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