Exploring a Link Between Spy1 and Hepatocellular Carcinoma Progression
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1 University of Windsor Scholarship at UWindsor UWill Discover Undergraduate Conference UWill Discover 2016 Mar 29th, 4:00 PM - 5:00 PM Exploring a Link Between Spy1 and Hepatocellular Carcinoma Progression Carlee Stoyanovich University of Windsor, stoyanoc@uwindsor.ca Bre-Anne Fifield University of Windsor, fifield@uwindsor.ca Lisa Porter University of Windsor, lporter@uwindsor.ca Follow this and additional works at: This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License Stoyanovich, Carlee; Fifield, Bre-Anne; and Porter, Lisa, "Exploring a Link Between Spy1 and Hepatocellular Carcinoma Progression" (2016). UWill Discover Undergraduate Conference This Event is brought to you for free and open access by the UWill Discover! at Scholarship at UWindsor. It has been accepted for inclusion in UWill Discover Undergraduate Conference by an authorized administrator of Scholarship at UWindsor. For more information, please contact scholarship@uwindsor.ca.
2 Spy1 and Hepatocellular Carcinoma Progression: Exploring a Link in a Murine Model Presented by : Carlee Stoyanovich Honors Thesis Project 2016
3 Hepatocellular Carcinoma (HCC) The most aggressive and prevalent form of primary liver cancer In men, it is the 5 th most common cancer and in women it is the 7 th worldwide Current treatments are invasive and include: transplantation, resection, ablation and chemotherapy The 5-year survival rate is 20%
4 The Progression Diet/Lifes tyle Factors Hepatitis B/C Fatty Liver Disease Steatohepatitis Fibrosis Cirrhosis? HCC Alcoholism + Fibrosis F
5 A Protective Mechanism G2 M G1 S p53 p53 Tumor suppressor Halts the cell cycle during unfavorable conditions Regulates cell death (apoptosis) Aids in DNA repair In HCC normally inactivated or mutated Spy1 Speed up cell division Override cell cycle barriers Enhance stemness in cell populations Known role in breast and brain cancers
6 MMTV-Spy1 Mouse B6CBAF1/J genetic background Designed to study breast cancer in mice models Constitutively overexpress Spy1 in the mammary gland
7 Percentage of Mice With Liver Tumours HCC in the Spy1 Mouse Model MMTV-Spy1 male mice with high levels of Spy1 have significantly more HCC than their male littermate controls. 45 * Cntl MMTV-Spy1 *p= MMTV-Spy1 n=23 B6CBAF1/J n=23 Fifield (2014)
8 A Potential Mechanism? M G1 p53 Fatty Liver Spy1 p53 G2 S Spy1 overrides p53 HCC Does an increase in Spy1 levels predispose the liver to HCC development?
9 Objectives Further characterize the MMTV-Spy1 liver phenotype. Develop a model to look at HCC progression in wildtype mice. Quantify Spy1 protein levels in the wild-type damaged mice livers. Monitor fat accumulation as well as p53 and TNFalpha levels in the mice livers.
10 Effects of Spy1 on Liver Morphology Normal hepatocytes Large vacuoles Disordered cell structure Healthy liver +1yr control mouse Fat accumulation +1yr MMTV-Spy1 mouse HCC +1yr MMTV-Spy1 mouse
11 Fat Accumulation in MMTV-Spy1 Mice 10 month control mouse 10 month MMTV-Spy1 mouse
12 Spy1 Increases Indices of Cell Division The percentage of bi-nucleated cells are significantly higher in control mice. +1 yr control mouse
13 The Progression in MMTV-Spy1 Mice + Fibrosis Increased fat accumulation? Increased HCC Decreased bi-nucleated cells
14 The Progression in MMTV-Spy1 Mice + Fibrosis Trichrome stain of +1yr MMTV-Spy1 mouse Collagen
15 The Methionine Choline Deficient (MCD) Diet Produces the most severe NASH phenotype in the shortest timeframe Causes increased fat accumulation in the hepatocytes Induces: Inflammation Apoptosis Oxidative damage Fibrosis Increased serum alanine aminotransferase levels
16 MCD Progression Oil Red O Staining TNF-alpha Levels Inflammatory Phase Fat Accumulation Trichrome Stain Fibrotic Phase Day 0 Day 2 1 week 6 weeks Stress Response When do endogenous Spy1 levels peak?
17 The MCD Diet Experiment Healthy Mouse Fat Accumulation Fibrosis Day 0- Mice are placed on diets Day 2 1 week 6 weeks Collect Liver Tissue MCD Diet Amino Acid Control Diet Same genetic background as MMTV-Spy1 mice Male mice between 8-12 weeks of age
18 Tissue Collection and Analysis Formalin Flash Frozen in 4% Liquid Nitrogen paraformaldehyde H+E Staining Immunohistochemistry Analyze gene expression Quantify protein levels Monitor fat accumulation
19 Area Stained Fat Accumulation in MCD Mice * Day 2 control Day 2 MCD * * week control 1 week MCD 0 Control Day 2 1 week 6 week MCD 6 week control 6 week MCD
20 Fibrosis in MCD Mice MCD mice had clear collagen deposition as compared to the controls. 6 week control 6 week MCD
21 p53 Protein (densitometry corrected for actin) Spy1 Protein (densitometry corrected for actin) Spy 1 and p53 Levels 3.5 MCD Diet (NASH) Cntl Diet MCD Diet Day 2 1 Week Day 2 1 Week Spy1 2 Activate p53 Actin Control Day 2 1 week 6 week MCD Halt the cell cycle Apoptosis Control Day 2 1 week 6 week MCD
22 TNF-alpha vs GAPDH TNF-alpha Gene Expression MCD Diet (NASH) Inflammation TNF-alpha activation Relative Quantification log Control Day 2 1 week 6 week MCD * -2.5
23 MCD Mice Progression Inflammatory Phase Fibrotic Phase Fat Accumulation Healthy Mouse Day 0 Day 2 1 week 6 weeks Stress Response Fat Accumulation Spy1 Protein p53 Protein TNF-alpha Gene Expression Collagen Deposition
24 Revised Timeline of Progression Spy1 as a prognostic indicator Healthy Liver NAFLD NASH Proliferation p53 Spy1 Fibrosis Cirrhosis HCC
25 Balance in the Face of Damage Proliferation/ Regeneration Fibrosis Stressed Liver Restores damaged hepatocytes Compensatory hyperplasia Allows for regeneration Maintains overall integrity of the organ Inflammation Formation of scar tissue Deposit collagen and fibrin
26 Does Proliferation Favor HCC Over Fibrosis? In response to an increase in fat accumulation and damage to hepatocytes, Spy1 will be up-regulated to increase regeneration and proliferative ability and decrease fibrosis. Spy1 Stressed Liver
27 Future Steps Developing a Spy1 driven mouse and follow it s progression on the MCD diet Spy1 Stressed Liver
28 Acknowledgements Thank you to my wonderful supervisor, Dr. Lisa Porter and to my mentor Dr. Bre-Anne Fifield! Thank you to the entire Porter Lab who have given me so much support and guidance! Thank you to our funding agencies:
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