Growth Factor Independence-1 Is Expressed in Primary Human Neuroendocrine Lung Carcinomas and Mediates the Differentiation of Murine Pulmonary Neuroendocrine Cells

[CANCER RESEARCH 64, 6874–6882, October 1, 2004] Growth Factor Independence-1 Is Expressed in Primary Human Neuroendocrine Lung Carcinomas and Mediates the Differentiation of Murine Pulmonary Neuroendocrine Cells

Avedis Kazanjian,1 Deeann Wallis,2 Nicholas Au,9 Rupesh Nigam,3 Koen J. T. Venken,4 Philip T. Cagle,5 Burton F. Dickey,6,8 Hugo J. Bellen,2,4,7 C. Blake Gilks,9 and H. Leighton Grimes1 1Institute for Cellular Therapeutics and Departments of Surgery, Biochemistry, and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky; 2Departments of Molecular and Human Genetics, 3Internal Medicine, 4Program in Developmental Biology, 5Pathology, 6Medicine and Molecular Physiology and Biophysics, and the 7Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas; 8Department of Pulmonary Medicine, M. D. Anderson Cancer Center, Houston, Texas; 9Department of Pathology, Vancouver General Hospital, Vancouver, British Columbia, Canada

ABSTRACT biochemical pathways underlying neuroendocrine differentiation have not been elucidated. Understanding the molecular basis of neuroen- Human small cell lung cancers might be derived from pulmonary cells docrine lung carcinomas, especially SCLC, should provide the oppor- with a neuroendocrine phenotype. They are driven to proliferate by tunity for novel therapeutic intervention with significant implications autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is for patients. relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcrip- Clues to the genetic components of the human neuroendocrine tion factor is critically required for the formation of pulmonary neuroen- tumor phenotype can be found in the genetic cascade that controls docrine cells and is a marker for human small cell lung cancers. The Drosophila peripheral nervous system development. Drosophila pe- Drosophila orthologues of ASH1 (Achaete and Scute) and the growth ripheral nervous system organ development is regulated by proneural factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact basic helix-loop-helix (bHLH) transcription factors atonal, amos, to inhibit Notch signaling and specify fly sensory organ development. scute, and achaete (7, 8). These proneural bHLH transcription factors Here, we show that GFI1, as with ASH1, is expressed in neuroendocrine dimerize with daughterless proteins to bind E-box DNA sequences lung cancer cell lines and that GFI1 in lung cancer cell lines functions as and transactivate target genes, including Delta, which activates Notch a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In signaling in adjacent cells (9). Activation of Notch signaling induces primary human lung cancer specimens, GFI1 expression strongly corre- Enhancer-of-split, a bHLH transcriptional repressor, which antago- lates with expression of ASH1, the neuroendocrine growth factor gastrin- nizes proneural bHLH factor expression and function (10). Proper releasing peptide, and neuroendocrine markers synaptophysin and chro- function of proneural genes is required in most tissues for the expres- mogranin A (P < 0.0000001). GFI1 colocalizes with chromogranin A and sion of senseless, which encodes a zinc-finger transcription factor calcitonin-gene–related peptide in embryonic and adult murine pulmo- (sens; ref. 7). Embryonic and adult peripheral nervous system devel- nary neuroendocrine cells. In addition, mice with a mutation in GFI1 opment requires sens: embryos that lack sens specify peripheral display abnormal development of pulmonary neuroendocrine cells, indi- nervous system cells, but most of the cells die through apoptosis, cating that GFI1 is important for neuroendocrine differentiation. whereas adult peripheral nervous system precursors that lack sens fail to become specified. In contrast, forced expression of Sens is sufficient to mediate peripheral nervous system development (7, 11). Thus, INTRODUCTION sens is a central target of proneural bHLH transcription factors in Lung cancer is the most common cancer in the world and is the peripheral nervous system development. leading fatal malignancy in both men and women in the United States The Achaete-Scute Homologue-1 (ASH1), the mammalian ortho- (1). It is classified into two major histopathological groups: small cell logue of Drosophila achaete and scute encoded proteins, is critical for lung carcinomas (SCLCs) and non–small-cell lung carcinomas murine pulmonary neuroendocrine cell development (5, 12). pulmo- (NSCLCs; ref. 2). SCLCs account for 15 to 20% of all human lung nary neuroendocrine cells are a minor cell population of the airway cancers and are aggressive lung tumors, which are virtually always epithelium, which function as oxygen sensors (13). Newborn mice fatal (2, 3). Lung tumors with a neuroendocrine phenotype include bearing a disruption of Ash1 completely lack detectable pulmonary SCLCs, large cell neuroendocrine carcinomas, and classical and atyp- neuroendocrine cells and die shortly after birth (5). Although the data ical carcinoid tumors. The neuroendocrine phenotype is associated are not conclusive, human neuroendocrine carcinomas might be de- with the production of neuropeptides that may stimulate transforma- rived from pulmonary neuroendocrine cells (14). Not surprisingly, tion and tumor growth through paracrine and autocrine signaling (4), human SCLC express ASH1 (15). Moreover, expression levels of and the maintenance of these signaling loops in lung tumors is ASH1 in SCLC tumors correlates with neuroendocrine markers such dependent on the neuroendocrine phenotype (5, 6). Although it is as gastrin-releasing peptide, L-dopa-decarboxylase activity and calci- clear that the growth factors secreted by neuroendocrine tumors tonin (16, 17). However, the role of ASH1 in SCLC is unclear. participate in tumorigenesis and tumor maintenance, the genetic and Transgenic expression of ASH1 in nonneuroendocrine murine lung Clara cells results in hyperplasia but not cancer (6). Dual transgenic Received 2/23/04; revised 7/23/04; accepted 7/28/04. expression of ASH1 and the polyoma large T antigen generates Grant support: Commonwealth of Kentucky Lung Cancer Research Program neuroendocrine lung tumors, but these are dissimilar to SCLC (6). It (H. Grimes), the Commonwealth of Kentucky Research Challenge Trust Fund, and the Jewish Hospital Foundation. is therefore uncertain whether the expression of ASH1 in SCLC is The costs of publication of this article were defrayed in part by the payment of page merely a marker of neuroendocrine differentiation or a driving force charges. This article must therefore be hereby marked advertisement in accordance with in neuroendocrine oncogenesis. 18 U.S.C. Section 1734 solely to indicate this fact. Note: A. Kazanjian, D. Wallis, and N. Au contributed equally to this work. The mammalian orthologue of Drosophila Sens is the growth factor Requests for reprints: H. Leighton Grimes, Institute for Cellular Therapeutics, University independence-1 (GFI1) oncoprotein. Gfi1 was cloned in an insertion of Louisville, Baxter Building, Room 404F, 570 South Preston Street, Louisville, KY 40202. Phone: (502) 852-2059; Fax: (502) 852-2085; E-mail: [email protected]. mutagenesis screen for targets of the Moloney murine leukemia virus, ©2004 American Association for Cancer Research. which could mediate leukemia progression from interleukin 2- 6874

