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 live-

cell 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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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 factor-

related 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 β-

naphthoflavone 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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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 MMP-

7, 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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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 Notch-

1+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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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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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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hours, cells treated with MMP-7 showed similar localization to untreated cells (Fig. 1, A

and C).

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Fig. 1A – Fixed COS-7 cells expressing GFP tagged full-length Notch-1 (FLN-1) in Opti-MEM for 2 hours. GFP is localized in the cytoplasmB – 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.

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.

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

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

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

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)

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Non-nuclear

Nuclear

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

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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.

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1 minute 168 minutes

GFP

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.

A B

C D

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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 full-

length 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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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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References

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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, GoldenringJR, Marino CR, Sandgren EP, Coffey RJ, Jr, Wright CV, et al. (1999) Expansion of Pdx1-expressing 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.

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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-to-ductal 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 TGFα-induced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 3:565-576.

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