Notch signaling

A long-term objective of the lab is to understand Notch signaling. Notch signaling is an important and highly significant topic because it represents one of the five major signal transduction pathways (Wnt/b-catenin, Hedgehog, receptor tyrosine kinases, nuclear receptors, and Notch) found in all metazoans responsible for growth, differentiation, and maintenance of cells. Misregulation of any of these pathways can result in disease states. Of the five signaling pathways, we know the least about Notch signaling.

The Notch receptor is a protease-activated transcription factor located on the cell membrane. Interaction with a ligand results in cleavage releasing the intracellular domain (NICD). The NICD enters the nucleus and forms a complex with CSL (CBF1, Suppressor of Hairless, and Lag-1) enhancer-binding proteins and Mastermind (MAM) to activate target genes. The ternary complex (CSL, NICD, and MAM) is believed essential for activation of target genes. The Notch signaling pathway has been best characterized genetically. However, the molecular mechanisms in the nucleus and molecular targets of Notch signaling remain poorly defined.

Notch is widely distributed across all studied metazoan organisms. The number of receptor types and MAM isoforms varies from higher metazoan (humans) to lower metazoans (C. elegans). For example, in C. elegans there are two Notch receptors and a single MAM protein, while in mammals, there are four Notch receptors and three MAM proteins. Notch1 and MAM1 remain the best characterized at the transcriptional level. The significance of the larger number of receptors in higher metazoan is currently unknown. We believe the increase in the number of Notch receptors and MAM proteins is due to increasing transcriptional complexity requiring a more refined signaling mechanism to be in place. We propose that the complexity of signaling is related to the various combinations of NICD and MAM proteins that interact forming different transcriptional complexes. These complexes can be characterized and measured in a quantitative way. Current projects in the lab are designed to identify novel genes that are regulated by Notch and Mastermind, understand the molecular architecture of Notch responsive promoters and identify novel factors that interact with Notch and Mastermind to activate target genes. 

Flavonoid induced cell death

A secondary research effort in the lab is to understand how small molecules that have been isolated from fruits and vegetables (i.e. bioactive compounds) alter signaling pathways in cancer cells. These bioactive compounds have shown a dual promise; they have been shown to act as both a treatment of cancer through cytotoxicity and potentially as a prevention of cancer yet their mechanisms of action are not fully characterized. We are currently working with a family of compounds called flavonoids. This family is the largest of the bioactive compounds which allows us to use structural relationships among the compounds to help characterize their molecular mechanisms of action.

We have undertaken a study using 14 flavonoids and screened them against 5 different human breast cancer cell lines. We selected these cell lines based upon various known signal transduction pathways that were either present or absent. We screened for cellular cytotoxicity and discovered that flavonoids worked equally well across cell lines suggesting that some signaling pathways are not absolutely required for flavonoid function. Further, we have recently published that flavonoids do not require caspase-3 or caspase-7 for activity. Our data has suggested to us that flavonoids function through mitochondrial decoupling. Interestingly, although the mitochondria are decoupled, we see a 2-3 fold increase in ATP production from the cells. We believe that this may be due to either through stimulation of AMPKinase pathway or through stimulation of fatty acid synthetase. Future studies will be designed to look at the role mitochondria play and if the AMPKinase signaling pathway is important.

Finally, in our initial screen, not all flavonoids were able to induce cytotoxicity and subsequent cell death. We believe that this is due to their ability to cross the cell membrane. Using mass spectrometry, we can show that only select flavonoids are able to enter cancer cells and are currently quantitating the amount of flavonoid present in the cell. Numerous studies have argued that flavonoids are metabolized by cells and that the metabolites may function in cell death. However, our data suggests that flavonoids are not highly metabolized in breast cancer cells. Further work is underway in the lab investigating this using mass spectrometry.

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