dependent to independent growth (18). Gfi1 is also a common proviral staining was performed with the biotin-streptavidin method. Sections were integration target in Moloney virus-induced leukemias (18–22). In- deparaffinized and antigen retrieval performed. Subsequently, slides were deed, Moloney virus insertions affecting Gfi1 in murine T-cell leuke- incubated with primary antibody according to a predetermined optimal con- mias are second in frequency only to those affecting Myc (23). centration overnight at 4°C or for 1 hour at 37°C. All slides were scored by the Deletion of Gfi1 results in hematopoietic abnormalities (24–26). same pathologist (C. Gilks). Staining in nontumor cells or in an inappropriate We have recently shown that GFI1 expression is not restricted to cellular compartment was not considered to be positive. The scores were the lymphoid lineage (27). Embryonic murine neurons and sensory entered into blank sector maps of the corresponding array at the time of Ϫ Ϫ scoring, and uninterpretable results were eliminated from additional consider- epithelia also express GFI1. Specifically, Gfi1 / embryos lose coch- ation. lear hair cells and display behavioral defects consistent with inner ear Data Analysis and Statistics. Data on the score sheets were converted into abnormalities (27). Thus, like its Drosophila orthologue Sens in a table format using the TMA-Deconvoluter program as described previously embryos (7, 11), GFI1 appears to play a role in the differentiation of (29). Fisher’s exact test using SPSS Graduate Pack 11.0 (SPSS, Inc., Chicago, sensory neurons. IL) was used to evaluate the correlation between different immunomarkers. Given the critical role of ASH1 in pulmonary neuroendocrine cell Kaplan-Meier curves and survival estimates were calculated for each outcome, development (5) and the clear association between GFI1 and ASH1 and a log-rank statistic was used to test for differences between groups. A orthologues in Drosophila (7, 11), we examined GFI1 expression in significant difference was declared if the P value from a two-tailed test was developing lungs and lung tumors. Here, we show that in human lung Ͻ0.05. tumors GFI1 is expressed in both neuroendocrine cell lines and in Cell Lines and Tissue Culture. SCLC cell lines NCI-H146, DMS53, and primary human tumor samples. Moreover, the correlation between the NCI-H209, the bronchial carcinoid cell line NCI-H727, and the NSCLC cell expression of GFI1, ASH1, and neuroendocrine markers is highly lines NCI-H1299, A549 (American Type Culture Collection, Manassas, VA), significant. We previously demonstrated that embryonic mouse lung and CALU1 were used for the experiments in this study. RPMI (Invitrogen, Carlsbad, CA) supplemented with 10% FCS was used to maintain NCI-H146, expresses Gfi1 mRNA in a clustered distribution similar to that of NCI-H727, and NCI-H1299. Waymouth MB 752/1 was used to maintain pulmonary neuroendocrine cells (27). Our new data indicate these DMS53 cells (Sigma, St. Louis, MO), Iscove’s modified Dulbecco’s medium GFI1-expressing cells in embryonic and adult murine lungs are indeed for NCI-H209 (Invitrogen), McCoy’s 5A-modified medium for CALU1, and Ϫ/Ϫ pulmonary neuroendocrine cells and that Gfi1 pulmonary neu- nutrient mixture F12 Ham Kaighn’s modification (F12K) medium for A549 roendocrine cells do not express or express aberrant levels of the (Sigma). The human kidney epithelial cell line 293T was maintained in markers synaptophysin (Syn) and calcitonin-gene-related peptide. DMEM supplemented with 10% fetal calf serum (Invitrogen). The human These data suggest that GFI1 has an important role in mediating acute T-cell leukemia cell line (Jurkat) was used as a positive control for GFI1 neuroendocrine differentiation and point to a proneural bHLH/GFI1 expression and was maintained in RPMI supplemented with 10% FCS (In- developmental pathway that is conserved from humans to Drosophila vitrogen). RNA from the NCI60 panel of tumors was provided by the Devel- and active in human lung cancer. opmental Therapeutics Program National Cancer Institute, NIH. Nuclear Extract Preparation and Western Blotting. Nuclear extracts were prepared using a modified procedure from Dignam et al. (30). Protein MATERIALS AND METHODS concentrations were determined by the BCA assay (Pierce, Rockford, IL), and ␮ Case Material. Archival paraffin-embedded tissue blocks of primary lung 25 g of protein extract were separated on 10% SDS-polyacrylamide gel and carcinoma were acquired from the pathology archives of Vancouver Hospital electroblotted onto an Immobilon-P membrane (Millipore, Billerica, MA). The and Health Sciences Centre and St. Paul’s Hospital (Vancouver, British Co- membranes were blocked with 5% milk in TBS [50 mmol/L Tris HCl, 150 lumbia, Canada). Tissue samples were fixed in formalin or Bouin’s fixative. mmol/L NaCl (pH 7.5)] at 4°C for 16 hours. The blocked membranes were Cases date from 1982 to 2001. A total of 441 cases was selected to build the incubated with primary antibodies in 5% milk in TTBS [0.05% Tween 20 in tissue microarrays (TMAs). Outcome data (1 day to 18.2 years, median ϭ 4.81 TBS] between 1 and 4 hours at room temperature. GFI1 was detected using years) for all of the cases was available. goat polyclonal antibody N-20 (Santa Cruz Biotechnology, Santa Cruz, CA), ␮ Tissue Microarray Construction. TMAs were constructed as described the 2.5D.17 monoclonal (0.3 g/mL), or a guinea pig antiserum. ASH1 was previously (28). Duplicate 0.6-mm tissue cores were used to construct TMAs. detected with mouse anti-mASH1 monoclonal antibody (BD PharMingen, San Array blocks were sectioned to produce 4-␮m sections, and the first section Diego, CA), and rabbit polyclonal antibody was used to detect Hes1 (13). The was stained with H&E to assess adequacy. A total of 408 cases was valid for respective secondary antibodies were added donkey antigoat horseradish per- final data analysis and included 123 squamous cell carcinomas, 93 adenocar- oxidase (HRP)-conjugated IgG (Santa Cruz Biotechnology), Sheep antimouse cinomas, 68 large cell undifferentiated carcinomas, 68 classical carcinoids, 31 immunoglobulin-HRP, and donkey antirabbit immunoglobulin-HRP (Amer- atypical carcinoids, 11 large cell neuroendocrine carcinomas, and 14 small cell sham, Piscataway, NJ). The secondary antibodies were prepared in 5% milk carcinomas. Fifty cases were omitted from data analysis because of technical TTBS and incubated at room temperature for 1 hour. The membranes were problems with immunohistochemistry for one or more markers. washed five times at room temperature, 5 minutes each wash, and the signals Immunohistochemistry on Human Tissues. Twelve molecular markers were detected using SuperSignal detection reagents according to the manufac- were chosen for investigation in this study (Table 1). Immunohistochemical turer’s instructions (Pierce).

