Identification of genes associated with the corticotroph phenotype in bronchial carcinoid tumors

The proopiomelanocortin (POMC) gene is occasionally expressed in nonpituitary tumors leading to Cushing's syndrome. Bronchial carcinoid tumors, one of the most frequent source for ectopic ACTH secretion, often display numerous features of the corticotroph phenotype. To identify new markers of corticotroph differentiation in these tumors, we compared the pattern of gene expression in ACTH-secreting (ACTH+) and nonsecreting (ACTH-) bronchial carcinoids by differential display/RT-PCR. Using groups of ACTH+ and ACTH- tumors, we initially selected approximately 300 differentially expressed genes. Fifteen were considered differentially expressed after further characterization by RT-PCR on a larger series of 8 ACTH+ and 12 ACTH- bronchial carcinoids; 11 were restricted to--or overexpressed in--ACTH+ and four in ACTH- tumors. In ACTH+, beside the expected POMC gene, we identified cFos, and KIAA1775, a large expressed sequence tag encoding a putative protocadherin-related protein. On the other hand, the tetraspanin TM4SF5 gene was specifically expressed in ACTH-. Dot blot analysis confirmed the specific expression of KIAA1775 in ACTH+ bronchial carcinoids. However, the expression of most of the differential genes, including KIAA1775, was detected by RT-PCR in pituitary or lung tumors, whether secreting ACTH or not, excepted for TM4SF5, which was only detected in some nonendocrine lung tumors. Our results show that corticotroph differentiation of bronchial carcinoid tumors is accompanied by induction and repression of specific genes. The nature of some of these genes, identified here, underlines the importance of cell-cell or cell-extracellular matrix interactions in the establishment of neoplastic corticotroph phenotype. These genes should help to better characterize ACTH+ bronchial carcinoids as well as other bronchial carcinoid subtypes.     

Integrated classification of lung tumors and cell lines by expression profiling

The utility of cancer cell lines depends largely on their accurate classification, commonly based on histopathological diagnosis of the cancers from which they were derived. However, because cancer is often heterogeneous, the cell line, which also has the opportunity to alter in vitro, may not be representative. Yet without the overall architecture used in histopathological diagnosis of fresh samples, reclassification of cell lines has been difficult. Gene-expression profiling accurately reproduces histopathological classification and is readily applicable to cell lines. Here, we compare the gene-expression profiles of 41 cell lines with 44 tumors from lung cancer. These profiles were generated after hybridization of samples to four replicate 7,685-element cDNA microarrays. After removal of genes that were uniformly up- or down-regulated in fresh compared with cell-line samples, cluster analysis produced four major branch groups. Within these major branches, fresh tumor samples essentially clustered according to pathological type, and further subclusters were seen for both adenocarcinoma (AC) and small cell lung carcinoma (SCLC). Four of eight squamous cell carcinoma (SCC) cell lines clustered with fresh SCC, and 11 of 13 SCLC cell lines grouped with fresh SCLC. In contrast, although none of the 11 AC cell lines clustered with AC tumors, three clustered with SCC tumors and six with SCLC tumors. Although it is possible that preexisting SCC or SCLC cells are being selected from AC tumors after establishment of cell lines, we propose that, even in situ, AC will ultimately progress toward one of two poorly differentiated phenotypes with expression profiles resembling SCC or SCLC.     

Atypical carcinoid of the lung. A distinct clinicopathologic entity

Eleven cases of atypical carcinoid (AC) of the lung were identified during an eight-year period. Their clinical features and treatment responses were contrasted with our experience at Vanderbilt with small cell lung cancer (SCLC) and a literature review of typical bronchial carcinoids (TC). Clinically, there were no features to distinguish AC from TC except for age at diagnosis (59 vs 49 years). Atypical carcinoid was similar to SCLC with respect to many clinical features, although female sex, absence of smoking history and localized disease at presentation were more common in AC. Pathologically, these tumors were distinguished by cellular atypia, necrosis, architectural disorder, or increased mitotic rate in the presence of a recognizable carcinoid pattern. Immunoperoxidase staining revealed no difference between AC and TC or SCLC. Atypical carcinoid of the lung represents a distinct clinicopathologic disease.     

MYC stimulates EZH2 expression by repression of its negative regulator miR-26a

The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.     

Nontumorigenic squamous cell carcinoma line converted to tumorigenicity with methyl methanesulfonate without activation of HRAS or MYC.

