ASCL1

Gene Summary

Gene:ASCL1; achaete-scute family bHLH transcription factor 1
Aliases: ASH1, HASH1, MASH1, bHLHa46
Location:12q23.2
Summary:This gene encodes a member of the basic helix-loop-helix (BHLH) family of transcription factors. The protein activates transcription by binding to the E box (5'-CANNTG-3'). Dimerization with other BHLH proteins is required for efficient DNA binding. This protein plays a role in the neuronal commitment and differentiation and in the generation of olfactory and autonomic neurons. Mutations in this gene may contribute to the congenital central hypoventilation syndrome (CCHS) phenotype in rare cases. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:achaete-scute homolog 1
HPRD
Source:NCBIAccessed: 26 August, 2015

Ontology:

What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 26 August 2015 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 26 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: ASCL1 (cancer-related)

Torchia J, Picard D, Lafay-Cousin L, et al.
Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis.
Lancet Oncol. 2015; 16(5):569-82 [PubMed] Related Publications
BACKGROUND: Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours.
METHODS: We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling.
FINDINGS: Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13-57, and 20%, 6-34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7-61, and 9%, 0-21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04-3·85; p=0·038) and 3·98 (1·71-9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes.
INTERPRETATION: An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours.
FUNDING: C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

Augustyn A, Borromeo M, Wang T, et al.
ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers.
Proc Natl Acad Sci U S A. 2014; 111(41):14788-93 [PubMed] Free Access to Full Article Related Publications
Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel "druggable" targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.

Huang C, Chan JA, Schuurmans C
Proneural bHLH genes in development and disease.
Curr Top Dev Biol. 2014; 110:75-127 [PubMed] Related Publications
Proneural genes encode evolutionarily conserved basic-helix-loop-helix transcription factors. In Drosophila, proneural genes are required and sufficient to confer a neural identity onto naïve ectodermal cells, inducing delamination and subsequent neuronal differentiation. In vertebrates, proneural genes are expressed in cells that already have a neural identity, but they are still required and sufficient to initiate neurogenesis. In all organisms, proneural genes control neurogenesis by regulating Notch-mediated lateral inhibition and initiating the expression of downstream differentiation genes. The general mode of proneural gene function has thus been elucidated. However, the regulatory mechanisms that spatially and temporally control proneural gene function are only beginning to be deciphered. Understanding how proneural gene function is regulated is essential, as aberrant proneural gene expression has recently been linked to a variety of human diseases-ranging from cancer to neuropsychiatric illnesses and diabetes. Recent insights into proneural gene function in development and disease are highlighted herein.

Endo T, Yazawa T, Shishido-Hara Y, et al.
Expression of developing neural transcription factors in lung carcinoid tumors.
Pathol Int. 2014; 64(8):365-74 [PubMed] Related Publications
In lung tumors, the association between carcinoids and high-grade neuroendocrine tumors (HGNETs) is controversial. To understand the phenotypic similarities/differences between lung carcinoids and HGNETs, we comparatively investigated the expression of three kinds of developing neural transcription factors (DNTFs: BRN2, TTF1 and ASCL1) and multiple endocrine neoplasia type 1 (MEN1) as well as RB1 and P53 using 18 carcinoids and 16 HGNETs. The DNTFs were expressed in 10 of the 18 carcinoids and in all the HGNETs, while normal neuroendocrine cells, which are considered the major cell origin of lung carcinoids and small cell carcinomas, did not express DNTFs. Both the DNTF(-) and DNTF(+) carcinoids contained typical and atypical carcinoids. All the DNTF(-) carcinoids examined were formed in the bronchial wall. All the MEN1(-) carcinoids examined were classified into the DNTF(-) carcinoids, while all the HGNETs expressed MEN1. This finding suggests that DNTF(-) MEN1(-) carcinoids are unlikely to be precursors of HGNETs. Although the status of RB1 and P53 between carcinoids and HGNETs were apparently different, the DNTF(+) carcinoids of two male patients and one female patient revealed morphologies resembling HGNET cells and relatively high Ki67 indices. Further investigation of DNTF expression in carcinoids might provide important clues to understand the association between carcinoids and HGNETs.

