The Ret receptor tyrosine kinase is overexpressed in approximately 30% of human breast cancers, many of which are estrogen receptor-alpha positive (ER+). Using the ER+ breast cancer models MCF7 and T47D, we have shown that RET is an ER target gene and that Ret signaling enhances estrogen-driven anchorage-dependent and -independent proliferation (Boulay et al 2008). In our current work we are investigating the potential role of Ret in endocrine therapy response using in vivo and in vitro models. Ret is activated by peptides of the glial derived neurotrophic factor (GDNF) family. GDNF treatment of ER+ T47D and MCF7 breast cancer cell lines stimulates activation of multiple intracellular pathways, increases anchorage-dependent and -independent growth, as well as migration. To test if Ret activation impacts on endocrine therapy response, we treated MCF7/Aromatase (Aro)-expressing cells for seven days with different endocrine agents in the presence or absence of GDNF to activate Ret. Activation of Ret signaling rescued the proliferative block that resulted from treatment of the MCF7/Aro cells with tamoxifen, fulvestrant or letrozole. A transcriptome analysis was performed and an Ingenuity Pathway Analysis of the transcripts altered in MCF7/Aro cells treated with fulvestrant or letrozole revealed that the highest pathways altered by addition of GDNF are related to interferon signaling and inflammation. Indeed, proinflammatory RNAs, including IL6, IL8 and CXCL10 are among the top transcripts upregulated in cultures treated with fulvestrant plus GDNF, or letrozole plus GDNF. Elevated IL6 serum levels in breast cancer patients correlate with poor prognosis. We have concentrated on IL6 in the work that will be presented based on this clinical data, as well as our finding that IL6 stimulates Ret expression and activation in ER+ breast cancer models. We verified that addition of GDNF to MDCF/Aro cultures treated with fulvestrant or with letrozole led to an increase of IL6 in the conditional medium of the cultures. Moreover, addition of GDNF to fulvestrant- or tamoxifen-treated tumor cells increased migration, which could be inhibited by an IL6 blocking antibody, showing the importance of this cytokine for tumor cell motility. Additional data will be presented showing there is a Ret-IL6 feed-forward loop present in ER+ tumor cell models and that this is important for migration. In order to examine the role of Ret in vivo, in a metastatic breast cancer model, we have used the ER+/Ret+ J110 tumor cell line, generated from mammary tumors of MMTV-AIB1 transgenic females. Ret is active in J110 mammary tumors and its activity can be blocked with a Ret selective kinase inhibitor. In vivo data will be presented showing the effect of blocking Ret alone, or in combination with endocrine agents, on metastatic spread of primary tumors to the lungs.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-06.