Identifying a Novel Diagnostic and Therapeutic Target for Metastatic Breast Cancer. Michele Vitolo, Ph.D.

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1 Identifying a Novel Diagnostic and Therapeutic Target for Metastatic Breast Cancer Michele Vitolo, Ph.D.

2 Clinical Problem: Breast Cancer #1 cancer (US women) Metastatic disease: 5-year survival 25% #2 cause of cancer-related death

3 Challenges of Metastasis Diagnostic Limited radiologic detection of early spreading Therapeutic Systemic agents have limited long-term success CTCs Key: Circulating Tumor Cells (CTCs)

4 Overview Tubulin-based membrane protrusions (microtentacles) observed in detached cells Role for microtentacles in cancer progression and metastasis α-tubulin acetylation, microtentacle formation, adhesion, and invasive migration

microtentacles in cancer progression and metastasis

5 Response of epithelial cells to detachment Membrane protrusions Frame / 1 sec.

6 Cytoskeletal support of membrane protrusions - Microtentacles Rebecca Whipple et al., Exp. Cell Res. 313: (2007). Control LA Inhibit actin polymerization Destroys Filopodia and invadopodia Phase Contrast -tubulin Actin

7 Microtentacles increase in invasive/metastatic breast tumor cell lines Rebecca Whipple et al., Cancer Research 68: (2008).

8 Example of a Patients CTC

9 Microtentacles promote binding to adjacent cells

10 Confocal imaging of live tumor cell attachment to endothelial cells

11 Confocal imaging of CTCs isolated from human breast cancer patients

12 Primary tumor Even small tumors shed millions of cells into bloodstream. Death from fragmentation Target microtentacles Fates of circulating cells? Death from apoptosis [1] ADHESION Microtentacles [2] EXTRAVASATION Actin-dependent Metastatic tumor

13 Microtubule stabilization and metastasis Matrone et al., Cancer Research 70(20): 7737:7741 (2010).

14 αtubulin Acetylation Acetylase ( TAT1) Deacetylases (HDAC6, SIRT2)

15 Metastatic breast tumor cell lines have high acetylation of α-tubulin Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

16 Metastatic breast tumor cell lines have high acetylation of α-tubulin that is enriched in more numerous McTNs. Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

17 Reduction of αtubulin Acetylation K40R acetylation resistant: ( Acetyl-Tub)

18 K40R α-tubulin mutant expression 2015 by American Association for Cancer Research Amanda E. Boggs et al. Cancer Res 2015;75:

19 K40R α-tubulin mutant decreases acetylation and McTN frequency Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

20 Increase in αtubulin Acetylation Acetylase ( TAT1) αtat overexpression: ( Acetyl-Tub)

21 Overexpression of αtat1 increases tubulin acetylation Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

22 αtat1 significantly increases α-tubulin acetylation and McTNs Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

23 α-tubulin acetylation significantly affects reattachment rates of suspended breast tumor cells 2015 by American Association for Cancer Research Amanda E. Boggs et al. Cancer Res 2015;75:

24 Chemotactic movement of breast tumor cells is affected by reducing acetylated α-tubulin Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

25 Chemotactic movement of breast tumor cells is affected by reducing acetylated α-tubulin Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

26 Acetylated α-tubulin is detected in patient primary and matched metastatic tumors 2015 by American Association for Cancer Research Amanda E. Boggs et al. Cancer Res 2015;75:

27 High acetylation of α-tubulin in patient primary tumors is linked to the basallike subtype and an increased risk of disease progression and death Amanda E. Boggs et al. Cancer Res 2015;75: by American Association for Cancer Research

28 Conclusions 1. Significant association between breast cancer cell lines and high acetylation of - tubulin that extends along the length of McTNs. 2. Mutation of the specific lysine 40 acetylation site on -tubulin as well as enzymatic modulation of this PTM has a significant impact on McTNs frequency and cancer cell attachment. -tubulin is necessary for migration. 4. Match primary and metastatic tissue arrays show tubulin acetylation is maintained or increased in nodal metastases. 5. Large proteomics study (390 patients) link high tubulin acetylation to the aggressive basal-like subype. 6. A trend of increased risk of disease progression and death when -tubulin is high in a patient s primary tumor.

