Project description:Lung cancer is the leading cause of cancer death in the United States. Among the various subtypes of lung cancers, pulmonary neuroendocrine (NE) cancer, including small cell lung cancer (SCLC) and NE-non-small cell lung cancer (NE-NSCLC), is a particularly aggressive malignancy that is distinct from classic non–small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Recently, the lineage-specific transcription factors Achaete-scute homolog 1 (ASCL1), NEUROD1, and POU2F3 have been reported to identify heterogeneity in pulmonary NE cancers. These transcription factors bind different genomic loci to regulate distinct gene programs in pulmonary NE cancers. However, the signaling pathways downstream of these transcription factors that distinguish these pulmonary NE cancer subtypes are not well characterized. Regulated protein secretion is critically involved in cell signaling and cell-cell communication events, and is known to be a hallmark associated with pulmonary NE tumors. Using a large-scale mass spectrometric approach, we performed quantitative secretomic analysis 13 cell lines including a pair of isogenic cell lines, i.e., an immortalized human bronchial epithelial cell and an ASCL1high NE NSCLC line. This panel also contained 6 additional ASCL1High and 5 NEUROD1High NE-lung cancer cell lines. From the conditioned media of the 13 cell lines, we identified and quantified 1,626 proteins. The NE-specific secretome is associated with a number of biological processes related to neurodevelopment. Further analysis of the upregulated proteins in ASCL1High subtype NE-lung cancer cells leads to the identification of IGFBP5 being a specific secreted marker for ASCL1High pulmonary NE cancer cells. Furthermore, IGFBP5 is also upregulated in the serum of a genetically modified mouse model of ASCL1High SCLC, as well as in human ASCL1High SCLC tumors. Mechanistically, ASCL1 binds to E-box elements in the IGFBP5 gene and directly regulates its transcription. Knockdown of ASCL1 in SCLC decreases IGFBP5 expression, which, in turn, leads to hyperactivation of the IGF-1R pathway. Pharmacological co-targeting of ASCL1 and IGF-1R signaling results in markedly synergistic, growth inhibitory effects in ASCL1High SCLC both in vitro and in vivo. Together, our quantitative proteomic analysis identifies a novel secreted marker and a new combination therapy for ASCL1High pulmonary NE cancer cells. In addition, we expect that the data sets will serve as an invaluable resource, providing the foundation for future mechanistic studies and biomarker discovery that helps delineate the molecular underpinnings of pulmonary NE tumors.

Project description:During colonization in the host gastrointestinal tract, the enteric bacteria Campylobacter jejuni is exposed to a variety of signaling molecules including the catecholamine hormones epinephrine (Epi) and norepinephrine (NE). NE has been determined to stimulate the growth of C. jejuni as well as increase its pathogenicity. To investigate the mechanisms of NE or Epi on the biology of C. jejuni, the global gene expression profiles of C. jejuni NCTC 11168 cultured in iron-restricted medium were analyzed in response to NE or Epi. Totally, 183 and 156 genes were differentially expressed by NE and Epi respectively, with 102 differentially expressed genes common between the two treatments. These genes are involved in diverse cellular functions including iron uptake systems, motility, virulence, oxidative stress response, nitrosative stress tolerance, enzyme metabolism, DNA repair and metabolism and ribosomal protein biosynthesis. Adherence to and invasion of Caco-2 cells by C. jejuni were enhanced upon exposure to NE or Epi. These results indicated that NE and Epi have similar effects on the gene expression of C. jejuni and that the effects on gene expression may contribute to elucidate the mechanisms on interaction between host and C. jejuni. Transcriptional profiles were analyzed using microarray to compared the epinephrine (Epi) or norepinephrine (NE) treated and untreated Campylobacter jejuni NCTC 11168. NE or Epi treated and untreated cultures of C. jejuni NCTC 11168 were collected at the mid-log phase (?36 h cultures). Three (for NE or Epi treated culture) or four (for untreated control culture) independent biological replicates were performed. Total RNA was extracted using RiboPure™-Bacteria Kit (Ambion, Life Technologies) according to the manufacturer’s instructions. The quality and quantity of total RNA were determined by an Agilent Bioanalyzer 2100. Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color, following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany). Each Slide was hybridized with 600ng Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent technologies, Santa Clara, CA, US) in Hybridization Oven, according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Slides were scanned by Agilent Microarray Scanner with default settings, Dye channel: Green, Scan resolution=5?m, PMT 100%, 10%, 16bit. Data were extracted with Feature Extraction software 10.7. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0. The genes with fold change ? 1.5 and P < 0.05 were selected as differentially expressed.

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Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry

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