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MUC1 as a biomarker for TGF- β1 inhibition: Investigating the role of MUC1 in the switch of TGF-β1 function from a tumor suppressor to a tumor promoter in pancreatic ductal adenocarcinoma.

Emily Ashkin

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Pancreatic cancer is the most lethal of all cancers.

•6% five-year-survival rate.•Median survival of 6 months.•4th leading cause of cancer related deaths

in the United States.

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Background: Pancreatic Ductal Adenocarcinoma

• Accounts for more than 95% of pancreatic cancer cases.

• Therapeutic interventions include surgical resection, radiation therapy, chemotherapy and immunotherapy

• Lack of specific symptoms and limitations in diagnostic methods enable cancer progression.

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Background: TGF-β1• Original member of the Transforming Growth

Factor Beta super family, consisting of three isoforms.

• Type of cytokine and secreted protein.

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Background: Dual Function of TGF-β1

• Tumor suppressor TGF-β1 induces apoptosis, or cell suicide, by signaling the SMAD pathway

• As the tumor progresses, genetic and/or biochemical changes allow TGF-β1 to stimulate tumor progression by its pleiotropic activities on the cancer cells.

• TGF-β1, binding to receptor II, induces epithelial-to-mesenchymal transition (EMT). TGF-β1-induced EMT leads to migration

and invasion of local epithelial cells

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Hypothesis Signaling through MUC1 inhibits TGF-β1-induced-apoptosis and supports TGF-β1-induced EMT and invasion.

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MUC1•A transmembrane mucin glycoprotein•Over-expressed in more than 90% of

pancreatic cancer•Plays a major role in EMT as well as drug

resistance, invasion, and metastasis in pancreatic cancer

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Methods and MaterialsExperiments Descriptions

Cell Culture All cell lines were previously established cell lines ordered from the American Type Culture Collection (ATCC).

Migration “Scratch” Assay The scratch assay is a simple, reproducible assay commonly used to measure basic cell migration parameters. This assay was performed in order to visibly see each cell's migratory capabilities and draw conclusions on metastasis.

BCA Assay Biochemical assay for determining the amount of protein present in a solution.

Western Blot Molecular weights: MUC1 (25 kDa), TGF-βR1 (56 kDa), TGF-βR2 (64 kDA), and β-actin (42 kDa)

TGF-β1 Binding MUC1+ and MUC1- cells were treated with pure recombinant human TGF-β1.

Flow Cytometry Cell cycle analysis was performed using Propidium Iodide DNA staining.

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Images

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Pancreatic Cancer Human and Mouse Cell Lines

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Results: Migration “Scratch” Assay

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Results: Migration “Scratch” Assay

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Results: Migration “Scratch” Assay•The most significant change in wound size

is between 0 and 14 hours•For both mouse and human cell lines, the

MUC1+ cells were more migratory than the MUC1- cell lines

•Using Image J programming, I was able to measure the width of the wound and analyze the decrease in size or lack thereof.

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Results: Western Blot

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Results: Western Blot

p<0.02

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Results: Western BlotAfter creating the loading dilutions based on the BCA Assay results,

the western blotting was performed to look for TGF-β RI, TGF-β RII, MUC1, and β-actin levels for the panel of human and mouse cell lines. The negative correlation between MUC1 and TGF-β1 RI levels from the Western blot results suggest that MUC1 could play a role in blocking TGF-β1-induced apoptosis. Based on these results, if MUC1 is up regulated, then TGF-β1 RI is down regulated, thus the apoptotic signaling pathway is not initiated. Also, the positive correlation between MUC1 and TGF-β1 RII with CFPAC, HPAC, and HPAF II is significant because the up regulation of TGF-β1 RII and down regulation of TGF-β1 RI indicates that TGF-β1-induced EMT is occurring. β-actin levels confirmed that the loading of all cell lines was equal.