29 Department of Physiology Stuart Martin Rebecca Whipple-Bettes Agnes Cheung Eric Balzer Jennifer Yoon Ed Cho Mike Matrone Keyata Thompson Monica Charpentier Amanda Boggs Lek Bhandary Kristi Chakrabarti Lindsay Hessler Olga Ioffe Kim Tuttle Toshi Yoneda (MD Anderson) Gordon Mills (MD Anderson) Jing Yang (UCSD) Wolfgang Losert (UM-College Park) Carol Otey (UNC-Chapel Hill) State of Maryland Cigarette Restitution Fund K01-CA Howard Temin Career Award (NCI) UMB Independent New Investigator Award (Dean s Office) Department of Defense Breast Cancer Concept Award S10-RR (NCRR, Xenogen) R01-CA (NCI) Department of Defense Breast Cancer Idea Award FAMRI Clinical Innovator Award DOD Breast Cancer Predoctoral (Balzer) DOD Breast Cancer Predoctoral (Cho) Maryland Stem Cell Research Foundation Susan G. Komen for the Cure Department of Defense Breast Cancer Era of Hope Scholar Award R01-CA (NCI) K01-CA (NCI)

30 Publications including data from the Xcelligence RTCA DP 1. Matrone, M.A., Whipple, R.A., Thompson, K., Cho, E.H., Vitolo, M.I., Balzer, E.M., Yoon, J.R., Ioffe, O.B., Tuttle, K.C., Tan, M. and Martin, S.S. (2010) Metastatic breast tumors express increased tau, which promotes microtentacle formation and the reattachment of detached breast tumor cells. Oncogene. Jun 3;29(22): Whipple, R.A., Cho, E.H., Balzer, E.M., Matrone, M.A., Vitolo, M.I., Yoon, J.R., Ioffe, O.B., Tuttle, K.C., Yang, J. and Martin, S.S. (2010) Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement. Cancer Res. Oct 15;70(20): Yoon, J.R., Whipple, R.A., Balzer, E.M., Cho, E.H., Matrone, M.A. and Martin, S.S. (2011). Local anesthetics inhibit kinesin motility and microtentacle protrusions of human epithelial and breast tumor cells. Breast Cancer Res.Treat. Oct;129(3): Vitolo MI, Boggs AE, Whipple RA, Yoon JR, Thompson K, Matrone MA, Cho EH, Balzer EM, Martin SS. (2013) Loss of PTEN induces microtentacles through PI3K-independent activation of cofilin. Oncogene. Apr 25;32(17): Whipple RA, Vitolo MI, Boggs AE, Charpentier MS, Thompson K, Martin SS. (2013) Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition. Breast Cancer Res.15(5):R Charpentier MS, Whipple RA, Vitolo MI, Boggs AE, Slovic J, Thompson KN, Bhandary L, Martin SS. (2014) Curcumin targets breast cancer stem-like cells with microtentacles that persist in mammospheres and promote reattachment. Cancer Res. Feb 15;74(4): Perry NA, Vitolo MI, Martin SS, Kontrogianni-Konstantopoulos A. (2014) Loss of the obscurin-rhogef downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells. Oncotarget. Sep 30;5(18): Boggs AE, Vitolo MI, Whipple RA, Charpentier MS, Goloubeva OG, Ioffe OB, Tuttle KC, Slovic J, Lu Y, Mills GB, Martin SS. (2015) α-tubulin acetylation elevated in metastatic and basal-like breast cancer cells promotes microtentacle formation, adhesion, and invasive migration. Cancer Res Jan 1;75(1): Bhandary L, Whipple RA, Vitolo MI, Charpentier MS, Boggs AE, Chakrabarti KR, Thompson KN, Martin SS. (2015) ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells. Oncotarget. Mar 20;6(8):

Department of BioScience Technology Chung Yuan Christian University 2015/08/13

Department of BioScience Technology Chung Yuan Christian University 2015/08/13 Cancer Cells Cancer, the 1st leading cause of death, is an example of a disease that arises from abnormalities in cell function