Table 1 Immunohistochemical reagents and procedures Antibody Clone Manufacturer Cellular compartment Dilution Antigen retrieval Synaptophysin Polyclonal Cell Marque Cytoplasmic 1:1000 Microwave Chromogranin A LK2H10 BioGenex Cytoplasmic 1:1000 None Bombesin 371 Incstar Cytoplasmic 1:2000 Microwave Neuron-specific enolase BBS/NC/VI-H14 Dako Cytoplasmic 1:100 None GFI1 2.5D.17 (HLG) Nuclear 1:20 Microwave ASH1 24B72D11.1 BD PharMingen Nuclear 1:50 Microwave p53 DO-7 Dako Nuclear 1:400 Microwave p63 4A4 Neomarkers Nuclear 1:500 Microwave p21 DCS-60.2 Neomarkers Nuclear 1:100 Microwave p27 DCS-72.F6 Neomarkers Nuclear 1:100 Microwave Carcinoembryonic antigen TF 3H8-1 Ventana Cytoplasmic 1:5 None Thyroid transcription factor-1 8G7G3/1 Dako Nuclear 1:100 Microwave 6875

Transient Transfections and Luciferase Assays. DMS53 seeded at normalized to give roughly equivalent GAPDH signal. The normalized templates 3.2 ϫ 104 cells/well or NCI-H146 seeded at 2.4 ϫ 104 cells per well in a were then analyzed for levels of human enhancer-of-split-1 (HES1), ASH1, and 96-well format were used for the transient transfection procedure. Lipo- GFI1 expression with the following primers: HES1 forward 5Ј-AGGCGGACAT- fectamine 2000 transfection reagent (Invitrogen) was used for the transfection TCTGGAAATG-3Ј and HES1 reverse 5Ј-CGGTACTTCCCCAGCACACTT-3Ј procedure per the manufacturer’s instructions. The click beetle luciferase (58°C annealing temperature), HES1 internal probe 5Ј-TGTGCTCAGCGCAGC- system (Promega, Madison, WI) was used essentially according to the manu- CATCTGC-3Ј (38); ASH1 forward 5Ј-GGGCTCGCCGGTCTCATCCT-3Ј and facturer’s instructions. The signals were monitored on a Wallac Victor 1420 ASH1 reverse 5Ј-CTCCCCCTCCCAACGCCACT-3Ј (59°C annealing tempera- Multichannel Reader using a 510/60 filter (red) and 610LP filter (green). A t ture), ASH1 internal probe 5Ј-CCCTGCTTCCAAAGTCCATTC-3Ј (39); statistic was calculated on the difference between the values of each measure- GAPDH forward 5Ј-CGGAGTCAACGGATTTGGTCGTAT-3Ј and GAPDH re- ment of luciferase activity to determine statistical significance for fold repres- verse 5Ј-AGCCTTCTCCATGGTGGTGAAGAC-3Ј (55°C annealing tempera- sion. ture), GAPDH internal probe 5Ј-GACCCCTTCATTGACCTCAA-3Ј (39); and Retroviral Transduction and Xenografts. A FLAG-epitope–tagged GFI1 GFI1 forward 5Ј-AAGGCGCTGGCGTCAAGGTGGAGT-3Ј and GFI1 reverse was cloned into the MIEV retroviral vector (31). Retroviral constructs were 5Ј-TGTGTTTGCGGCTGTGGGTGATG-3Ј (60°C annealing temperature), GFI1 transfected into Phoenix cells for virus productions (32). NCI-H146 or A549 internal probe 5Ј- ACAGCGGTACCAGACCCTTT-3Ј. were infected (33). Cells were tested for green fluorescent protein (GFP) Embryo Staging and Tissue Preparation. Gfi1-mutant mice were gener- fluorescence by flow cytometric analysis. GFI1 protein expression was as- ated as described previously (24). Embryos were considered to be E 0.5 days sessed by FLAG-HRP (Sigma) and anti-GFI1 2.5D.17 monoclonal antibodies on the morning the vaginal plug was observed. To harvest the embryos, by Western analysis. Cell cycle analysis was performed by the method pregnant females were sacrificed by cervical dislocation and the embryos of Vindelov (34). For each experiment, 5 ϫ 106 MIEV- or MIEV-GFI1– dissected out of the uterus. Portions of the yolk sac or tail were saved for transduced NCI-H146 cells were injected s.c. into five 4 to 6-week-old genotyping. Embryos were fixed overnight in 4% paraformaldehyde, dehy- NCRNU-M male mice (Taconic farms, Germantown, NY) and measured for drated in an ethanol series, and embedded in paraffin for sectioning according tumor diameter and volume. No differences were noted between trends for to standard histologic protocols. Ten-micron sections were collected and volume or diameter. The experiment was repeated twice with independently analyzed by in situ hybridization or immunohistochemistry. Lung tissue for transduced cells. postnatal stages was collected by harvesting the lungs of appropriately aged Mapping the Binding Site of the Anti-Growth Factor Independence-1 mice, fixing overnight in 4% paraformaldehyde, dehydrating in an ethanol mAb 2.5D.17. The TGA stop codons of GFI1, SV40SwapGFI1 (35), GFI1B, series, and embedding in paraffin for sectioning. Ten-micron sections were and SNAIL were mutated to a BamHI restriction endonuclease site by PCR and collected and analyzed by immunohistochemistry. cloned into the CMV14 vector (Sigma). The constructs were transiently Immunohistochemistry on Mouse Tissues. We used antibodies to transfected into the human kidney epithelium 293T cell line using Lipo- PGP9.5 (1:2000 dilution; Affiniti Research Products Limited, Plymouth Meet- fectamine 2000 (Invitrogen) according to the manufacturer’s instructions. ing, PA), ASH1 (1:100 dilution; BD PharMingen), CGRP (1:8000 dilution; Nuclear extracts were prepared 48 hours after transfection as described above. Sigma), and GFI1 (1:2000 dilution). Guinea pig antisera to murine GFI1 was The protein extracts were quantified (BCA; Pierce), and 