Plasticity of human tumor populations could account for the reason why many tumorigenic human cell lines lose this feature when grown in culture. Methyl methanesulfonate (MMS) was used to convert premalignant squamous cell carcinoma (SCC) cell line SCC-83-01-82 to a malignant phenotype. The MMS-treated SCC-83-01-82 cells (MMS-SCC-83-01-82) produced progressively growing tumors in 5 of 11 splenectomized BALB/c nude mice within 3-5 months. A cell line, designated SCC-83-01-82 CA, was established in vitro from one of the mouse tumors and was repassaged successively. This SCC-83-01-82 CA cell line was aggressively tumorigenic. A tumor greater than or equal to 2.0 cm in size was present within a month, as opposed to the 3-5 months required for the tumors produced by the MMS-SCC-83-01-82 cells. Examination of frozen cross sections by in situ hybridization revealed that focal areas of the tumor produced by the MMS-SCC-83-01-82 cells expressed MYC and HRAS mRNA. However, by the third passage in vivo, the levels of expression of the corresponding genes in the mouse tumors were undetectable. Blot-hybridization analysis of the RNA from the MMS-SCC-83-01-82 cells and the subsequently derived tumors and cells did not indicate any consistent overexpression of MYC, HRAS, or KRAS. Restriction fragment length polymorphism analysis of both MYC and HRAS genes revealed neither rearrangement nor amplification of MYC nor point mutation in the 11th or 12th codon of HRAS. The data suggest that alterations in MYC and HRAS were not directly involved in either the initial transformation or MMS-induced tumorigenic conversion of the SCC-83-01-82 cell line. Persistence of tumorigenicity after reisolation of the MMS-converted premalignant SCC-83-01-82 cells did not disappear immediately following the treatment with MMS.     

Gene analysis of K-, H-ras,p53, and retinoblastoma susceptibility genes in human lung cancer cell lines by the polymerase chain reaction/single-strand conformation polymorphism method

In order to know the involvement of multiple gene alterations in the pathogenesis of human lung cancer, we examined the genes of K-, H-ras (codons 12, 13, 61), p53(exons 5–9) and the retinoblastoma susceptibility gene (RB)(exons 20–22) using the polymerase chain reaction/single-strand conformation polymorphism method in 32 human lung cancer cell lines (5 squamous-cell carcinomas, 10 adenocarcinomas, 3 large-cell carcinomas, 14 small-cell carcinomas). In 18 non-small-cell lung cancer lines, gene alterations were found in 4 for K-ras (22%), none for H-ras (0%), 4 for p53 (22%) and none for the RB (0%) gene. In 14 small-cell lung cancer (SCLC) lines, no gene alterations were found in K-ras (0%), or H-ras (0%), but 6 were found for p53 (43%) and 3 for the RB (21%) gene. Coincident abnormalities of K-ras and p53, or K-ras and RB genes were not found in any cell lines, and those of the p53 and RB genes were found in only 2 SCLC lines. No association was observed between these three gene alterations and N-myc amplification. Although the above three genes may be involved to some extent in the pathogenesis of lung cancer, more factors are required for its development.Abbreviation PCR-SSCP polymerase chain reaction/single-strand conformation polymorphism     

Etiology-dependent molecular mechanisms in human hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is characterized by aggressive tumor behavior coupled with poor prognosis. Various etiologies have been linked to HCC development, most prominently chronic hepatitis B and C virus infections as well as chronic alcohol consumption. In approximately 10% of HCCs, the etiology remains cryptic; however, recent epidemiological data suggest that most of these cryptogenic HCCs develop due to nonalcoholic steatohepatitis. To identify etiology-dependent DNA copy number aberrations and genes relevant to hepatocarcinogenesis, we performed array-based comparative genomic hybridization of 63 HCCs of well-defined etiology and 4 HCC cell lines followed by gene expression profiling and functional analyses of candidate genes. For a 10-megabase chromosome region on 8q24, we observed etiology-dependent copy number gains and MYC overexpression in viral and alcohol-related HCCs, resulting in up-regulation of MYC target genes. Cryptogenic HCCs showed neither 8q24 gains, nor MYC overexpression, nor target gene activation, suggesting that tumors of this etiology develop by way of a distinct MYC-independent pathomechanism. Furthermore, we detected several etiology-independent small chromosome aberrations, including amplification of MDM4 on 1q32.1 and frequent gains of EEF1A2 on 20q13.33. Both genes were overexpressed in approximately half the HCCs examined, and gene silencing reduced cell viability as well as proliferation and increased apoptosis rates in HCC cell lines. CONCLUSION: Our findings suggest that MDM4 and EEF1A2 act as etiology-independent oncogenes in a significant percentage of HCCs.     

Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis

BACKGROUND & AIMS: To define the genetic reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. METHODS: Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. RESULTS: A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. CONCLUSIONS: This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.     

Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines

Small cell lung cancer (SCLC) is a fatal malignancy due to its propensity to metastasize widely and to reoccur after chemotherapy in a drug-resistant form. While most SCLC cell lines are anchorage independent for growth, laminin induced the attachment of five of six SCLC cell lines tested (NCI-N417, NCI-H345, NCI-H146, NCI-H187, NCI-H510, and NCI-H209). NCI-N417 SCLC cells adopted a flattened morphology on laminin, and a classic SCLC cell line (NCI-H345) demonstrated a neuron-like appearance while the other SCLC cell lines except NCI-H187 cells, attached but did not spread. Adhesion to laminin was associated with increased resistance to several cytotoxic drugs. Matrigel, an extract of basement membrane proteins, greatly accelerated tumor growth when coinjected with SCLC cells in athymic mice. A synthetic peptide from the B1 chain of laminin, cyclic-YIGSR (Tyr-Ile-Gly-Ser-Arg), inhibited laminin-induced SCLC cell adhesion and migration in vitro and reduced the size of the tumors they formed when coinjected with matrigel and YIGSR. These results suggest that the interaction of SCLC cells with laminin and possibly with other basement membrane proteins can enhance their tumorigenicity and drug resistance.     

