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Oncogenic PI3K/mTOR activation is frequently observed in human cancers and activates cell motility via p27 phosphorylations at T157 and T198. Here we explored the potential for a novel PI3K/mTOR inhibitor to inhibit tumor invasion and metastasis. An MDA-MB-231 breast cancer line variant, MDA-MB-231-1833, with high metastatic bone tropism, was treated with a novel catalytic PI3K/mTOR inhibitor, PF-04691502, at nM doses that did not impair proliferation. Effects on tumor cell motility, invasion, p27 phosphorylation, localization, and bone metastatic outgrowth were assayed. MDA-MB-231-1833 showed increased PI3K/mTOR activation, high levels of cytoplasmic p27pT157pT198 and increased cell motility and invasion in vitro versus parental. PF-04691502 treatment, at a dose that did not affect proliferation, reduced total and cytoplasmic p27, decreased p27pT157pT198 and restored cell motility and invasion to levels seen in MDA-MB-231. p27 knockdown in MDA-MB-231-1833 phenocopied PI3K/mTOR inhibition, whilst overexpression of the phosphomimetic mutant p27T157DT198D caused resistance to the anti-invasive effects of PF-04691502. Pre-treatment of MDA-MB-231-1833 with PF-04691502 significantly impaired metastatic tumor formation in vivo, despite lack of antiproliferative effects in culture and little effect on primary orthotopic tumor growth. A further link between cytoplasmic p27 and metastasis was provided by a study of primary human breast cancers which showed cytoplasmic p27 is associated with increased lymph nodal metastasis and reduced survival. Novel PI3K/mTOR inhibitors may oppose tumor metastasis independent of their growth inhibitory effects, providing a rationale for clinical investigation of PI3K/mTOR inhibitors in settings to prevent micrometastasis. In primary human breast cancers, cytoplasmic p27 is associated with worse outcomes and increased nodal metastasis, and may prove useful as a marker of both PI3K/mTOR activation and PI3K/mTOR inhibitor efficacy.

Antiestrogen therapies arrest susceptible estrogen receptor (ER)-positive breast cancers by increasing p27. Since Src phosphorylates p27 to promote p27 proteolysis, Src activation observed in up to 40% of ER-positive cancers may contribute to antiestrogen resistance. In this article, we show that treatment with the Src-inhibitor saracatinib (AZD0530) together with ER-blocking drugs increased breast cancer cell cycle arrest via p27. Saracatinib and fulvestrant together more effectively increased p27, reduced Ki67, and impaired MDA-MB-361 xenograft tumor growth in vivo than either of the drugs alone. In contrast, saracatinib monotherapy rapidly gave rise to drug resistance. Since combined ER and Src inhibition delays development of resistance in vivo, these data support further clinical investigation of saracatinib in combination with fulvestrant for women with ER-positive breast cancer. Proteomic analysis revealed striking bypass activation of the mTOR pathway in saracatinib-resistant tumors. mTORC1 activation also arose following long-term culture of ER-positive breast cancer lines in the presence of saracatinib. These data indicate the utility of proteomic analysis of drug-resistant tumors to identify potential means of drug resistance. The use of mTOR kinase inhibitors with saracatinib may subvert drug resistance and prove to be more effective than saracatinib alone.