Ethan Solomon

Characterization of the Notch-1 signaling pathway in pancreatic ductal adenocarcinoma
Abstract Notch-1 is a cell surface protein shown to direct certain cell fate decisions. Consisting of a small intracellular domain, a transmembrane component, and a larger extracellular domain, the Notch protein family acts as a “signal” to alter gene expression. It has been found that activation of the Notch signaling pathway requires cleavage of the extracellular domain by a metalloproteinase (MMP) endopeptidase family. Studies of molecular genetics in mice have shown MMPs influence multiple stages of tumor progression, including tumorigenesis and tumor growth. This experiment characterizes the response of the Notch-1 signaling pathway upon activation by MMP-7 through livecell study of human pancreatic ductal adenocarcinoma (PDA) cells, the most common type of pancreatic tumor and one of the most fatal human cancers. The effects of Notch-1 inhibitors (alone and in conjunction with MMP-7) are also tested in an in vitro context. An understanding of the Notch response to activating and inhibitory pharmacological treatments provides a vital background to developing treatments for a genuinely in vivo situation. Introduction/Background Premalignant lesions known as pancreatic intraepithelial neoplasia (PanIN) are widely held to give rise to pancreatic ductal adenocarcinoma (PDA), one of the most fatal human cancers (1). The metaplastic duct lesion (MDL) is prominently associated with PDA as well as with chronic pancreatitis (CP), a risk factor for PDA (2). MDLs, in turn, arise from acinar-to-ductal metaplasia (ADM), in which acinar cells are progressively

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Ethan Solomon replaced by duct-like epithelia that are hypothesized to possess progenitor cell-like properties (3). The Notch receptor family plays a crucial role in determining cell fates in animals (4). The receptor itself consists of a large extracellular domain, a transmembrane component, and a smaller cytoplasmic domain. Vertebrates express 4 Notch proteins (-1, -2, -3, -4). The extracellular domain of Notch, composed of epidermal growth factorrelated molecules (EGFR), is ligated by a member of the DSL family of ligands, which are also transmembrane proteins containing EGFRs in the extracellular domain (in vertebrates, these ligands are Jagged -1 and -2, and Delta-like-1, -3, -4) (5). Following cleavage of the extracellular domain, the enzyme γ-secretase cleaves the cytoplasmic domain from the transmembrane component, allowing the cytoplasmic domain to translocate to the nucleus where it can regulate gene expression (6) by binding to the DNA binding protein RBP-J and subsequently activating target gene transcription, which in turn represses expression of various downstream genes (5). Recent studies have shown that Notch may play a role in tumor development and progression in the intestine. Disruption of the Notch signaling pathway with βnaphthoflavone and inhibition of γ-secretase activity by dibenzazipene (DBZ) caused proliferative tumor cells in the small intestine to differentiate into goblet cells, a normal but rare intestinal tissue (7). 20% of the adenomas from Apcmin (disposed to develop multiple intestinal neoplasia) mice observed after treatment with DBZ showed up to 50% conversion to goblet cells; less than half showed absolutely no conversion. In each of the 100 untreated adenomas, fewer than 1% goblet cells were observed. This study clearly demonstrated the potential for Notch inhibitors to be used as a pharmacological basis for

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Ethan Solomon cancer treatment. Its constitual activation in PDA suggests it may play a similar role in this cancer. The matrix metalloproteinases constitute a family of zinc-dependent proteinases which are frequently expressed in cancer. The ability of MMPs to collectively degrade all components of the extracellular matrix suggest a role in tumor progression, and mouse genetics studies have shown that MMPs contribute to multiple stages of tumor progression, including tumorigenesis and tumor growth. The MMP family member MMP-7 is expressed by the tumor cells of many adenomas and adenocarcinomas, including in the colon (8), stomach (8), and a majority of PDAs. MMP-7 deficiencies in Apcmin mice have been shown to inhibit intestinal tumor formation, similar to the effects seen by treating these mice with DBZ and interrupting the Notch signaling process. Through a mouse model of chronic pancreatitis, an MMP-7 deficiency was also shown to inhibit all stages of disease progression, including MDL formation (9). Recently, our laboratory has shown that MMP-7 activates Notch. This suggests that the interaction of MMP-7 and Notch plays a crucial role in the growth and development of tumors, possibly including PDA. This study aims to establish a stable system for the testing and analysis of MMP7, various inhibitors, and a combination of both on the Notch signaling pathway in PDA. The study focuses on the treatment of the disease in a pathological context, including late-stage PDA. A working system demonstrating the effectiveness of various pharmacological approaches to treating PDA is an essential step in order to determine the validity and effectiveness of results of future studies in a clinical setting.