Kasim M, Benko E, Winkelmann A, et al.
Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia.
J Biol Chem. 2014; 289(39):26973-88 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, whereas additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pulldown experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease.

Beyeler S, Joly S, Fries M, et al.
Targeting the bHLH transcriptional networks by mutated E proteins in experimental glioma.
Stem Cells. 2014; 32(10):2583-95 [PubMed] Related Publications
Glioblastomas (GB) are aggressive primary brain tumors. Helix-loop-helix (HLH, ID proteins) and basic HLH (bHLH, e.g., Olig2) proteins are transcription factors that regulate stem cell proliferation and differentiation throughout development and into adulthood. Their convergence on many oncogenic signaling pathways combined with the observation that their overexpression in GB correlates with poor clinical outcome identifies these transcription factors as promising therapeutic targets. Important dimerization partners of HLH/bHLH proteins are E proteins that are necessary for nuclear translocation and DNA binding. Here, we overexpressed a wild type or a dominant negative form of E47 (dnE47) that lacks its nuclear localization signal thus preventing nuclear translocation of bHLH proteins in long-term glioma cell lines and in glioma-initiating cell lines and analyzed the effects in vitro and in vivo. While overexpression of E47 was sufficient to induce apoptosis in absence of bHLH proteins, dnE47 was necessary to prevent nuclear translocation of Olig2 and to achieve similar proapoptotic responses. Transcriptional analyses revealed downregulation of the antiapoptotic gene BCL2L1 and the proproliferative gene CDC25A as underlying mechanisms. Overexpression of dnE47 in glioma-initiating cell lines with high HLH and bHLH protein levels reduced sphere formation capacities and expression levels of Nestin, BCL2L1, and CDC25A. Finally, the in vivo induction of dnE47 expression in established xenografts prolonged survival. In conclusion, our data introduce a novel approach to jointly neutralize HLH and bHLH transcriptional networks activities, and identify these transcription factors as potential targets in glioma.

Li S, Mattar P, Dixit R, et al.
RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis.
J Neurosci. 2014; 34(6):2169-90 [PubMed] Related Publications
Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1's fate specification properties in murine cortical progenitors--at higher RAS/ERK levels, Ascl1(+) progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1's GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.

Tesfazghi S, Eide J, Dammalapati A, et al.
Thiocoraline alters neuroendocrine phenotype and activates the Notch pathway in MTC-TT cell line.
Cancer Med. 2013; 2(5):734-43 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC.

Teicher BA
Targets in small cell lung cancer.
Biochem Pharmacol. 2014; 87(2):211-9 [PubMed] Related Publications
Recurrent small cell lung cancer is a recalcitrant malgnancy. The application of genomic technologies has begun to elucidate the large number of genetic abnormalities in SCLC. Several cell surface receptors are known to be overexpressed by SCLC in clinic specimens and cell in culture including GPCRs such as the bradykinin receptor, the chemokine receptor CXCR4, the vasopression receeptor and the three bomebsin receptors. The glucose transporter GLUT1, the tetraspanin family member PETA/CD151 and the immunoglobulin superfamily member ALCAM/CD166 are also overexpressed by SCLC. NCAM/CD56 is overexpressed by nearly all SCLC and is currently the target for an antibody drug conjugate in Phase II trial. Although SCLC is not considered a RTK driven disease, IGF1R and FGFRs are often overexpressed by SCLC. SCLC abberantly expresses several developmental transcription factors including ASCL1, SOX2, 4, and 11, OCT4, NANOG, PAX5; however, overexpression of MYC may be a driver in SCLC. Like other cancers, SCLC expresses survival factors and uses aerobic glycolysis as a major source of ATP. The drawback of many potential targets overexpressed by SCLC is expression of the same proteins by normal tissues. We are slowly learning more about the molecular abnormalities that occur in SCLC; however, therapeutic impact from new findings remains a goal to work toward.

