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Transcript of Ligon Lab Presentation New
Expression of -Tubulin Post-Translational Modifications in
Breast Cancer Cell Lines
By: David VanHoute
Dr. Lee Ligon Laboratory
Department of Biology
Rensselaer Polytechnic Institute
2.14.2011
What is the Epithelial-Mesenchymal Transition (EMT) Pathway?
• An event first described during embryonic development• Transition by which epithelial cells differentiate into
mesenchymal cells• Is not a single event/process, rather varying degrees of
modification. • Recently observed in cancer pathology and tumor
phenotypes
*Raghu Kalluri, Robert A. Weinberg. The basics of epithelial-mesenchymal transition. Published in Volume 119, Issue 6J Clin Invest. 2009;119(6):1420–1428 doi:10.1172/JCI39104
Structural Changes During an EMT-like Event
Epithelial• Polarity• Adhesion to Basal membrane• Characteristic epithelial
markers (E-cadherins, Integrins, etc.)
Mesenchymal• Loss of polarity• Fibroblast shape Motility epithelial markers N-Cadherin, Vimentin,
Fibronectin mesenchymal markers
• Increase invasiveness• Cytoskeleton
rearrangement
*Cell Research (2009) 19:156–172. doi: 10.1038/cr.2009.5; published online 20 January 2009
Why Study EMT-like Events in our Lab?
• Recently, EMT-like transitions have been implicated during tumor progression (PubMed search yielded 3194 search results)
• Are epithelial cancer cells exploiting an EMT pathway?
• Do cancer cell lines exhibit EMT-like characteristics, specific to Microtubule organization that give insight into metastatic capability?
• Which characteristics?
*J Clin Invest. 2009;119(6):1438–1449 doi:10.1172/JCI38019
Post-Translational Modifications (PTMs) to -Tubulin
• Detyrosinated -tubulin– Typically tyrosinated by Tubulin tyrosine ligase (down-
regulated in cancerous cells; accumulation of detyrosinated -tubulin).
– Recruits Kinesins-1 to preferentially transport Vimentin and Transferrin (mesenchymal markers).
• Acetylated -tubulin– Regulator of cell motility, associated with stable MTs.– Acetylation enzyme still unknown but occurs on K40– Access site is on interior of MT.
• Poly-glu tubulin– Prevalent in neurons, motility, mitotic spindle– Addition of glutamate to C-terminal end of both and
tubulin Glutamates– Up to 20 residues– Increased sensitivity of Kinesins on both and Tubulin
*Curr Opin Cell Biol. 2008 Feb;20(1):71-6. Epub 2008 Jan 15.
Most PTMs occur on microtubules and not on unpolymerized tubulin
Problem: Are there significant differences in PTMs at various stages of cancer progression?
Rotation Hypothesis: Do phenotypic differences in 3 breast cancer cell lines express significant amounts of post-translational modified microtubules and therefore exhibit tumor progression through an EMT-like event?
• Examining 3 PTMs of alpha Tubulin using immunocytochemistry and Western Blot analysis:
• Detyrosination• Acetylation• Poly-glutamylation
3 Cell lines: MCF7, MCF10a, and MDA-MB-231. A “Snapshot” of Metastatic Capacity
Cell Line
Phenotype Motility Adhesiveness Other Characteristics
Representative stage during
EMT
MCF10a Phenotypically normal Little motility
Highly adhesive Non-tumorgenic, fast growing
epithelial cancer cell line
Characteristic luminal ductal
cells
Epithelial
MCF7 Moderately normal epithelial Moderate Moderate Mildly invasive
Slow Growing
Grows in mono-layers
Epithelial-like
MDA-MB-231
Phenotypically non-epithelial
Fibroblast phenotypeHighly motile
Less adhesive Fast-growing
Highly invasive
Highly aggressive
Mesenchymal
-Tubulin55 kDa
GAPDH37kDa
MDCK MCF7 MCF10a MDA-MB-231 MDCK MCF7 MCF10a MDA-MB-231
MDCK MCF7 MCF10a MDA-MB-231
-Tubulin55 kDa
GAPDH37kDa
-tubulin
Poly-glu -tubulin
Western Blot Results:
-Tubulin Acetylated -Tubulin
Poly-glu -Tubulin
Microscopy: Acetylated -
Tubulin
MCF10a
MCF7
Acetylated-tubulinDAPI
MDA-MB-231
MCF10a MCF7 MDA-MB-231Detyrosinated-tubulinDAPI
Detyrosinated -Tubulin
Poly-Glutamylation Poly-glu-tubulinDAPI
MCF10a MCF7 MDA-MB-231
Conclusions• Microscopy shows
increased acetylated and detyrosinated PTM in MCF7 and MDA-MB-231
• Western Blot and Microscopy exhibit significant acetylation in MCF7s and 231.
• Less in MCF10a• Little to no organization
• PTMs are associated, not regulatory, with cancer progression
MDA-MB-231 MCF7
MDCK MCF7 MCF10a MDA-MB-231
-Tubulin
GAPDH
55 kDa
37kDa
Detyrosination
Further Studies
• Western Blot for detyrosination
• Develop ways in which PTM may be used as diagnostic tools for different stages of cancer metastatic capacity.
• Look into the reverse of EMT, as metastatic cells inhabit new environments and become sessile.
Acknowledgements
• Special Thanks to:
• Dr. Lee Ligon
• Gerri Quinones
• Maria Apostolopoulou
• Vimla Singh
• Josh McLane
References• Nature Reviews Molecular Cell Biology 4, 938-948 (December 2003) |
doi:10.1038/nrm1260• Cancer Research, Epithelial-to-Mesenchymal Transition Promotes
Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement. Doi 10.1158/0008-5472.Can-09-4613.
• American Journal of Pathology, Epithelial-Mesenchymal Transition A Cancer Researcher's Conceptual Friend and Foe. Doi 10.2353/Ajpath.2009.080545
• Raghu Kalluri, Robert A. Weinberg. The basics of epithelial-mesenchymal transition. Published in Volume 119, Issue 6J Clin Invest. 2009;119(6):1420–1428 doi:10.1172/JCI39104
• Hammond, Jennetta. Curr Opin Cell Biol. 2008 Feb;20(1):71-6. Epub 2008 Jan 15
• Whipple R. A., Matrone M. A., Cho E. H., Balzer E. M., Vitolo M. I., Yoon J. R., Ioffe O. B., Tuttle K. C., Yang J. and Martin S. S., "Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement," Cancer Res 70(20):8127-37 (2010)
• J Clin Invest. 2009;119(6):1438–1449 doi:10.1172/JCI38019