15 ␮g were separated generated previously with an immunogen encoding murine GFI1 between the on 10% SDS-polyacrylamide gel and electroblotted onto an Immobilon-P SNAG domain and zinc fingers (27). For single antibody localization, primary membrane (Millipore). Western blotting was performed as described above antibodies were followed with the ABC Vectastain kit directions with bioti- using the 2.5D.17 monoclonal antibody (0.3 ␮g/mL) and HRP-conjugated nylated secondary antirabbit antibody (PGP9.5 and CGRP) antimouse antibody anti-FLAG antibody (Sigma). For peptide competition, the 2.5D.17 antibody (mASH1), or antiguinea pig antibody (GFI1) followed by 3,3Ј-diaminobenzi- was incubated either by itself or with 1.5 ␮g/mL peptides encoding GFI1 dine staining. Briefly, paraffin sections were blocked in 1% H2O2 in methanol amino acids 7–26 (SKKAHSYHQPRSPGPDYS) or a control peptide from an for 20 minutes at room temperature, rehydrated in a series of ethanols, boiled unrelated protein (SNEDRGEVADEEKR; Research Genetics, Huntsville, AL) in citrate antigen retrieval solution in a microwave for 5 to 10 minutes, and in 0.5 mL of PBS overnight at 4°C. The mix was added to the membranes and blocked with serum in PBS 30 minutes at room temperature. Primary antibody development of the Western blotting was performed as described above. was diluted in blocking solution and incubated on the section overnight at 4°C. Electrophoretic Mobility Shift Assay. Nuclear extract from DMS53 and Slides were rinsed, incubated in Vectastain ABC solution, rinsed again, and the Ј A549 cell lines were prepared as described above. Double-stranded oligonu- signal was detected with 2 mg/mL 3,3 -diaminobenzidine, 0.02% H2O2 in cleotide containing the B30 GFI1-consensus binding site (5Ј-GGAC- PBS. Some slides were counterstained with para-1-hemotoxylin. The slides CGGGGTGCAGTGATTTGGTGTGGCGATCCC-3Ј) and a GFI1-binding were then dehydrated in a series of alcohols and mounted. For colocalization mutant (5Ј-GGACCGGGGTGCAGTGACCTGGTGTGGCGATCCC-3Ј) were of GFI1 with neuroendocrine markers CGRP and chromogranin A, both biotin labeled with [32P]dCTP using Klenow Exo- (New England Biolabs, Beverly, MA). and alkaline phosphatase-conjugated secondary antibodies were used. The A total of 2.5 ␮g of each nuclear extract was incubated with GFI1-specific alkaline phosphatase signal detected with nitroblue tetrazolium/5-bromo-4- antibodies on ice for 1 hour before the rest of the components were added in the chloro-3-indolyl phosphate (50 mg/mL each in 70% dimethylformamide) binding buffer [10 mmol/L HEPES (pH 7.9), 10% (v/v) glycerol, 0.2% v/v NP40, followed by methanol treatment. The slides were then mounted. Numbers of 50 ng/␮L poly(deoxyinosinic-deoxycytidylic acid), and 2.5 mg/mL nonfat milk, neuroendocrine cells are expressed as number of CGRP-positive cells per final NaCl concentration was adjusted to 60 mmol/L NaCl; adopted from ref. microscopic field of view. Low-power images of each mouse lung were taken (36)]. Probe (5.0 ϫ 104 cpm) was added, and the reaction mix was incubated for (n ϭ 3 wild-type and 3 null mice), and all positive cells per image were tallied. an additional 20 minutes at room temperature. The samples were separated on 6% For each mouse, the total number of positive cells was divided by the total nondenaturing polyacrylamide gel (37), dried, and exposed to BiomaxMS film number of images or fields of view (n ϭ 9 to 15) to determine the cells per field (Kodak). of view. Reverse Transcription-PCR. Total RNA extraction was performed using the ULTRASPEC RNA isolation system (Biotecx Laboratories, Houston, TX), RESULTS according to the manufacturer’s recommendations. RNA and genomic DNA extracted from the human Jurkat cell line was used as a control for the reverse Growth Factor Independence-1 Is Expressed in Neuroendo- ␮ transcription-PCR. Total RNA (5 g) from each sample was DNase treated crine Human Lung Tumor Cell Lines. ASH1 is expressed in human (Ambion, Austin, TX) and then used for cDNA synthesis with SuperScript II SCLC (12), and the orthologues of ASH1 and GFI1 interact to inhibit reverse transcriptase kit (Invitrogen). Random hexamer oligos were used for Notch signaling in Drosophila peripheral nervous system develop- cDNA synthesis per manufacturer’s instructions. PCR reactions were performed for 20 cycles. The cDNA templates were first analyzed for the levels of the ment (7). To determine whether an interaction between ASH1 and D-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) housekeeping gene. GFI1 may occur in human SCLC, we first examined a panel of human Products of the reaction were analyzed by Southern with 32P-labeled oligonucleo- lung tumor cell lines for the expression of ASH1, the Notch effector tides internal to the PCR primer sequences. The resulting signal was quantified on human enhancer-of-split-1 (HES1), and GFI1. As expected (39), the a STORM phosphorimager (PE Biosystems, Foster City, CA) and templates were neuroendocrine SCLC cell lines DMS53, H209, and NCI-H146 and 6876

