The Notch pathway is important in maintaining the cancer stem cell population in pancreatic cancer.

BackgroundPancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized.MethodsExpression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth.ResultsNotch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population.ConclusionThe Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer

N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) Interaction with LYS 3.28(192) Is Crucial for Its Inverse Agonism at the Cannabinoid CB1 Receptor

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca2+ current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K→A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via arom. stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a mutant thermodn. cycle was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodn. cycle calcns. indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism

The Notch Pathway Is Important in Maintaining the Cancer Stem Cell Population in Pancreatic Cancer

<div><p>Background</p><p>Pancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized.</p><p>Methods</p><p>Expression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth.</p><p>Results</p><p>Notch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population.</p><p>Conclusion</p><p>The Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.</p></div

GSI treatment slowed tumor growth in an orthotopic model of primary pancreatic cancer.

<p><b>A</b>. Representative images of tumors monitored using bioluminescent imaging. Fold change in bioluminescence of tumors over time from day of implantation. <b>B</b>. Images of tumors excised at the completion of 4 weeks of treatment for each treatment group. Final tumor weights (in grams) of the excised tumors (*p<.01 vs. control). <b>C</b>. (upper panel) mRNA harvested from tumors excised at completion of 4 weeks of treatment was analyzed by qRT-PCR for Hes1, shown normalized to GAPDH (*p<.02 vs. control). (Lower panel) Single cells were isolated from tumors excised at completion of treatment were analyzed for percentage of CSC by FACS analysis of cells stained with DAPI and antibodies to H2k, ESA, CD44, and CD24. Percentage of CSC represents ESA+/CD44+/CD24+ cells as a percentage of live DAPI-negative and human H2k-negative cells (*p<.01 vs. control). <b>D</b>. H+E stained sections from tumors treated with GSI, gemcitabine, or combination of GSI and gemcitabine. Proliferative index calculated as number of Ki67+ stained cells from formalin-fixed sections of tumor treated with drug. Graph representing mean number of Ki67+ cells per 40x field from 5 random sections from each of 3 tumors per treatment group (*p<.01 vs. control). TUNEL assay performed on sections of tumor treated <i>in vivo</i> and tabulated mean number of TUNEL+ apoptotic cells (*p<.001 vs. control).</p

Notch pathway activation with DSL peptide stimulates tumorsphere formation and increased CSC population.

<p><b>A</b>. Pancreatic cancer cell tumorspheres were cultured in sphere medium containing increasing concentration of DSL peptide as indicated for 5 days. mRNA levels of Hes1 from treated tumorspheres were analyzed by qRT-PCR and normalized to internal control GAPDH (*p<.001 vs. control). <b>B</b>. Tumorspheres cultured as in <b>A</b>. were Western blotted for cleaved-Notch1, Notch1, Hes1 or β-actin. <b>C</b>. Pancreatic cancer cells cultured in sphere medium containing increasing concentration of DSL analyzed for percentage of CSC by FACS analysis of cells stained with DAPI and antibodies to ESA, CD44, and CD24. Percentage of CSC represents ESA+/CD44+/CD24+ cells as a percentage of live DAPI-negative cells (*p<.02 vs. control). <b>D+E</b>. Tumorsphere formation assay performed on pancreatic cancer cells cultured in increasing concentration of DSL peptide. Representative images shown in <b>D</b>. with quantitation of number of tumorspheres per 1000 cells plated shown in <b>E</b>. (*p<.01 vs. control).</p

Hes1 shRNA treatment effect on pancreatic tumorspheres.

<p><b>A</b>. Pancreatic cancer cells transduced with either control or Hes1 shRNA harvested after culturing for 4 days. Hes1 transcript levels quantitated by qRT-PCR, normalized to GAPDH (*p<.001 vs. control). <b>B</b>. Cell lysates were Western blotted for Hes1, Notch1, cleaved Notch1, or β-actin. <b>C</b>. Representative images of tumorspheres generated from pancreatic cancer cells transduced with either control scramble sequence or shRNA to Hes1 cultured for 5 days. <b>D</b>. Quantitation of number of tumorspheres generated per 1000 cells plated (*p<.001 vs. control).</p

Expression profile of Notch pathway components in cancer stem cells (CSC).

<p><b>A</b>. Flow cytometry analysis. Dissociated low-passage human pancreatic cancer xenograft cells were stained with DAPI and antibodies to H2K, ESA, CD44 and CD24. DAPI positive dead cells and H2K positive mouse cells were both eliminated from the analysis. The CSC population (with surface marker ESA⁺/CD44⁺/CD24⁺) was gated by both P5 and P7, and the non-CSC population from P6. <b>B</b>. Transcript levels of Notch pathway components in CSC compared to in non-CSC obtained by qPCR, normalized to GAPDH. The mean fold change between tumors is represented by a horizontal bar.</p