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Neurod1 Regulates Survival and Migration of Neuroendocrine Lung Carcinomas Via Signaling Molecules Trkb and NCAM

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Neurod1 Regulates Survival and Migration of Neuroendocrine Lung Carcinomas Via Signaling Molecules Trkb and NCAM NeuroD1 regulates survival and migration of neuroendocrine lung carcinomas via signaling molecules TrkB and NCAM Jihan K. Osbornea, Jill E. Larsenb, Misty D. Shieldsb, Joshua X. Gonzalesa, David S. Shamesb,1, Mitsuo Satob,2, Ashwinikumar Kulkarnia,3, Ignacio I. Wistubac, Luc Girarda,b, John D. Minnaa,b, and Melanie H. Cobba,4 aDepartment of Pharmacology and bHamon Cancer Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041; and cDepartment of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030 Contributed by Melanie H. Cobb, March 4, 2013 (sent for review February 8, 2013) Small-cell lung cancer and other aggressive neuroendocrine can- and prostate cancers, and pituitary adenomas (10–16). To charac- cers are often associated with early dissemination and frequent terize the mechanisms of NeuroD1 action in lung tumor patho- metastases. We demonstrate that neurogenic differentiation genesis, we analyzed a panel of lung cell lines. HBEC cell lines, 1 (NeuroD1) is a regulatory hub securing cross talk among survival assigned a number to distinguish lines from different individuals, and migratory-inducing signaling pathways in neuroendocrine are immortalized by overexpression of cyclin-dependent kinase 4, lung carcinomas. We find that NeuroD1 promotes tumor cell and human telomerase reverse transcriptase (e.g., HBEC3KT) (17). survival and metastasis in aggressive neuroendocrine lung tumors The immortalized HBEC3KT cell line was sequentially trans- through regulation of the receptor tyrosine kinase tropomyosin- formed by knockdown of the tumor suppressor p53 and expression related kinase B (TrkB). Like TrkB, the prometastatic signaling of K-RasV12 (HBEC3KTRL53) (Table S1) (18, 19). Microarray analysis of lung cell lines revealed that 11 of the 20 SCLC and 3 of molecule neural cell adhesion molecule (NCAM) is a downstream the 5 neuroendocrine NSCLC (NSCLC-NE) had significantly target of NeuroD1, whose impaired expression mirrors loss of Neu- higher expression of NEUROD1 compared with HBEC and roD1. TrkB and NCAM may be therapeutic targets for aggressive NSCLC (Fig. 1A and Fig. S1A). We confirmed the neuroendo- neuroendocrine cancers that express NeuroD1. crine cell lines generally expressed high levels of NeuroD1 com- pared with HBEC and other lung cancer cell lines (Fig. 1 B and bHLH | SCLC C). We conducted further mechanistic studies in three SCLC cell lines (H69, H82, and H2171) and one NSCLC-NE (H1155). ased on histology, 15–20% of lung carcinomas are catego- To investigate the indication that various NSCLC tumors Brized as small-cell lung cancer (SCLC) and 80–85% as non– spontaneously undergo neuroendocrine differentiation, micro- small-cell lung cancer (NSCLC) (1–4). Lung cancers with neu- array analysis was performed on an additional 275 NSCLC pri- roendocrine features represent nearly 25% of all lung cancer mary resected patient samples not previously annotated as cases including all SCLC, a subset of NSCLC, and typical and neuroendocrine (Fig. S1B). Validation of a subset of the NSCLC atypical carcinoids. SCLC is the deadliest histological subtype tumor samples revealed several had relatively high NeuroD1 because it is associated with high rates of metastatic disease at expression compared with H69, whereas the majority of tumor D time of diagnosis (1, 2). Mixed histological variants containing samples expressed more NeuroD1 than HBEC (Fig. 1 ). As we fi both SCLC and NSCLC components have previously been linked suspected, several tumors annotated as NSCLC (speci cally to a lower overall survival than SCLC alone (5). With neuro- adenocarcinomas and squamous cell carcinomas) may have un- endocrine differentiation thought to occur spontaneously in dergone neuroendocrine differentiation. 10–30% of all NSCLC (6), a subset of cells within a tumor may promote poorer prognoses and responses than predicted by NeuroD1 Regulates Survival and Metastasis of Neuroendocrine Lung original pathology. Cancers. To test whether NeuroD1 was essential for tumorigen- esis, we established SCLC and NSCLC-NE lines that stably The neuronal transcription factor neurogenic differentiation C 1 (NeuroD1) is overexpressed in a variety of aggressive neural/ expressed shRNAs against NeuroD1 (Fig. S1 ). Depletion of neuroendocrine carcinomas. NeuroD1 is important for the de- NeuroD1 prevented survival of the neuroendocrine lung cancer cells as measured by reduction of colonies in soft agar (Fig. 1E velopment and function of several neural/neuroendocrine tis- D sues, the fate of specific neurons in the central and peripheral and Fig. S1 ). The residual soft agar colonies were less than 25% of the control colony size, suggesting defects in sustained nervous system and for insulin gene transcription in adult pan- growth (Fig. S1E). This phenotype could be partially rescued by creatic beta cells (7, 8). NeuroD1 mutations