Date of Graduation
5-2012
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Khandan Keyomarsi, Ph.D.
Committee Member
Michelle Barton, Ph.D.
Committee Member
Mong-Hong Lee, Ph.D.
Committee Member
Sendurai Mani, Ph.D.
Committee Member
Frank Marini, Ph.D.
Abstract
Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that
mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in
breast cancer generates two low molecular weight (LMW) isoforms that exhibit both
enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress
LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E
disrupts normal mammary acinar morphogenesis and serves as the initial route into
breast tumor development. We first demonstrate that LMW-E overexpression in
non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice
significantly more than overexpression of full-length cyclin E and requires CDK2-
associated kinase activity. Further in vivo passaging of these tumors augments
LMW-E expression and tumorigenic potential. When subjected to acinar
morphogenesis in vitro, LMW-E mediates significant morphological disruption by
generating hyperproliferative and multi-acinar complexes. Proteomic analysis of
patient tissues and tumor cells with high LMW-E expression reveals that the
activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E
expression predicts poor patient survival. Combination treatment using roscovitine
(CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor)
effectively prevented aberrant acinar formation in LMW-E-expressing cells by
inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells
exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness.
These tumor cells also enrich for cells with CSC phenotypes such as increased
CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of
ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo
population also shows positive correlation with LMW-E expression in both the tumor
cell line model and breast cancer patient samples (p<0.0001 & p=0.0435,
respectively). Combination treatment using doxorubicin and salinomycin
demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in
those with full-length cyclin E expression. Finally, ProtoArray microarray identifies
Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression
results in enrichment for CSCs. Collectively, these data emphasize the strong
oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible
therapeutic strategies to treat breast cancer patients with high LMW-E expression.
Keywords
cyclin E, breast cancer, acinar morphogenesis, roscovitine, b-Raf-ERK1/2-mTOR pathway, cancer stem cells, epithelial-mesenchymal transition, salinomycin, Hbo1