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. GFI1 IN NEUROENDOCRINE LUNG CANCER the bronchial carcinoid H727 express ASH1 RNA (Fig. 1A) and protein (Fig. 1B), whereas NSCLC cell lines H1299, A549, and CALU1 lack ASH1 expression (Fig. 1). In contrast, HES1 was expressed in some NSCLC and SCLC cell lines. HES1 is generally absent in cells with a neuroendocrine phenotype (39). We find that HES1 is expressed in DMS53 and at a low level in H209 (Fig. 1). These data confirm previous reports of HES1 expression in pheno- typically neuroendocrine DMS53 cells (40, 41). HES1 in DMS53 cells may not be functional because Notch signaling or HES1 overexpression in these cells induced significant phenotypic changes (41). We next examined GFI1 expression. The presence of GFI1 RNA (Fig. 1A) and protein (Fig. 1B) was concordant with that of ASH1. NSCLC cell lines Calu1, A549, H1299 (Fig. 1A) and seven other NSCLC cell lines from the NCI60 panel of tumor cell lines (data not shown) lacked GFI1 expression. The pattern of GFI1 expression in SCLC cell lines was determined with both commercial antiserum (N20) and a monoclonal antibody (2.5D.17; Fig. 1), as well as with a guinea pig antiserum (data not shown). The doublet seen in Western analysis of NCI-H209 nuclear extract may represent an alternatively processed form of GFI1 but was not seen with the commercial or guinea pig antiserum. In conclusion, the expression of GFI1 in human lung tumor cell lines was restricted to those with a neuroendocrine phenotype. Growth Factor Independence-1 Is a Functional Transcriptional Repressor in Neuroendocrine Human Lung Tumor Cell Lines. We next determined the presence of GFI1 DNA-binding activity in human SCLC cell lines. Nuclear extracts from SCLC cell lines DMS53, NCI-H146, and H209 and the NSCLC cell line A549 were evaluated by electrophoretic mobility shift analysis using an oligonucleotide encoding a canonical high-affinity GFI1-binding site (B30; refs. 35, 37). Nuclear extracts from the DMS53 SCLC cell line show multiple complexes that retard the migration of the B30 oligonucleotide, only one of which is unique in comparison to the A549 NSCLC cell line (Fig. 2A, compare Lanes 2 and 12). The presence of GFI1 in

Fig. 2. A, electrophoretic mobility shift analysis of nuclear extracts from the A549 and DMS53 cell lines using an oligonucleotide encoding a high-affinity GFI1 binding site B30. Arrows indicate putative GFI1 complexes. B, transient transcription analysis of GFI1 transcriptional repression activity in the indicated SCLC cell lines. Fold repression represents the activity of a control reporter divided by the activity of a GFI1-responsive reporter. The endogenous activity of GFI1 was significantly augmented by cotransfection of GFI1 expression constructs. Results shown are representative of three separate exper- DMS53 P ϭ 0.0000007. C, MIEV and ,ءء ,NCI-H146, P ϭ 0.00004 ,ءء .iments MIEV-GFI1 retroviral-vector transduced NCI-H146 cell line xenograft growth in NCI- nude mice. The average size of each tumor per day for MIEV (Ⅺ) and MIEV-GFI1 (f)–transduced cells was divided by the number of mice with tumors then plotted as a Fig. 1. Expression of GFI1, ASH1, and HES1 in human lung cancer cell lines. A. line, whereas a scatter plot illustrates individual measurements used to generate the Products of the reverse transcription-PCR analysis of the indicated lung cancer cell line average. Western analysis (inset) of transduced NCI-H146 SCLC cells. Results shown are 32 RNA were Southern blotted and probed with an internal P-radiolabelled oligonucleo- the sum of two independent experiments. W ϭ wild type B30 oligonucleotide probe, tide. B, Western analysis of nuclear extracts from the same cell lines using antibodies to M ϭ mutant B30 probe, N ϭ N20 anti-GFI1 goat antisera, Cn ϭ Goat polyclonal antisera ASH1, HES1 and GFI1. GFI1 expression was detected with both commercially available control, D ϭ 2.5D.17 monoclonal antibody, Cd ϭ Isotype matched control mouse IgG. antisera (N20) and a monoclonal antibody (2.5D.17). 6877

