Regulation of lung cancer cell growth and differentiation by notch signaling

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 homolog-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 cell growth and NE differentiation of SCLC, we used recombinant adenoviruses to overexpress active forms of Notch or the Notch effector protein human hairy enhancer of split-1 (HES1) in SCLC cells. Notch proteins, but not HES1 or control adenoviruses, caused a profound growth arrest, associated with a G1 cell cycle block and up-regulation of p21waf1/cip1 and p27kip1. Active Notch proteins also led to dramatic reduction in hASH1 expression, as well as marked activation of phosphorylated ERK1 and ERK2, findings which have been shown to be associated with cell cycle arrest in SCLC cells. 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.; Classically, Notch signaling negatively regulates the expression of bHLH factors through the transactivation of HES genes which leads to transcriptional repression of bHLH genes. In addition to its known transcriptional mechanism, overexpression of active Notch1 also leads to rapid degradation of hASH1. The initial appearance of active Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of downregulation hASH1 mRNA, but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was co-infected 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 hASH1 protein and increased the fraction of hASH1 which was poly-ubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1 dimerizing partner E12 could protect hASH1 from degradation. Substituting exogenous hASH1 with another class II bHLH protein MyoD resulted in a similar reduction of MyoD protein abundance caused by Notch1. This novel function of activated Notch to rapidly degrade class II bHLH proteins may prove to be important in additional contexts in development and in cancer.