene and squalene in vivo. This is the first report describing a bifunctional terpene cyclase, which biosynthesizes two classes of cyclic terpenes with different numbers of carbons as natural products in the organism.

Sesquarterpenes (C35 terpenes) biosynthesized via the cyclization of a linear C35 isoprenoid by a tetraprenyl-ß-curcumene synthase and a tetraprenyl-ß-curcumene cyclase: identification of a new terpene cyclase.

In this study, mono- and pentacyclic C(35) terpenes from Bacillus subtilis were biosynthesized via the cyclization of C(35) isoprenoid using purified enzymes, including the first identified new terpene cyclase that shows no sequence homology to any of the known terpene cyclases. On the basis of these findings, we propose that these C(35) terpenes should be called the new family of "sesquarterpenes."

Differential gene expression between flat adenoma and normal mucosa in the colon in a microarray analysis.

BACKGROUND: Flat adenomas in the colon are associated with a relatively higher potential for malignancy. Distinct genes may be involved in the development of flat adenoma. The aim of this study was to profile gene expression changes in flat adenomas in the colon. METHODS: A genomewide expression analysis was carried out by using flat adenoma and adjacent normal mucosa in the colon to detect differences in gene expression. Because the right and left colon have different embryonic origins, each sample was classified according to its location, and the gene expression levels between flat adenoma and adjacent normal mucosa were also compared among samples derived from the right or left colon. RESULTS: A total of 180 genes were differentially expressed between flat adenoma and normal mucosa in the colon, including matrix metalloproteinase 7 (MMP7), cadherin 3 (CDH3), S100P, and dual oxidase 2 (DUOX2). In addition, a total of 89 and 49 genes were differentially expressed between flat adenoma and normal mucosa among the samples from the right and left colon, respectively. Subsequent quantitative real-time reverse transcriptase-polymerase chain reaction supported the reliability of the expression analysis. Immunohistochemical analysis confirmed differential CDH3 and MMP7 protein expression. CONCLUSIONS: This is the first report characterizing the genes differentially expressed in flat adenomas using a microarray analysis. Considerable differences in the gene expression profiles of flat adenomas also exist between the right and left colon. These data should lead to new insights into the pathogenesis of flat adenomas in the colon as well as to new therapeutic strategies.

Changes in plasma ghrelin levels, gastric ghrelin production, and body weight after Helicobacter pylori cure.

BACKGROUND: Ghrelin is a body weight-regulating peptide produced and secreted primarily by the gastric mucosa. Helicobacter pylori infection impairs gastric ghrelin production, leading to a lower plasma ghrelin concentration. However, the effect of H. pylori eradication on plasma ghrelin levels and its relation to body weight change after H. pylori cure are still uncertain. We examined the association of plasma ghrelin levels with gastric ghrelin production and body weight change before and after H. pylori eradication. METHODS: Plasma ghrelin concentrations, gastric ghrelin expression, and body weight were determined in a total of 134 consecutive individuals before and 12 weeks after successful H. pylori eradication. Gastric ghrelin expression was evaluated by determining mRNA expression levels and the number of ghrelin-producing cells in gastric mucosa biopsy specimens by real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: Plasma ghrelin concentration increased in 50 patients and decreased in 84 patients after H. pylori eradication. After H. pylori cure, however, gastric preproghrelin mRNA expression was increased nearly fourfold (P < 0.0001), and the number of ghrelin-positive cells was increased or unchanged. In contrast, plasma ghrelin changes after H. pylori cure were inversely correlated with both body weight change (P < 0.0001) and initial plasma ghrelin levels (P < 0.0001). CONCLUSIONS: Changes in plasma ghrelin concentrations before and after H. pylori cure were inversely correlated with body weight change and initial plasma ghrelin levels but not with gastric ghrelin production in Japanese patients.

Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration.

Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca2+]i). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca2+]i, activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system.