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. GFI1 IN NEUROENDOCRINE LUNG CANCER these protein complexes was determined by supershift using two not shown): however, subtle differences may be occluded by cells that different antibodies: a commercially available goat polyclonal anti- were not transduced. We determined that 10 million NCI-H146 cells serum and a monoclonal antibody (Fig. 2A). Both reagents clearly generate a 1-cm tumor in NCI nude mice within 45 days. To increase resulted in retarded migration of the DMS53-unique complex; how- environmental pressures on the xenograft, we restricted the injection ever, none of the complexes were affected by the addition of respec- of transduced cells to 5 million transduced NCI-H146 cells. Fifteen tive isotype controls (Fig. 2A). Thus, these complexes appear to days after injection, 40% of the mice injected with GFI1-overexpress- contain GFI1 (Fig. 2A, bottom arrow). ing NCI-H146 cells began to show signs of an established tumor GFI1 binds to a consensus 12-bp DNA sequence with an absolute burden (Fig. 2C). In contrast, empty vector transduced cells did not requirement for AATC (37). Mutation of the AATC core of the begin to establish a tumor until day 24 (Fig. 2C). At all points consensus sequence ablates DNA binding (35, 37). To determine measured, the difference in tumor diameter (Fig. 2C) and volume whether the DMS53-unique complex has similar requirements we (data not shown) was greater on average for MIEV-GFI1 tumors. performed cold competition. Although excess unlabeled B30 oligo- Because retroviral vectors can alter the genome by insertion mutagen- nucleotides competed with the labeled B30 oligonucleotides for bind- esis, we repeated this experiment with independently transduced cells, ing to the DMS53-unique complex (Fig. 2A), an excess of unlabeled which generated essentially the same results. In both experiments, the oligonucleotides mutated to eliminate the AATC core of the GFI1 xenografted MIEV-GFI1–transduced NCI-H146 tumors continue to binding site did not abolish binding to the same complex (Fig. 2A). express FLAG-tagged GFI1 (data not shown). Taken together, these Moreover, labeled mutant-B30 oligonucleotides displayed a similar data make it unlikely that an insertion mutagenesis event explains the binding pattern with NSCLC and SCLC nuclear extracts (Fig. 2A, increase in xenograft establishment and growth and indicate that GFI1 compare Lanes 10 and 20). Importantly, the DMS53-unique complex has oncogenic activity in nonhematopoietic cells. was not observed (Fig. 2A, bottom arrow). We note that the mutant Forced Growth Factor Independence-1 Expression Alters B30 oligonucleotide was not bound by a larger complex and that this NSCLC Cell Size and Cell Cycle Progression. To determine complex was also decreased but not disrupted by the addition of whether the oncogenic effect of GFI1 overexpression is singular to GFI1-specific antiserum (Fig. 2A, top arrow). Thus, this larger com- SCLC, we examined NSCLC cells. The NSCLC cell line A549 was plex depends on an intact AATC sequence and may contain GFI1. transduced with MIEV-GFI1 retrovirus and characterized four days Complexes similar to those found in DMS53 were observed using later by flow cytometry to determine the extent of infection. An nuclear extracts from NCI-H146 and H209 SCLC cell lines (data not average of 90% showed GFP expression (data not shown) and West- shown). Thus, human SCLC cell lines contain GFI1-specific DNA- ern analysis demonstrated the expression of the FLAG-epitope– binding activity. tagged GFI1 protein (Fig. 3A). However, cell cycle analysis demon- Ͻ We have previously shown that DNA binding by GFI1 zinc fingers strated a significant G1 arrest (Fig. 3, B and C; P 0.05). Cell cycle is required to repress transcription of target genes (35). To address the changes were coincident with a reduction in cell size (Fig. 3D) and a regulatory function of GFI1 in lung tumor cell lines we performed failure of the cultures to propagate (data not shown). We repeated this transient transcription assays. The activity of the herpes-simplex-virus experiment in Calu1 and in A549 by both retroviral transduction and minimal-thymidine-kinase promoter was analyzed with and without transient transfection of GFI1 expression vectors with similar results B30 high-affinity GFI1-binding sites to determine the activity of (data not shown). GFI1. Similar reporters were previously used to monitor the activity Growth Factor Independence-1 Expression Correlates with Ex- of endogenous GFI1 in lymphocytes (35). In SCLC cell lines, the pression of Neuroendocrine Markers in Primary Human Lung activity of the GFI1 reporter was mildly repressed in comparison to Tumor Specimens. We have generated a murine monoclonal anti- the control reporter activity (Fig. 2B). Much greater differences were body against GFI1 (2.5D.17). The specificity of this monoclonal noted in T lymphocyte cell lines (35). The discrepancy between reagent was determined using Western and immunohistochemical lymphoid and SCLC cells in endogenous GFI1 activity could either be analyses with peptide competition (Supplementary Fig. 1). These data due to ϳ10-fold lower levels of GFI1 in SCLC cell lines (data not indicate that the epitope of the 2.5D.17 monoclonal is within the shown) or to deficiencies in the transcriptional machinery necessary 20-amino acid GFI1 SNAG transcriptional-repression domain. for GFI1 function. To delineate between these two possibilities, we Immunoreactivity with the 2.5D.17 monoclonal may be associated cotransfected GFI1 expression vectors with both control and GFI1- with the neuroendocrine phenotype of these lung tumors. We analyzed responsive reporters. Increased transcriptional repression was induced GFI1 expression on a tissue array containing 358 primary human lung by cotransfection of GFI1 expression vector (Fig. 2B). Thus, the tumor specimens. Examples of positive Neuroendocrine tumors are quantity but not the function of GFI1 is lower in SCLC cell lines in shown (Fig. 4A and Supplementary Fig. 2). We noted that the intensity comparison to T lymphocytes. Therefore, GFI1 probably functions as of the stain is greatest in SCLC tumor specimens (Fig. 4A and a transcriptional repressor in lung cancer cell lines. Supplementary Fig. 2) and that all SCLC examined stained intensely Growth Factor Independence-1 Overexpression Potentiates with the 2.5D.17 monoclonal antibody (data not shown). Although SCLC Xenografts in NCI Nude Mice. Given the ability of GFI1 to less than half of nonneuroendocrine tumors were positive for GFI1, function as an oncoprotein in T lymphocytes, we next determined the nearly all neuroendocrine tumors stained positively for GFI1 (Fig. 4B activity of GFI1 in SCLC xenografts. The MIEV retroviral vector and Supplementary Fig. 2). The increased likelihood of a neuroendo- transcribes a bicistronic message encoding the gene of interest and an crine (versus a nonneuroendocrine) tumor staining positively for GFI1 enhanced jellyfish GFP (31). High-titer VSVg-pseudotyped retroviral was highly significant (P Ͻ 0.001). Because both less aggressive supernatants were generated and used to transduce the NCI-H146 carcinoids and aggressive SCLC stained positively, GFI1 expression SCLC cell line with MIEV-GFI1 constructs and empty vector control. could not be assigned a prognostic value in neuroendocrine lung Three days later, the cells were analyzed by flow cytometry for GFP tumors. In nonneuroendocrine tumors, sample size limited assessment fluorescence. In general, ϳ60% of NCI-H146 cells were transduced of the prognostic significance of GFI1 immunostaining. (data not shown). Western analysis with either a monoclonal reagent We next analyzed the same lung cancer tissue array with antibodies against GFI1 (2.5D.17), or FLAG-specific antiserum illustrate the against bHLH transcription factors, tumor suppressors, cell cycle expression of FLAG-tagged GFI1 in transduced NCI-H146 cells (Fig. regulators, neuroendocrine markers, and markers purported to have 2C, inset). No differences in cell cycle progression were noted (data prognostic value in lung cancers (Table 1). GFI1 expression, as 6878