I performed a densitometric analysis using Image J programming to quantify the densities of each Western Blot. Based on statistical analysis, using a hypothesis test, I was able to analyze the efficacy of MUC1 levels as a screening tool. In relationship to MUC1 levels, TGF-β1 R1 levels varied inversely with MUC1 levels. The higher the MUC1 levels correspond to lower TGF-β1 R1 levels (indicated by box). However, for low MUC1, TGF-β1 R1 levels varied widely (indicated by circle). This data has a significance of a p-value less than .02.

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Results: Flow Cytometry Cell Cycle Analysis using Propodium Iodide Staining

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Results: Flow Cytometry Cell Cycle Analysis using Propodium Iodide Staining

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Results: Flow Cytometry Cell Cycle Analysis using Propodium Iodide StainingLastly, I performed a Flow Cytometry Cell Cycle Analysis using Propodium Iodide Staining on three cell lines: KCM (MUC1+), KCKO (MUC1-), and KC (MUC1 spontaneous). I analyzed them both treated and untreated with pure recombinant human TGF-b1. Based on the apoptotic data, KCKO (MUC1-) had higher apoptotic levels after TGF-β1 treatment than before treatment. KCM (MUC1+) had lower apoptotic levels after TGF-β1 treatment. The Post-Mitosis analysis showed that KCM had much higher post-mitotic levels than KCKO and KC, meaning that more KCM cells proliferated after treatment.

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Conclusions•So far, data indicates that MUC1 does

play a role in the switch of TGF-β1 function from a tumor suppressor to a tumor promoter.

Negative correlation between MUC1 and TGF-β1 RI levels. Positive correlation between MUC1 and TGF-β1 RII levels. Scratch assay results show that there is a relationship

between MUC1 levels and the metastasis of the cells. KCKO has higher apoptotic levels than KCM after TGF-β1

treatment.

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Future Experimentation

•Analysis of other types of cancer i.e. breast, lung, lymphoma, etc.

•Development of a way to inhibit MUC1 levels

•Development and implementation of a detector that ascertains TGF- β1 function

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Real World Application and Impact• How and why TGF-β1 function switches from a tumor suppressor to

a tumor promoter has been researched extensively with little success• First study to find a potential player in this functional transition • Indentifies a screening tool to predict whether inhibition would be

an effective treatment for a particular patient• Not only does this study open up a whole new field of study using

TGF-β1 inhibition as an effective treatment, but this study can also take us one step closer to understanding pancreatic cancer and cancer in general.

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Acknowledgements and Dedications

•I want to thank Dr. Pinku Mukherjee, Dr. Sritama Nath, and Mohammad Ahmad at the University of North Carolina at Charlotte Biology Department for allowing me to use their facilities to conduct my research.

•My research project is dedicated to my family and friends who have battled cancer and inspired me to make a difference.

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Primary References:• Besmer, DM, JM Curry, LD Roy, TL Tinder, M. Sahraei, J. Schettini, SI Hwang,

YY Lee, SJ Gendler, and P. Mukherjee. "Pancreatic Ductal Adenocarcinoma Mice Lacking Mucin 1 Have a Profound Defect in Tumor Growth and Metastasis." PubMed- US National Library of Medicine. National Institute of Health, 10 May 2011. Web. 9 July 2013.

• Deer, Emily L., Jessica Gonzalez-Hernandez, Jill D. Coursen, Jill E. Shea, Josephat Ngatia, Courtney L. Scaife, Matthew A. Firpo, and Sean J. Mulvihill. "Phenotype and Genotype of Pancreatic Cancer Cell Lines." NIH Public Access. National Institute of Health, 1 May 2011. Web. 29 June 2013.

• Gendler, Sandra J. "MUC1, the Renaissance Molecule." Journal of Mammary Gland Biology and Neoplasia. Plenum Publishing Corporation, 2001. Web. 18 July 2013.

• All other references can be found in my research paper*

• All images were either produced by me or adapted from the Nature Reviews: Cancer*