DEC1 (STRA13) protein expression relates to hypoxia- inducible factor 1-alpha and carbonic anhydrase-9 overexpression in non-small cell lung cancer

Differentiated embryo-chondrocyte expressed gene 1 (DEC1) is involved in cell differentiation, proliferation, and apoptosis, and was recently shown to be regulated by hypoxia. The present immunohistochemical study demonstrates extensive nuclear expression of the protein in 38% of a series of 115 non-small cell lung carcinomas using a polyclonal antibody (Ab) recognizing DEC1 protein. Such expression was directly related to the expression of two hypoxia-regulated proteins, namely the hypoxia-inducible factor (HIF) 1alpha and carbonic anhydrase-9. Although DEC1 was not related to angiogenesis or to the expression of VEGF and thymidine phosphorylase, a direct association with up-regulated bFGF receptors was noted. DEC1 was persistently expressed in the nuclei of normal bronchial and alveolar tissue. It is suggested that loss of DEC1 expression is an early event in the development of lung cancer, while DEC1 gene expression occurs in a subset of tumours and parallels the overexpression of other hypoxia-regulated proteins.     

Abl expression in human fetal and adult tissues,tumours and tumour microvessels

Abl kinases encoded by the abl oncogenes inhibit apoptosis without affecting cell proliferation. The aim of this study was to examine a wide range of normal fetal and adult human tissues and a variety of tumour types for Abl immunoreactivity. Sections from 193 paraffin blocks of normal fetal and adult tissues and 72 blocks from representative tumours were stained immunohistochemically using a polyclonal antibody to c-Abl/Bcr–Abl oncoprotein. Weak Abl immunoreactivity was observed in many adult tissues. Moderately intense or strong staining (cytoplasmic, nuclear or membranous) was consistently seen in hyaline cartilage, adipocytes, and ciliated epithelium. In fetal tissues, there was a broadly similar staining pattern, but Abl expression was also seen in muscle (all types) and occasionally in endothelial cells. The most intense staining was seen in sites of endochondral ossification and in the umbilical cord stroma. Negatively staining tissues included epidermis and squamous mucosa, lymph nodes, tonsil, spleen, hepatocytes, and adrenals. Most tumours showed focal or weak Abl immunoreactivity. The most intense staining was seen in chondrosarcoma, liposarcoma, and diffuse gastric (signet ring) adenocarcinoma. In the latter two tumour types, Abl expression was also observed in tumour microvessels. These results suggest that Abl not only functions as an apoptosis inhibitor, but also may have a role in connective tissue maturation and differentiation and in tumour growth and angiogenesis. © 1997 John Wiley & Sons, Ltd.     

Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1)

The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.     

Discovered on gastrointestinal stromal tumours 1 (DOG1) expression in non-gastrointestinal stromal tumour (GIST) neoplasms

Hemminger J & Iwenofu O H
(2012) Histopathology  61, 170–177 Discovered on gastrointestinal stromal tumours 1 (DOG1) expression in non‐gastrointestinal stromal tumour (GIST) neoplasms Aims: To further characterize discovered on GIST1 (DOG1) antibody clone K9 expression in a broad range of mesenchymal and epithelial tumours. Methods and results: Formalin‐fixed paraffin‐embedded sections of various tumours were stained with the anti‐DOG1 monoclonal antibody clone K9. The tumours (n = 187) included: gastrointestinal stromal tumours (GISTs) (n = 20); malignant melanoma (n = 19); schwannoma (n = 10); neurofibroma (n = 10); leiomyosarcoma (n = 10); low‐grade fibromyxoid sarcoma (n = 5); angiosarcoma, (n = 10); epithelioid sarcoma (n = 5); clear cell sarcoma (n = 3); synovial sarcoma (n = 10); malignant peripheral nerve sheath tumour (MPNST) (n = 12); alveolar soft part sarcoma (n = 3); chordoma (n = 5); pleomorphic undifferentiated sarcoma (n = 5); perineurioma (n = 4); granular cell tumour (n = 6); acinic cell carcinoma (n = 5); adenocarcinoma, lung (n = 5), colon (n = 10), endometrioid (n = 10), prostate (n = 10) and renal cell (n = 10). Nineteen of 20 GISTs expressed DOG‐1 and 12 of 20 were diffusely positive (≥95%) with moderate to strong intensity. There was focal, predominantly luminal staining of colorectal (three of 10), endometrioid (four of 10) and acinic cell carcinomas (four of five). One case each of spindle cell/desmoplastic melanoma (2+), schwannoma (trace) and MPNST (2+) showed DOG‐1 expression. Conclusions: Our study supports that DOG‐1 is a highly sensitive and specific marker for GISTs and also highlights hitherto unrecognized and unusual patterns of expression in non‐mesenchymal neoplasms.     