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Ethan Solomon Methods MIA PaCa-2 and COS-7 cells MIA PaCa-2 cells were purchased commercially from the American Type Culture Collection. The cell line originated from a Caucasian male 65 years of age with pancreatic cancer. MIA PaCa-2 cells are representative of PDA and are entirely undifferentiated, highly invasive, and characterize the late stages of pancreatic cancer. The cells are morphologically epithelial. COS-7 cells were also purchased from the American Type Culture Collection. COS-7 is an African green monkey kidney fibroblast-like cell line suitable for transfection by vectors requiring expression of SV40 T antigen. All cells were stored, thawed, and cultured according to manufacturer’s instructions. Transfection and Selection for Notch-1 positive cells MIA PaCa-2 cells were cultured in DMEM (Invitrogen) + 10% FBS at 37°C in a humidified 95% air/5% CO2 incubator. Initial transfection with Notch-1 was performed in a six-well tissue culture plate using 3µg of FLN1-GFP, a plasmid vector encoding for green fluorescent protein and Notch-1, and Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Forty-eight hours after transfection, cells positive for Notch1+GFP were selected using 50µg/mL G418 disulfate dissolved in water and added to DMEM+10% FBS cell media. To further increase the yield of GFP+FLN-1 positive cells, approximately 1 cell/300µL cell media dilutions were seeded into three 96-well plates. Forty-eight hours later, wells seeded by individual GFP+Notch-1 positive cells were visually identified by observation of GFP. Wells with 90-100% observable

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Ethan Solomon positivity were re-seeded into 24-well tissue culture plates, and after appropriate levels of cell growth, seeded into individual 10cm tissue culture plates. Western Blots Cells were washed in ice-cold PBS and lysed in ice-cold RIPA buffer (1% NP-40, 1% sodium deoxycholic acid, 0.1% SDS, 150mM NaCl, 1 mM NaHPO4, 0.2 mM EDTA, plus protease inhibitors). Lysates were resolved on a 5% acrylamide gel and transferred to nitrocellulose. Blots were blocked in 5% milk/TBST and probed with 2mg/mL primary GFP monoclonal antibody (Living Colors®) overnight at 4°C, followed by secondary and tertiary antibodies, and visualized via chemiluminescence. Confocal Analysis and Immunohistochemistry GFP tagged full length Notch-1 transfected MIA PaCa-2 and COS-7 cells were cultured in DMEM (Invitrogen) + 10% FBS at 37°C in a humidified 95% air/5% CO2 incubator on an 8-well chamber slide. For treatment, DMEM media was aspirated and cells were washed with Opti-MEM I Reduced Serum Media (Invitrogen). Cells were treated with media alone and media + .18µg/mL MMP-7 or other reagents. At appropriate durations after treatment, cells were washed in PBS and fixed with paraformaldehyde. For immunohistochemistry, cells were washed with PBS and incubated in goat block for 1 hour, and then treated with GFP monoclonal antibody (Living Colors®) in goat block overnight at 4°C. The secondary antibody, biotinylated anti-mouse IgG (H+L) (Vector Laboratories, Inc.), was applied overnight under similar conditions. After washing with PBS, ABC Kit (Vectastain) reagents were applied according to manufacturer’s instructions. Cells were visualized with diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and counter-stained (a purely nuclear stain) with

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Ethan Solomon Mayer’s Hematoxylin Solution for 2 minutes, followed by a wash with 1x tris-buffered saline solution (crystallized NaCl is required for the nuclear stain). Cells were dehydrated with ethanol and a coverslip was mounted with Permount (SP15-100 Toluene Solution UN1294). Stains were analyzed under a microscope.

For confocal analysis, cells were visualized immediately after fixing with paraformaldehyde on a Zeiss LSM-510 Meta confocal microscope. In-vitro Analysis After transfected cells were plated into 30mm tissue culture dishes, media was aspirated and cells were washed with warm Opti-MEM (Invitrogen). MMP-7 was diluted to .18µg/mL in Opti-MEM and added to cells. After 2 hours, cells were observed on a Zeiss LSM-510 Meta confocal microscope. Still images were captured at 1-minute intervals for 3 hours. Images were later compiled into a time-lapse video.

