hes1-protein--human and Carcinoma--Small-Cell

In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Small Cell; Cell Line; Cysteine Endopeptidases; Dimerization; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Half-Life; Homeodomain Proteins; Humans; Lung Neoplasms; Membrane Proteins; Multienzyme Complexes; Muscle Proteins; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Structure, Tertiary; Receptor, Notch1; Receptors, Cell Surface; RNA, Messenger; Signal Transduction; TCF Transcription Factors; Transcription Factor 7-Like 1 Protein; Transcription Factor HES-1; Transcription Factors; Tumor Cells, Cultured; Ubiquitin

Among the various forms of human lung cancer, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype. Neural and NE differentiation in SCLC depend, in part, on the action of the basic-helix-loop-helix (bHLH) transcription factor human achaete-scute homologue-1 (hASH1). In nervous system development, the Notch signaling pathway is a critical negative regulator of bHLH factors, including hASH1, controlling cell fate commitment and differentiation. To characterize Notch pathway function in SCLC, we explored the consequences of constitutively active Notch signaling in cultured SCLC cells. Recombinant adenoviruses were used to overexpress active forms of Notch1, Notch2, or the Notch effector protein human hairy enhancer of split-1 (HES1) in DMS53 and NCI-H209 SCLC cells. Notch proteins, but not HES1 or control adenoviruses, caused a profound growth arrest, associated with a G1 cell cycle block. We found up-regulation of p21(waf1/cip1) and p27kip1 in concert with the cell cycle changes. Active Notch proteins also led to dramatic reduction in hASH1 expression, as well as marked activation of phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2, findings that have been shown to be associated with cell cycle arrest in SCLC cells. These data suggest that the previously described function of Notch proteins as proto-oncogenes is highly context-dependent. Notch activation, in the setting of a highly proliferative hASH1-dependent NE neoplasm, can be associated with growth arrest and apparent reduction in neoplastic potential.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Small Cell; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; DNA-Binding Proteins; Enzyme Activation; G1 Phase; Helix-Loop-Helix Motifs; Homeodomain Proteins; Humans; Lung Neoplasms; MAP Kinase Signaling System; Membrane Proteins; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Muscle Proteins; Receptor, Notch1; Receptor, Notch2; Receptors, Cell Surface; Signal Transduction; Transcription Factor HES-1; Transcription Factors; Tumor Suppressor Proteins; Up-Regulation

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

Topics: Animals; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; beta-Galactosidase; Carcinoma, Small Cell; Cell Line; Conserved Sequence; DNA-Binding Proteins; Drosophila; Gene Expression Regulation, Neoplastic; Helix-Loop-Helix Motifs; Homeodomain Proteins; Humans; Lung Neoplasms; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transcription Factor HES-1; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vertebrates