Date of Graduation

5-2015

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Khandan Keyomarsi, Ph.D.

Committee Member

Jill Schumacher, Ph.D.

Committee Member

Wei Zhang, Ph.D.

Committee Member

Jeffrey Frost, Ph.D.

Committee Member

Zhimin Lu, Ph.D.

Abstract

Cyclin E is altered or overexpressed in approximately one-third of tumors from patients with invasive breast cancer and is a powerful independent predictor for survival in women with stage I-III breast cancer. Full-length cyclin E (EL) is post-translationally cleaved into two low-molecular-weight isoforms, LMW-E (T1) and LMW-E (T2). LMW-E have been shown to exhibit greater binding affinity for cyclin-dependent kinase 2 (CDK2) , cyclin dependent kinase inhibitors (CKIs), p21 and p27, but are resistant to p21 and p27 inhibition. In addition, transgenic mice expressing LMW-E have increased mammary tumor development and metastasis compared to EL transgenic mice. Therefore, LMW-E are more aggressive in cell cycle abrogation and mammary tumor development. The LMW-E isoforms are tumor specific and accumulate in the cytoplasm due to lack of a nuclear localization sequence (NLS). Therefore, we hypothesized that aberrant localization of LMW-E isoforms leads to molecular events that ultimately contribute to LMW-E breast cancer tumorigenicity. To address this hypothesis, we used a retrovirus-based protein complementation assay (RePCA) to identify LMW-E (T1) protein-protein interactions in breast cancer. Using this methodology, we found ATP-citrate lyase (ACLY) as a novel interacting protein of LMW-E (T1) in the cytoplasm. ACLY is a 125kDa homotetrameric enzyme that catalyzes cytoplasmic citrate to acetyl-CoA and oxaloacetate in the de novo lipogenesis pathway. End products of this pathway consist of complex fatty acids that fuel membrane production of highly proliferating cells and lipid-based post-translational modifications that mediate protein-protein interactions. Additionally, we found that LMW-E upregulates ACLY enzymatic activity which leads to lipid droplet formation; thereby providing cells with essential building blocks to support growth. ACLY is also required for LMW-E mediated transformation, migration and invasion in vitro, as well as tumor growth in vivo. Taken together these data suggest a novel interplay between LMW-E and ACLY and how metabolic pathways and the cell cycle are linked in breast cancer tumorigenesis.

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