Kosari F, Ida CM, Aubry MC, et al.
ASCL1 and RET expression defines a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation.
Oncogene. 2014; 33(29):3776-83 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine (NE) cell development, but its value as a biomarker of NE differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1 expression in lung cancer samples of varied histologic subtype, clinical outcome and smoking status and compared with expression of traditional NE markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers and other lung cancer subtypes. ASCL1 protein expression by immunohistochemical (IHC) analysis correlated best with synaptophysin compared with chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1-positive tumors (ASCL1(+)) compared with ASCL1(-) tumors (q-value <10(-9)). High levels of RET expression in ASCL1(+) but not in ASCL1(-) tumors was associated with significantly shorter overall survival (OS) in stage 1 (P=0.007) and in all AD (P=0.037). RET protein expression by IHC had an association with OS in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression defines a clinically relevant subgroup of ∼10% of AD characterized by NE differentiation.

Wang P, Zhang HL, Li W, et al.
Generation of patient-specific induced neuronal cells using a direct reprogramming strategy.
Stem Cells Dev. 2014; 23(1):16-23 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Direct reprogramming of human fibroblasts into functional neurons in vitro by defined factors provides an invaluable resource for regenerative medicine. However, clinical applications must consider the risk of immune rejection, thus patient-specific induced neuronal cells (iNCs) may serve as an ideal source for autologous cell replacement. In this study, we report a robust process for functional neuronal cells from the patients' scalp by lentiviral gene delivery of Ascl1, Myt1l, and Sox2. These three-factor iNCs are similar to human neuronal cells in morphology, surface antigens, gene expression, and electrophysiological characteristics. Our findings might provide a source of patient-specific functional neurons for cell therapy.

Jin C, Yu D, Čančer M, et al.
Tat-PTD-modified oncolytic adenovirus driven by the SCG3 promoter and ASH1 enhancer for neuroblastoma therapy.
Hum Gene Ther. 2013; 24(8):766-75 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Secretogranin III (SGC3) belongs to the granin family and is highly expressed in endocrine and neural tissues. The human SCG3 promoter has not yet been characterized. We identified that a 0.5-kb DNA fragment upstream of the SCG3 gene can selectively drive transgene expression in neuroblastoma cell lines. The strength of transgene expression was further increased, with specificity maintained, by addition of the human achaete-scute complex homolog 1 (ASH1) enhancer. We developed an oncolytic serotype 5-based adenovirus, in which the SCG3 promoter and ASH1 enhancer drive E1A gene expression. The virus was further modified with a cell-penetrating peptide (Tat-PTD) in the viral capsid, which we have previously shown results in increased adenovirus transduction efficiency of many neuroblastoma cell lines. The virus, Ad5PTD(ASH1-SCG3-E1A), shows selective and efficient killing of neuroblastoma cell lines in vitro, including cisplatin-, etoposide-, and doxorubicin-insensitive neuroblastoma cells. Furthermore, it delays tumor growth and thereby prolonged survival for nude mice harboring subcutaneous human neuroblastoma xenograft. In conclusion, we report a novel oncolytic adenovirus with potential use for neuroblastoma therapy.

