Patients with metastatic carcinoid tumors, rare neuroendocrine (NE) neoplasms characterized by the secretion of amines and polypeptide hormones like serotonin (5-HT) and histamine, often have debilitating symptoms of the carcinoid syndrome, including diarrhea, bronchospasm, flushing, and thickening of the right-sided heart valves 47–50
. Widespread metastases render conventional surgical treatment palliative at best and therefore there exists a great need to develop novel targeted therapeutic strategies both to reduce tumor burden and control symptoms of the carcinoid syndrome.
Recently, Notch signaling has been shown to play an essential role in the neuroendocrine (NE) differentiation of cells in the lung and GI tract 6, 51
. In the developing fetal lung epithelium, Ito et al demonstrated strong immunohistochemical staining for Notch1, Notch2, Notch3, and the immediate Notch effector HES-1 in non-NE pulmonary cells, whereas the mammalian homologue of achaete-scute complex-like 1 (ASCL1) was found only in clusters of cells belonging to the pulmonary NE cells (PNECs) 25
. Thus, the overall effect of Notch activation is to limit the number of cells that can differentiate into enteroendocrine cells.
In the present study, we identified multiple small molecule activators of the Notch pathway by HTS and validated the strongest hit, RESV, in a series of studies. Treatment of BON GI carcinoid cells and NCI-H727 bronchopulmonary cells with RESV resulted in marked induction of the Notch signaling pathway, and profound growth inhibition associated with S-phase cell cycle arrest. Western blot analysis revealed upregulation of p21WAF1/Cip1 and phosphorylated cdc2 (tyr15). Active Notch signaling led to a dramatic reduction in ASCL1 expression, as well as marked inhibition of CgA and serotonin. Transfection of Notch antisenseRNAs into GI carcinoid cells blocked the effects of RESV on Notch expression, ACSL1 suppression, and the NE markers CgA and serotonin. This study is the first to report the results of a large-scale HTS to identify activators of the Notch signaling pathway and validates the strongest hit from this screen.
The effects of RESV on other cancer cells have been extensively described. Broadly, in in vitro
models, RESV inhibited the growth of tumor cell lines derived from various human cancers. This effect has been associated with the ability of RESV to arrest cell cycle progression, to promote cell differentiation and to induce programmed cell death by caspase-independent or caspase-dependent apoptosis or by autophagocytosis. Specifically, in human colorectal cancer cells, RESV has been shown to induce cell death by a novel pathway involving lysosomal cathepsin D 52
. Similarly, using microarray gene expression profiling and high-throughput immunoblotting (PowerBlot) methodologies, Whyte et al. showed the arrest of lung cancer cells in the G1 phase of the cell cycle and demonstrated this growth arrest is mediated via the transforming growth factor pathway, particularly through the Smad proteins53
. While these and other studies intimate that RESV possesses cancer chemopreventive properties in vivo, several phase I pharmacokinetic dose-escalation studies and our own data support the hypothesis that RESV is safe and results in minimal adverse effects on normal cells. In a phase I study of oral RESV (single doses of 0.5, 1, 2.5, or 5 g), Boocock et al. showed that RESV was well tolerated with follow-up of over 40 healthy volunteers failing to reveal any serious adverse reaction either clinically or by biochemical and hematologic analyses54
. Similarly, our mouse xenograft studies with i.p injections failed to demonstrate any adverse effects in either the control or RESV-treated mice over the 2-week treatment period.
The mechanism by which RESV regulates ASCL1 expression is a subject of ongoing investigation, as the dietary polyphenol is known to influence diverse biologic pathways. Several studies have demonstrated that an early target of RESV is the sirtuin (Sirt) class of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases 55
. It has been shown that under oxidizing conditions in neuronal precursor cells, induced Sirt1 and Hes1 form a complex that binds to and deacetylates histones at the mASH1
promoter, while recruiting co-repressors such as TLE1 56
. Together, these events cause downregulation of ASCL1 expression and block neuronal differentation; furthermore, the influence of the redox state on cell-fate decisions was eliminated by removal of Sirt1 activity by siRNA or through the use of Sirt1 inhibitors. Interestingly, RESV-induced suppression of ASCL1 in human carcinoid cells appears to be independent of Sirt1 activity, as blockade of RESV-mediated Sirt1 induction did not reverse the RESV-mediated changes in ASCL1 protein expression (data not shown).
RESV-induced regulation of ASCL1 expression, and subsequently the NE phenotype in human carcinoid tumor cells, more certainly appears to be regulated by Notch signaling. In medullary thyroid cancer (MTC), a NE tumor derived from the calcitonin-producing C-cells of the thyroid gland, Notch1-mediated silencing of ASCL1
gene transcription results in decreased expression of CgA and calcitonin 20
. Similarly, stable expression of Notch1 in BON cells has been shown to result in decreased expression of CgA, 5-HT, SYP, and NSE 19
. In the present study, we clearly demonstrate that RESV induces functional Notch signaling, the first time that this has been shown in human carcinoid cells. More importantly, it is the relative contribution of Notch2 rather than Notch1 induction which seems to be responsible for RESV-induced regulation of ASCL1 protein expression, as demonstrated by Notch1 and Notch2 siRNA knockdown studies. Based on these findings it is suggested that if Notch1 and Notch2 ICDs exert the same downstream effects; therefore, they may be used interchangeably in scenarios where the goal is to exogenously activate the Notch signaling pathway for the purpose of controlling cell fate.
Utilization of cell based HTS systems should be done with caution and represent a limitation of these data. The current cell based HTS is based on the NCI-60 cell line screen system, an anticancer drug screen developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumors in anticancer drug screening57
. However, such cell line screen systems remain in their relative infancy and are not mature enough to utilize as primary therapeutic indicators. In cell based, high-throughput molecular-targeted screens, compounds can potentially function in various ways to activate cell based reporters. Molecular action in any of several 'upstream' pathways could result in activity. Therefore, it is essential to identify the detailed mechanism of action of selected compounds, often a very challenging laboratory task. In the present study, RESV was identified as a potential Notch activator based on the preliminary HTS screen; furthermore, extensive laboratory testing confirmed that RESV does, in fact, activate functional Notch signaling and that this activation is responsible for the phenotypic changes seen in vitro.
In summary, we described the use of a novel, laboratory-derived cell based HTS for the identification of Notch activating compounds. We analyzed 7,264 compounds and confirmed that the top HTS hit, RESV, activates the Notch signaling pathway in human carcinoid cells in vitro. Importantly, this dietary polyphenol induces functionally active Notch which represses ASCL1 protein expression and directly inhibits the NE phenotype characterized by the production of bioactive hormones and peptides like CgA and 5-HT. Similarly, RESV-induced Notch activation is associated with S phase cell cycle arrest in vitro and growth inhibition. As RESV is a nontoxic, naturally occurring compound with an established safety profile, it is an attractive target for further in vivo studies utilizing a murine model of metastatic carcinoid cancer. Based on the data presented here, RESV may additionally be an attractive target for the development of a limited phase II clinical trial in patients with unresectable carcinoid disease. Thus, this HTS method could be used to screen for other modulators of the Notch signaling pathway.