Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin β1. Rab11b-mediated control of integrin β1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin β1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.

Manuscript Summary:Rab11b upregulation in the brain microenvironment promotes recycling of cargo proteins required for breast cancer brain metastasis, including increased surface expression of integrin β1, which allows brain extracellular matrix attachment and mechanotransduction. Inhibition of the mevalonate pathway with statins prevents geranylgeranylation of Rab11b, decreasing cargo recycling, and inhibiting brain metastasis. Declaration of Conflict of Interest:The authors declare no competing interests. 6/10/2019Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth -Google Docs https://docs.google.com/document/d/133ky5YwP8HxilKMV1nLO0styFXb_a77AufdN8Q_jytI/edit 2/27 GRAPHICAL ABSTRACT SUMMARYBreast cancer brain metastases (BCBM) have a 520 year latency and account for up to 30% of mortality.Developing new therapeutics requires a molecular understanding of adaptation to the brain microenvironment.Here, we combined RNAsequencing of BCBM development with a reverse genetic screen in Drosophila melanogaster and identified Rab11b, an endosomal recycling protein, as a mediator of metastatic adaptation.We show that disseminated cells upregulate Rab11b early after arrival in the brain, allowing control of the cell surface proteome through recycling of proteins required for successful interaction with the microenvironment, including integrin β1. Rab11bmediated control of integrin β1 surface expression allows ligation to the brain ECM, activating mechanotransduction signaling to allow survival and proliferation. We propose a model in which upregulation of Rab11b allows disseminated cells to recycle needed proteins during metastatic adaptation, without strictly requiring transcription and translation, to allow for metastatic outgrowth. 6/10/2019 Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth -Google Docs https://docs.google.com/document/d/133ky5YwP8HxilKMV1nLO0styFXb_a77AufdN8Q_jytI/edit 3/27 AUTHOR