express these markers. Thus, GFI1 is specifically expressed in the pulmonary neuroendocrine cells of embryonic and adult mouse lungs. Gfi1 Is Required for the Differentiation of Murine Pulmonary Neuroendocrine Cells. ASH1 is critically required for the formation of murine pulmonary neuroendocrine cells (5). Drosophila proteins orthologous to GFI1 and ASH1 act in a mutually dependent pathway to specify nervous system organ development (7, 11). To determine the relationship between ASH1 and GFI1 in mice, we examined Gfi1Ϫ/Ϫ lungs by immunohistochemistry for ASH1 expression. Pul- monary neuroendocrine cells are most abundant in the days surround- ing birth. Hence, we characterized pulmonary neuroendocrine cells at E17.5. Neither the number of ASH1-positive cells nor the level of ASH1 expression was altered in Gfi1Ϫ/Ϫ lungs (Fig. 5C). Thus, GFI1 is not required for the expression of ASH1 in pulmonary neuroendocrine cells. We next examined Gfi1Ϫ/Ϫ pulmonary neuroendocrine cells for the expression of proteins indicative of neuroendocrine differentiation. In murine auditory hair cell development, GFI1 is not required for the expression of bHLH factors; however, Gfi1Ϫ/Ϫ hair cells do not differentiate properly, many of the cochlear hair cells die, and Gfi1Ϫ/Ϫ mice are deaf (27). Because ASH1-expressing cells are formed in Gfi1Ϫ/Ϫ mice, we next examined the extent of their differentiation. Protein gene product 9.5 (PGP9.5) stains both neurons and pulmonary neuroendocrine cells. Like for ASH1, we detected no change in level or abundance of wild-type and Gfi1Ϫ/Ϫ cells positive for PGP9.5 (data not shown). However, CGRP expression is a specific marker in lungs for pulmonary neuroendocrine cells. The number of single pulmonary neuroendocrine cells, clusters of pulmonary neuroendocrine cells (NEB), as well as the number of pulmonary neuroendocrine cells per NEB, was significantly reduced in Gfi1Ϫ/Ϫ mice (Fig. 5, D and E; P Ͻ 0.05). As shown in Fig. 5G, immunohistochemical analysis revealed a 3.4-fold reduction in the number of CGRP-positive cells in Gfi1Ϫ/Ϫ mice as compared with wild type (Fig. 5, F and G; Fig. 3. A, Western analysis of MIEV retroviral vector-mediated expression of FLAG- epitope–tagged GFI1 in the A549 NSCLC cell line. B, an example of the cell cycle P Ͻ 0.0038). Moreover, the cells which stain positively for CGRP analysis of MIEV and MIEV-GFI1–transduced A549 NSCLC cells. C, average percent of were strongly reduced in intensity in E17.5 Gfi1Ϫ/Ϫ mice compared three independent MIEV- versus three independent MIEV-GFI–transduced cell cultures in with littermate controls (Fig. 5F). In addition, the number of cells ء cell-cycle phases G1, S, and G2-M. , The difference between MIEV versus MIEV-GFI1– transduced cells in each phase of the cell cycle is significant. P Ͻ 0.05. D, an example of positive for the pulmonary neuroendocrine cell-specific marker syn- the forward versus side scatter flow cytometry plots of the same cell lines illustrates aptophysin in Gfi1Ϫ/Ϫ mice was roughly half that of wild type (data changes in cell size and granularity in MIEV-GFI1–transduced A549 cells. not shown). Other lung epithelial cell types were not affected (data not shown). We therefore propose that GFI1 is not necessary for the detected by immunohistochemistry, showed strong correlations with specification of pulmonary neuroendocrine cells but is required for expression of ASH1 and the neuroendocrine markers chromogranin their differentiation and proper maturation. A, synaptophysin, and gastrin-releasing peptide. Significantly, the majority of tumors positive for ASH1, synaptophysin, chromogranin DISCUSSION A, and gastrin-releasing peptide were also positive for GFI1 (Fig. 4C; P Ͻ 0.0000001). None of the other markers studied showed a statis- Our analysis of GFI1 expression in human lung cancer reveals a tically significant relationship to GFI1 staining. Thus, GFI1 is pro- previously unknown link between GFI1 and the neuroendocrine phe- foundly associated with ASH1 and the neuroendocrine phenotype in notype. Nearly all of the neuroendocrine tumors analyzed express primary human lung cancers. GFI1, indicating that GFI1 may have an important function in the Growth Factor Independence-1 Is Expressed in Murine Pulmo- generation and/or maintenance of the neuroendocrine phenotype. nary Neuroendocrine Cells. We previously determined that Gfi1 GFI1 expression is not specific to neuroendocrine tumors, as a sig- mRNA is expressed in embryonic mouse lung tissue (27). At E12.5, nificant minority of nonneuroendocrine large cell carcinomas express Gfi1 is expressed in regional patches of cells, but by E15.5, Gfi1 low levels of GFI1. On the other hand, high levels of GFI1 activity expression is confined to a punctate pattern resembling pulmonary may be required for the expression of neuroendocrine-specific genes neuroendocrine cells (27). To determine whether Gfi1-expressing by other transcription factors. The defects found in Gfi1Ϫ/Ϫ lungs cells are indeed pulmonary neuroendocrine cells, we performed im- support this hypothesis. Specifically, the expression of the neuroen- munohistochemical analysis for pulmonary neuroendocrine cell- docrine-specific genes synaptophysin and CGRP are reduced in specific markers. At E18.5, GFI1 can be visualized in specific clusters Gfi1Ϫ/Ϫ lungs. GFI1 is therefore required to mediate the normal of embryonic mouse lung cells and colocalizes with the pulmonary differentiation of lung cells with a neuroendocrine phenotype. neuroendocrine cell-specific protein calcitonin-gene–related peptide ASH1 is a proneural bHLH transcription factor that is critically (CGRP; Fig. 5A). Moreover, CGRP and chromogranin A colocalize required for the formation of pulmonary neuroendocrine cells (5). with GFI1 in the pulmonary neuroendocrine cells of adult mouse ASH1 functions through dimerization with E2A gene products, and lungs (Fig. 5B). GFI1 expression was not detected in cells that do not these dimers bind to E-box DNA sequences to transactivate target 6879

Fig. 4. A, positive primary human neuroendocrine lung cancer specimens analyzed by immunohistochemistry with the 2.5D.17 monoclonal antibody. B, enumeration of GFI1-negative, GFI1-low, and GFI1-high expressing samples from immunohistochemical analysis of a 358 sample human lung cancer tissue array. C, cross-tabulation analysis of GFI1 versus stains for ASH1, synaptophysin, chromogranin A, and gastrin-releasing-peptide (GRP). f ϭ positive for ASH1, synaptophysin, chromogranin A, or gastrin-releasing-peptide. Ⅺ ϭ negative for ASH1, synaptophysin, chro- The ,ءء .mogranin A, or gastrin-releasing-peptide relationship between staining for GFI1 and these markers are significant (P Ͻ 0.0000001).