Differential expression of vesicular monoamine transporter (VMAT) 1 and 2 in gastrointestinal endocrine tumours.

Neuroendocrine tumours are characterized by their capacity to produce hormones, which are stored in vesicles and secretory granules. Demonstration of granule/vesicle proteins in tumours is taken as evidence of neuroendocrine differentiation. Vesicular monoamine transporters (VMAT1 and VMAT2) mediate the transport of amines into vesicles of neurons and endocrine cells. The expression of VMAT1 and VMAT2 and the usefulness of VMAT1 and VMAT2 in the histopathological diagnosis of gastrointestinal endocrine tumours have not been fully explored. This study therefore investigated the expression of VMAT1 and VMAT2 in 211 human gastrointestinal tumours by immunocytochemistry and western blotting. VMAT1 and/or VMAT2 were demonstrated in the majority of amine-producing endocrine tumours of gastric, ileal, and appendiceal origin. Serotonin-producing endocrine tumours (ileal and appendiceal carcinoids) expressed predominantly VMAT1, while histamine-producing endocrine tumours (gastric carcinoids) expressed VMAT2 almost exclusively. In peptide-producing endocrine tumours such as rectal carcinoids and endocrine pancreatic tumours, only a small number of immunopositive tumour cells were observed. No labelling was found in non-endocrine tumours, including gastric, colorectal and pancreatic adenocarcinomas and gastrointestinal stromal tumours. In conclusion, VMAT1 and VMAT2 are differentially expressed by gastrointestinal endocrine tumours, with a pattern specific for each tumour type, reflecting their neuroendocrine differentiation and origin. VMAT1 and VMAT2 may therefore become valuable markers in the classification of neuroendocrine tumours and may also indicate patients suitable for radioisotope treatment operating via these transporter systems.     

Obestatin in human neuroendocrine tissues and tumours: expression and effect on tumour growth

The hormone obestatin, which is derived from the same precursor as ghrelin and whose receptor(s) is still unrecognized, possesses a variety of metabolic/modulatory functions mostly related to food intake suppression and reduction of gastrointestinal motility. The distribution of obestatin in normal and neoplastic human tissues is poorly understoood. We report that in fetal tissue samples, obestatin peptide was detected in the thyroid, pituitary, lung, pancreas and gastrointestinal tract, usually being co‐localized with chromogranin A. In adult tissues, obestatin protein expression was restricted to pituitary, lung, pancreas and gastrointestinal tract and was co‐localized strictly with ghrelin. By contrast, in endocrine tumours obestatin was expressed in a small fraction of thyroid, parathyroid, gastrointestinal and pancreatic neoplasms, in most cases with a focal immunoreactivity and co‐localized with ghrelin. Messenger RNA levels of the specific fragments of ghrelin and obestatin were comparable in both normal and tumour samples, confirming that post‐translational mechanisms rather than alternative splicing events lead to ghrelin/obestatin production. Finally, in TT and BON‐1 cell lines obestatin induced antiproliferative effects at pharmacological doses, opposite to those observed with ghrelin. In summary, our data demonstrate that obestatin is produced by the same endocrine cells that express ghrelin in normal tissues from fetal to adult life, whereas, as compared to ghrelin, in neoplastic conditions it is down‐regulated by post‐translational modulation and shows potential antiproliferative properties in vitro. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Frequent expression of 75 kDa nerve growth factor receptor and phosphotyrosine in human peripheral nerve tumours: an immunohistochemical study on paraffin-embedded tissues

One hundred and three benign, and 10 malignant peripheral nerve tumours were examined immunohistochemically for expression of 75 kDa nerve growth factor receptor (NGFR). In benign tumours NGFR was demonstrated at 61% in neurinoma, 71% in neurofibroma, 93% in neurofibromatosis and 90% in traumatic neuroma. Malignant neurogenic tumours were 100% positive for NGFR. Phosphotyrosine-immunoreactivity was detected in 76% of NGFR-positive tumours but the frequency of immunostained tumour cells was low. These results suggest that both benign and malignant peripheral nerve tumours express 75 kDa NGFR. The receptor seems to serve as growth signal transduction of the tumour cells in terms of phosphorylation of the tyrosine residue of the receptor or the target protein of the NGFR protein tyrosine kinase.     