Pei D, Luther W, Wang W, et al.
Distinct neuroblastoma-associated alterations of PHOX2B impair sympathetic neuronal differentiation in zebrafish models.
PLoS Genet. 2013; 9(6):e1003533 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b(+), ascl1(+), elavl3(-) cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

Rheinbay E, Suvà ML, Gillespie SM, et al.
An aberrant transcription factor network essential for Wnt signaling and stem cell maintenance in glioblastoma.
Cell Rep. 2013; 3(5):1567-79 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Glioblastoma (GBM) is thought to be driven by a subpopulation of cancer stem cells (CSCs) that self-renew and recapitulate tumor heterogeneity yet remain poorly understood. Here, we present a comparative analysis of chromatin state in GBM CSCs that reveals widespread activation of genes normally held in check by Polycomb repressors. These activated targets include a large set of developmental transcription factors (TFs) whose coordinated activation is unique to the CSCs. We demonstrate that a critical factor in the set, ASCL1, activates Wnt signaling by repressing the negative regulator DKK1. We show that ASCL1 is essential for the maintenance and in vivo tumorigenicity of GBM CSCs. Genome-wide binding profiles for ASCL1 and the Wnt effector LEF-1 provide mechanistic insight and suggest widespread interactions between the TF module and the signaling pathway. Our findings demonstrate regulatory connections among ASCL1, Wnt signaling, and collaborating TFs that are essential for the maintenance and tumorigenicity of GBM CSCs.

Li J, Wang Y, Zhu Y, Oupický D
Recent advances in delivery of drug-nucleic acid combinations for cancer treatment.
J Control Release. 2013; 172(2):589-600 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations.

Ishii J, Sato H, Sakaeda M, et al.
POU domain transcription factor BRN2 is crucial for expression of ASCL1, ND1 and neuroendocrine marker molecules and cell growth in small cell lung cancer.
Pathol Int. 2013; 63(3):158-68 [PubMed] Related Publications
BRN2 is a developmental neural cell-specific POU domain transcription factor and is crucial for cell lineage determination. We investigated the importance of BRN2 in the expression of the lineage-specific transcription factors (achaete-scute homolog-like 1 (ASCL1) and NeuroD1 (ND1)) and neural/neuroendocrine marker molecules (neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYP) and chromogranin A (CHGA)) in small cell lung cancer (SCLC) using cultured lung cancer cells. All examined SCLC cell lines expressed BRN2, as well as ASCL1, ND1, NCAM1, SYP and CHGA. The expression levels of ASCL1, ND1, NCAM1, SYP and CHGA considerably decreased when BRN2 was knocked down in SCLC cells, and the addition of a BRN2 transgene into non-SCLC (NSCLC) cells induced the expression of ASCL1, ND1, NCAM1, SYP and CHGA. However, the BRN2 gene was not activated by the forced expression of ASCL1 or ND1 in NSCLC cells. The knockdown of BRN2 caused significant growth retardation with decrease of S to G2 phase population and mitotic cell rates and unaltered Ki-67-labeled or apoptotic cell rates in SCLC cells, indicating increase of G1 phase population. These findings suggest that BRN2 is a higher level regulator than ASCL1 and ND1 and BRN2 might be involved in aggressiveness of SCLC.

Wang XY, Jensen-Taubman SM, Keefe KM, et al.
Achaete-scute complex homolog-1 promotes DNA repair in the lung carcinogenesis through matrix metalloproteinase-7 and O(6)-methylguanine-DNA methyltransferase.
PLoS One. 2012; 7(12):e52832 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1) is a member of the basic helix-loop-helix (bHLH) transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor-initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7) and O(6)-methylguanine-DNA methyltransferase (MGMT). Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

