Background: Chemoprevention of breast cancer, which continues to be a leading cause of cancer-related mortality among women globally, is feasible as exemplified by clinical success of selective estrogen receptor (ER) modulators (SERM) and aromatase inhibitors. However, the SERMs are ineffective against ER- negative breast cancers and have serious side effects. The aromatase inhibitors appear promising for prevention of breast cancer in high-risk postmenopausal women. Even though the safety of aromatase inhibitors in a preventative setting is still under study, their use raises concerns for increased risk of cardiovascular events and bone fracture. Therefore, a non-toxic preventive intervention effective against both ER-positive and ER-negative breast cancers is highly desirable. In the previous funding period, we demonstrated that a small-molecule (withaferin A; hereafter abbreviated as WA) derived from an Ayurvedic medicine plant (Withania somnifera) not only prevents ER-negative mammary cancer development in a clinically-relevant transgenic mouse model (MMTV-neu) without any toxicity, but also inhibits ER- ? expression and activity at pharmacologic doses in cultured human breast cancer cells. At the molecular level, we have identified novel mechanistic targets of WA, including tubulin (downregulation of ?- and ?- tubulin as well as covalent modification of Cys-303 of ? -tubulin in MCF-7 cells) and complex III of the mitochondrial electron transport chain (activity inhibition), potentially contributing to its chemopreventive activity by eliciting mitotic arrest and reactive oxygen species (ROS)-dependent apoptotic cell death. These effects of WA are observed in mammary cancer cells representing major subtypes, including ER-positive, ER-negative, and triple-negative breast cancer. More importantly, normal mammary epithelial cells are significantly more resistant to tubulin and complex III targeting by WA compared with breast cancer cells. Despite these exciting mechanistic findings, the functional significance of down-regulation or Cys-303 modification of tubulin in growth arrest by WA is still unclear. Likewise, the molecular basis for WA-mediated inhibition of complex III activity remains elusive. Hypothesis: The present renewal application logically builds upon these novel and largely published observations to test an exciting hypothesis that WA administration prevents both ER-positive and ER-negative (already shown in the previous funding period) breast cancers in relevant animal models in association with post-translational modifications of tubulin(s) and complex III subunit(s) leading to mitotic arrest and ultimately apoptotic cell death selectively in cancerous cells.
Specific Aims : The proposed research utilizes relevant cellular and in vivo animal models of breast cancer and cutting-edge technologies to: (1) determine the efficacy of dietary WA administration for prevention of ER-positive mammary cancer in a well-established rat model of chemically-induced (N-methyl- N-nitrosourea) breast cancer; (2) study the functional significance of downregulation and post-translational modification of tubulins in mitotic arrest by WA; and (3) determine the molecular mechanism by which WA inhibits complex III activity. Translational Impact: The progress in the previous funding period exceeded our own expectations as evidenced by completion and publication of the proposed work as well as identification of novel mechanistic targets of WA. The translational impact of the studies proposed in this renewal application is ultimately realized by: (a) rational design of a pilot biomarker-driven trial in a neoadjuvant window setting, which is beyond the scope of this application because clinical trial design without a full appreciation of the molecular pharmacology of WA is premature; (b) identification of mechanistic biomarker(s) predictive of WA exposure, and possibly response, which is critical for its clinical development because primary cancer incidence is too demanding of an end point; and (c) identification of a non-toxic regimen for targeting tubulin/microtubule network selectively in cancer cells as currently available anti-mitotics (e.g., taxanes) have side effects. In this era of targeted therapies and personalized medicine, the tubulin/microtubule network still remains an attractive therapeutic target for breast and other cancers.

Public Health Relevance

Breast cancer affects thousands of families worldwide. In the United States alone, nearly 40,000 women succumb to breast cancer every year. Innovative approaches for chemoprevention of breast cancer effective against both hormone-dependent and hormone-independent disease are needed to diminish disease-related cost, morbidity, and mortality associated with this illness. The long-term objective of this research project is to develop a preventive intervention against breast cancer using withaferin A (WA), which is a non-toxic small-molecule derived from a medicinal plant.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA142604-08
Application #
9189592
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Seifried, Harold E
Project Start
2009-12-04
Project End
2019-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
8
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Sehrawat, Anuradha; Kim, Su-Hyeong; Hahm, Eun-Ryeong et al. (2017) Cancer-selective death of human breast cancer cells by leelamine is mediated by bax and bak activation. Mol Carcinog 56:337-348
Samanta, Suman K; Sehrawat, Anuradha; Kim, Su-Hyeong et al. (2017) Disease Subtype-Independent Biomarkers of Breast Cancer Chemoprevention by the Ayurvedic Medicine Phytochemical Withaferin A. J Natl Cancer Inst 109:
Kim, Su-Hyeong; Kaschula, Catherine H; Priedigkeit, Nolan et al. (2016) Forkhead Box Q1 Is a Novel Target of Breast Cancer Stem Cell Inhibition by Diallyl Trisulfide. J Biol Chem 291:13495-508
Palliyaguru, Dushani L; Chartoumpekis, Dionysios V; Wakabayashi, Nobunao et al. (2016) Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms. Free Radic Biol Med 101:116-128
Sehrawat, Anuradha; Roy, Ruchi; Pore, Subrata K et al. (2016) Mitochondrial dysfunction in cancer chemoprevention by phytochemicals from dietary and medicinal plants. Semin Cancer Biol :
Lee, Joomin; Hahm, Eun-Ryeong; Marcus, Adam I et al. (2015) Withaferin A inhibits experimental epithelial-mesenchymal transition in MCF-10A cells and suppresses vimentin protein level in vivo in breast tumors. Mol Carcinog 54:417-29
Antony, Marie L; Lee, Joomin; Hahm, Eun-Ryeong et al. (2014) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ?-tubulin. J Biol Chem 289:1852-65
Sehrawat, Anuradha; Sakao, Kozue; Singh, Shivendra V (2014) Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells. Breast Cancer Res Treat 146:543-55
Hahm, Eun-Ryeong; Lee, Joomin; Singh, Shivendra V (2014) Role of mitogen-activated protein kinases and Mcl-1 in apoptosis induction by withaferin A in human breast cancer cells. Mol Carcinog 53:907-16
Vyas, Avani R; Singh, Shivendra V (2014) Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone. AAPS J 16:1-10

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