[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 Grimes 1 1 Institute for Cellular Therapeutics and Departments of Surgery, Biochemistry, and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky; 2 Departments of Molecular and Human Genetics, 3 Internal Medicine, 4 Program in Developmental Biology, 5 Pathology, 6 Medicine and Molecular Physiology and Biophysics, and the 7 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas; 8 Department of Pulmonary Medicine, M. D. Anderson Cancer Center, Houston, Texas; 9 Department of Pathology, Vancouver General Hospital, Vancouver, British Columbia, Canada ABSTRACT Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcrip- tion factor is critically required for the formation of pulmonary neuroen- docrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. Here, we show that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly corre- lates with expression of ASH1, the neuroendocrine growth factor gastrin- releasing peptide, and neuroendocrine markers synaptophysin and chro- mogranin A (P < 0.0000001). GFI1 colocalizes with chromogranin A and calcitonin-gene–related peptide in embryonic and adult murine pulmo- nary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indi- cating that GFI1 is important for neuroendocrine differentiation. INTRODUCTION Lung cancer is the most common cancer in the world and is the leading fatal malignancy in both men and women in the United States (1). It is classified into two major histopathological groups: small cell lung carcinomas (SCLCs) and non–small-cell lung carcinomas (NSCLCs; ref. 2). SCLCs account for 15 to 20% of all human lung cancers and are aggressive lung tumors, which are virtually always fatal (2, 3). Lung tumors with a neuroendocrine phenotype include SCLCs, large cell neuroendocrine carcinomas, and classical and atyp- ical carcinoid tumors. The neuroendocrine phenotype is associated with the production of neuropeptides that may stimulate transforma- tion and tumor growth through paracrine and autocrine signaling (4), and the maintenance of these signaling loops in lung tumors is dependent on the neuroendocrine phenotype (5, 6). Although it is clear that the growth factors secreted by neuroendocrine tumors participate in tumorigenesis and tumor maintenance, the genetic and biochemical pathways underlying neuroendocrine differentiation have not been elucidated. Understanding the molecular basis of neuroen- docrine lung carcinomas, especially SCLC, should provide the oppor- tunity for novel therapeutic intervention with significant implications for patients. Clues to the genetic components of the human neuroendocrine tumor phenotype can be found in the genetic cascade that controls Drosophila peripheral nervous system development. Drosophila pe- ripheral nervous system organ development is regulated by proneural basic helix-loop-helix (bHLH) transcription factors atonal, amos, scute, and achaete (7, 8). These proneural bHLH transcription factors dimerize with daughterless proteins to bind E-box DNA sequences and transactivate target genes, including Delta, which activates Notch signaling in adjacent cells (9). Activation of Notch signaling induces Enhancer-of-split, a bHLH transcriptional repressor, which antago- nizes proneural bHLH factor expression and function (10). Proper function of proneural genes is required in most tissues for the expres- sion of senseless, which encodes a zinc-finger transcription factor (sens; ref. 7). Embryonic and adult peripheral nervous system devel- opment requires sens: embryos that lack sens specify peripheral nervous system cells, but most of the cells die through apoptosis, whereas adult peripheral nervous system precursors that lack sens fail to become specified. In contrast, forced expression of Sens is suffi- cient to mediate peripheral nervous system development (7, 11). Thus, sens is a central target of proneural bHLH transcription factors in peripheral nervous system development. The Achaete-Scute Homologue-1 (ASH1), the mammalian ortho- logue of Drosophila achaete and scute encoded proteins, is critical for murine pulmonary neuroendocrine cell development (5, 12). pulmo- nary neuroendocrine cells are a minor cell population of the airway epithelium, which function as oxygen sensors (13). Newborn mice bearing a disruption of Ash1 completely lack detectable pulmonary neuroendocrine cells and die shortly after birth (5). Although the data are not conclusive, human neuroendocrine carcinomas might be de- rived from pulmonary neuroendocrine cells (14). Not surprisingly, human SCLC express ASH1 (15). Moreover, expression levels of ASH1 in SCLC tumors correlates with neuroendocrine markers such as gastrin-releasing peptide, L-dopa-decarboxylase activity and calci- tonin (16, 17). However, the role of ASH1 in SCLC is unclear. Transgenic expression of ASH1 in nonneuroendocrine murine lung Clara cells results in hyperplasia but not cancer (6). Dual transgenic expression of ASH1 and the polyoma large T antigen generates neuroendocrine lung tumors, but these are dissimilar to SCLC (6). It is therefore uncertain whether the expression of ASH1 in SCLC is merely a marker of neuroendocrine differentiation or a driving force in neuroendocrine oncogenesis. The mammalian orthologue of Drosophila Sens is the growth factor independence-1 (GFI1) oncoprotein. Gfi1 was cloned in an insertion mutagenesis screen for targets of the Moloney murine leukemia virus, which could mediate leukemia progression from interleukin 2- Received 2/23/04; revised 7/23/04; accepted 7/28/04. Grant support: Commonwealth of Kentucky Lung Cancer Research Program (H. Grimes), the Commonwealth of Kentucky Research Challenge Trust Fund, and the Jewish Hospital Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: A. Kazanjian, D. Wallis, and N. Au contributed equally to this work. Requests for reprints: H. Leighton Grimes, Institute for Cellular Therapeutics, University of Louisville, Baxter Building, Room 404F, 570 South Preston Street, Louisville, KY 40202. Phone: (502) 852-2059; Fax: (502) 852-2085; E-mail: lee.grimes@louisville.edu. ©2004 American Association for Cancer Research. 6874 Research. on October 14, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from