Conconi D, Panzeri E, Redaelli S, et al.
DNA copy number alterations and PPARG amplification in a patient with multifocal bladder urothelial carcinoma.
BMC Res Notes. 2012; 5:607 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
BACKGROUND: Bladder cancer is the seventh most common cancer worldwide and over 90% are transitional cell carcinoma (TCC). At the first time of diagnosis at least 70% of TCC present as superficial bladder cancer. Because the clinical outcome of superficial bladder tumors is relatively unpredictable, there is a pressing need to identify markers that may predict tumor recurrence and progression and new treatment strategies.
CASE PRESENTATION: We present a unique case of a 67-year old male who underwent total cystectomy after repeated trans-urethral resections of the bladder for multifocal non-muscle invasive bladder cancer. The first and the third tumor were diagnosed as high grade non-infiltrating (HGNI), while the second as carcinoma in situ (CIS). We performed both array comparative genomic hybridization and a targeted chromosomal profile by UroVysion in order to detect copy number variations (CNVs) that may be involved with tumor recurrence and progression. The overall data from this study provide new evidence for the monoclonal origin of urothelial tumor multifocality as several genetic changes were found in different tumors of the same patient. From the analysis of shared CNVs two gained regions emerged at 3p25.2 and 12q23.2, including PPARG and ASCL1 genes, respectively. The copy number level of these genes would seem inversely mutually correlated and highly dependent on histological grade, because the highest level of amplification at 3p25.2 was evidenced in the two HGNI samples, while the highest level of copy number gain at 12q23.2 was reported in the CIS.
CONCLUSION: We provide new evidence on the role of PPARG in initiation and maintenance of bladder cancer. For the first time we also suggest a possible explanation for the elevated expression of PPARG in this type of tumor through a focal high level amplification at 3p25.2. Furthermore, a new gene, ASCL1, emerged as a potential candidate to assist PPARG in bladder carcinogenesis.

Xiao Y, Jaskula-Sztul R, Javadi A, et al.
Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy.
Nanoscale. 2012; 4(22):7185-93 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.

Demelash A, Rudrabhatla P, Pant HC, et al.
Achaete-scute homologue-1 (ASH1) stimulates migration of lung cancer cells through Cdk5/p35 pathway.
Mol Biol Cell. 2012; 23(15):2856-66 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
Our previous data suggested that the human basic helix-loop-helix transcription factor achaete-scute homologue-1 (hASH1) may stimulate both proliferation and migration in the lung. In the CNS, cyclin-dependent kinase 5 (Cdk5) and its activator p35 are important for neuronal migration that is regulated by basic helix-loop-helix transcription factors. Cdk5/p35 may also play a role in carcinogenesis. In this study, we found that the neuronal activator p35 was commonly expressed in primary human lung cancers. Cdk5 and p35 were also expressed by several human lung cancer cell lines and coupled with migration and invasion. When the kinase activity was inhibited by the Cdk5 inhibitor roscovitine or dominant-negative (dn) Cdk5, the migration of lung cancer cells was reduced. In neuroendocrine cells expressing hASH1, such as a pulmonary carcinoid cell line, knocking down the gene expression by short hairpin RNA reduced the levels of Cdk5/p35, nuclear p35 protein, and migration. Furthermore, expression of hASH1 in lung adenocarcinoma cells normally lacking hASH1 increased p35/Cdk5 activity and enhanced cellular migration. We were also able to show that p35 was a direct target for hASH1. In conclusion, induction of Cdk5 activity is a novel mechanism through which hASH1 may regulate migration in lung carcinogenesis.

Kashiwagi K, Ishii J, Sakaeda M, et al.
Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin, and chromogranin A in lung cancer cells.
Pathol Int. 2012; 62(4):232-45 [PubMed] Related Publications
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.

