Notch signaling controls the balance of ciliated and secretory cell fates in developing airways

Although there is accumulated evidence of a role for Notch in the developing lung, it is still unclear how disruption of Notch signaling affects lung progenitor cell fate and differentiation events in the airway epithelium. To address this issue, we inactivated Notch signaling conditionally in the endoderm using a Shh-Cre deleter mouse line and mice carrying floxed alleles of the Pofut1 gene, which encodes an O-fucosyltransferase essential for Notch-ligand binding. We also took the same conditional approach to inactivate expression of Rbpjk, which encodes the transcriptional effector of canonical Notch signaling. Strikingly, these mutants showed an almost identical lung phenotype characterized by an absence of secretory Clara cells without evidence of cell death, and showed airways populated essentially by ciliated cells, with an increase in neuroendocrine cells. This phenotype could be further replicated in cultured wild-type lungs by disrupting Notch signaling with a gamma-secretase inhibitor. Our data suggest that Notch acts when commitment to a ciliated or non-ciliated cell fate occurs in proximal progenitors, silencing the ciliated program in the cells that will continue to expand and differentiate into secretory cells. This mechanism may be crucial to define the balance of differentiated cell profiles in different generations of the developing airways. It might also be relevant to mediate the metaplastic changes in the respiratory epithelium that occur in pathological conditions, such as asthma and chronic obstructive pulmonary disease.

Hyperplasia to neoplasia sequence of duodenal and pancreatic neuroendocrine diseases and pseudohyperplasia of the PP-cells in the pancreas.

Hyperplastic changes of the neuroendocrine cell system may have the potential to evolve into neoplastic diseases. This is particularly the case in the setting of genetically determined and hereditary neuroendocrine tumor syndromes such as MEN1. The review discusses the MEN1-associated hyperplasia-neoplasia sequence in the development of gastrinomas in the duodenum and glucagon-producing tumors in the pancreas. It also presents other newly described diseases (e.g., glucagon cell adenomatosis and insulinomatosis) in which the tumors are (or most likely) also preceded by islet cell hyperplasia. Finally, the pseudohyperplasia of PP-rich islets in the pancreatic head is defined as a physiologic condition clearly differing from other hyperplastic-neoplastic neuroendocrine diseases.

A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.

Transformation to "high grade" neuroendocrine carcinoma as an acquired drug resistance mechanism in EGFR-mutant lung adenocarcinoma.

Several different acquired resistance mechanisms of EGFR mutant lung adenocarcinoma to EGFR-tyrosine kinase inhibitor (TKI) therapy have been described, most recently transformation to small cell lung carcinoma (SCLC). We describe the case of a 46-year-old female with relapsed EGFR exon 19 deletion lung adenocarcinoma treated with erlotinib, and on resistance, cisplatin-pemetrexed. Liver rebiopsy identified an afatinib-resistant combined SCLC and non-small cell carcinoma with neuroendocrine morphology, retaining the EGFR exon 19 deletion. This case highlights acquired EGFR-TKI resistance through transformation to the high-grade neuroendocrine carcinoma spectrum and that that such transformation may not be evident at time of progression on TKI therapy.

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.

Cox-2 Promotes Chromogranin A Expression and Bioactivity: Evidence for a PGE2-Dependent Mechanism and the Involvement of a Proximal cAMP-Responsive Element.

Abstract The prostanoid biosynthetic enzyme cyclooxygenase-2 (Cox-2) is upregulated in several neuroendocrine tumors. The aim of the current study was to employ a neuroendocrine cell (PC12) model of Cox-2 over-expression to identify gene products that might be implicated in the oncogenic and/or inflammatory actions of this enzyme in the setting of neuroendocrine neoplasia. Expression array and real-time PCR analysis demonstrated that levels of the neuroendocrine marker chromogranin A (CGA) were 2-fold and 3.2-fold higher, respectively, in Cox-2 over-expressing cells (PCXII) vs their control (PCMT) counterparts. Immunocytochemical and immunoblotting analyses confirmed that both intracellular and secreted levels of CGA were elevated in response to Cox-2 induction. Moreover, exogenous addition of prostaglandin E2 (1u�­M), mimicked this effect in PCMT cells, while treatment of PCXII cells with the Cox-2 selective inhibitor NS-398 (100 nM) reduced CGA expression levels, thereby confirming the biospecificity of this finding. Levels of neurone specific enolase (NSE) were similar in the two cell lines, suggesting that the effect of Cox-2 on CGA expression was specific and not due to a global enhancement of neuroendocrine marker expression/differentiation. Cox-2-dependent CGA upregulation was associated with significantly increased chromaffin granule number and intracellular and secreted levels of dopamine. CGA promoter-driven reporter gene expression studies provided evidence that prostaglandin E2-dependent upregulation required a proximal cAMP-responsive element (CRE; -71 - -64 bp). This study is the first to demonstrate that Cox-2 upregulates both CGA expression and bioactivity in a neuroendocrine cell line and has major implications for the role of this polypeptide in the pathogenesis of neuroendocrine cancers in which Cox-2 is upregulated.