can lead to maturity- onset diabetes of the young 6 (MODY6) (Online Mendelian Inheritance in Man 606394), the only setting in which NeuroD1 is thus far known to be critical to disease (9). Author contributions: J.K.O., J.E.L., J.D.M., and M.H.C. designed research; J.K.O., J.E.L., M.D.S., and J.X.G. performed research; D.S.S., M.S., and I.I.W. contributed new reagents/ To investigate the role of NeuroD1 in tumorigenesis, we focused analytic tools; J.K.O., J.E.L., A.K., and L.G. analyzed data; and J.K.O., J.E.L., J.D.M., and on neuroendocrine lung cancer cells lines and isogenic normal M.H.C. wrote the paper. and tumorigenic immortalized human bronchial epithelial cells The authors declare no conflict of interest. (HBEC). We observed that down-regulation of NeuroD1 prevents Data deposition: The data reported in this paper have been deposited in the Gene Ex- survival, invasion, and metastasis of several neuroendocrine lung pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession nos. GSE4824 cancer cell lines. Tropomyosin-related kinase B (TrkB) and neural and GSE32036). cell adhesion molecule (NCAM) are prometastatic signaling mol- 1Present address: Oncology Diagnostic, Genentech, Inc., South San Francisco, CA 94080. ecules downstream of NeuroD1 and are responsible at least in part 2Present address: Department of Respiratory Medicine, Nagoya University Graduate for the phenotypic consequences of NeuroD1 expression. School of Medicine, Nagoya 466-8550, Japan. 3Present address: Department of Molecular and Cell Biology, University of Texas at Dallas, Results Richardson, TX 75080. NeuroD1 Is Highly Expressed in Aggressive Neuroendocrine Lung 4To whom correspondence should be addressed. E-mail: [email protected]. Cancers. NeuroD1 is overexpressed in several aggressive neural/ This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. neuroendocrine cancers including SCLC, medulloblastoma, gastric 1073/pnas.1303932110/-/DCSupplemental. 6524–6529 | PNAS | April 16, 2013 | vol. 110 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1303932110 Downloaded by guest on September 26, 2021 A ) 2 NS B *** Neuroendocrine Non-Neuroendocrine 16 ** ** 2 8 4 2 1 1 0.5 0.25 0 0.125 mRNA Relative NeuroD1 NeuroD1 expression (log C D 10 1 0.1 0.01 0.001 0.0001 mRNA NeuroD1 0.00001 Fig. 1. NeuroD1 regulates survival and metastasis Relative NeuroD1 Relative NeuroD1 0.000001 of neuroendocrine lung cancers. (A) mRNA expres- Loading NSCLC Patient Tumor Samples sion in 86 cell lines, 8 HBEC, 56 NSCLC, and 22 SCLC, Control was assessed using Affymetrix HG-U133A and B GeneChips. Cell lines were categorized histopatho- E Control shRNA logically and by NEUROD1 expression (**P < 0.001, NeuroD1 shRNA ***P < 0.0001; two-tailed t test). (B and C) NeuroD1 150 expression was validated via quantitative RT-PCR 100 (qRT-PCR) and immunoblotting in cell lines from 50 μ ** **** ** each type noted above; 50 g of protein was loaded 0 per lane, and NFAT was used as loading control. H69 H82 H2171 H1155 Pancreatic beta-cell lines are positive controls. HBEC3KT colonies % control F (D) Thirty-five adenocarcinoma and squamous pa- Control Neurod1 fi Control tient samples analyzed via qRT-PCR to con rm H 40 Vector NEUROD1 expression. Values were normalized to 30 Neurod1 H69 values. The arbitrary line compares expression 0 hr Vector 20 of NEUROD1 in the normal bronchial epithelial cell line HBEC30KT. (E) Soft agar assays of H69, H82, 10 and H2171 and H1155 infected with shControl or Cells migrating 0 H358 shNeuroD1. Cells were sorted by expression for GFP 8 hr Control Neurod1 on the GIPZ plasmids. Plotted are average number ± NeuroD1 of colonies after 2 wk. Error bars represent SD of four independent experiments in triplicate (**P < GAPDH 0.001; one-way ANOVA). (F–H) HBEC3KT and H358 G cells were transfected with pCMV-Neurod1, and Control I then subjected to Transwell or wound-healing as- 500 Vector 100 Control shRNA say. Graph represents mean ± SD of three in- 400 Neurod1 80 ** NeuroD1 dependent experiments in duplicate for HBEC3KT 300 Vector 60 shRNA < 200 (**P 0.001; one-way ANOVA). (I) Clone 5 cells 40 100 infected with shNeuroD1 or shControl were sub- 20 Cells migrating ** ± 0 jected to Transwell assay. Graph represents mean 0 HBEC3KT SD of three independent experiments in triplicate Cells migrating HBEC3KTRL53- Clone 5 (**P < 0.001; one-way ANOVA). a shRNA-resistant mouse NeuroD1 plasmid (Fig. S1 F and G). colonization and metastasis (Fig. 2B and Fig. S2C). These results As many patients present with metastases at the time of di- provide evidence that NeuroD1 is necessary and sufficient for agnosis, we investigated the ability of NeuroD1 to regulate mi- not only anchorage-independent growth and motility but also gration. Overexpression of NeuroD1 in HBEC3KT resulted in tumorigenic and metastatic potential. a significant increase in the motility of the cells (Fig. 1 F and G and Fig. S1H), whereas reduction of NeuroD1 in clone 5 resulted NeuroD1 Downstream Targets, TrkB and NCAM, Regulate Survival and in a significant decrease in motility, as well as decreased soft agar Migration in Neuroendocrine Lung Cancer.
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