Righi L, Rapa I, Votta A, et al.
Human achaete-scute homolog-1 expression in neuroendocrine breast carcinoma.
Virchows Arch. 2012; 460(4):415-21 [PubMed] Related Publications
Neuroendocrine (NE) breast carcinoma is defined by morphological features similar to those of NE tumors of other organs and NE marker expression in at least 50 % of neoplastic cells. However, a NE morphology may be observed even in breast carcinomas lacking NE markers. Human achaete-scute homolog-1 (hASH-1) is a transcription factor that plays a key role in the regulation of mammalian neural and NE cell development and has been identified in several human NE tumors. The aim of this study was to investigate hASH-1 expression in human breast cancers. hASH-1 expression was evaluated in 482 consecutive non-NE invasive breast carcinomas, in a series of 84 breast cancers with >50 % NE marker expression (high NE differentiation) and 21 carcinomas with NE histology but negative or focally (<50 %) positive for NE markers (low NE differentiation). hASH-1 protein was evaluated by a specific monoclonal antibody using immunohistochemistry and gene expression by real-time polymerase chain reaction. None of the non-NE invasive breast carcinomas expressed hASH-1 at any levels. hASH-1 was expressed in tumor cell nuclei of 63 and 38 % of cases with high and low NE differentiation, respectively. Strong correlation with protein and gene expression levels was observed (p < 0.0001). hASH-1 expression was correlated to a low mitotic count (p = 0.02) and a low Ki67 proliferative index (p = 0.0062). hASH-1 expression occurs in breast cancers with NE differentiation regardless of the extent of the NE cell population, and it is restricted to a subset of tumor cells having a low proliferative potential.

Zhao H, Zhu L, Jin Y, et al.
miR-375 is highly expressed and possibly transactivated by achaete-scute complex homolog 1 in small-cell lung cancer cells.
Acta Biochim Biophys Sin (Shanghai). 2012; 44(2):177-82 [PubMed] Related Publications
In this study, we identified five miRNAs highly expressed in the small-cell lung cancer (SCLC) cell line NCI-H209. Among them, the expression levels of miR-375 were dramatically elevated in all SCLC cell lines examined, coincident with the expression of the transcription factor achaete-scute complex homolog 1 (ASCL1). Moreover, miR-375 was upregulated and correlated with ASCL1 in the cell lines generated from mouse SCLC-like tumors as well. Dual-luciferase assays further showed that ASCL1 activated the expression of miR-375 by binding to the three E-box elements in the miR-375 promoter. These results imply a role of ASCL1 in SCLC via the upregulation of miR-375.

Hou J, Lambers M, den Hamer B, et al.
Expression profiling-based subtyping identifies novel non-small cell lung cancer subgroups and implicates putative resistance to pemetrexed therapy.
J Thorac Oncol. 2012; 7(1):105-14 [PubMed] Related Publications
INTRODUCTION: A challenge of cancer therapy is to optimize therapeutical options to individual patients. Cancers with similar histology may show dramatically different responses to therapy, indicating that a refined approach needs to be developed to classify tumors by intrinsic characteristics that may predict response to chemotherapy. Global expression profile-based classification has the potential to identify such tumor-intrinsic subclasses. Pemetrexed effectiveness has been related to the expression of its target thymidylate synthase. The relatively frequent resistance of squamous cell carcinoma to Pemetrexed is correlated with high levels of thymidylate synthase expression.
METHODS: A global expression profile-based molecular classification of non-small cell lung cancer (NSCLC) was performed. Gene expression was used to predict Pemetrexed responsiveness. The distinct molecular attributes of NSCLCs predicted likely to be resistant to Pemetrexed were bioinformatically characterized. We tested if routine immunohistochemical markers can be used to distinguish putative Pemetrexed responders, predicted by gene signatures, from nonresponders.
RESULTS: Ninety NSCLCs were divided into six subclasses by gene expression signatures. The relevance of this novel phenotyping was linked to other tumor characteristics. Two of the subclasses correlated to putative Pemetrexed resistance. In addition, the identified signature genes characterizing putative Pemetrexed responsiveness predicted therapeutic benefit in a subset of squamous cell carcinoma.
CONCLUSIONS: Gene expression signatures can be used to identify NSCLC subgroups and have potential to predict resistance to Pemetrexed therapy. We suggest that a combination of classical pathological markers can be used to identify molecular tumor subclasses associated with predicted Pemetrexed response.