Results MMP-7 Causes Nuclear Translocation of Notch Through immunohistochemistry and in-vitro analysis, MMP-7 appears to induce translocation of Notch to the cell nucleus. To test this, COS-7 cells were transfected with GFP tagged full-length Notch-1. Cells were treated with MMP-7 or left untreated in media, fixed in paraformaldehyde and analyzed under a confocal microscope. After 4 hours of treatment with MMP-7, cells demonstrated an almost complete nuclear translation compared to cells in media alone (Figure 1, B and D). However, translocation was determined to take place between 2 hours and 4 hours after treatment began. At 2

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Ethan Solomon hours, cells treated with MMP-7 showed similar localization to untreated cells (Fig. 1, A and C).
Fig. 1 A – Fixed COS-7 cells expressing GFP tagged fulllength Notch-1 (FLN-1) in Opti-MEM for 2 hours. GFP is localized in the cytoplasm B – Fixed COS-7 cells expressing GFP tagged FLN-1 in Opti-MEM for 4 hours. GFP is localized in the cytoplasm. C – Fixed COS-7 cells expressing GFP tagged FLN-1 in Opti-MEM + MMP-7 for 2 hours. GFP is localized in the cytoplasm. D – Fixed COS-7 cells expressing GFP tagged FLN-1 in Opti-MEM + MMP-7 for 4 hours. GFP has localized to the nucleus.

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Ethan Solomon In vitro observation of cells on a confocal microscope after 4 hours of treatment further indicated that MMP-7 causes nuclear translocation. At 4 hours, untreated COS-7 cells were not processing nuclear GFP (Fig. 2A), but cells treated with MMP-7 clearly demonstrated a nearly complete nuclear translocation of GFP tagged Notch-1 (Fig. 2B). In vitro, nuclear translocation also seems to take place between 2 and 4 hours after treatment.

Fig. 2A Media – after 4 hours of treatment, cells in regular media exhibited no signs of cytoplasmic to nuclear translocation of GFP.

Fig. 2B MMP-7 – after 4 hours of treatment, cells treated with media + MMP-7 (.18µg/mL) showed evidence of nuclear translocation of Notch-1 + GFP.

Photographs courtesy of Eric Sawey, Dept. of Pharmacology, Stony Brook University

Establishment of Viable System for In-vivo Testing A stable line of MIA PaCa-2 cells expressing GFP+Notch-1 was established by transfection and demonstrated validity as a viable system for testing MMP-7/Notch inhibitors in a pharmacological context. Western blotting clearly showed sufficient GFP

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Ethan Solomon tagged Notch-1 present in the MIA PaCa-2 cell line after transfection to confirm the protein was being processed effectively by the cells. GFP processing was present in cells treated with MMP-7 for 4 hours as well as in untreated cells.

Immunohistochemistry further supports that the transfected MIA PaCa-2 cell line is useful for testing MMP-7 and Notch inhibitors on the Notch signaling pathway in a pharmacological context. Cell staining revealed the presence of purely nuclear and purely non-nuclear localization of Notch in untreated cells. Cells treated with MMP-7 alone for 4 hours tended to express greater nuclear localization, while cells treated with Notch pathway inhibitors and MMP-7, γ-secretase inhibitor and GM6001 (an MMP inhibitor), tended to express less definite nuclear translocation. The presence of entirely nuclear and entirely non-nuclear Notch-1 representative cells in the untreated group makes this particular model useful for inhibitor studies (Fig. 3)

Nuclear

Non-nuclear Fig. 3 – MIA PaCa-2 GFP+FLN-1 Immunohistochemistry

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Ethan Solomon To test the validity of the MIA PaCa-2 model as a means to test pharmacological inhibitors in an in vitro context, a time-lapsed video of MIA PaCa-2 cells was created after treatment with MMP-7. Using the video, it was possible to see the nuclear translocation of Notch in one cell in real time. The visible overlay clarifies that the localization of GFP in the cell shifted solely to the nuclear region after 168 minutes of treatment (Fig. 4D). This same model could be used to test Notch inhibitors by demonstrating movement of Notch out of the nuclear region. 1 minute 168 minutes

A

B

GFP

C

D

GFP + overlay

Fig. 4 – in vitro analysis of Notch localization in MIA PaCa-2. Arrows indicate cell with observable translocation. Note in 4D the dark visible outline of the cell body and the comparatively smaller nuclear region.

