Neural lineage-specific homeoprotein BRN2 is directly involved in TTF1 expression in small-cell lung cancer.

Thyroid transcription factor 1 (TTF1) plays crucial roles in thyroid, lung, and developing brain morphogenesis. Because TTF1-expressing neoplasms are generated from organs and tissues that normally express TTF1, such as the thyroid follicular epithelium and peripheral lung airway epithelium, TTF1 is widely used as a cell lineage-specific and diagnostic marker for thyroid carcinomas and for lung adenocarcinomas with terminal respiratory unit (TRU) differentiation. However, among lung neuroendocrine tumors, small-cell carcinomas (small-cell lung cancers (SCLCs)), most of which are generated from the central airway, also frequently express TTF1 at high levels. To clarify how SCLCs express TTF1, we investigated the molecular mechanisms of its expression using cultivated lung cancer cells and focusing upon neural cell-specific transcription factors. Both SCLC cells and lung adenocarcinoma cells predominantly expressed isoform 2 of TTF1, and TTF1 promoter assays in SCLC cells revealed that the crucial region for activation of the promoter, which is adjacent to the transcription start site of TTF1 isoform 2, has potent FOX-, LHX-, and BRN2-binding sites. Transfection experiments using expression vectors for FOXA1, FOXA2, LHX2, LHX6, and BRN2 showed that BRN2 substantially upregulated TTF1 expression, whereas FOXA1/2 weakly upregulated TTF1 expression. BRN2 and FOXA1/2 binding to the TTF1 promoter was confirmed through chromatin immunoprecipitation experiments, and TTF1 expression in SCLC cells was considerably downregulated after BRN2 knockdown. Furthermore, the TTF1 promoter in SCLC cells was scarcely methylated, and immunohistochemical examinations using a series of primary lung tumors indicated that TTF1 and BRN2 were coexpressed only in SCLC cells. These findings suggest that TTF1 expression in SCLC is a cell lineage-specific phenomenon that involves the developing neural cell-specific homeoprotein BRN2.

Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin, and chromogranin A in lung cancer cells.

Neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYPT), and chromogranin A (CGA) are immunohistochemical markers for diagnosing lung neuroendocrine tumors (LNETs). However, the precise expression mechanisms have not been studied in enough detail. The purpose of the present study is to define the molecular mechanisms of NCAM1, SYPT, and CGA gene expressions, using cultivated lung cancer cells and focusing upon NeuroD1 (ND1), achaete-scute homolog-like 1 (ASCL1), and known transcription factors, repressor element 1 (RE1)-silencing transcription factor (REST) and c-AMP responsive element-binding protein (CREB). Promoter assays, chromatin immunoprecipitation, and transfection experiments revealed that ND1 activated NCAM1, that ASCL1 weakly upregulated SYPT expression, and that CGA expression was not regulated by ND1 or ASCL1. REST expression was restricted in non-small cell lung cancer (NSCLC) cells, and knockdown of REST could cause as much SYPT expression as in SCLC cells and weak CGA expression in NSCLC cells. However, CGA gene upregulation via CREB activation was not found in REST-lacking NSCLC cells, indicating the requirement of some additional mechanism for sufficient expression. These results suggest that NCAM1, SYPT and CGA expressions are differently regulated by neuroendocrine phenotype-specific transcription factors and provide a reason why NCAM1 and SYPT are frequently expressed in LNETs, irrespective of malignancy grade.

Neuroendocrine cancer-specific up-regulating mechanism of insulin-like growth factor binding protein-2 in small cell lung cancer.

Small cell lung cancer (SCLC) exhibits insulin-like growth factor-dependent growth. SCLC is the most aggressive among known in vivo lung cancers, whereas in vitro growth of SCLC is paradoxically slow as compared with that of non-SCLC (NSCLC). In this study, we demonstrate that SCLC cells overexpress insulin-like growth factor binding protein (IGFBP)-2 via NeuroD, a neuroendocrine cell-specific transcription factor. Chromatin immunoprecipitation, electrophoretic mobility shift, and IGFBP-2 promoter assays all revealed that NeuroD binds to the E-box in the 5'-untranslated region of IGFBP-2. A NeuroD transgene in both airway epithelial and NSCLC cells up-regulated the transcription of IGFBP-2 and retarded cell growth. Recombinant IGFBP-2 repressed the growth of both airway epithelial and NSCLC cells in a dose-dependent manner. A NeuroD-specific small interfering RNA repressed IGFBP-2 expression in SCLC, and neutralization of IGFBP-2 and an IGFBP-2-specific small interfering RNA increased SCLC cell growth. Pathological samples of SCLC also expressed IGFBP-2 abundantly, as compared with NSCLC, and showed only rare (8%) IGFBP-2 promoter methylation, whereas the IGFBP-2 promoter was methylated in 71% of adenocarcinomas and 29% of squamous cell carcinomas. These findings suggest that 1) SCLC has an IGFBP-2 overexpression mechanism distinct from NSCLC, 2) secreted IGFBP-2 contributes to the slow growth of SCLC in vitro, and 3) the epigenetic alterations in the IGFBP-2 promoter contribute to the striking differences in IGFBP-2 expression between SCLC and NSCLC in vivo.

Dioxin-induced up-regulation of the active form of vitamin D is the main cause for its inhibitory action on osteoblast activities, leading to developmental bone toxicity.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is known to cause bone toxicity, particularly during animal development, although its action mechanism to cause this toxicity has yet to be elucidated. Mouse pups were exposed to TCDD via dam's milk that were administered orally with 15 microg TCDD/kg b.w. on postnatal day 1. Here we report that TCDD causes up-regulation of vitamin D 1alpha-hydroxylase in kidney, resulting in a 2-fold increase in the active form of vitamin D, 1,25-dihydroxyvitamin D3, in serum. This action of TCDD is not caused by changes in parathyroid hormone, a decrease in vitamin D degrading enzyme, vitamin D 24-hydroxylase, or alterations in serum Ca2+ concentration. Vitamin D is known to affect bone mineralization. Our data clearly show that TCDD-exposed mice exhibit a marked decrease in osteocalcin and collagen type 1 as well as alkaline phosphatase gene expression in tibia by postnatal day 21, which is accompanied with a mineralization defect in the tibia, lowered activity of osteoblastic bone formation, and an increase in fibroblastic growth factor-23, a sign of increased vitamin D effect. Despite these significant effects of TCDD on osteoblast activities, none of the markers of osteoclast activities was found to be affected. Histomorphometry confirmed that osteoblastic activity, but not bone resorption activity, was altered by TCDD. A prominent lesion commonly observed in these TCDD-treated mice was impaired bone mineralization that is characterized by an increased volume and thickness of osteoids lining both the endosteum of the cortical bone and trabeculae. Together, these data suggest that the impaired mineralization resulting from reduction of the osteoblastic activity, which is caused by TCDD-induced up-regulation of vitamin D, is responsible for its bone developmental toxicity.