Immunocytochemical demonstration of intermediate filament cytoskeleton proteins in human endocrine tissues and (neuro-) endocrine tumours

Summary The presence and distribution of intermediate filament proteins, such as cytokeratins, vimentin, neurofilament proteins and glial fibrillary acidic protein were assessed immunohistochemically in pituitary adenomas, medullary thyroid carcinomas, endocrine pancreatic tumours, gastric, intestinal and bronchial carcinoids, parathyroid adenomas, pheochromocytomas, paragangliomas and related non-neoplastic tissues. In some cases, immunohistochemical results were correlated with cytoskeletal proteins as analysed by SDS-polyacrylamide gel electrophoresis. Cytokeratin antibodies with broad range of immunoreactivity (i.e. to murine liver cytokeratin component D) reacted with epithelial cells in all non-neoplastic endocrine tissues and related neuroendocrine tumours studied, except for adrenal medulla, pheochromocytoma and paraganglioma, independently of hormone production and biological behaviour. In contrast, antibodies to epidermis-derived cytokeratins failed to stain endocrine tissues and tumours. Paranuclear cytokeratin accumulations were seen in bronchial, gastric, and intestinal carcinoids and seem to be a common feature of neuroendocrine tumours. One-and two-dimensional SDS-polyacrylamide gel electrophoresis of non-neoplastic endocrine tissues and related tumours revealed two major keratin polypeptides corresponding to cytokeratins No. 8 and 18 of the cytokeratin catalog of human cells (Moll et al. 1982). According to this cytokeratin polypeptide composition, endocrine tissues and related tumours conform to the simple type of epithelia. Vimentin-related immunoreactivity was restricted to stromal cells and to folliculo-stellate cells in normal pituitary gland, Schwann cells in carcinoids and satellite cells in normal adrenal medulla and in pheochromocytomas. Neurofilament protein- (70 kD)-antibodies only stained nerve fibers in normal tissues and at the periphery of carcinoid tumour cell complexes, and, to a variable degree, cells in nontumorous adrenal medulla, pheochromocytomas and paragangliomas. Furthermore, neurofilament reactivity was observed along with cytokeratin expression in two bronchial carcinoids.     

Expression of cortistatin and MrgX2, a specific cortistatin receptor, in human neuroendocrine tissues and related tumours

Cortistatin (CST), a novel hormone originally described in the rat, mouse, and human cerebral cortex, displays structural and functional similarities to somatostatin (SRIF). CST binds to all five somatostatin receptors and, differently from SRIF, also binds to MrgX2, which has recently been identified as its specific receptor. Little is known about the distribution of CST and MrgX2 in peripheral non-tumour and neoplastic tissues. The aim of the present study was therefore to determine by immunohistochemistry and mRNA analysis (RT-PCR) the distribution of CST and MrgX2 in 56 human non-tumour and 108 tumour tissues, with special reference to neuroendocrine tissue types. Despite the high level of CST mRNA expression in non-tumour and tumour (both neuroendocrine and non-neuroendocrine) tissues, the presence of immunoreactive CST was confirmed in a subset of gastroenteropancreatic, parathyroid, and pituitary non-tumour cells only, and showed a predominantly focal pattern in most neuroendocrine tumours. Co-localization experiments in the gastroenteropancreatic system demonstrated that the normal CST-producing cells are delta cells, while in the adenohypophysis no preferential co-localization of CST with any of the pituitary hormones was observed. MrgX2 mRNA was variably detected in the hypothalamus, pituitary, thyroid, lung, gastroenteropancreatic tract, testis, and ovary, and was negative in the cerebral cortex, parathyroid, and adrenal, as well as in a variety of tumour types. Conversely, immunolocalization of MrgX2 protein was restricted to neurohypophysis and testis, whilst all tumours analysed were negative. A possible explanation for the discrepancy between RT-PCR and immunohistochemistry is that MrgX2 protein was widely detected in blood vessels, scattered lymphocytes, and gastrointestinal ganglia in both normal and neoplastic samples. The findings demonstrate a selective distribution of CST in normal and neoplastic neuroendocrine tissues, suggesting that CST might have a broader functional role than previously assumed, whereas possible autocrine/paracrine actions via its recently described specific receptor MrgX2 are restricted to selected tissues.     

