Abstract 1972: Silencing kinase interacting stathmin gene enhances erlotinib sensitivity by inhibiting serine 10 p27 phosphorylation in EGFR-expressing breast cancer
Authors: Dongwei Zhang, Ana M. Tari, Ugur Akar, Banu K. Arun, Tiffany A. LaFortune, Rene Nieves-Alicea, Gabriel N. Hortobagyi, Naoto T. Ueno
Journal: Cancer Research (2010)

Abstract The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, have been approved for lung cancer treatment but have induced limited clinical responses in breast cancer patients even though EGFR is frequently overexpressed in breast cancer. The purpose of this study was to evaluate whether erlotinib sensitivity in EGFR-expressing breast cancer is affected by phosphorylation of p27Kip1 (p27) and depletion of kinase interacting stathmin (KIS), a nuclear protein that phosphorylates p27. We previously reported that the cytoplasmic localization of p27 is associated with erlotinib resistance. In the present study, we tested the expression levels of phosphorylated p27 after erlotinib treatment in EGFR-expressing breast cancer cell lines such as BT-474 and SK-BR-3. Expression of p27 phosphorylated at serine (S) 10 was significantly increased in response to erlotinib. We then transfected phosphoinhibitory S10A-p27 mutant or phosphomimetic S10D-p27 mutant into those cells and found that S10A-p27-transfected cells were more sensitive to erlotinib and S10D-p27-transfected cells were more resistant to erlotinib. KIS binds the C-terminal domain of p27 and phosphorylates it at S10 in vitro and in vivo, resulting in export of p27 from the nucleus to the cytoplasm. To study the role of KIS in erlotinib sensitivity, we tested the basal expression level of KIS in a panel of breast cancer cell lines and found that KIS expression level is low in erlotinib-sensitive cells and high in erlotinib-resistant cells, suggesting that KIS expression level is associated with erlotinib sensitivity. To test whether inhibition of S10-phosphorylated p27 enhances erlotinib's antiproliferative effect, we performed KIS siRNA knockdown in BT-474 and SK-BR-3 cells. KIS siRNA knockdown led to p27 nucleus accumulation and enhanced erlotinib-induced G1 arrest. Furthermore, KIS depletion enhanced the inhibitory effects of erlotinib on proliferation and anchorage-independent growth in breast cancer cells. Most importantly, in an orthotopic breast cancer xenograft model, KIS gene silencing by KIS siRNA intravenous injection led to p27 nuclear accumulation and enhanced the antitumor activity of erlotinib. Further, KIS depletion enhanced erlotinib sensitivity in erlotinib-resistant EGFR-expressing triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468. These results demonstrate that erlotinib-mediated cytotoxicity is regulated by phosphorylation of p27 at S10 and that inhibiting S10 p27 phosphorylation by KIS gene silencing enhances erlotinib sensitivity. Our study provides a rationale for developing combinations of erlotinib with KIS inhibition to overcome EGFR TKI resistance in EGFR-expressing breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1972.