Abstract P3-05-25: Efficacy of everolimus on multiple mechanisms of AI-resistance in vitro and xenograft, and characterization of their everolimus-resistance
Authors: Kimura Mariko , Fujiki Natsu , Hanamura Toru , Niwa Toshifumi , Yamaguchi Yuri , Ishikawa Takashi , Endo Itaru , Hayashi Shin-ichi 
Published: May 2015

Abstract
Background: mTOR inhibitor, everolimus showed remarkable clinical efficacy and has been considered as a promising agent for breast cancer treatment especially among hormone receptor positive (HR+) postmenopausal advanced breast cancer. Now Affinitor (everolimus) is approved for HR+ breast cancer in Japan. However, definitive biomarker has been unclear so far. In addition, the subsequent treatment after refractory to everolimus has not been defined. In preclinical study, it has been reported there are several resistant models of endocrine therapy and we also reported several types of models established from MCF-7-E10, in which estrogen receptor (ER) activity could be monitored by transfected-ERE-GFP. Those EDR cells are characterized as the followings: 1) Type 1 shows constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 shows low expression of ER (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated (Fujiki, 2014, J Steroid Biochem Mol Biol). 3) Type4, which was cultured under the condition of estrogen depletion and addition of testosterone. It showed using androgen metabolite as ligand and both PI3K/Akt/mTOR and MAPK signaling pathway upregulated (Hanamura, 2013, Breast Cancer Res Tr). Methods: Efficacy of everolimus was analyzed in those cell lines in vitro and in vivo about inhibition of cell proliferation. In vivo study, ovarectomized mice were inoculated with Type1 or Type4. Treatments were performed as single agent letrozole, everolimus, or combination and tumor volume changes were compared. Then everolimus resistant cells were generated from those EDR cells after long term exposure to everolimus in vitro. Though we could not acquire everolimus resistant cell from Type4, Type1 and 2 showed its resistance. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy between AI and everolimus after the first line AI treatment failure. Using those cell lines, we investigated the different mechanism of resistance to everolimus. Results: Everolimus extremely inhibited cell proliferation in each EDR type in vitro and in vivo. Besides, Type4 was more remarkably sensitive than parental MCF-7-E10 under testosterone supplemented condition in vitro. Everolimus or combination treatments reached significant tumor volume reduction around 50 percent after 21 days treatment period, in contrast to increase of tumor volume in placebo or letrozole group. In vitro study of developing resistance to everolimus, Type1-EVR cells no longer responded to everolimus and surprisingly fulvestrant also lost sensitivity to EVR cell in spite of keeping ERE activity. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than parental cell, however, MEK inhibitor or PI3K inhibitor alone was not effective but combination with fulvestrant significantly effective more than parental cell. In type2, JNK inhibitor was effective for parental cell but EVR cell lost sensitivity to JNK inhibitor, however, Src inhibitor was much more effective in Type2-EVR than in parental cell. Conclusion: Everolimus showed remarkable efficacy to any types of EDR cells, however, the mechanism of acquired resistance to everolimus was likely to be different between each EVR cell. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance.
Citation Format: Mariko Kimura, Natsu Fujiki, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Takashi Ishikawa, Itaru Endo, Shin-ichi Hayashi. Efficacy of everolimus on multiple mechanisms of AI-resistance in vitro and xenograft, and characterization of their everolimus-resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-25.