Abstract P5-05-04: Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer
Authors: O'Brien Neil A , Conklin Dylan , Ching Michaela , Tong Luo , Ayala Raul , Issakhanian Shawnt , Hurvitz Sara A , di Tomaso Emmanuelle , Linnartz Ronald , Finn Richard S , Hirawat Samit , Slamon Dennis J 
Published: May 2015

Abstract
Background: The addition of the selective mTORC1 inhibitor everolimus (RAD001/Afinitor®), to exemestane significantly prolongs progression free survival in patients with advanced hormone receptor (ER+) positive breast cancer. However, despite these improved outcomes, the majority of patients that achieve an initial clinical benefit will eventually go on to develop progressive disease. In order to prevent or delay the onset of this acquired resistance, it is essential to identify the mechanisms by which continued proliferation is being driven in these resistant tumors. In this study, we used reverse phase protein array (RPPA) technology to compare cell signaling in ER+ breast cancer cell line xenografts that were responsive to everolimus versus those that had acquired resistance to everolimus.
Materials and Methods: Xenograft models were established from three ER+ breast cancer cell lines containing different molecular alterations that confer activated PI3K/mTOR signaling; MCF7 and KPL-1 (both PIK3CA mutant) and ZR75-1 (PTEN-null). Mice were treated daily with either vehicle or 10 mg/kg everolimus until progression. Snap frozen tissue samples (n = 3) were collected from vehicle control tumors, from everolimus resistant tumors and from everolimus responsive tumors. Whole cell lysates were prepared from tumor tissues and processed for RPPA analysis. A customized panel of 27 known cancer/PI3K pathway associated signaling proteins were selected for the RPPA analysis.
Results: In the vehicle controls, significantly higher levels of pAKT, pPRAS40 and p4EBP1 were detected in the PTEN-null ZR75-1 tumors compared to the PIK3CA mutant tumors, indicating potential variations in intensity of PI3K/AKT pathway signaling depending on the initiating molecular alteration. Complete inhibition of tumor proliferation was observed in response to everolimus in all 3 xenograft models for up to 7-weeks of treatment, until tumors showed signs of progression. Significantly lower S6 phosphorylation was observed in tumors responding to everolimus, which was accompanied by feedback activation of pAKT. Significant inhibition of S6 phosphorylation was also observed in each of the ZR75-1 and MCF7 tumors that were progressing on everolimus, indicating that although the tumor cells have acquired resistance to the anti-proliferative activity of everolimus, mTORC1 signaling is still being successfully blocked. Feedback activation of pAKT was observed in the ZR75-1 resistant model, however no feedback activation of AKT occurred in the MCF7 resistant model. The KPL-1 model showed the expected loss of S6 pathway inhibition upon resistance. No significant signaling changes were observed in any of the other signaling proteins measured, including ER, HER2, EGFR, MEK and ERK. Ongoing studies to measure the mRNA expression changes in these tumors using mRNA microarrays may identify signaling pathways that are driving the proliferation of these resistant tumors.
Discussion: These preclinical data show that multiple pathways of resistance can develop in response to long-term everolimus treatment and resistance can occur despite continued inhibition of PI3K/mTOR signaling. Data from mRNA microarray experiments will be presented at the time of the meeting.
Citation Format: Neil A O'Brien, Dylan Conklin, Michaela Ching, Luo Tong, Raul Ayala, Shawnt Issakhanian, Sara A Hurvitz, Emmanuelle di Tomaso, Ronald Linnartz, Richard S Finn, Samit Hirawat, Dennis J Slamon. Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer [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 P5-05-04.