Background: Brain metastasis (BM) develops in ~10-30% of breast cancer (BC) patients; triple negative breast cancer (TNBC), the most aggressive subtype of BC, exhibits the highest incidence of BM. Treatment options for BCBM are extremely limited due to the poor biological understanding. Hence, there is an urgent need to elucidate molecular mechanisms driving BCBM. We aim to study BCBM pathogenesis by dissecting the role of tumorigenic fucosylated proteins secreted by BM-associated fibroblasts (bmCAFs). Methods: We assessed global fucosylation (post-translational protein modification by L-fucose) patterns by fucose-binding lectin pulldown and immunoblot (IB) analysis. Conditioned media (CM) derived from CAFs that were depleted or not of fucosylated proteins were used to treat BC cells to assess motility and invasion. Fuco-proteomics and phosphoproteomic profiling identified bmCAF-secreted fucosylated (sf) proteins in bmCAF-derived CM and associated global signaling changes induced in BC cells, respectively. qRT-PCR was performed to analyze FUT11 levels under normoxia and hypoxia. Immunofluorescence and IB analyses of BC cells treated ± with bmCAF-derived CM was performed to validate PVR downstream signaling changes. Stereotactic intracranial implantation of BC cells alone or with ctrl/PVR knocked-down- bmCAFs was used for the BCBM mouse model. Results: We discovered that fucosylated proteins secreted uniquely by bmCAFs but not by normal-breast fibroblasts (NBF) or primary tumor-CAFs (tCAF), potently drives BC proliferation and invasiveness. Fucosylated proteomic profiling of the bmCAF secretome identified a soluble Polio Virus Receptor (PVR) isoform as uniquely upregulated, fucosylated, and secreted by bmCAFs. Of the 13 fucosyltransferases (FUTs), we found that bmCAFs upregulate FUT11, a key hypoxia-related gene. FUT11 and secreted fucosylated PVR (sfPVR) are significantly increased in bmCAFs exposed to hypoxia, consistent with the hypoxic environment of the brain. PVR can exist as transmembrane and secreted isoforms. Whereas transmembrane PVR is known to contribute to poor prognosis in a number of cancers by facilitating tumorigenic cell:matrix interactions and attenuating anti-tumor immunity, roles of secreted fucosylated PVR (sfPVR) in cancer are unknown. Our phosphoproteomics analyses of BC cells identified cellular adhesion/cytoskeletal and EPHA2 signaling as significantly modulated by bmCAF sfPVR to drive BC cell motility and invasiveness. Indeed, PVR knockdown abrogates the ability of bmCAFs to enhance BC growth/spread in the brain in a mouse model. Conclusion: Our data demonstrate that pathological hypoxia drives FUT11-mediated fucosylation and secretion of PVR by bmCAFs, which potently drives BC cells motility and invasiveness, representing a mechanism by which bmCAFs can promote BCBM. We expect our ongoing and further studies to advance our understanding of how sfPVR from bmCAFs promotes BCBM and to establish a basis for therapeutic targeting of PVR and/or use of fucosylated PVR and its signaling effectors as biomarkers.

Citation Format: Emma Adhikari, Qian Liu, Viktoriya Marusyk, Victoria Lzumi, John M. Koomen, Andriy Marusyk, Eric Lau. Hypoxia-induced secretion of fucosylated PVR/CD155 from brain met-associated fibroblasts drives breast cancer invasive capacity by altering cell-cell contacts & focal adhesion [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B003.