Nishikawa E, Osada H, Okazaki Y, et al.
miR-375 is activated by ASH1 and inhibits YAP1 in a lineage-dependent manner in lung cancer.
Cancer Res. 2011; 71(19):6165-73 [PubMed] Related Publications
Lung cancers with neuroendocrine (NE) features are often very aggressive but the underlying molecular mechanisms remain elusive. The transcription factor ASH1/ASCL1 is a master regulator of pulmonary NE cell development that is involved in the pathogenesis of lung cancers with NE features (NE-lung cancers). Here we report the definition of the microRNA miR-375 as a key downstream effector of ASH1 function in NE-lung cancer cells. miR-375 was markedly induced by ASH1 in lung cancer cells where it was sufficient to induce NE differentiation. miR-375 upregulation was a prerequisite for ASH1-mediated induction of NE features. The transcriptional coactivator YAP1 was determined to be a direct target of miR-375. YAP1 showed a negative correlation with miR-375 in a panel of lung cancer cell lines and growth inhibitory activities in NE-lung cancer cells. Our results elucidate an ASH1 effector axis in NE-lung cancers that is functionally pivotal in controlling NE features and the alleviation from YAP1-mediated growth inhibition.

Fujiwara T, Hiramatsu M, Isagawa T, et al.
ASCL1-coexpression profiling but not single gene expression profiling defines lung adenocarcinomas of neuroendocrine nature with poor prognosis.
Lung Cancer. 2012; 75(1):119-25 [PubMed] Related Publications
BACKGROUND: Lung adenocarcinoma is heterogeneous regarding histology, etiology and prognosis. Although there have been several attempts to find a subgroup with poor prognosis, it is unclear whether or not adenocarcinoma with neuroendocrine (NE) nature has unfavorable prognosis.
MATERIALS AND METHODS: To elucidate whether a subtype of adenocarcinoma with NE nature has poor prognosis, we performed gene expression profiling by cDNA microarray for 262 Japanese lung cancer and 30 normal lung samples, including 171 adenocarcinomas, 56 squamous cell carcinomas and 35 NE tumors. A co-expression gene set with ASCL1, an NE master gene, was utilized to classify tumors by non-negative matrix factorization, followed by validation using an ASCL1 knock-down gene set in DMS79 cells as well as an independent cohort (n=139) derived from public microarray databases as a test set.
RESULTS: The co-expression gene set classified the adenocarcinomas into alveolar cell (AL), squamoid, and NE subtypes. The NE subtype, which clustered together almost all the NE tumors, had significantly poorer prognosis than the AL subtype that clustered with normal lung samples (p=0.0075). The knock-down gene set also classified the 171 adenocarcinomas into three subtypes and this NE subtype also had the poorest prognosis. The co-expression gene set classified the independent database-derived American cohort into two subtypes, with the NE subtype having poorer prognosis. None of the single NE gene expression was found to be linked to survival difference.
CONCLUSION: Co-expression gene set with ASCL1, rather than single NE gene expression, successfully identifies an NE subtype of lung adenocarcinoma with poor prognosis.

Miki M, Ball DW, Linnoila RI
Insights into the achaete-scute homolog-1 gene (hASH1) in normal and neoplastic human lung.
Lung Cancer. 2012; 75(1):58-65 [PubMed] Related Publications
Achaete-scute homolog-1 (ASH1) is pivotal for the development of pulmonary neuroendocrine (NE) cells. We examined human ASH1 (hASH1) expression across a comprehensive panel of human lung cancer cell lines, primary human lung tumors and normal fetal and post-natal lungs. While hASH1 was a cardinal feature of NE carcinomas, a subgroup of non-NE lung cancers also exhibited expression of this factor. Twenty lung cancer cell lines out of 33 were positive for hASH1 mRNA by reverse transcription PCR, including 6/6 small cell carcinomas (SCLC), 5/5 carcinoids, 6/7 non-SCLC with NE features, and 3/14 other non-SCLC. Among human primary tumors, 2/2 SCLC, 5/5 pulmonary carcinoids, and 10/41 non-SCLC (only 4 of which had NE features) were positive for hASH1 by immunohistochemistry and RNA-RNA in situ hybridization. In normal human fetal lung, the expression of hASH1 and the neural marker synaptophysin was highly concordant in neuroepithelial bodies and solitary NE cells, while the rest of the epithelium was negative. In childhood and adulthood, the markers became progressively discordant, with a majority of hASH1-immunoreactive foci (69%) being negative for synaptophysin in adults, potentially representing dormant NE cell progenitors. We conclude that hASH1 provides an early indication of NE program in human lung.