Altered pattern of Cul-1 protein expression and neddylation in human lung tumours: relationships with CAND1 and cyclin E protein levels

The Cul-1 protein is the scaffold element of SCF complexes that are involved in the proteasomal degradation of numerous proteins regulating cell cycle progression. Owing to this central role in cell growth control, aberrant expression of the components of SCF is thought to play a role during tumourigenesis. Nothing is known about Cul-1 expression in human tumours. In this study, we have analysed its status in a series of 128 human lung carcinomas, comprising 50 non-small cell lung cancers (NSCLCs; 29 squamous cell carcinomas and 21 adenocarcinomas) and 78 neuroendocrine (NE) lung tumours (24 typical and atypical carcinoids, 19 large cell NE carcinomas and 35 small cell lung carcinomas), using immunohistochemistry. We report for the first time an altered pattern of Cul-1 expression in human tumours; indeed, we show that Cul-1 expression is up-regulated in 40% (51/128) of all lung tumours as compared to normal lung tissues, including 34% (17/50), 75% (18/24) and 30% (16/54) of NSCLCs, carcinoids and high grade neuroendocrine lung carcinomas, respectively. Furthermore, we demonstrate that high levels of Cul-1 protein are associated with a low KI67 proliferative index (p = 0.005) and with a decrease in the cyclin E oncoprotein (p = 0.0003), one of the major targets of SCF complexes. These data suggest that up-regulation of Cul-1 could protect cells from hyperproliferative signals through cyclin E down-regulation. Cul-1 is modified by neddylation, a post-translational modification that grafts ubiquitin-like Nedd8/Rub1 residues and controls Cul-1 activity. We also provide evidence that neddylated forms of Cul-1 are specifically expressed in high-grade NE lung tumours and are associated with down-regulation of the Cul-1 inhibitor CAND1 (p = 0.03) and a high level of cyclin E (p = 0.0002). These data support the notion that alterations in the Cul-1 neddylation/deneddylation pathway could contribute to the development of these highly aggressive lung tumours.     

转录因子 Elf-1在上皮性卵巢癌中的表达及其意义

目的 探讨转录因子Elf-1在上皮性卵巢癌中的表达及其临床意义,并探讨Elf-1与卵巢癌细胞增殖的相关性.方法 收集31例上皮性卵巢肿瘤组织及3例正常卵巢组织标本,采用免疫组化S-P法测定Elf-1在上皮性卵巢癌中的表达并对其表达情况进行分析.结果 在23例上皮性卵巢癌组织中Elf-1阳性表达15例(65.22%).Elf-1的表达与组织学类型有关(χ2=6.54,P＜0.05),与患者的年龄、FIGO临床分期差异无统计学意义(χ2=0.25和0.08,P＞0.05).Elf-1在上皮性卵巢癌中的表达与c-Fos在上皮性卵巢癌中的表达呈正相关(Kappar=0.39,P＞0.05).结论 Elf-1在上皮性卵巢癌组织中呈高水平表达,且与原癌基因c-Fos的表达呈正相关,提示Elf-1可能通过影响上皮性卵巢癌细胞的增殖而参与其发生和发展.     

Mutational analysis of hypoxia‐related genes HIF1α and CUL2 in common human cancers

Park SW, Chung NG, Hur SY, Kim HS, Yoo NJ, Lee SH. Mutational analysis of hypoxia‐related genes HIF1α and CUL2 in common human cancers. APMIS 2009; 117: 880–5. Hypoxia is a general feature of solid cancer tissues. Hypoxia upregulates hypoxia‐inducible factor 1α (HIF1α) that transactivates downstream genes and contributes to cancer pathogenesis. HIF1α is upregulated not only by hypoxia but also by genetic alterations in HIF1α‐related genes, including VHL. Cullin 2 (CUL2) interacts with the trimeric VHL‐elongin B‐elongin C complex and plays an essential role in the ubiquitinated degradation of HIF1α. The aim of this study was to explore whether HIF1α and CUL2 genes are somatically mutated, and contribute to HIF1α activation in common human cancers. For this, we have analyzed the coding region of oxygen‐dependent degradation domain of HIF1α in 47 colon, 47 gastric, 47 breast, 47 lung, and 47 hepatocellular carcinomas, and 47 acute leukemias by a single‐strand conformation polymorphism assay. In addition, we analyzed mononucleotide repeat sequences (A8) in CUL2 in 55 colorectal and 45 gastric carcinomas with microsatellite instability (MSI). We found one HIF1α mutation (p.Ala593Pro) in the hepatocellular carcinomas (1/47; 2.1%), but none in other cancers. We found two CUL2 frameshift mutations in colon cancers (p.Asn292MetfsX20), which were exclusively detected in high MSI cancers (4.9%; 2/41). Our data indicate that somatic mutation of HIF1α is rare in common cancers, and somatic mutation of CUL2 occurs in a fraction of colorectal cancers (colorectal cancers with high MSI). The data suggest that neither HIF1α nor CUL2 mutation may play a central role in HIF1α activation in gastric, colorectal, breast, lung and hepatocellular carcinomas, and acute leukemias.