Morphologic changes in the urethral epithelium in an ethanol-drinking rat strain (UChA and UChB)

The extreme use of ethanol causes metabolic and pathologic changes in testes and urogenital system in different animal species. The enzyme alcohol dehydrogenase (ADH) catalyses the conversion of ethanol into carcinogenic metabolite acetaldehyde which is partly excreted into the urine. However, papers relating the chronic ethanol consumption to the urethral morphology are unknown. This work evaluates the toxic effect of the chronic ethanol ingestion on the urethral epithelium of UChA and UChB rats. Conventional techniques of histology, histochemistry, immunohistochemistry and ultrastructural analysis were used. The analysis showed the presence of lipid drops and intercellular spaces in the epithelial cells in the urethra of UChA and UChB rats compared to control rats. Urethral neuroendocrine cell were observed and characterized for presenting vesicles containing electron-dense granules associated with nervous fibers. We conclude that the chronic consumption of ethanol induces the presence lipid drops in the epithelial cells of the urethra of UChA and UChB rats. The NE cells of the urethra of UChA and UChB rats did not show alterations under chronic effect of the ethanol. (C) 2007 Elsevier Ltd. All rights reserved.

Cysteine String Protein Functions Directly in Regulated Exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPγS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.

Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a receptor-mediated mechanism.

Pulmonary neuroendocrine cells are localized predominantly at airway branchpoints. Previous work showed that gastrin-releasing peptide (GRP), a major pulmonary bombesin-like peptide, occurred in neuroendocrine cells exclusively in branching human fetal airways. We now demonstrate that GRP and GRP receptor genes are expressed in fetal mouse lung as early as embryonic day 12 (E12), when lung buds are beginning to branch. By in situ hybridization, GRP receptor transcripts were at highest levels in mesenchymal cells at cleft regions of branching airways and blood vessels. To explore the possibility that bombesin-like peptides might play a role in branching morphogenesis, E12 lung buds were cultured for 48 hr in serum-free medium. In the presence of 0.10-10 microM bombesin, branching was significantly augmented as compared with control cultures, with a peak of 94% above control values at 1 microM (P < 0.005). The bombesin receptor antagonist [Leu13- psi(CH2NH)Leu14]bombesin alone (100 nM) had no effect on baseline branching but completely abolished bombesin-induced branching. A bombesin-related peptide, [Leu8]phyllolitorin also increased branching (65% above control values at 10 nM, P < 0.005). [Leu8]Phyllolitorin also significantly augmented thymidine incorporation in cultured lung buds. Fibronectin, which is abundant at branchpoints, induces GRP gene expression in undifferentiated cell lines. These observations suggest that BLPs secreted by pulmonary neuroendocrine cells may contribute to lung branching morphogenesis. Furthermore, components of branchpoints may induce pulmonary neuroendocrine cell differentiation as part of a positive feedback loop, which could account in part for the high prevalence of these cells at branchpoints.

CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

"Leiomyomatoid angiomatous neuroendocrine tumor" (LANT) of the pituitary reflects idiosyncratic angiogenesis in adenomas of the gonadotroph cell lineage.

Based on a single-case observation, the descriptive label "leiomyomatoid angiomatous neuroendocrine tumor" (LANT) has been tentatively applied to what was perceived as a possible novel type of dual-lineage pituitary neoplasm with biphasic architecture. We report on two additional examples of an analogous phenomenon encountered in male patients, aged 59 years (Case 1) and 91 years (Case 2). Both tumors were intra- and suprasellar masses, measuring 5.6 cm × 4.4 cm × 3.4 cm, and 2.7 cm × 2 cm × 1.7 cm, respectively. Histologically, Case 1 was an FSH-cell adenoma interwoven by vascularized connective tissue septa that tended to exhibit incremental stages of adventitial overgrowth. The epithelial component of Case 2 corresponded to an LH-cell adenoma, and lay partitioned by a maze of paucicellular to hyalinized vascular axes. Irrespective of architectural variations, perivascular spindle cells exhibited immunopositivity for vimentin, muscular actin, and smooth muscle actin. Conversely, negative results were obtained for CD34, EMA, S100 protein, GFAP, and TTF-1. Ultrastructural study failed to reveal metaplastic cell forms involving transitional features between adenohypophyseal-epithelial and mesenchymal-contractile phenotype. We propose that LANT be regarded as a peculiar reflection of maladaptive angiogenesis in some pituitary adenomas, rather than a genuine hybrid neoplasm. While no mechanistic clue is forthcoming to account for this distinctive pattern, hemodynamic strain through direct arterial - rather than portal - supply of the adenoma's capillary bed may be one such explanatory factor. The apparent predilection of the LANT pattern for macroadenomas of the gonadotroph cell lineage remains unexplained.