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Ethan Solomon Discussion MMP-7 has been shown to effectively induce nuclear translocation of Notch in the COS-7 cell line and the MIA PaCa-2 cell line has proven to be viable as a system to test the effects of MMP-7 and Notch inhibitors on the Notch signaling pathway in an in vitro context using cells representative of pancreatic ductal adenocarcinoma. As Notch has been shown to cause cellular dedifferentiation and result in MDLs, closely associated with PDA (10), it is crucial to develop a stable system with which to test pharmacological influences on Notch signaling and processing. Recent studies also have shown Notch can regulate gene expression and may play a role in intestinal tumor development (5, 6). Our lab has recently shown that MMP-7, found frequently in PDA, cleaves the extracellular domain of Notch, precipitating the Notch signaling action. Thus, MMP-7 and its inhibitors, and Notch pathway inhibitors form a promising basis for pharmacological treatment of PDA in a truly in vivo context. MIA PaCa-2 highly undifferentiated cells were effectively transfected with fulllength Notch-1 and were shown to process the protein in the cytoplasm and nucleus and were also shown to be viable for in vitro analysis of the Notch pathway. Since cells developed which expressed nuclear and non-nuclear Notch before treatment, this particular model would be useful to test Notch and MMP inhibitors, such as γ-secretase inhibitors and GM6001, an MMP inhibitor. γ-secretase has been shown to be essential for the intracellular cleavage of the Notch protein (6). Pancreatic ductal adenocarcinoma is one of the most fatal human cancers (1). Crucial to the development of viable pharmacological treatments for PDA is an in vitro model of cells representative of this cancer. The MIA PaCa-2 cell line, derived from a

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Ethan Solomon late-stage pancreatic tumor, can effectively constitute such a model. Future studies examining in vitro or in vivo responses of highly undifferentiated cells to treatment by MMPs or Notch/MMP inhibitors would be valuable in determining methods of treatment in a clinical setting. In vitro studies such as this provide a setting to observe the response of the Notch signaling pathway in real time, highly relevant and essential to assess the viability of pre-clinical pharmacological trials.

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Ethan Solomon References

1. Klein WM, Hruban RH, Klein-Szanto AJ, Wilentz, R (2002) Direct correlation between proliferative activity and dysplasia in pancreatic intraepithelial neoplasia (PanIN): additional evidence for a recently proposed model of progression. Mod Pathol 15:441-447. 2. Lowenfels AB, Maisonneuve P (2006) Epidemiology and risk factors for pancreatic cancer. Best Pract Res Clin Gastroenterol 20:197-209. 3. Song SY, Gannon M, Washington MK, Scoggins CR, Meszoely IM, Goldenring JR, Marino CR, Sandgren EP, Coffey RJ, Jr, Wright CV, et al. (1999) Expansion of Pdx1expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor alpha. Gastroenterology 117:1416-1426. 4. Artavanis-Tsakonas, S. et al. (1999) Notch signaling: cell fate control and signal integration in development. Science 284:770-776. 5. Johan H. van Es and Hans Clevers (2005) Notch and Wnt inhibitors as potential new drugs for intestinal neoplastic disease, Trends in Molecular Medicine 11:496-502. 6. Weinmaster, G. (1997) The Ins and Outs of Notch Signaling. Molecular and Cellular Neuroscience 9:91-102. 7. Johan H. can Es, Marielle E van Gijn, Orbicia Riccio, Maaike van den Born, Marc Vooijs, Harry Begthel, Miranda Cozijnsen, Sylvia Robine, Doug J. Winton, Freddy Radtke and Hans Clevers (2005) Notch/γ-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435:959-963. 8. McDonnell S, Navre M, Coffey RJ, Jr, Matrisian LM (1991) Expression and localization of the matrix metalloproteinase Pump-1 (MMP-7) in human gastric and colon carcinomas. Mol Carcinog 4:527-533. 9. Crawford HC, Scoggins CR, Washington MK, Matrisian LM, Leach SD (2002) Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates acinar-toductal metaplasia in exocrine pancreas. J Clin Invest 109:1437-1444. 10. Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, Iacobuzio-Donahue CA, Sriuranpong V, Iso T, Meszoely IM, Wolfe MS, et al. (2003) Notch mediates TGFαinduced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 3:565-576.

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