Targeting RET in Brain Metastases from Estrogen Receptor Positive Breast Cancer

Abstract

Brain metastases (BrM) occur in 10-15% of patients with estrogen receptor (ER)-positive breast cancer, and remain a significant clinical challenge. While current therapeutic paradigms, including surgical resection and systemic therapy, have efficacy in the management of BrM from ER+BC, median overall survival following the diagnosis of BrM is approximately 18 months. The limited efficacy of current therapies, along with a relative paucity of therapeutic options, highlight the urgent clinical need to identify druggable targets for BrM from ER+BC. We previously identified recurrent overexpression of the receptor tyrosine kinase RET in breast cancer BrM relative to patient-matched primary tumors. The principal ligand for RET, Glial Cell-Derived Neurotrophic Factor (GDNF), is predominately expressed in the brain, where it functions in the maintenance of neurons and multiple glial cell types. Here, we show that increased RET expression and activation correlates with brain metastasis in breast cancer patients. Further, we confirm the role of RET signaling in the promotion of metastatic properties in vitro, including tumor spheroid formation, migration, invasion, and transformation. Using RET-overexpressing and RET-deficient models in four ER+BC cell lines, we demonstrate that RET overexpression enhances activation of RET downstream targets, such as ERK and AKT, and promotes metastatic phenotypes in in vitro, ex vivo and in vivo assays. Importantly, we demonstrate that the phenotypic effects of GDNF-mediated RET signaling can be abrogated by administration of Pralsetinib, a highly potent RET-selective kinase inhibitor. Using an ex vivo mouse brain slice co-culture model, we further demonstrate that RET overexpression in breast cancer cell lines potentiates their colonization and invasion into the brain tissues, which are reduced in RET-knockdown cell lines. RET overexpression facilitates brain colonization of breast cancer cells in vivo and is associated with reduced survival. Using proteomic analyses, we identify novel GDNF-mediated signaling pathways in breast cancer cell line models, including PLC{gamma}, P70S6K, STAT3 and CREB. RET-mediated activation of this range of downstream targets is sensitive to inhibition by Pralsetinib. In sum, this study identifies and characterizes RET as a targetable driver of breast cancer brain metastasis, and further describes the action of Pralsetinib in ER-positive breast cancer cell lines, providing insights to improve current targeted therapies for patients with breast cancer brain metastasis. Bullet pointsO_LIRET expression and activation are increased in breast cancer brain metastases. C_LIO_LIPralsetinib potently inhibits RET activation in RET overexpressing and in non-overexpressing breast cancer cell line models. C_LIO_LIRET overexpression and activation confers pro-metastatic properties including cell proliferation, colonization, migration and invasion in vitro and in ex vivo brain co-culture and enhances brain colonization of breast cancer cells in vivo. C_LIO_LIGDNF increases activation of ERK1/2, AKT, PCL{gamma}, P70S6K, STAT3, and CREB in RET overexpressing MCF-7 cells. C_LIO_LICREB or P70S6K knockdown attenuates GDNF stimulated activation of RET downstream targets and cell migration. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/647085v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1628735org.highwire.dtl.DTLVardef@19ad565org.highwire.dtl.DTLVardef@2fdcc2org.highwire.dtl.DTLVardef@468233_HPS_FORMAT_FIGEXP M_FIG C_FIG