Publication Date

2011-12-05

Availability

Embargoed

Embargo Period

2013-12-04

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2011-09-16

First Committee Member

Joyce M. Slingerland

Second Committee Member

Thomas K. Harris

Third Committee Member

Mark D. Pegram

Fourth Committee Member

Enrique A. Mesri

Fifth Committee Member

Sharon J. Elliot

Abstract

The complex PI3K/mTOR pathway regulates tumor progression via effects on cellular proliferation, apoptosis, autophagy, and motility. New drugs that inhibit the catalytic site of both PI3K and mTOR have shown promise in clinical trials. Here, we report the first use of a novel, dual PI3K/mTOR catalytic site inhibitor (PF-04691502, PF1502) in a xenograft model of breast cancer metastasis to bone. Metastatic MDA-MB-1833 cells showed PI3K/mTOR activation relative to parental MDA-MB-231. Low-dose PF1502 significantly impaired tumor cell motility and invasion in vitro without causing cell cycle arrest, apoptosis, or reduced proliferation. Pre-treatment of tumor cells at this dose reduced bone metastatic outgrowth in vivo. The atypical tumor suppressor, p27KIP1, is phosphorylated in its C-terminal region by multiple AGC kinases downstream of PI3K/mTOR. These phosphorylation events promote cytoplasmic mislocalzation of p27 which, in turn, facilitates inhibition of the RhoA cytoskeletal regulatory protein. The resulting turnover of the actin cytoskeleton is thought to underlie the increased cellular motility attributed to cytoplasmic p27. In MDA-MB-1833 cells, PI3K/mTOR inhibition reduced p27 C-terminal phosphorylation at T157 and T198 and reduced cytoplasmic p27 levels. Overexpression of a p27T157D/T198D phospho-mimetic mutant conferred resistance to the anti-motility effects of PF1502 in vitro. MDA-MB-1833 cells demonstrate p27-dependent inhibition of RhoA-ROCK signaling, as well as p27-dependent motility and invasion in vitro, however, RhoA knockdown did not confer resistance to the anti-motility effects of PF1502. p27shRNA dramatically impaired the bone metastatic outgrowth of MDA-MB-1833 in vivo. In an effort to explore potentially novel RhoA-independent mechanisms whereby cytoplasmic p27 might drive tumor cell motility and metastasis, we turned to the process known as epithelial-to-mesenchymal transition (EMT). The EMT program has been implicated as a critical driver of tumor metastasis in a variety of cancer models. PI3K/mTOR inhibition and shRNA p27 treatment both reversed expression of EMT markers in MDA-MB-1833. Thus, PI3K/mTOR appears to drive p27-dependent motility and metastasis at least in part by induction of an EMT-like phenotype, a novel mechanism through which p27 might act to promote tumor progression. These results provide an important new clinical rationale supporting the use of PI3K/mTOR inhibitors as anticancer agents via their inhibition of tumor invasion and metastasis.

Keywords

p27; metastasis; mTOR/PI3K; cancer

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