In vivo and in vitro propagation of intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are one of the three known curable precursor lesions of invasive pancreatic ductal adenocarcinoma, an almost uniformly fatal disease. Cell lines from IPMNs and their invasive counterparts should be valuable to identify gene mutations critical to IPMN carcinogenesis, and permit high-throughput screening to identify drugs that cause regression of these lesions. To advance the study of the biological features of IPMNs, we attempted in vivo and in vitro growth of selected IPMNs based on the hypothesis that IPMNs could be grown in the most severely immunodeficient mice. We examined 14 cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rgamma(null) (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines. One sample was directly cultured only. Thirteen tumors were implanted into the three types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, in which the majority (8 of 10) grew. This included five IPMNs lacking an invasive component. One of the explanted IPMNs, with an associated invasive carcinoma, was successfully established as a cell line. Tumorigenicity was confirmed by growth in soft agar, growth in immunodeficient mice, and the homozygous deletion of p16/cdkn2a. Epithelial differentiation of the cell line was documented by cytokeratin expression. Patient origin was confirmed using DNA fingerprinting. Most non-invasive IPMNs grow in NOG mice. We successfully established one IPMN cell line, and plan to use it to clarify the molecular pathogenesis of IPMNs.

Regulation of neuroendocrine differentiation in gastrointestinal carcinoid tumor cells by notch signaling.

CONTEXT: Gastrointestinal (GI) carcinoid tumors elaborate serotonin and other vasoactive substances, causing the carcinoid syndrome. Based on developmental biology data, we hypothesized that basic helix-loop-helix transcription factors, including achaete-scute complex homolog-like 1 (Ascl1)/hASH1, and the Notch signaling pathway might regulate the neuroendocrine phenotype in GI carcinoids. OBJECTIVE: The aim of this study was to evaluate expression of developmental transcription factors and Notch signaling components in GI carcinoids and model their interaction in a relevant GI carcinoid cell line. DESIGN: Fourteen GI carcinoid tumor specimens, five paired adjacent normal tissues, fetal tissues, and tumor cell lines were analyzed by RT-PCR and immunoblot. BON carcinoid cells were further analyzed after Notch overexpression for neuroendocrine marker expression, serotonin production, and growth. SETTING: The study was conducted in an academic referral center. PATIENTS OR OTHER PARTICIPANTS: Deidentified archival pathology specimens were examined. RESULTS: Among a panel of six developmental transcription factors tested, only Ascl1 mRNA was overexpressed compared with surrounding normal tissue (seven of 10 GI carcinoid tumors and in BON cells, none of five normal tissues). Ascl1 protein was also expressed in four of four carcinoid tumors and BON cells). Notch pathway ligands, receptors, and downstream effectors were widely expressed in tumor and normal specimens. Overexpression of activated Notch1 in BON cells led to induction of the Notch effector hairy and enhancer of split 1 (Hes1), loss of Ascl1, reductions in neuron-specific enolase, synaptophysin, and chromogranin A, and most significantly, an 89% decrease in serotonin concentration and equivalent reductions in serotonin-reactive cells and repression of tryptophan hydroxylase 1 mRNA. CONCLUSIONS: The Notch signaling pathway is a significant regulator of neuroendocrine differentiation and serotonin production in GI carcinoid tumors.

Notch signaling induces rapid degradation of achaete-scute homolog 1.

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.