The gastroenteropancreatic endocrine system and related tumors

Up to 16 types of endocrine cells have been characterized morphologically (and most of them also functionally) in the gastroenteropancreatic area. Four main groups of pancreatic endocrine tumors (with several subtypes) have been identified: islet cell, ectopic, nonfunctioning, and poorly differentiated tumors. A detailed classification system that combines cytologic and clinicopathologic patterns has been developed for the study of 132 pancreatic tumors. Among a large series (more than 120 cases) of endocrine tumors arising in the gastrointestinal tract, serotonin-producing argentaffin carcinoids have been separated from hindgut trabecular carcinoids, producing glucagon- and pancreatic polypeptide-related peptides, paragangliomas, somatostatin cell tumors, gastrinomas, and argyrophil ECL cell carcinoids. The clinicopathologic profile of the various pancreatic and gastrointestinal tumor entities has been delineated and involvement in the multiple endocrine neoplasia syndrome has been analyzed in detail.     

Expression of cyclo-oxygenase-2 in gastrointestinal carcinoid tumors

BACKGROUND: Cyclo-oxygenase (COX)-2 overexpression is observed in various neoplasms and COX-2 inhibition has been attempted as prevention and/or therapy in these neoplasms. Carcinoid tumors are thought to arise from neuroendocrine cells and originate mainly in the gastrointestinal tract. Cyclo-oxygenase-2 is reportedly expressed in neuroendocrine cells of normal colorectal mucosa. The role of COX in carcinoids has not previously been investigated. The aim of the present paper was to clarify the expression of COX-1 and -2, and their role in human gastrointestinal carcinoids. METHODS: Expression of COX-1 and -2 was studied immunohistochemically in 38 gastrointestinal carcinoids. Five bronchopulmonary and seven metastatic carcinoids were also examined, for comparison with gastrointestinal carcinoids. The immunohistochemical score (IHS) was calculated from staining intensity and immunoreactive cell population, and ranked according to four grades (negative to strong). RESULTS: Cyclo-oxygenase-2 was expressed in all gastrointestinal carcinoids (weak, 1; moderate, 13; strong, 24) and bronchopulmonary carcinoids (weak, 1; moderate, 4), as well as their metastases (moderate, 3; strong, 4). The IHS of COX-2 in larger tumors was significantly lower than that in smaller tumors. However, the IHS of COX-2 at the advancing tumor edge was significantly higher than that at the centers of tumors >or=10 mm in size. Faint COX-1 expression was detected in only one duodenal, one rectal and four bronchopulmonary carcinoids. CONCLUSIONS: Enhanced COX-2 expression was observed in gastrointestinal as well as bronchopulmonary carcinoids and their metastases, especially at the advancing edges of the tumors. Cyclo-oxygenase-2 may play a role in carcinoid progression.     

Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion

MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21(Cip1) was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.     

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.     

A Retrospective Analysis of 1570 Appendiceal Carcinoids

Objective : Information about the management and outcome of appendiceal carcinoids is sparse, because few series comprise more than 100 cases. In this study we have analyzed the epidemiology of 1570 appendiceal carcinoids, to compare outcome with other gastrointestinal carcinoid tumors. Methods : We evaluated 1570 appendiceal carcinoids in a series of 8305 carcinoid tumors from the SEER, the End Results Group, and the Third National Cancer Survey programs of the National Cancer Institute over the time period 1950–1991. Results : Appendiceal carcinoids comprised 18.9% of all carcinoid tumors and exhibited a marked female predominance (M/F ratio: 0.47). Age-adjusted incidence rates were 1.7-fold higher in women compared to men. Appendiceal carcinoids present earlier (average age: 42.2 yr) than other gastrointestinal carcinoids (62.9 yr) or noncarcinoid appendiceal tumors (61.9 yr). At the time of diagnosis 35.4% were nonlocalized. The overall 5-yr survival for localized lesions was 94%, for regional invasion 84.6%, and for distant metastases 33.7%. The 5-yr survival of appendiceal carcinoids (85.9%) was the highest among all types of carcinoid tumors. In 14.6% noncarcinoid tumors at other sites were also evident. Conclusion : The high relative incidence of carcinoid tumors in the appendix is still poorly understood. The good overall 5-yr survival rates of appendiceal carcinoids as opposed to other carcinoids represents either a different biological behavior, earlier diagnosis, or expeditious management (appendectomy). However, the increased likelihood of coexisting neoplasms and the not uncommon presentation of metastatic disease should warrant careful evaluation and postoperative follow-up of such lesions.