Xiong F, Wu C, Chang J, et al.
Genetic variation in an miRNA-1827 binding site in MYCL1 alters susceptibility to small-cell lung cancer.
Cancer Res. 2011; 71(15):5175-81 [PubMed] Related Publications
Genetic variations in microRNAs (miRNA) that affect control of their target genes may alter individual susceptibilities to cancer. In this study, we took an in silico approach to identify single-nucleotide polymorphisms (SNP) within the 3'-untranslated region (UTR) of miRNA genes deregulated in human small-cell lung cancer (SCLC), and then investigated their associations with SCLC susceptibility in 666 SCLC patients and 758 controls. Odds ratios (OR) were estimated by multivariate logistic regression, and biochemical assays were conducted to investigate SNP functions. We identified 2 SNPs, rs3134615 and rs2291854, which were located in the 3'-UTR of the L-MYC gene MYCL1 and the neuronal development Achaete-Scute Complex homolog ASCL1. Case-control analyses showed that the rs3134615T allele was associated with a significantly increased risk of SCLC, with the OR for carrying the GT or TT genotype being 2.08 (95% confidence interval, 1.39-3.21; P = 0.0004) compared with the GG genotype. In support of the likelihood that these 3'-UTR SNPs may directly affect miRNA-binding sites, reporter gene assays indicated MYCL1 as the target of hsa-miR-1827 and the rs3134615 G>T change resulted in altered regulation of MYCL1 expression. Our findings define a 3'-UTR SNP in the human L-MYC oncogene that may increase susceptibility to SCLC, possibly resulting from attenuated interaction with the miRNA hsa-miR-1827.

Huang HS, Turner DL, Thompson RC, Uhler MD
Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cyclic AMP-dependent protein kinase.
J Neurochem. 2012; 120(5):667-83 [PubMed] Article available free on PMC after 26/09/2015 Related Publications
cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβ protein. PKIβ played a very specific role in the Ascl1-mediated differentiation process as other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells.

Nasgashio R, Sato Y, Matsumoto T, et al.
The balance between the expressions of hASH1 and HES1 differs between large cell neuroendocrine carcinoma and small cell carcinoma of the lung.
Lung Cancer. 2011; 74(3):405-10 [PubMed] Related Publications
To clarify the biological differences between small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), we investigated the expression of two bHLH type transcription factors, human achaete-scute homolog 1 (hASH1) and hairy/enhancer of split 1 (HES1), which positively and negatively regulate the neuroendocrine differentiation of respiratory epithelial cells, respectively. Eighty-eight formalin-fixed and paraffin-embedded pulmonary carcinomas (32 SCLC, 32 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas) and 14 SCLC and 1 LCNEC derived cell lines were used. hASH1 and HES1 mRNA were detected using a highly sensitive in situ hybridization method with digoxigenin-labeled cRNA probes and biotinylated tyramide. The staining results were scored from 0 to 12 by multiplying the staining intensity by the percentage of positive tumor cells. The mean staining score of hASH1 mRNA was significantly higher in SCLC than in LCNEC (p<0.01); conversely, that of HES1 mRNA was lower in SCLC than in LCNEC (p<0.01). These findings reveal that SCLC more strongly expresses the neuroendocrine phenotype, while LCNEC shows characteristics more similar to the ciliated epithelium phenotype, suggesting that the biological characteristics of these two tumors are different.

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