genes (42). Inhibitors of ASH1 function sequester binding partners or GFI1 and ASH1 expression. Indeed, our tissue array analyses indicate compete for binding to E-box sequences (42). HES1, the downstream that GFI1 expression not only correlated with that of ASH1 but also effector of Notch signaling, sequesters binding partners of proneural with putative transcriptional targets of proneural bHLH factors and bHLH genes and represses the transcription of both proneural bHLH thus a neuroendocrine phenotype. Conversely, mice without GFI1 genes and their transcriptional targets (40). Hence, HES1 is an ASH1 demonstrate reduced expression of neuroendocrine markers but not a antagonist. Although Ash1Ϫ/Ϫ mice lack pulmonary neuroendocrine reduction in the number of cells expressing ASH1. In a manner similar cells, in Hes1Ϫ/Ϫ mice the number of ASH1-expressing cells and to the requirement for Sens in Drosophila proneural bHLH protein pulmonary neuroendocrine cells are increased (13). Thus, the level of activity (7), it is thus possible that the optimal expression of neuroen- functional ASH1 is determined by the corresponding levels of antag- docrine-specific bHLH target genes depends on GFI1 function. onists such as HES1. Recently, the GFI1-related protein GFI1B was suggested to have The very strong statistical correlation between ASH1 and GFI1 transcriptional activating properties in erythroid lineage cells (43). expression in lung cancers may indicate a functional relationship. The Moreover, the Drosophila orthologue of GFI1 can directly repress Drosophila orthologue of GFI1, Sens, functions to antagonize the genes or induce transactivation of genes (11). Here, we show that Drosophila orthologues of HES1 (7, 11). Specifically, Sens directly GFI1 in SCLC cell lines functions as a DNA-binding transcrip- binds to orthologues of HES1, resulting in the transactivation of genes tional repressor. Therefore, lack of GFI1 would be expected to encoding proneural bHLH factors (11). Although GFI1 does not result in the activation of GFI1 target genes. Thus, GFI1 loss may contain the protein sequences that mediate Sens protein binding to indirectly result in lower expression of synaptophysin and CGRP, HES1 orthologues, these data indicate that the functional role of Sens perhaps because the bHLH factors are impaired. In fact, the levels in Drosophila development is to reinforce the developmental program of the ASH1 antagonists, Inhibitor of DNA-binding-1 (ID1) and induced by proneural bHLH factors. GFI1 may have a similar function ID2, were recently demonstrated to be dramatically up-regulated in in lung epithelium, which could explain the close association between Gfi1Ϫ/Ϫ thymocytes (26). ID1 and ID2 sequester the E2A gene 6880

Fig. 5. GFI1 in murine pulmonary neuroendocrine cells (PNECs). A, GFI1 colocalization with the PNEC-marker chromogranin A (CgA) in wild-type embryonic lung. B, GFI1 colocalization with the PNEC markers chromogranin A and CGRP in adult lung. C, ASH1 expression in wild-type and Gfi1-mutant lung. D, enumeration of single PNEC in -The difference in number is signif ,ء .wild-type and Gfi1-mutant lung icant (P ϭ 0.048). E, enumeration of clusters of PNEC (NEB) in -The difference in number is signif ,ءء .wild-type and Gfi1-mutant lung icant (P ϭ 0.0059). F, enumeration of PNECs within NEB in wild-type The difference in number is significant ,ء .and Gfi1-mutant lung (P ϭ 0.05). F, CGRP expression in wild-type and Gfi1-mutant lung. G, quantification of CGRP expression in wild-type and Gfi1-mutant lung is .(P ϭ 0.0038 ,ءء) reduced in Gfi1-mutant lung by ϳ3.4-fold

products that are required for ASH1 to bind DNA. It is possible (45). In human SCLC cell lines, antisense oligonucleotides targeting that repression of ID1 and ID2 increases the cellular pool of E2A ASH1 or activated Notch signaling leads to lower ASH1 levels, loss of gene products and thus transcriptionally active ASH1. This possi- neuroendocrine target gene expression, and subsequent cell cycle bility requires additional investigation. arrest (5, 41). Thus, SCLC may rely on proneural bHLH factors such GFI1 functions as an oncoprotein in human SCLC. In Moloney as ASH1 for autocrine and paracrine stimulation by neuroendocrine murine leukemia virus-induced tumors, activation of the Gfi1 locus by growth factors. If GFI1 functions in a manner similar to Sens, it would proviral insertion is potently selected (23); however, transgenic ex- reinforce the action of bHLH factors and thus provide growth factor pression of GFI1 alone is not overtly oncogenic (44). Conversely, support for SCLC tumors. Indeed, the level of GFI1 immunohisto- transgenic GFI1 rapidly accelerates leukemogenesis induced by other chemical stain was intense in all SCLC tumor samples examined. This oncoproteins (44). It is therefore likely that the context of GFI1 was true in three separate laboratories and a total of 18 SCLC expression determines GFI1 oncogenic activity. specimens (data not shown). Thus, unlike NSCLC tumors, neuro- Here, for the first time, we demonstrate GFI1 oncogenic activity endocrine tumors may provide an epithelial cellular context in which in nonhematopoietic malignancies. Specifically, SCLC tumor xe- GFI1 functions as an oncoprotein. By analogy to Sens, we would nograft establishment and growth was accelerated by the forced predict that GFI1 action enforces the activity of proneural bHLH expression of GFI1. GFI1 activity may therefore be limiting for transcription factors that are essential for the expression of neuroen- SCLC growth. Interestingly, although GFI1 overexpression is well docrine growth factors and therefore autocrine and paracrine stimu- tolerated in SCLC cell lines, it impaired growth of NSCLC cell lines. lation of the tumor. As such, GFI1 may provide a new molecular Indeed, the effect of GFI1 overexpression in NSCLC cell lines is target for the detection, diagnosis, and treatment of neuroendocrine similar to the effect of activated Notch expression in SCLC cell lines tumors of the lung. 6881

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. Growth Factor Independence-1 Is Expressed in Primary Human Neuroendocrine Lung Carcinomas and Mediates the Differentiation of Murine Pulmonary Neuroendocrine Cells

Avedis Kazanjian, Deeann Wallis, Nicholas Au, et al.

Cancer Res 2004;64:6874-6882.

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